Ligand-Based Fluorine NMR Screening: Principles and Applications in

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Ligand-based Fluorine NMR Screening: Principles and Applications in Drug Discovery Projects Claudio Dalvit, and Anna Vulpetti J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01210 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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Journal of Medicinal Chemistry

Ligand-based Fluorine NMR Screening: Principles and Applications in Drug Discovery Projects

Claudio Dalvit*1 and Anna Vulpetti*2

Corresponding authors:

1

C. Dalvit

Lavis (Trento) Italy E-mail: [email protected] 2

A. Vulpetti

Novartis Institutes for Biomedical Research, Global Discovery Chemistry, 4002 Basel, Switzerland E-mail: [email protected] 1 ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Ligand-based fluorine NMR screening has gained popularity in drug discovery projects during the past decade and has become a powerful methodology to produce high quality hits. Its high sensitivity to protein binding makes it particularly suitable for fragment screening allowing detection and binding strength measurement of very weak affinity ligands. The screening can be performed in direct or competition format and its versatility allows application to complex biological and chemical systems. As the potential of the methodology has now been recognized and successfully demonstrated in several relevant medicinal chemistry projects, is now an appropriate time to report the learned lessons and point the way to the future. In this perspective the principles of the methodology along with several applications to pharmaceutical projects are presented.

INTRODUCTION Fluorine substituents are very popular in the pharmaceutical industry and approximately 20% of all drugs sold on the market contain at least one fluorine atom. It is frequently introduced in a lead molecule for improving its physico-chemical and adsorption, distribution, metabolism, and excretion properties.1-13 This has been made possible by the advancement of new, selective and safe fluorinating reagents and novel chemical and enzymatic reactions14-24 which allow the synthesis of new fluorinated structural motifs that are capable to emulate many functionalities (bioisosteres).25 Another useful application of fluorine in drug discovery projects, which recently emerged, is in combination with NMR screening.26-32 These approaches have now been recognized as powerful tools in the lead identification and optimization phases of drug discovery projects. Several 2 ACS Paragon Plus Environment

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academic laboratories and pharmaceutical companies have invested in them and successfully applied to different relevant pharmaceutical projects. Over the years the techniques have been optimized and important technological advancements have improved their sensitivity and throughput. The methods are divided in protein-, substrate/cofactor- and ligand-based fluorine NMR screening. The protein observed technique, also known as Protein-Observed Fluorine NMR (PrOF NMR),33-36 is a binding assay and requires the selective labelling of the biological target with fluorinated amino acids.33-45 Molecules are screened with PrOF and the fluorine chemical shifts ( 19F) of the fluorinated protein amino acids are monitored. In the presence of a binding event changes in  19F are observed. Titration experiments performed at different concentrations of the identified binder allows the measurement of the dissociation binding constant (KD) for weak affinity ligands.46 Similar screening experiments can be performed with 19F labelled RNA or DNA (see for example references47-51 and references cited in). The substrate/cofactor observed technique, also known as n-Fluorine Atoms for Biochemical Screening (n-FABS),52-57 is a functional assay and requires the selective labelling of the cofactor or substrate of an enzymatic reaction with a fluorinated group.58-62 The principles and several applications of the n-FABS to simple and complex biological and chemical systems have been described in a comprehensive review article.63 Recently, the versatility of the n-FABS has allowed its application to the screening and IC50 measurements in intact human living cells.64 The ligand observed technique, also known as Fluorine chemical shift Anisotropy and eXchange for Screening (FAXS), is a binding assay.65, 66 The screening can be performed in direct65-67 or competition mode65, 66 and allows the measurement of the binding affinity of the identified hits. Over the last decade it has become a proven method to generate bona fide hits. While its inherent sensitivity is lower when compared to other biophysical methods, its high relative sensitivity to protein binding makes it

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particularly suitable for fragment screening allowing identification of very low binding affinity ligands. The two mechanisms accountable for the great protein binding sensitivity are the large 19F chemical shift anisotropy (CSA) and the large exchange term deriving from the different

19F

isotropic chemical shifts for the free and bound state δf and δb, respectively. The principles of this powerful and versatile binding assay along with selected reported applications in pharmaceutical projects are presented and discussed. The detailed NMR theory of the FAXS experiments along with NMR technical aspects are intentionally reported in the supporting information for facilitating the reading of the article to a broad audience.

THEORY Fluorine NMR spectroscopy is a powerful tool to screen small molecules against a biological target. The intrinsic sensitivity of fluorine NMR fluorine is comparable to proton sensitivity (83% of proton sensitivity) and the NMR active isotope, 19F, is present at 100% natural abundance.68-70 The

19F

NMR-based screening is performed in the buffered solution most appropriate for the

protein stability and solubility. This is possible because buffers, or detergents do not contain 19F NMR signals and thus do not interfere with the 19F NMR spectral acquisition. The large spreading in 19F chemical shift70 reduces the signal overlap problem thus allowing the assay of large chemical mixtures and automated analysis of the spectra.71 The paramagnetic contribution to the chemical shift is mainly responsible for the large chemical shift dispersion and it originates from the availability of energetically low-lying p atomic orbitals. Different measured

19F

NMR parameters can be used for the binding screening: (1) transverse

relaxation, (2) isotropic chemical shift, (3) Double-Quantum (DQ) coherence relaxation, (4) LongLived State (LLS) relaxation, (5) longitudinal relaxation, (6) 1H19F Saturation Transfer


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Difference (STD) and (7) translational diffusion. Another experiment with 19F detection, but that reports indirectly the 1H 1H STD effect, has also been reported in the literature.72, 73 The dynamic range, defined as the ratio of the observable parameter in the presence and absence of protein, for (5) to (7) is small thus requiring a larger amount of protein for the screening.72, 74, 75 The most sensitive observable parameters to protein binding are (1) to (4). However, the measured parameters (3) and (4) are limited to molecules containing two fluorine atoms which are scalar coupled through bond or through space. Therefore, their application is most suitable in the competition format of the assay.76,

77

The

19F

LLS experiments can display, in appropriate

experimental conditions, a very large dynamic range.77 An application of the relaxation of 19F DQ coherence and translational diffusion to the screening against the serine protease trypsin is reported in Figure S1 and S2, respectively. The principle of the FAXS that uses (1) and (2) parameters, performed in direct mode, is represented in the schematic diagram of Figure 1(left).



Direct Binding

F

F

Mol S

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Competition Binding

Mol S

F

Mol S

Ligand

Receptor

Receptor

Mol C

Mol C

F

F Mol S

Mol C

Mol C Mol S + Receptor

- Receptor

Mol S

Mol C Mol S

+ Receptor + Ligand

+ Receptor

 19F

Mol C

 19F

Figure 1. Schematic diagram of the FAXS experiment performed in direct (left) and competition (right) format. The 19F NMR signal of the ligand S becomes broad and sometimes undergoes a shift in frequency in the presence of the receptor while the signal of molecule C that does not bind to the receptor remains sharp and does not change its chemical shift (left). Molecules S and C can then be used as spy and control molecule, respectively, in the competition format of the experiment (right). The broad signal of the spy (reporter) molecule S becomes sharp and returns to the chemical shift of the free state due to its displacement from the receptor in the presence of a competitive ligand.


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The fluorinated molecule C which does not bind to the receptor displays a sharp 19F signal in the absence and presence of the biological target that does not change in intensity and chemical shift. The fluorinated molecule S, which is a binder to the receptor, displays a

19F

signal that in the

presence of the protein becomes broader or, in other words, its transverse relaxation rate (R2) becomes larger. In addition to the signal broadening, a change in the

19F

chemical shift can

sometimes be observed. The high relative sensitivity of the method to binding (see Annex S3 in Supporting Information) makes it particularly suitable for fragment screening in Fragment Based Drug Discovery (FBDD) projects. In this approach, libraries of low molecular weight chemical fragments are initially screened against the target of interest. These fragments are then chemically combined or grown resulting into potent ligands.78-82 Fragments, due to their small size, interact only very weakly with the receptor and typically have a short residence time (res) on the biological target. Molecule S, identified in the FAXS experiment in direct format, or another fluorinated ligand identified with other biophysical techniques, can be used in the FAXS performed in competition mode for screening single molecules or mixtures of molecules containing or not fluorine as shown in the schematic diagram of Figure 1(right). Molecule C can also be used in this assay as an internal reference. The 19F signal intensity and isotropic chemical shift of the molecule S are partially or fully restored in the presence of a binder due to displacement of molecule S from the protein through a competition mechanism or, sometimes, through an allosteric mechanism.

FAXS IN DIRECT MODE It is often stated in books and review articles on FBDD that fragments need to be highly soluble in water because they have to be tested at high concentration i.e. close to their KD for the 7 ACS Paragon Plus Environment


detection.83, 84 Although this statement is most of the time correct, it does not apply to the FAXS experiments performed in the direct mode. According to the equation (2) reported in Annex S3, the sensitivity to binding of the FAXS in the direct mode is directly proportional to the fraction of bound ligand pb = [EL]/[LT] (with

[EL] and [LT] the protein bound and total fragment

concentration, respectively) that is given by the equation: [𝐸𝐿]

[𝐿𝑇]

=

[𝐸𝑇] + [𝐿𝑇] + 𝐾𝐷 ― ([𝐸𝑇] + [𝐿𝑇] + 𝐾𝐷)2 ― 4[𝐸𝑇][𝐿𝑇] 2[𝐿𝑇] (1)

with [ET] the total protein concentration. This is different with respect to most of the biophysical techniques such as for example Surface Plasmon Resonance (SPR), Fluorescence Polarization (FP), Fluorescence Lifetime (FLT), Microscale Thermophoresis (MST), Isothermal Titration Calorimetry (ITC), Thermal Shift Assay, protein observed NMR for which the sensitivity to protein binding is directly proportional to the fraction of bound protein i.e. [EL]/[ET].85 The fraction of bound protein is given by the equation: [𝐸𝐿]

[𝐸𝑇]

=

[𝐸𝑇] + [𝐿𝑇] + 𝐾𝐷 ― ([𝐸𝑇] + [𝐿𝑇] + 𝐾𝐷)2 ― 4[𝐸𝑇][𝐿𝑇] 2[𝐸𝑇] (2)

Simulations of the behaviour of the fractions of bound protein and bound ligand vs. the concentration of the tested fragments are reported in Figure 2 (left and right, respectively). 8 ACS Paragon Plus Environment

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1.0 0.0020

0.015

0.0018

[EL]/[LT]

0.8

0.0016 0.0014

0.6

[EL]/[LT]

[EL]/[ET]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4

0.010

0.0012 100

200

300

400

500

600

[LT] in M

0.005

0.2 0.0 10 µM

200

400

600

800

0.000

1000

[LT] in M

10 µM

200

400

600

800

1000

[LT] in M

Figure 2. Plot of the fraction of bound protein (left) and bound ligand (right) as a function of the concentration of the tested fragment [LT] (from 10 to 1010 µM) and for four values of dissociation binding constant KD (values reported close to the respective functions). The protein concentration is 2 µM. The insert (right) represents the vertically zoomed region with the KD value of 1 mM.

The simulations were performed for four different values of dissociation binding constants ranging from 100 to 1000 µM which are typically values found for small molecules interacting with the receptor. As it can be appreciated from the plots of Figure 2, the two fractions have opposite behavior. The fraction of bound protein [EL]/[ET] increases by testing the fragments at high concentration.85 On the contrary, the fraction of bound ligand [EL]/[LT] increases by screening the fragments at low concentration. The range of displayed [LT] is from 10 to 1010 µM which is typically used in fragment screening. At very low [LT] concentration (not displayed on Figure 2 9 ACS Paragon Plus Environment


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right) a plateau with the maximum value of fraction of bound ligand corresponding to [ET]/KD is reached.85 Therefore the FAXS experiments have to be performed by using a low concentration of the tested molecules for maximizing the value [EL]/[LT]. Despite the small fraction of bound ligand achievable, as shown on the Y axis of the graph of Figure 2(right), FAXS is able to detect these binders. For the detection of very low binding affinity ligands, the protein concentration can also be augmented for increasing the fraction of bound fragment.85 The fragment concentration selected for the screening is determined by the use of 5 or 3 mm NMR tubes and by the sensitivity of the instrument. With the advent of the cryoprobe technology for

19F

detection86, 87 it is now

possible to use a concentration in the low µM (10-20 µM for CF3 and 30-50 µM for CF containing molecules) using 3 mm NMR tubes. In addition to the increased sensitivity to protein binding, the use of low fragment concentration has several other advantages: i) Screening of fragments with limited solubility. ii) Avoiding the formation of aggregates which often are the cause of false positives. iii) Testing of large fluorinated mixtures (e.g. > 30 fluorinated fragments in each mixture) thus increasing the throughput and decreasing the protein consumption. iv) Simultaneous identification of various fragments present in the same mixture and binding to the same pocket on the receptor. This last point has been demonstrated theoretically and experimentally.88 The 19F NMR experiments are recorded without (or with a very short transverse relaxation (T2) filter e.g. 3-4 ms for achieving a flat spectral baseline) and with a T2 filter before the acquisition period. The T2 filter is used to enhance the sensitivity of the method to protein binding. It can be achieved using a Carr Purcell Meiboom Gill (CPMG) pulse sequence,89,

90

a CPMG with the

perfect-echo scheme91-93 or using a spin-echo with a broadband adiabatic 180o refocusing pulse without phase distortion for covering a broad spectral bandwidth which is often encountered with the screening of fluorinated fragments with different fingerprints.74, 76, 94-96 In our experience this 10 ACS Paragon Plus Environment

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last pulse sequence is robust and easy to set up. The CPMG sequence is typically performed using a long period (40-80 ms) between the train of 180o pulses for benefiting from the exchange contribution between the free and bound state to the observed transverse relaxation rate (R2,obs).26 However, there are special cases where the period is kept very short.74 In addition, this period can be varied in order to extract, in suitable cases, the residence time τres of the fragment on the receptor. The different pulse sequences that can be used for recording the spectra are reported in Figure S4. Several initial steps are required for the proper set-up of the methodology. We describe here the procedure based on our experience that we have followed in our laboratories.

FLUORINATED LIBRARY DESIGN The

19F

NMR ligand-based screening approach requires the design of a library of fluorinated

fragments. While fluorine is the most abundant halogen and the thirteenth most abundant element in the earth’s crust, fluorinated secondary metabolites are extremely rare.97 Indeed, until now, only about a dozen of naturally occurring fluorinated organic compounds are known. However, due to the popularity of fluorine in the pharmaceutical, agro and material science areas there are now millions of fluorinated molecules or building blocks which are available for generating libraries of fluorinated molecules. Different criteria can be used for generating such libraries27 as shown in Figure 3.



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Available collections Diversity Oriented Synthesis (DOS)

In-house collection Commercial collections

Scaffolds with F for parallel chemistry Scaffolds without F decorated with fluorinated BB

Natural/drug like inspired Fn

3D shaped fragments Fn

Electrochemistry/ photochemistry Dynamic combinatorial chemistry

Natural product like

Fn

Fragmentation of drugs

Fn Fn Fn

Hypothesis driven

Peptide chemistry

Target-based selection

Fluorinated peptides or peptidomimetics

Family-based selection

Figure 3. Different sources and approaches for the generation of the 19F NMR fragment library.

Some of the approaches described in Figure 3 are discussed in detail below. Fragments containing a single CF3 or CF or CF2 (with equivalent fluorine atoms) are ideal for the 19F

NMR fragment library. The 19F NMR spectra display one singlet resonance for each molecule

with no signal intensity reduction as instead is often observed for poly-fluorinated fragments. However, poly-fluorinated fragments are also included in the library.76 Often, the 19F signals of these molecules are doublets or of higher multiplicity due to the splitting resulting from the homonuclear through bond or through space coupling constants JFF. These molecules can be screened either using the 19F DQ98 (see Figure S1) or LLS77 or the so-called CPMG with perfect echo93 (see Figure S4) experiments. Therefore, it is preferable to put all the poly-fluorinated fragments in separated mixtures for screening them with these pulse sequences.


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In addition to the selection of the type of fluorine tag (CF/CF2/CF3), the physico-chemical properties, the tractability of the fragments for follow-up medicinal chemistry activities and the evaluation of molecular diversity are additional considerations that are taken into account in the library design workflow. The molecular weight and clogP criteria, as part of the popular ‘rule of three’ (MW ≤300 Da; clogP ≤3)99 are widely used in the fragment library design. However, in this context we have extended the MW to 350 for the CF3 containing fragments due to the large weight of the constant CF3 group (69 Da). Molecules containing highly chemically reactive functional groups and aggregating molecules should be removed during the library design workflow by using in silico filters100, 101 and during the extensive quality control process. LEF Descriptor. The fragments containing different fluorine motifs can be chosen from proprietary, commercial collections or ad hoc synthesized as shown in Figure 3. Various descriptors and metrics can be used to ensure maximal global chemical diversity102-105 during the fragments selection process. Descriptors can be 2D or also 3D to include differently shaped fragments.106 In addition, we have proposed a novel local fingerprints descriptor to better characterize the chemical topologies around the fluorinated motif and have them all represented in the 19F NMR fragment library. We called this novel local fingerprints descriptor concept LEF, which stands for Local Environment of Fluorine.88, 107 These fluorine environment fingerprints are based on the topological torsion descriptors of Nilakantan et al.108 The fluorine environment fingerprints include paths consisting of between one and five bonds and only paths starting from the fluorinated motif (i.e., the fluorine atom or CF3 group). We favor the idea of incorporating in the library all the diverse fluorinated motifs showing different LEF and a large spread in 19F NMR chemical shifts. This is supported by the observation that the



dual character of fluorine, i.e. its ability to act as an hydrophobic moiety or as a weak hydrogen bond acceptor, can be related to its 19F NMR chemical shift.109 In-house collection. A database of fluorinated fragments can be compiled from the large in-house pharma compounds collection. This constitutes the “fluorine fragment space” from which are selected those to include in the

19F

NMR fragment library. The availability of the fragments in

sufficient amount in powder (e.g. ≥ 20 mg) is another important requirement to avoid rapid depletion and allow multiple screening and additional characterizations. Commercial collections. Due to the increased interest in this methodology nowadays there are several commercial vendors selling fluorinated fragments libraries. Table 1 reports a list of these vendors. The size of these libraries ranges from few hundreds to few thousands fluorinated fragments. Vendor

Web site

Charles River

https://www.criver.com

Enamine

https://www.enamine.net

Key Organics

https://www.keyorganics.net

Life Chemicals

http://www.lifechemicals.com

Maybridge

https://www.maybridge.com

Otava Chemicals

http://www.otavachemicals.com

FCH

http://fchgroup.net/fragment-libraries.php

Table 1. Major vendors selling fluorinated fragment libraries.

Although the fluorinated compounds have been well characterized and are pure we suggest to perform also on these purchased molecules the NMR-based quality control experiments in aqueous


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solution described below. The fluorinated molecule in water could not fulfill one or more of the requirements needed for the screening. Diversity-Oriented Synthesis (DOS). The synthesis of ad hoc designed fluorinated fragments is mostly valuable for enhancing the fragment library with scaffolds that can be effectively expanded by chemical follow-up. We have reported the incorporation of small diverse designed libraries constructed from chosen fluorinated scaffolds or from non fluorinated scaffolds decorated with fluorinated substituents as a useful strategy to augment the fragment library built from purchased or in-house selected fragments.27 In addition, the synthesis of ad hoc specific fluorinated molecules enables the generation of fluorinated fragments that are not sufficiently represented in the available proprietary/commercial “fluorine fragment space”. An analysis of this chemical space has indicated that the CF2 and the Csp3-F motif, i.e. the mono-alkyl fluorinated motif, are currently under populated in the “Novartis fluorine fragment space”, as well in different databases (such as for example Integrity, PDB, ChEMBL).76 Moreover, as recently reported, the CH2F motif offers also interesting properties, such as lower logP, better solubility and opportunity to form weak hydrogen bond (HB) interactions.110-112 The molecular matched pair (MMP) analysis performed on the Novartis archive shows the impact on experimental logP (lipophilicity) and logS (solubility) (both in a favorable direction) when CF3 or CH3 or CHF2 is exchanged with CH2F.113 Despite the paucity of molecules with the Csp3-F motif there are some drugs containing it. Examples of Csp3-F containing molecules that are actively progressing through the drug R&D pipeline or have been already registered (Source: Integrity Thomson Reuters, accessed on June 2018) are: Sofosbuvir (launched in 2013) and Adafosbuvir (phase II), Dexamethasone (launched in 1958) and Solithera (pre-registered) to name few. No molecules under active development were found with a XCH2F motif (with X=N, O, S) while few contain OCHF2 or SCHF2. Other interesting CF2 motifs are the



OCF2Cl group present in Asciminib (phase III), the CF2Cl group present in Padsevonil (phase II) and the CF2CF3 group present in Fulvestrant (launched 2002), Pefcalcitol (pre-registered) and Vilaprisan (phase III). Maximization of 3D character of the fluorinated fragments can also be considered as an additional task achievable by using an ad hoc DOS-based library expansion strategy. Fluorinated peptides and peptidomimetics. It is worth mentioning that 19F NMR screening is not limited to fluorinated fragments, but can also be applied to the screening of libraries containing larger fluorinated molecules. As recently proposed,27 libraries of fluorinated peptides or peptidomimetics of various dimensions and amino acid composition can also be efficiently screened. These libraries are particularly appropriate for the detection of protein–protein interaction inhibitors. Sometimes, if the peptides are sufficiently soluble in water, it is worth the effort to chemically introduce into the peptide sequence a fluorinated amino acid with multiple equivalent CF3 groups. This results in an enhancement in sensitivity because the detected 19F signal originates from 6 up to 12 fluorine atoms depending on the type of amino acid introduced into the peptide.114-118 The combination of this chemical approach with the cryoprobe technology makes possible to screen the peptides at concentrations in the few hundreds nM range.87 An example with three short peptides differing only by a CH2 group and containing a fluorinated moiety with two equivalent CF3 is reported in Figure 4.


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Figure 4. 19F NMR spectrum recorded at 20 oC, 50 mM Tris pH 7.5, 0.001% Triton X-100, and 8% D2O for the three peptides shown in the figure. The three peptides differ only by the number (n) of CH2 on the sidechain of the polyfluorinated amino acid. The concentration of the peptides was 20 µM. The recording time of each spectrum was 4 minutes. The 19F NMR signal of TFA, set at 0 ppm, was used as reference for the chemical shift. Figure adapted from reference.115



The intense

19F

signals of the three peptides originating from six fluorine atoms each are well

dispersed despite the very small structural difference.115 It should be pointed out that the screening of a fluorinated set of molecules does not aim only at providing starting points for optimization, but also at identifying suitable fluorinated reporters which do not necessarily possess ideal physico-chemical properties.

QUALITY CONTROL SPAM NMR filter. Before being incorporated into in the fluorinated library all the selected compounds have to pass an NMR quality control filter known also as Solubility, Purity and Aggregation of the Molecule (SPAM) NMR filter.119, 120 The method requires the acquisition of two 1H NMR spectra i.e. a One Dimensional (1D) reference spectrum and a Water Ligand Observed by Gradient SpectroscopY (WaterLOGSY)121 spectrum and two 19F NMR spectra i.e. 1D without and with a T2 filter spectra for each selected fluorinated fragment prepared in aqueous buffered solution and containing a water soluble reference fluorinated fragment for which the exact concentration is known. This process is time consuming, but it is certainly worth the effort for avoiding afterwards wasting time and precious resources in the follow-up of unsuitable hits. A typical buffered solution used for the SPAM NMR filter is phosphate-buffered saline solution (PBS) with 8-10% D2O, pH 7.4 and with 5-10 µM ethylenediaminetetraacetic acid (EDTA) for chelating possible present paramagnetic impurities. The concentration of the fluorinated fragment and reference molecule is usually in the range 200 to 500 µM. A typical SPAM NMR filter performed on a fluorinated fragment is reported in Figure 5.


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*

b

*

c

F

H 2N

NH

d

1

*

δ 19F S

F

O

N

*

N H

a

2

δ 1H

Figure 5. SPAM NMR filter carried out at 25 oC in PBS pH 7.4, 5 µM EDTA and 9% D2O on a 400 MHz NMR instrument for the molecule 2 (labelled with a filled circle) in the presence of the reference molecule 3-fluoro-4-methylbenzamidine 1 (labelled with an asterisk). The nominal concentration of the tested molecule 2 and the real concentration of the reference molecule 1 were 200 µM. 1H NMR spectrum recorded with

19F

decoupling during the acquisition period (a),

aliphatic region of the WaterLOGSY spectrum (b), 19F NMR spectra recorded without (c) and with (d) a T2 filter of 280 ms and with 1H decoupling during the acquisition period. The signals originating from 2 and reference molecule 1 are indicated accordingly.



This combination of experiments allows the rapid evaluation of the identity, purity, solubility and aggregation state of the fragments for their inclusion or not in the library. The analysis of the 1H NMR reference spectrum allows confirmation of the structure, identification of impurities and measurement of the real concentration in aqueous solution using the known concentration of the reference molecule. The analysis of the WaterLOGSY spectrum permits the identification of fragments that aggregate. The LOGSY effect for the reference and tested molecule are analyzed for this purpose. Negative LOGSY effects (positive NOE), as shown in Figure 5, are indicative of absence of aggregation whereas positive LOGSY effects (negative NOE) are indicative of aggregation.

Recently, this experiment has been used to study aggregation

mechanisms as a function of time and to derive the kinetics of the aggregating process.122 The close inspection of the 19F NMR spectrum allows the identification of fluorinated impurities, instability of the molecule, presence of conformers, the measurement of the real concentration in aqueous solution using the known concentration of the reference molecule and the determination of the 19F chemical shift of the signal(s). This last point is important for generating the mixtures avoiding signal overlap and for performing on-the-fly deconvolution. The referencing of the 19F chemical shift is an aspect that becomes relevant when the chemical shifts among different laboratories are compared or when a chemical shift prediction tool based on the 19F chemical shift spectral database is applied in different laboratories. In our experience, the 19F chemical shift value  reported for a molecule recorded in the same experimental conditions in various laboratories can vary up to 1 ppm.76 Similar findings have been reported in a recent paper which discusses in depth different ways to reference the 19F chemical shift and proposes a method for proper referencing.123 A consensus among the different laboratories should be reached on this issue. Nevertheless, it should be specified the method used for referencing so that proper corrections can be performed


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using Table 1 contained in reference123 for allowing comparison of the data originating from different laboratories. In our quality control process we add either CFCl3 as an internal reference or, alternatively, use the 19F chemical shift of the fluorinated reference molecule for which a 19F spectrum in the same experimental conditions was recorded in the presence of CFCl3. Multiple 19F signals. Sometimes multiple 19F signals are observed despite the presence of only one CF or CF3 in the molecule. This may be due to different reasons: i) Presence of impurities such as for example the starting fluorinated building block used for the chemical reaction. If the % of these impurities is not high the fragment is retained. ii) Presence of diastereoisomers for the molecule. iii) Presence of conformers in slow exchange on the NMR time scale, such as for example those originating from hindered rotation around a tertiary amide bond. The two 19F signals can have very different chemical shifts and often their integrals are different reflecting the different energy levels of the two conformers. These molecules should not be rejected, but should be incorporated into the fluorinated library. iv) Instability of the fluorinated molecule in aqueous solution, e.g. nucleophilic substitution or cleavage due to chemical hydrolysis results in the presence of additional signals to the one(s) presumed. In order to assess this situation, additional 19F

NMR spectra are recorded after several days from the preparation of the NMR sample and the

acquisition of the first spectra. If the intensity of the additional 19F peak(s) increases over time and the initial 19F signal decreases in intensity then this represents an indication that the molecule is not stable and therefore not suitable for the library. For example, a chemical fluorinated motif that is not stable in aqueous solution at neutral pH is the trifluoroacetamide group, which undergoes chemical hydrolysis over time generating trifluoroacetate, as seen in Figure 6.



* * F

H 2N

NH

δ 1H O F3C

N H

N N

N

H 2N N

O +

F3C

OH

TFA

δ 19F Figure 6. SPAM NMR filter performed at 25 oC in PBS pH 7.4, 5 µM EDTA and 9% D2O on a 400 MHz NMR instrument for the tested molecule in the presence of the reference molecule 3fluoro-4-methylbenzamidine. The nominal concentration of the tested molecule and the real concentration of the reference molecule were 200 µM. Aromatic region of the 1H NMR spectrum recorded with 19F decoupling during the acquisition period (top) and expanded region of the 19F NMR spectrum recorded without the T2 filter and with 1H decoupling during the acquisition period (bottom). The 1H signals originating from the reference molecule, the initial molecule and the cleaved molecule are indicated with an asterisk, a filled circle and an open circle symbol, respectively (top). The 19F signal of the initial molecule is indicated with a filled circle whereas the signal of trifluoroacetate generated by the hydrolysis is indicated with TFA (bottom).


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This process is slow and requires several hours or days to go to completion. These molecules are not suitable for their incorporation into the library designed with the scope of testing it against many targets and at different times. However, freshly prepared mixtures containing the moderately unstable molecules could be tested separately with

19F

NMR FAXS in a direct or competition

format against the desired protein as a function of the incubation time. In our experience we identified binders among the cleaved molecules whereas the starting molecules were not binders.26 v) Additional 19F signals are those originating from the ion F- which resonates at ~ –119 ppm (at low concentration, in phosphate-buffered saline, pH 7.4, and 25o is at -118.63 ppm using Dolbier’s system for referencing70 and at -119.45 ppm using the recently proposed method for referencing123) or trifluoroacetate (CF3COO-) which resonates at ~ –75 ppm (in phosphate-buffered saline, pH 7.4, and 25oC is at -74.42 ppm using Dolbier’s system for referencing70 and at -75.25 ppm using the recently proposed system for referencing123). These additional signals should not be mistaken with other impurities. 19F

signal relaxation properties. Finally the 19F T2 filter NMR experiment is used to assess the

R2 of the

19F

signal(s) of the fragment. A T2 filter of 100 to 300 ms is typically used in these

experiments. Some fragments, while displaying 1H NMR spectra with sharp resonances and being compatible with the structure, can display broad

19F

signals (large R2) resulting in a complete

signal loss after the T2 filter (or in some extreme cases even in the absence of T2 filter) as it can be realized for the molecule in Figure 7.



Page 24 of 93

* * F

c

H 2N

NH 1

d F

b

O O S N H

δ 19F

3

*

*

a δ 1H

Figure 7. SPAM NMR filter performed at 25 oC in PBS pH 7.4, 5 µM EDTA and 9% D2O on a 400 MHz NMR instrument for the molecule 3 (labelled with a filled circle) in the presence of the reference molecule 1 (labelled with an asterisk). The nominal concentration of the tested molecule 3 and the real concentration of the reference molecule 1 were 200 µM. 1H NMR spectrum recorded with

19F

decoupling during the acquisition period (a), aliphatic region of the WaterLOGSY

spectrum (b), 19F NMR spectra recorded without (c) and with (d) a T2 filter of 280 ms and with 1H decoupling during the acquisition period. The signals originating from 3 and the reference molecule 1 are indicated accordingly. The two lowest energy conformations of molecule 3 calculated in water using the DFT/B3LYP method, the 6–31G** basis set and the SM8 solvation model in Jaguar (version 11.5.011; Schrödinger, LLC, New York, USA) are shown in ball and sticks. 24 ACS Paragon Plus Environment

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This signal broadening (large R2) is often mistakenly attributed to aggregation of the molecule. However, as it can be appreciated in Figure 7, the LOGSY effects on the methyl resonances of 3 and 1 are negative, thus excluding the formation of aggregates. In truth, it is the exchange mechanism between two or more different states of the molecule that is responsible for the observed broadening. Protonation state, conformational exchange due to partially hindered rotation, transient intramolecular and intermolecular HB formation and tautomerization can cause broad

19F

signals.113 It should be pointed out that the formation of transient intramolecular and

intermolecular HB could involve directly the fluorine atom or other functional groups which are close or also distant from the fluorine atom. The broadening effect appears more noticeable with the fluorine in the ortho or para positions to a functional group involved in intramolecular HB due to a significant resonance and field/inductive effect on the fluorine electronic cloud and consequently on the 19F NMR chemical shift. The 19F chemical shift difference for the fluorine signal between the two different states of the molecule can be very large. When the exchange rate is comparable to this chemical shift difference (exchange rate in the range of 105 to 103 s-1) then a broadening of the signal is observed. In some rare cases the 19F signal is so broad (very large R2) that can hardly be detected. It is worth noting that such broadening can be observed even if one of the two or more states of the molecule is very low populated.113 For example the molecule 3 can assume the two conformations shown in Figure 7 among the lowest energy conformations in water (as calculated by quantum mechanics calculation). These two conformations have a difference in energy ∆G of 1.45 kcal/mol, with the conformation showing the fluorine close to the hydrogen of the NH of the sulfonamide being the lowest in energy (92% populated). The other conformation in which the NH points away from the fluorine is significantly less populated (8%). The chemical



Page 26 of 93

shift difference between the two 19F signals deriving from the two conformers would be very large. Other two examples for molecules displaying a 19F broad signal are reported in Figures S5 and S6. In these cases the fluorine is located in para position to a functional group involved in a transient intramolecular HB. The resonance and inductive contribution is responsible for the large chemical shift difference of the fluorine signal between the conformer with and without the intramolecular HB. For protons the chemical shift difference is very small and consequently the exchange rate is often larger than this difference resulting in sharp signals. It should be pointed out that this phenomenon is significantly less frequently observed with the CF3 containing molecules. This is solely due to the smaller chemical shift difference of the CF3 signals between the two states of the molecules. Therefore, similarly to the protons, the exchange rate will be larger than the

19F

chemical shift

difference, resulting in no broadening for the CF3 signal. Although the broadening effect observed for the CF signal of some molecules is not helpful for the 19F NMR screening purpose it is valuable for studying and characterizing relevant dynamics processes of the molecules in solution that escape detection with 1H or 13C NMR spectroscopy. The molecules displaying 19F broad signals, if the R2 is not very large, are put in a separated mixture(s) and tested against the receptor with a short 19F T2 filter or by using the 19F combined T1 filter and short T2 filter, or the 19F T1 filter or the 19F T1 filter experiments, as previously discussed.74 An alternative solution, which however would require a special setup, is the use of

19F

NMR dissolution dynamic nuclear polarization

(DNP)124, a technique for increasing the NMR sensitivity, coupled with the acquisition of the 19F T2 filter spectra on low field NMR instruments (e.g. 60 MHz). In this case the exchange between the two states of the molecule will be often larger than their 19F chemical shift difference resulting in a sharp 19F signal. These mixtures should be tested using a larger protein concentration with


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respect to that used for the screening of the other mixtures due to the smaller dynamic range of the utilized experiments. It is clear that these mixtures can always be tested in the competition format of the assay. Finally, another approach is the selection of an isomer of the molecule displaying broad

19F

signal with the fluorine in another position (for example with the F in meta position

instead of ortho or para position to the functional moiety involved in HB) and displaying a sharp 19F

signal. If this isomer is a binder then it can be used as reporter in the competition format of the

assay for testing if the isomer displaying broad 19F signal is also a binder and for measuring its binding affinity. The solubility requirement of the fluorinated molecules does not need to be too stringent: usually 50 -100 μM is enough when working with a 600 MHz NMR instrument. Its sensitivity allows the screening to be performed at concentrations which are ≤ 50 μM. Sometimes solubility can be augmented after the identification of the hit(s) during the Structure-Activity Relationship (SAR) by archive process by screening close analogs for increasing the chance of getting a crystal structure of the protein ligand complex. Mixtures generation. All the fluorinated fragments that pass the quality control are then used for the generation of the mixtures. Typically mixtures of only CF3 containing fragments and mixtures of only CF/CF2 containing fragments are generated. This is due to practical reasons: i) the CF3 containing fragments are tested at lower concentration with respect to the CF/CF2 containing fragments. ii) The spectral window for the CF3 signals is different with respect to most of the CF/CF2 signals. Therefore, the experimental conditions are optimized for the two types of mixtures. The

19F

NMR chemical shifts of the fragments measured in the quality control

experiments are used to generate the mixtures for avoiding signal overlap. The number of fragments per mixture can range from 25 to 40 or even higher. This large number is possible due 27 ACS Paragon Plus Environment


to the large fluorine chemical shift dispersion and the low fragment concentration used for the testing resulting only in a very small amount of DMSO (~ 0.5 % to 2%) present in the NMR sample. Another nice feature is that the 19F NMR library mixture design is trivial when compared to 1H NMR library mixture design where special algorithms are used for minimizing the severe overlap.125, 126 The total number of fluorinated fragments of the library can range from few hundreds to few thousands. It should be pointed out that new entries, with the goal of increasing the chemical space, can be added at later stage to the library. The selected fragments, after passing the quality control, are then used to generate additional new mixtures.

SCREENING IN DIRECT MODE A typical screening run with FAXS in direct mode against the serine protease trypsin is shown in Figure 8.88


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CF3 N NH

H 2N 4

- protein

+ protein

+ protein + known ligand

δ 19F Figure 8. Expanded region of the

19F

564 MHz NMR T2 filter spectra recorded with proton

decoupling for a 36 CF3-containing molecules (18 µM concentration) mixture without (upper trace) with bovine pancreas trypsin (middle trace) and with bovine pancreas trypsin plus a high affinity binder (KD in the nM range) (lower trace).88 The protein concentration was 9 µM. The T2 filter length was 0.4 s and 160 scans were acquired with a repetition time of 3.8 s resulting in 12 min recording time for each spectrum. The arrow shows the CF3 resonance of the identified binder 5-(trifluoromethyl)-2-pyridylamidine 4. Figure adapted from reference.88



The mixture contained 36 different CF3 fragments at a concentration of 18 µM. All the resonances are well resolved despite the large number of fragments present in the mixture. The detection of the binding fragments in the mixture is a simple process. The ligands are identified as those molecules whose signals are reduced in intensity or even vanished due to the increased linewidth, i.e. larger R2, in the presence of the protein. The

19F

chemical shift of each molecule is known

from the quality control experiments and consequently the perturbed signal in the presence of the protein is directly associated to the molecule that interacts with the protein. In the example of Figure 8 the signal at -61.79 ppm vanishes upon addition of the protein (middle trace). According to the chemical shift table of the fragments of the mixture the signal corresponds to 5(trifluoromethyl)-2-pyridylamidine 4 (MW = 189.14) thus identifying this fragment as a binder to trypsin. Typically, if available, a known ligand is then added into the samples displaying active mixtures. This is performed for validating the identified hits, as shown in the lower trace of Figure 8. The known ligand in this example is a potent binder to the S1 and S4 pockets of trypsin. The resonance of 4, missing in the spectrum in presence of the protein (middle trace), reemerges in the spectrum upon addition of the known ligand (lower trace). This observation convincingly suggests that 4 is a binder for trypsin and, furthermore, suggests that the molecule might bind to the same protein site occupied by the known ligand. Sometimes, in addition to the change of the R2, a change in the fluorine chemical shift (∆δobs) defined as ∆δobs = (obs - f ) with obs and f the chemical shifts in the presence and absence of protein, respectively, is also observed for the fragment binding to the protein in the spectrum recorded in presence of the protein. This is not always the case and it depends on the residence time of the fragment on the protein and the difference |b - f| where b is the chemical shift of the ligand in the bound state, as shown in the simulation of Figure S3(2).


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The ∆δobs 19F can efficiently be utilized to the screening of fluorinated fragments that are in racemic form. Fluorinated racemates have been used as sensitive NMR probes for monitoring the structure and dynamic of RNA.127 More recently they have been proposed for the screening of fluorinated mixtures against proteins.128 This can be realized with the example of Figure 9 describing the 19F NMR screening of a 15 fluorinated molecules mixture against the bromodomain 1 of the bromodomain containing protein 4 i.e. BRD4(1).

F3C

N O NH2 5

- protein

+ protein

+ protein + bromosporine

δ19F


O


Figure 9.

19F

NMR screening of a mixture of 15 fluorinated fragments against the protein

BRD4(1). Expanded region of the 19F NMR spectra recorded without T2 filter displaying the CF3 signal of the molecule 5 (chemical structure reported on the top). The spectra were recorded without (upper trace), with BRD4(1) (middle trace) and with BRD4(1) plus 100 µM bromosporine (lower trace). Figure taken from reference.128

The 19F NMR spectrum for molecule 5 in the absence of the protein displays only one sharp 19F signal originating from both enantiomers because these are undistinguishable. The addition of the protein results in a splitting of the

19F

signal in two resonances of equal integral. One signal is

sharp and has nearly the same chemical shift of the fragment in the absence of protein whereas the other signal is broad and is downfield shifted. These experimental observations indicate that the molecule is a binder for BRD4(1), but only one of the two enantiomers. Upon addition of bromosporine129 which is a known potent ligand for BRD4(1) (see Figure 9 lower trace) the broad and downfield shifted signal disappears and complete signal intensity is reestablished at the chemical shift of the free molecule. This originates from the total displacement of the bound enantiomer by bromosporine. It is worth noting that both 19F NMR spectra without (or with a very short T2 filter) and with T2 filter should be recorded. In the example of Figure 9 (middle trace) the downfield broad signal would not be visible in the spectrum recorded with the T2 filter thus not allowing to differentiate the relative binding strengths of the two enantiomers. Another important point, according to the simulations of Figure S3(2) and reference113, is that both NMR observed parameters i.e. 19F chemical shift and linewidth should be analyzed for establishing the relative protein binding affinity of the two enantiomers. The opposite situation to that displayed in Figure 9 can also be encountered where one signal is not shifted or very little, but becomes


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broad and the other is shifted and remains sharp. In this case it is the former that originates from the enantiomer with the better binding affinity. When available, priority should be given to the selection of the fluorinated molecules in their racemic form as representatives of defined chemical scaffolds. 76, 128 The selection of only one of the enantiomers for the fluorinated screening library could result in failure at identifying relevant binding chemical scaffold motifs. This issue becomes particularly important when screening fluorinated fragment libraries with a larger degree of three dimensionality.106 The presence of one or more asymmetric carbon atoms in these fragments increases the chances of having chiral centers. Finally, the 19F ligand-based NMR screening is particularly useful to characterize the binding of diastereisomers130 and conformers that are in slow exchange on the NMR time scale.128

DYNAMIC RANGE The method has a very large dynamic range (see Annex S3 for the theory) thus permitting the detection of very weak affinity binders. The dynamic range can be augmented by going to higher magnetic fields due to the larger |b - f| value and the increased transverse relaxation rate in the bound state (R2,b) due to the significant contribution originating from the large 19F chemical shift anisotropy (CSA). Figure 10 shows the detection limits can be achieved by properly optimizing the experimental conditions.



Page 34 of 93

CF3 N NH

H 2N

Non binder

4

-protein

+protein

δ 19F Figure 10. 19F 564 MHz NMR T2 filter spectra acquired with proton decoupling for a non binding molecule and the molecule 4 without (upper trace) and with (lower trace) 360 nM bovine pancreas trypsin.88 The molecules concentration was 18 µM. The length of the T2 filter was 0.8 s and 96 scans were acquired with a repetition time of 3.8 s resulting in 9 min recording time for each spectrum. Figure adapted from reference.88


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The T2 filter length was augmented in this example to 0.8 s for increasing the protein binding sensitivity of the assay. This is feasible because the transverse relaxation rate R2 value of the 19F signal of fragment 4 in the absence of protein is small (0.93 s-1). The addition of the protein results in the complete disappearance of the 19F signal. Similarly to what reported in the literature131 one can define the % of contrast C as: 𝐶=

100(𝐼𝑓 ― 𝐼𝑜𝑏𝑠) 𝐼𝑓

(3)

with Iobs and If the 19F signal intensity of the binder in the presence and absence of the protein, respectively. The KD of the fragment measured with ITC is 621 +/- 60 M. The protein and fragment concentrations are 360 nM and 18 µM, respectively, corresponding to concentrations that are, respectively, 1725 and 34.5times lower than the KD value of the fragment. This corresponds to a pb for the binding fragment of only 0.000563 (i.e. 0.0563% bound and 99.9437% free) or, differently stated, to 1 molecule bound and 1775 free. This value of pb is very close to the maximum pb i.e. [ET]/KD of 0.000580 that can be achieved with the KD of 621 µM and the protein concentration of 360 nM, according to the simulations of Figure 2(right). The contrast, defined by equation (3), achieved in this example is 100% despite the very small fraction of bound ligand and the small size of the protein. It is possible to detect even weaker binders by keeping the concentration of the fragment low and by increasing the concentration of the protein as discussed above. Doubling the protein concentration results in doubling the fraction of bound ligand. This can be useful when only a small reduction of the 19F fluorine signal is observed upon addition of the protein. In this case, the addition of another aliquot of protein would result in a more pronounced signal reduction thus confirming the fragment as a potential binder to the protein. It should be pointed out that, in our experience, CF containing fragments are typically more sensitive



Page 36 of 93

than CF3 containing fragments to protein binding despite their 2-3 fold higher tested concentration. This is due to the exchange contribution to the R2,obs which is larger for CF containing fragments because the chemical shift difference 19F NMR |b - f| is greater for the CF vs. CF3 containing fragments (see Annex S3). .

THROUGHPUT AND DECONVOLUTION Typically, if large mixtures are used, few thousand fragments can be rapidly screened and the hits validated in few days. The protein consumption is low (e.g. ~ 1-3 mg for a 30 kDa protein is needed for the screening and validation of the hits).27 The absence in the

19F

NMR spectra of strong

interfering signals makes the method particularly suitable for screening membrane proteins which require the presence of large amount of detergents for their solubilisation.132 The deconvolution is carried out on-the-fly as each 19F signal is assigned to a specific molecule of the mixture. The flat baseline of the spectra, the large chemical shift dispersion of the signals and the presence of only one (or two) 19F signals for each molecule allows for an automated analysis of the data.71 Software modules from different vendors (ACDLab, Bruker and Mestrelab) are now available for this task thus reducing significantly the time required for the analysis of all the spectra. Mixtures of fluorinated compounds produced by parallel chemical synthesis or mixtures of fluorinated peptides can be screened directly with the FAXS without previous purification. The precise constitution of these mixtures is often undefined. Therefore, the presence of one or more hits detected with the FAXS would require, for the identification of the binder(s), the purification, isolation, characterization and retesting for all the molecules of the mixture. However, this process is time consuming and can result in significant material loss. The same concerns apply to molecules 36 ACS Paragon Plus Environment

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produced via chemical reactions performed in the NMR tube and tested with FAXS as a function of the incubation time, as it has been previously shown.26 A combined approach which is based on an algorithm for 19F NMR chemical shift prediction, TwoDimensional (2D)

19F

NMR Diffusion-Ordered SpectroscopY (DOSY) and 2D

19F-1H

NMR

heteronuclear long-range (COrrelation SpectroscopY) COSY experiments has been proposed for the deconvolution of these complex mixtures.98 The first method uses the

19F

chemical shift prediction algorithm based on the fingerprint

descriptor as previously reported.107 This method is particularly useful for the characterization of chemical mixtures containing molecules with fluorine atoms having different fingerprints. It makes use of an available representative list of chemical shifts for fluorines with different local chemical environments. As shown in Figure 11, the method predicts quite accurately the chemical shifts of the four displayed fragments.98


19F


CF3 OH

Page 38 of 93

CF3

CF3 O

O

61

Entry

MW

Predicted δ 19F (ppm)

Measured δ 19F (ppm)

6

190.12

-58.53

-58.55

7

204.15

-58.78

-58.99

8

237.61

-61.42

-61.39

9

261.20

-61.59

-61.41

CF3

OH

O

Cl

27

N H

O

83

9 4

94

OH

N H

O

–9.22

D –9.18

83

27

–9.14

1 6

δ 19F

11

Figure 11. (Left) MW, predicted

19F

chemical shift obtained from the fingerprints descriptor

algorithm and measured 19F chemical shift for the four CF3 molecules contained in the mixture. (Right) 2D 19F NMR DOSY spectrum recorded for the mixture. The chemical structures of the fragments are shown above their corresponding 19F resonance. Figure adapted from reference.98

However, the two molecules 8 and 9 are described by the same fingerprint and their chemical shifts are indeed very similar. While the method predicts with accuracy the

19F

chemical shifts of

molecules 8 and 9 the unambiguous assignment is not possible. The use of 2D 19F NMR DOSY experiments76,

133-135

is capable of distinguishing the two molecules based on their different

diffusion coefficients which depend on the molecular weights of the molecules, as shown in Figure


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11. The pulse sequence of the 2D 19F NMR DOSY was modified76 in order to cover a large spectral bandwidth which is often encountered with fragments having different fingerprints. In our experience we found the 2D

19F

NMR DOSY experiments particularly useful in the

deconvolution of mixtures of fluorinated peptides or peptidomimetics. One synthetic approach consists in producing mixtures of small peptides with different amino acid composition and different length and including one amino acid with a fluorinated moiety.27 Long range three dimensional effects will result in different

19F

chemical shifts for the fluorinated amino acid

inserted in the different peptides. These mixtures are tested with the 19F NMR direct binding assay for identifying peptidomimetics or peptides which bind to the receptor. The

19F

2D DOSY

experiment allows, if no aggregation is present, the separation of the different fluorinated peptides according to their MW, and consequently the identification of the peptide of the mixture that interacts with the receptor. The third approach used for deconvolution a mixture makes use of the 2D NMR 19F-1H long-range COSY experiments.98, 136, 137 Fluorine displays many heteronuclear scalar couplings with protons (JFH). Small nJFH coupling constants can be observed with protons which are separated from the fluorine by more than 5 bonds (n > 5). An application of this experiment to the structural characterization of a hit against trypsin is shown in Figure 12.



Page 40 of 93

– protein + protein difference

δ 19F δ 19F *

* **

* CF3

H

H

H NH2

H

H H 10

δ 1H Figure 12. 19F NMR screening and deconvolution of the active mixture for the identification and characterization of the binder to trypsin. (Top) Expanded region of the 19F NMR spectra recorded with a T2 filter of 1.28 s and with 1H decoupling during the acquisition period without and with 5 μM bovine pancreas trypsin. The only observable signal in the difference spectrum, obtained with the subtraction of the spectra with and without trypsin, corresponds to the signal of the binder. (Bottom) Expanded regions of the 2D 19F - 1H NMR long-range COSY spectrum displaying the long range connectivities between the CF3 groups and the aromatic and aliphatic protons. The five cross peaks on the dashed line deriving from the long range connectivities of the CF3 of 10 to all the protons of the fragment are indicated with asterisks. Figure adapted from reference.98 40 ACS Paragon Plus Environment

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The intensity of the signal at -58.1 ppm is reduced in the presence of trypsin and it is the only signal visible in the difference spectrum thus representing a hit (Figure 12 top). The connectivities between this fluorine signal and all the protons of the fragment are observed in the 2D NMR 19F1H

long-range COSY spectrum (Figure 12 bottom) thus allowing the structural identification of

the binder as the 2-(trifluoromethyl)benzylamine molecule 10.98 If these experiments were not sufficient for the unambiguous assignments additional heteronuclear experiments such as for example the selective

19F-1H

TOCSY can also be employed for this purpose.138

FAXS IN COMPETITION MODE Screening. The validated fluorinated ligands identified with the direct FAXS binding assay or with other biophysical techniques can be utilized as “spy molecules” or reporters in the FAXS in competition mode for screening fragments and large molecules and for calculating the dissociation binding constants of the identified binders.139 The experimental parameters, such as selection of the spy molecule, protein and spy molecule concentration and length of the T2 filter have to be first optimized. Then these selected parameters will be used for the screening. For the screening in competition mode the concentration of the spy molecule is the same as for the assay in direct mode. This low concentration ensures the detection of low affinity binders that compete with the spy molecule. It should be pointed out that the sensitivity to protein binding of the FAXS in competition mode is directly proportional to the fraction of bound protein. Therefore, the screening is performed at a concentration of the fragments ≥ 100 µM. If instead the purpose of the competition experiments is to perform KI measurements and affinity ranking of the compounds then one should perform the experiments at different 41 ACS Paragon Plus Environment


concentrations of the competing molecule and starting from low concentration. With an appropriate spy molecule it is possible to reduce the measuring time for the screening by using a combined pulse sequence which permits the acquisition of the 19F NMR spectra without and with T2 filter in a single experiment (see Figure S4).140 It is worth noting that the tested molecules do not require the presence of fluorine. Only the

19F

NMR signal of the spy molecule and control

molecule (if used) will be monitored in this assay. Molecules can be screened in single or in mixes for throughput increase. For the latter the deconvolution of the active mixtures will be necessary for identifying the binder(s). Some of the applications of this assay27 include: i) SAR by archive / SAR by catalog (hit optimization). In this process close analogues or bigger molecules containing the identified binding chemical scaffold are gathered from the proprietary compound collection or purchased. Binding affinity ranking or KI measurement, as described below, of the selected molecules is possible with the FAXS in competition. The goal of this process is to improve potency, as well as solubility, if necessary, in order to enhance the probability of obtaining the protein-ligand complex X-ray structure. ii) Fluorine scan i.e., testing isomers of the original hits with the fluorine in different positions for spotting the possible existence of one or more fluorophilic protein environments. iii) Secondary screening i.e. screening of additional available chemical libraries, e.g. specifically constructed for the target or libraries generated from virtual screening. iv) Natural product extracts screening. The FAXS in competition mode is very suitable for screening different fractions of natural product extracts as there is no signal interference due to the lack of fluorine in natural products. v) Hit list triaging for the validation of hits obtained from High Throughput Screening (HTS) or from focused screening performed with other biophysical techniques. vi) Lead


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optimization. The method represents a rapid tool for characterizing the potency of the newly synthesized molecules with respect to the original fragment. KD and KI measurements. NMR spectroscopy has been extensively applied to the measurement of the binding constants (see reference46 and references cited in it). In order to not generate confusion, we define KD and KI as the dissociation binding constant of the spy and competing molecule, respectively. The calculated KD and KI are dissociation binding constants only in a truly competition mechanism i.e. when spy and competing molecules bind to the same site. The

19F

NMR competition experiments can be used for rapidly measuring the KD of the spy molecule if the KI value of the competing molecule is known or the KI of the competing molecule if the KD of the spy molecule is known.26, 66, 77, 139, 141, 142 We report here a novel and rapid method for measuring these binding constants. This knowledge is important for classifying the fragment hits according to their binding affinity and for prioritizing early on structure based and medicinal chemistry activities. In the experimental condition of [LT] >> [ET] which applies to the NMR ligand based screening the equation (1) simplifies to

𝑝𝑏 =

[𝐸𝐿]

[𝐿𝑇]

=

[𝐸𝑇] 𝐾𝐷 + [𝐿𝑇]

(4) It is worth noting that pb is directly proportional to [ET] and, according to the equation (2) reported in Annex S3, the R2,obs will also be directly proportional to [ET]. In other words, by doubling the protein concentration will result in doubling the fraction of bound ligand and, owing to the fact that [LT] is constant, will result in doubling the concentration of bound ligand [EL]. In the conditions for which the equation (2) reported in Annex S3 is valid i.e. [LT] >> [ET], the plot of 43 ACS Paragon Plus Environment


Page 44 of 93

[ET] vs. ln[Is(T2)/Is(0)] or vs. ln[Is(T2)/Ic(T2)] is linear, with ln the natural logarithm, Is(0) the spy molecule signal integral or intensity in the spectrum recorded without T2 filter (or with a very short T2 filter for achieving a flat baseline) and Is(T2) and Ic(T2) the spy and control molecule signal integral or intensity, respectively, in the spectrum recorded with the T2 filter. This can be appreciated in Figure 13 for the molecule 2-hydroxy 3-fluorobenzoic acid 11 interacting with Human Serum Albumin (HSA) where the data could be fitted nicely with a straight line (R2 = 0.99). The control molecule i.e. the molecule that does not interact with the protein is 1-[5(trifluoromethyl)1,3,4 thiadiazol-2-yl]piperazine 12.66 I

N N

OH O F

OH

N

S

F F F

OH

HN spy (s) 11

O control (c)

O

O

competitor (I)

12

13


Page 45 of 93

0.6

[ET] in M

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4

0.2

0.0 0.3

0.6

0.9

1.2

-ln(s/c) Figure 13. Plot of the -ln[Is(T2)/Ic(T2)] (abbreviated as –ln(s/c)) with I the signal intensities (x axis) as a function of the protein (HSA) concentration (y axis). The spy molecule (s) is 11 and the control molecule (c) is 12.The spy molecule concentration was 50 µM. The intensities were extracted from the 19F T2 filter (80 ms long) spectra. The straight line represents the best fit of the experimental points. If instead the protein concentration is not known it is sufficient to put on the y axis the different small volumes of protein concentrated stock solution added to the NMR sample divided by the total volume of the NMR sample solution after each addition.



Alternatively, one could plot on the X axis the difference of the measured values in the presence of the different protein concentrations from the measured value in the absence of protein. In this case the straight line will pass through the origin. If the ratios of the signal integral or intensity are not used in their ln form then the data would be fitted with an exponential function.66 The graph of Figure 13 can always be used afterwards to calculate the concentration of novel prepared batches of protein. A defined protein concentration, within the range of concentrations used for the protein titration experiments, is then selected for the competition experiments. The measured values ln[Is(T2)/Is(0)] or ln[Is(T2)/Ic(T2)] in the absence and presence of the protein correspond to the percentage displacement value F for the spy molecule of 100% and 0%, respectively, as shown in Figure 14. The displacement F is defined as:

(

𝐹 = 100 1 ―

[𝐸𝐿]𝐼 [𝐸𝐿]

)

(5) with [EL]I and [EL] the concentration of the spy-protein complex in the presence and absence of competitor, respectively. Due to the linear dependence of ln[Is(T2)/Is(0)] or ln[Is(T2)/Ic(T2)] with [ET] (see Figure 13) and consequently, according to equation (4) with [EL] (because [LT] is constant) the two data points in Figure 14 are fitted with a straight line. In the presence of a competing molecule the spy molecule signal is partially restored due to the partial displacement of the spy molecule from the protein. From the measured value ln[Is(T2)/Is(0)] or ln[Is(T2)/Ic(T2)] in the presence of the competing molecule I it is straightforward to calculate the F value as shown visually in the example of Figure 14. 46 ACS Paragon Plus Environment

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Alternatively the F value is simply given by the equation:

{

𝐹 = 100 ∗ 1 ―

𝐼𝑠(𝑇2)

𝐼𝑠(𝑇2)

[ ] [ ] [ ] [ ]

𝑙𝑛

𝐼𝑐(𝑇2)

𝑙𝑛

― 𝑙𝑛

―𝐸

𝐼𝑠(𝑇2) 𝐼𝑐(𝑇2)

𝐼𝑐(𝑇2)

― 𝑙𝑛

―𝐸

}

+𝐸 + 𝐼

𝐼𝑠(𝑇2) 𝐼𝑐(𝑇2)

+𝐸

(6) if the method with the control molecule is used, and by the equation:

{

𝐹 = 100 ∗ 1 ―

𝐼𝑠(𝑇2)

𝐼𝑠(𝑇2)

[ ] [ ] ( ) ( ) [ ] [ ]

𝑙𝑛

𝑙𝑛

𝐼𝑠(0)

― 𝑙𝑛

―𝐸

𝐼𝑠 𝑇2

𝐼𝑠(0)

― 𝑙𝑛

―𝐸

𝐼𝑠(0)

𝐼𝑠 𝑇2

𝐼𝑠(0)

}

+𝐸 + 𝐼

+𝐸

(7) if the method with only the spy molecule is used. The subscripts –E, +E and +E+I indicate absence of protein, presence of protein and presence of protein and competing molecule, respectively whereas the other terms have been defined above.



- protein

- protein

+ protein + competing molecule

+ protein + competing molecule

100

+ protein

80

F in %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 93

60 40

δ 19F + protein

20 0 0.3

0.6

0.9

1.2

-ln(s/c) [LT ] 50 µM

[I]

(-ln(s/c)) -E

25 µM 0.06188

F (%)

(-ln(s/c)) +E

F (%)

(-ln(s/c)) +E+I

F (%)

KD (NMR)

KD (ITC)

KI (NMR)

100

1.38629

0

0.35954

77.5

41.8 µM 41.0 µM 3.3 µM

KI (Fluorescence) 3.3 µM

Figure 14. Measurement of the displacement value F (top left) and the KI or KD values (bottom) of the competing and spy molecules, respectively, with the FAXS performed in competition mode using only one concentration of the competing molecule [I]. The protein was HSA (600 nM), the spy (s) and control (c) molecules were 11 and 12, respectively, and the competing molecule (I) was the warfarin derivative 4-hydroxy-3-[1-(p-iodophenyl)-3-oxobutyl]coumarin (13). In the graph the -ln[Is(T2)/Ic(T2)] abbreviated as –ln(s/c) with I the signal intensities (x axis) is plotted as a function of the displacement value F of the spy molecule (y axis). The intensities were extracted from the

19F

T2 filter (80 ms long) spectra. The expanded region of the

19F

T2 filter

spectra containing the signal of the reporter 11 without HSA, with HSA and with HSA and 25 µM of 13 are shown on the top right. The F values of 0 and 100% corresponds to the value in the absence of competing molecule and in the presence and absence of protein (E), respectively. Due 48 ACS Paragon Plus Environment

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to the linearity of –ln(s/c) with respect to the protein concentration used for the competition experiments (600 nM) the two points can be fitted with a straight line. The measured –ln(s/c) in the presence of the competing molecule allows the calculation of the F value as shown on the graph. The fitting straight line function is used to calculate the F value. (Bottom) The extracted F value is then used to calculate, using equation (8) or (9), the KD or KI, respectively. The NMR KD of the spy molecule was calculated using the KI of the competing molecule from fluorescence measurements whereas the NMR KI of the competing molecule was calculated using the KD of the spy molecule from ITC measurements.

The F value extracted with either one of the two methods can then be used to calculate with a single competing molecule concentration either the KD of the spy molecule, if the KI of the competing molecule is known, or the KI of the competing molecule if the KD of the spy molecule is known, in the assumption of a competitive mechanism, using equation (8) for KD and equation (9) for KI 𝐾𝐷 =

𝐹𝐾𝐼[𝐿𝑇]

{100[𝐼] ― 𝐹(𝐾𝐼 + [𝐼])} (8)

𝐾𝐼 =

(100 ― 𝐹)[𝐼] 𝐾𝐷 𝐹{[𝐿𝑇] + 𝐾𝐷} (9)

with [LT] and [I] the concentrations of the spy and competing molecule, respectively. Application of this method to the protein HSA, with 11 as spy molecule (s), 12 as control molecule (c) and 13 as competing molecule (I) is reported in Figure 14.



It is worth noting, according to equations (8) and (9), that the concentration of the protein does not enter into the calculations and therefore the experiments can be recorded without knowing the protein concentration. However, one has to make sure that [ET] is significantly smaller than [LT] to guarantee a linear dependence of the ln[Is(T2)/Is(0)] or ln[Is(T2)/Ic(T2)] with [ET], as shown in Figure 13. In addition, [ET] has to be smaller than the concentration of the competing molecule [I]. However, it is worth noting that even if the competition experiments are performed at an amount of protein concentration for which there is no any longer linear dependence, it is always possible to derive the displacement value F. In this case, the monotonic fitting function of the data derived from the protein titration experiments performed with different protein concentrations or volumes of added protein stock solution, if the protein concentration is not known, is used for extracting F. As it can be appreciated in Figure 14, the NMR derived binding constants extracted with a single competing molecule concentration and using equations (8) and (9) compare favourable with those obtained from full titration fluorescence spectroscopy and ITC measurements. This NMR method is particularly useful in the SAR process for rapidly assessing and ranking similar molecules to the original hit obtained from the in-house archive, vendors or newly synthesized molecules for their affinity to the receptor. In this process it is recommended to record also the 1D 1H NMR spectrum for determining the purity and exact concentration of the selected analogue for a more accurate measurement of its KI. It is also possible to derive the binding constants KI and KD from a full titration analysis. The 19F NMR experiments are performed at different [I] values and the % of displacement F values for each [I] concentration are extracted with the procedure described above. The displacement F of the spy molecule in a competition mechanism as a function of the concentrations and binding constants of the spy and competing molecules is given by the equation:


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𝐹=

100[𝐼]

(

[𝐿𝑇]𝐾𝐼 𝐾𝐷

)

+ 𝐾 + [𝐼] 𝐼

(10) Derivation of equations (8-10) are reported in the Annex S7 of the Supporting Information. Therefore by plotting the F values vs. the different [I] values and using equation (10) for the data fitting it is possible to derive, in the assumption of a pure competition mechanism, KI if the KD is known or KD if the KI is known, as shown in the example of Figure 15. In the first case KD and [LT] are fixed in equation (10) and the parameter KI is varied for obtaining the best fit of the experimental points, whereas in the second case KI and [LT] are fixed in equation (10) and the parameter KD is varied for obtaining the best fit of the experimental points. In addition, the value 100 in equation (10) can be replaced with a parameter that is allowed to float during the fitting procedure in order to test if the non linear curve fitting of the data results in a value for the parameter close to 100. Significant deviation from this value could be due to different causes. One common cause is the low solubility of the competing molecule at the higher concentrations [I] used to generate the F vs. [I] plot.143



- HSA + 25 µM (I)

+ 52 µM (I)

+ 10 µM (I) + 0 µM (I)

+ HSA

δ 19F 100 80

F in %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

60 40 20 0 0

10

20

30

40

50

60

[I] in M

Figure 15. (top) Expanded region of the 19F T2 filter (80 ms long) spectra containing the signal of the spy molecule (s) 11without HSA, with HSA and with HSA and three different concentrations of 13 i.e. the competing molecule I. The concentrations of HSA and spy molecule 11 were 0.6 and 50 µM, respectively. (Bottom) Plot of the F values vs. the concentration of the competing molecule [I] and best fit of the experimental data using equation (10). Three different concentrations of (I), reported on the top, were used and the respective F values were extracted with the procedure


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described above. The control molecule (c) used in these experiments is 12. The fitting of the data with equation (10) and [LT] and KI fixed at 50 and 3.3 µM, respectively, results in a KD = 31 ± 4 µM whereas the fitting of the data with equation (10) and [LT] and KD fixed at 50 and 41 µM, respectively, results in a KI = 3.9 ± 0.3 µM.

Sometimes, full titration experiments could be useful for identifying and characterizing allosteric mechanisms. The competition mechanism is characterized by an apparent change of the KD of the spy molecule whereas the allosteric mechanism is characterized by a real change of KD. The spy molecule could either increase or decrease its binding affinity in the presence of I. In the first case the displacement F value, as defined by equation (5), will become negative whereas in the second case the displacement will not necessarily reach the 100% value at high competing molecule concentration. It should be pointed out that the approach described here for KD and KI measurements with a single competing molecule concentration or with full titration is not restricted to the 19F T2 filter NMR experiments, but can also be applied in the same way to the 1H T1 , T2 and selective T1 filter experiments for which the observed parameter has a linear dependence on pb. Furthermore, it can also be applied in the same way to the STD and WaterLOGSY experiments for which the observed effect is proportional to [EL]. The high sensitivity to protein binding of the FAXS in direct mode allows detection of weak affinity binders with KD in the high µM to low mM range. These molecules sometimes cannot be identified as binders with other biophysical techniques due to their limited solubility. Nevertheless, even these molecules can be used as suitable reporters in the FAXS in competition mode. Their low concentration used in the FAXS in competition mode allows the approximation



Page 54 of 93

[LT] + KD ≈ KD and KI{([LT]/ KD ) + 1)} ≈ KI. For a competition mechanism, then equation (9) simplifies to:

𝐾𝐼 =

(100 ― 𝐹)[𝐼] 𝐹 (11)

and equation (10) simplifies to:

𝐹=

100[𝐼] 𝐾𝐼 + [𝐼]

(12) It is worth noting that in the particular case with [LT] 1 mM. The same fluorinated library was also screened against additional six targets with hit rates ranging from zero/very low (0.0 and 0.08% for a cytokine receptor and a Wnt signaling protein, respectively) to high (2.2%, 7.3% and 7.8% for Derp7, an apoptotic protein and a kinase protein, respectively). Interestingly, the experimental fragment hit rate correlated very well with the estimated ligandability from commonly accepted computational methods. In a second paper the same group highlighted the utility and the strength of 19F NMR based fragment screening to identify fragments binding to BACE-1 in the presence of a bound blocking potent molecule.145 This approach is also known as second-site screening and is used to probe regions of the target’s binding pocket not occupied by the known binder used to block one or more regions of the protein. Compound 14 (KD ~ 16 nM) was used in the screening as a blocking molecule (see Figure 16). It occupies most of the binding site of BACE-1 while leaving the S3 and the S3subpocket regions accessible. Seven fragments exhibited binding in the presence of compound 14, three of these with affinities in the 100-300 µM range, as assessed by SPR. Interestingly, the highest affinity compound 15 (KD = 114 μM) did not bind in the absence of the blocking molecule 14. The identification of molecule 15 as second-site binding fragment made feasible the fragment-linking approach, leading to the designed molecule 17 with increase in potency and improved selectivity (~2000-fold) over cathepsin-D in both enzymatic and cellular assays.



F

O N H

X N

R NH2

Page 56 of 93

N

S1’

O

Cl

14: X=N, R=Me KD SPR = 16 nM 16: X=C, R=H KD SPR = 140 nM

H N

S3sp

15: KD SPR = 114 M F

S3

H N

N

O

S1

S2’

O N H

N

NH2

17: KD SPR = 0.4 nM

Figure 16. X-ray crystal structure of molecule 16 bound to BACE-1 (PDB ID 5IE1, orange) overlaid with X-ray crystal structure of molecule 17 bound to BACE-1 (PDB ID 5I3Y, green). It is worth noting that the toluyl methyl group of molecule 16 aims directly to the S3 pocket and the fluorophenyl motif of molecule 17 extends deeply into the S3subpocket (labelled as S3sp), affording increased potency against BACE-1 and additional selectivity against cathepsin-D.

HSP90. Pharmacological inhibition of Heat shock protein 90 (HSP90) ATPase activity causes the degradation of several client proteins in vivo, and represents a favorable approach for the development of novel cancer drugs. Many fragment-based screening campaigns against this target have been reported in literature. In one of these, 19F NMR was extensively used to identify binders and suitable reporters.146 First, a subset of 300 fluorinated fragments was screened using the FAXS in direct format against the N-terminal domain of human HSP90α leading to the detection of several fluorinated binders. Two of these binders, i.e. compounds 18 and 19, competitive with ATP, had KI of 29.6 and 7.5 μM, respectively, as measured in a fluorescence polarization (FP) assay.


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F

CF3 NH2

N

N O

N O

N

NH2

O 19: Ki FP = 7.5 M

18: Ki FP = 29.6 M NH2 N

N N

NH2

NH2

N

R N

N

N

N R

N

20: Ki FP = 37.2 M Ki FAXS NMR = 32.2 M KD ITC = 32.4 M

HO

23: R=OH Ki FAXS NMR= 6.1 M 24: R=OCH3 Ki FAXS NMR = 2.6 M

N H

N

N

O O

25: Ki FP = 1 nM

OH

OH

NO2

Cl HO

N

O

Cl

21: Ki FAXS NMR = 0.6 M

O

HO O N

22: KD SPR = 0.346 nM

NH O N

Compound 19 was finally selected as reporter and used to screen a library of ~ 1200 fragments in mixtures of ten with the FAXS competitive binding assay. 23 ligands were identified (1.9% hit rate) and X-ray crystal structures were obtained for four of them. It is worth noting that the solubility of fragment 19 in aqueous buffer was only ~ 15 µM. Despite this low solubility it could be identified in the FAXS in direct mode and served as a well suited reporter for the FAXS in competition mode. These experiments were performed using only a 6 µM concentration of compound 19 i.e. the spy molecule and a protein concentration of 0.6 µM. The screening identified compound 20 with a KI value of ~ 32 μM (by FP, FAXS-NMR and ITC methods), and compound 21 with a KI value of 0.6 μM calculated by FAXS-NMR (the KI value by FP was not measurable due to interference with fluorescence) which were further pursued for structure-based 57 ACS Paragon Plus Environment


Page 58 of 93

optimization. Compound 21 was optimized to compound 22 (see Figure 17a for their X-ray bound structures to HSP90), which showed subnanomolar affinity and high discrimination towards kinases and the ATPase Hsc70.147 It showed potent anti-proliferative action in tumor cell lines and a satisfactory pharmacokinetic profile, with selective retention in tumor tissue. Furthermore the compound displayed tumor shrinkage in several human tumor xenografts and, thanks to its ability to cross the blood brain barrier, was very active in an intracranially implanted melanoma model.148 The X-ray crystal structure of the aminotriazoloquinazoline fragment 20 (see Figure 17b) guided structure-based designs leading to the promising compound 25 with a > 30000-fold gain in binding affinity. Intriguingly, the crystal structure determination of the synthetic intermediate 24 showed an unexpected flipped binding mode (see Figure 17c) opening the opportunities to decorate the same initial scaffold in different directions, thus covering different chemical space. It is worth mentioning that FAXS was extensively used in the lead optimization of both chemical classes for rapidly measuring the binding affinity of the newly synthesized molecules. Furthermore, it is worth noting that, although the initial fragment used as spy molecule contained a fluorine group, the fragments originating from the

19F

NMR FAXS in competition mode and the optimized potent

ligands do not contain fluorine atoms. a

b

c

Figure 17. (a) X-ray crystal structure of compound 21 (PDB ID 4BQG, cyan) and 22 (PDB ID 4B7P, white) bound to HSP90. (b) X-ray crystal structure of compound 23 (PDB ID 4CWO,


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yellow) and 21 (PDB ID 4BQG, cyan) bound to HSP90. Notice that the resorcinol moieties of both compounds are nicely overlaid in the adenonise pocket. (c) X-ray crystal structure of compound 23 (PDB ID 4CWO, yellow) and 24 (PDB ID 4CWN, purple) bound to HSP90. Notice that the compounds, despite having very similar structures, assume a different binding mode. Waters molecules are not shown, for clarity.

TERRA.

19F

NMR screening was also reported for the identification of chemical diverse

compounds binding to telomeric repeat-containing RNA (TERRA).149 Telomeric RNA is an important constituent of the telomere apparatus. There is growing evidence that TERRA molecules form G-quadruplex structures and play a pivotal function in telomere protection and control. The screening of the diverse Spanish National Cancer Research Center (CNIO) fluorinated library of 355 fragments against a telomeric RNA composed of 16 r(UUAGGG) repeats (TERRA16) provided 20 hits (5.6% hit rate). The library was screened in mixtures of eight fluorinated molecules each. Seven hits were chosen for further analysis. These were confirmed to bind against TERRA16 by 1H NMR STD experiments and were also screened by target-observed NMR method against a shorter construct i.e. the telomeric RNA sequence 5′-r(UAGGGUUAGGGU)-3′ (TERRA2). Six of the seven ligands resulted to bind to TERRA2 with KD values in the 120-1900 μM range. Circular dicroism melting curves of TERRA2 alone and in the presence of the seven selected hits confirmed the interaction. Only one of the seven hits tested against phenylalanine tRNA (tRNAPhe) was positive (later shown to be an aggregator, false positive), while all the seven interacted with the DNA analogue of TERRA2 5’-d(TAGGGTTAGGGT)-3′ (dTEL2). dTEL2 has been extensively characterized by NMR and crystallography showing the existence of two interconverting G-quadruplex conformers: a parallel propeller-like structure and an antiparallel structure.150 Interestingly, the presence of the fluorinated ligands appeared to move the equilibrium 59 ACS Paragon Plus Environment


of the DNA to the parallel structure, which is the conformer observed in the RNA G-quadruplexes, implying a specific recognition of these binders towards this structural fold. Factor D. 19F NMR screening of the Novartis fluorinated fragment library, known as LEF library, was carried out against Factor D (FD), a serine protease of the S1 family implicated in the alternative complement pathway. Five very weak binding affinity fragments were identified (0.35% hit rate) which could not be validated by other biophysical techniques.151 As explained above, the experimental conditions and the sensitivity to protein binding of the various biophysical techniques are different with respect to the FAXS in direct format. The lack of validation of the 19F

NMR identified hits by other biophysical methods should be judiciously examined. Therefore,

the initial hits of which two (26 and 27) are shown in Figure 18 were pursued by SAR by archive activities with the aim of improving their potency. About 50 close analogs were screened in the 19F

NMR FAXS in competition mode using compound 28 as the fluorinated reporter. Compound

29, a racemic mixture of trans/cis diastereoisomers, was the only one for which the FD crystal structure could be obtained, showing the trans (1S,2S) isomer bound. The racemic mixture showed a KD value of ~ 500 μM obtained from the 19F NMR reporter assay and its binding could also be detected in the 2D 1H-15N HSQC measurement. The key interactions between FD and compound 29 perfectly mimic the interactions observed for the more potent compound 28 (see Figure 18): HBs to the backbone of Gly193 and Leu41 and occupancy of the hydrophobic hot spot located at the S2’ region. A fragment hopping approach was consecutively pursed by using molecular-fields 3D similarity search against the Novartis archive. The carefully selection of five analogs resembling the described key pharmacophoric binding elements provided an additional fragment (compound 30) with similar binding affinity, but belonging to a different chemical series. The key binding element of compound 29 was then chemically merged with the proline-lead series (as


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exemplified by compound 28) resulting in very potent non-covalent and orally bioavailable FD inhibitors.152 NH2

F

N F

N N H 26

HN

O

N

N H N

N

O

OH

H N

O

OCF3

O

28: IC50 = 14 M

27 F O HN

HO

29: KI 19F NMR ~500 µM

S215

L41

O N H

N H O

S1’

O

HO

G193

N H

S1

O

S2’

30: KI 19F NMR ~500 µM

Figure 18. X-ray crystal structures of compound 28 (PDB ID 5FBE, cyan) and 29 (PDB ID 5MT0, yellow) bound to Factor D. Notice that the two molecules share the same key pharmacophoric features: HB with the NH of Gly193, HB with the C=O of Leu41 and occupancy of the hydrophobic hot spot at the S2’ sub-pocket.

Trypsin. The Novartis fluorinated fragment library, known as LEF, was also screened against several other protease targets always resulting in significantly higher hit rates with respect to the low Factor D hit rate. For example, the 19F NMR screening of this library against bovine pancreas trypsin resulted in 31 hits (2.2% hit rate) of which 40% were solved by X-ray crystal structure determination.153 Only partial 19F signal attenuation was observed for molecule 31 in the presence of the protein due to its very weak binding affinity. As a consequence, molecule 31 could not be validated by other biophysical techniques. However, despite its very weak affinity, it could be detected with the FAXS experiment and its binding mode elucidated by X-ray crystallography (see Figure 19) revealing the usefulness of the approach in delivering weak but novel binders.



Page 62 of 93

S214 S195

O HN

CF3

31: 0% Inhibition at 1 mM

G218

Figure 19. X-ray crystal structure of compound 31 bound to trypsin (PDB ID 3NK8). The CF3 moiety is involved into multipolar interaction with the carbonyl of the backbone of Ser214. In addition, it is close to the OH of Ser195. The nitrogen on the lactam ring is at HB distance to the carbonyl of Gly218, indicating that is the oxo tautomer of 2-pyridone and not the hydroxyl form bound to trypsin, in agreement with previous findings.154

C-type lectin receptors (CTLRs). CTLRs play an important role in recognizing pathogen associated molecules and in regulating the immune response. However, only few drug-like compounds have been developed for this protein family. The screening of a library of 281 fluorinated fragments by

19F

NMR in eight mixtures of maximum 36 compounds against the

Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) target resulted in a high hit rate (13.5%) of binders in Ca2+-associated sites.155 SPR confirmed 18 of these (47%). Hits not validated by SPR were either super-stoichiometric binders, not competitive with Ca2+ or had binding affinities below the detection threshold of SPR. Interestingly, 62 ACS Paragon Plus Environment

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three of the identified hits (32-34) are analogs to substructures of the previously described DCSIGN inhibitor 35.156 CF3

O

NH

H N

N HN 32: KD DC-SIGN = 2.6 mM

F

33: KD DC-SIGN = 2.2 mM O N

O N

H N

F

O

O

34: KD DC-SIGN = 2.1 mM

O S

N H 35: KD DC-SIGN = 2 M

19F

NMR screening towards other CTLRs (Langerin and MCL) revealed analogously high hit rates

(15.7% and 10.0%, respectively).157 N-acetylmannosamine kinase (MNK). This key enzyme, involved in the sialic acid biosynthesis, is an important regulator of cell surface sialylation in mammals, a mechanism found in several diseases from infection to tumor immune evasion. The enzyme catalyzes the phosphorylation of ManNAc to ManNAc-6-phosphate in the presence of ATP. The same library used towards the previous reported example on CTLRs was screened by 19F NMR against MNK.158 The addition of ManNAc and ATP revealed eight fragments to be competitive (2.8% hit rate). Of these, 4(trifluoromethyl)picolinic acid 36, was confirmed in a biochemical assay with 900 μM activity. More potent inhibitors were generated, see for example compound 37, in the absence of crystallographic information.



Page 64 of 93

CF3 N N

COOH

N

COOH

O

36: KD MNK = 900 M

37: KD MNK = 80 M

Nuclear receptor Nurr1. This receptor is essential in the development of midbrain dopaminergic neurons in the central nervous system and could represent a relevant target for the treatment of Parkinson’s disease. A library of approximately 1000 fluorinated compounds (with MW up to 500 Da) was screened by 19F NMR and the hits were further evaluated for their binding strength using 1H

STD and 2D 1H-15N HSQC NMR experiments. Four compounds showed a KD < 500 µM by

1H

STD. Follow up of the best hit (compound 38, KD = 100 µM) by using a second site screening

approach resulted in the discovery of a ligand, molecule 39, with a 5-fold improvement in binding affinity.159 OH N N

N

NH N

N H

N N H

F

F 38: KD Nurr1 = 100 M

39: KD Nurr1 = 20 M

The following examples describe some applications of the FAXS in competition mode. This strategy is broadly applicable to many drug discovery efforts. Phosphopantetheine adenylyltransferase (PPAT) This enzyme is an attractive antibacterial target, responsible for catalyzing the penultimate step in coenzyme A (CoA) biosynthesis, i.e. the transfer of an AMP motif from ATP to phosphopantetheine (PhP) generating dephosphocoenzyme A (dPCoA) and inorganic pyrophosphate. HTS and STD-NMR fragment screen were carried out 64 ACS Paragon Plus Environment

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against this target and the hits were validated and characterized using

19F

NMR as biophysical

triage tool.160 Two weakly binding fluorinated fragments derived from the STD-NMR fragment screen, were selected as spy molecules to categorize the HTS hits as ATP site binders versus no ATP site binders. Fragments 40 and 41 had PPAT IC50 > 100 µM and showed a CF3 19F NMR signals at 76.56 and -76.08 ppm, respectively. In the presence of PPAT protein, both 19F signals disappeared. The addition of ATP resulted in the selective reappearance of the 19F signal at -76.56 ppm thus suggesting that compound 40 was an ATP site binder whereas compound 41 was binding on another site of the protein. This knowledge was then used in the 19F FAXS experiments performed in competition mode to classify HTS hits according to their protein binding site. For example compound 42 was selectively competing with the ATP probe fragment 40 thus classifying it as an ATP site binder. O O

Cl

O N

O

O

CF3

40: IC50 PPAT > 100 M  19F = -76.56 ppm

O

N H

Br

N N

CF3

41: IC50 PPAT > 100 M  19F = -76.08 ppm

COOH

N H 42: IC50 E. coli PPAT = 6.2 M

Choline Kinase. Choline kinase (ChoK) is a cytosolic enzyme that catalyzes the MgATPdependent phosphorylation of choline to phosphocholine (pCho). This represents the first step in the Kennedy pathway responsible for the biosynthesis of phosphatidylcholine (PtdCho), one of the major lipid component of biological membranes. ChoK is regarded as an attractive cancer target. FAXS in competition mode was used with either the 2-fluoroadenosine (43, KD = 165 μM) or fluorocholine chloride (44, KD = 113 μM) as spy molecules to monitor binding to ATP and 65 ACS Paragon Plus Environment


substrate sites, respectively.161 55 fragment hits were examined with 13 showing competition with the choline site reporter 44 and 21 displaying competition with the ATP site reporter 43. One of these fragments i.e. compound 45 (KD = 132 μM) predicted by the FAXS competition experiment to bind to the choline site was selected for crystallization. The X-ray structure confirmed the binding at the choline site situated in the predominantly helical C-lobe and provided key structural information (see Figure 20). Optimization of this fragment using structure-guided approaches led to highly potent inhibitors of ChoK with KD in the nM range (e.g. compound 46). In cancer cell lines, the lead molecules show a dose-dependent drop of phosphocholine, cell growth inhibition and initiation of apoptosis at low micromolar concentrations.161

NH2 N HO

O N

N N

F HO

N+

N F

N

NH2

HO OH 43: KD ChoK = 165 M

44: KD ChoK = 113 M

N

45: KD ChoK = 132 M

CN

N

N N

46: KD ChoK = 0.01 M

Figure 20. X-ray crystal structures of ChoK in complex with compound 45 (PDB ID 5EQE, cyan) and 46 (PDB ID 5EQY, yellow).

Abelson kinase. Abelson kinase (Abl1) is a non-receptor tyrosine kinase with two druggable pockets: the ATP and the myristate pocket. Abl1 kinase inhibitors targeting the ATP binding pocket are currently used to treat chronic myelogenous leukemia (CML), but their use can be 66 ACS Paragon Plus Environment

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significantly limited due to drug resistance (especially due to the T315I mutation). As an alternative, the inactive kinase conformation can be induced with molecules binding at the myristate pocket. Suitable fluorinated reporters for both pockets were generated by screening the subset of the Novartis LEF library of fragments containing a CF3 moiety (540 fragments) against the SH3SH2-SH1 Abl1(83-534) construct.162 The binding of fragment 47 and 48 was detected by the 19F

signal intensity reduction in the presence of the target. The selective recovery of the CF3

19F

signal of fragment 47 and 48 upon addition of the potent allosteric inhibitor Asciminib

(ABL001) and then Imatinib indicated their exclusive binding to either the myristate or ATP pocket, respectively. The two reporters in the

19F

NMR competition assay were used to characterize known Abl1

inhibitors, such as Crizotinib and Ingolimod against wild type and mutated Abl1 (T315I). O N F3C

N H

N

47: IC50 Abl1 = 43 M  19F = -61.06 ppm

NH2 N

N N

N H

CF3

48: IC50 Abl1 = 380 M  19F = -64.15 ppm

MAP kinase extracellular regulated kinase 2 (ERK2).

19F

NMR screening against ERK2, a

target for anticancer drugs development, provided a high hit rate.163 19 fluorinated hits were identified by screening a fluorinated library of ~ 500 fragments. These were subsequently triaged using two non-fluorinated binders of ERK2 to get insight about the binding sites occupied by the hit fragments. 15 out of the 19 fluorinated hits compete with both or just one of the two ATP ERK2 known binders whereas four compounds resulted as non-competitive binders. 67 ACS Paragon Plus Environment


Ribonucleic acid. Fluorinated reporters in competition binding experiments were applied to the detection and quantifications of the binding of non-fluorinated ligands to Ribonucleic acid (RNA).164 Two fluorinated reporters were assessed against 16S23 RNA: compound 49, a racemic analog of Desoxystreptamine and compound 50, a fluorinated truncated analog of Paromomycin. The two used fluorinated reporters have 2 mM and 300 μM affinity, thus covering a broad spectrum of affinity. The ranking of the KD values obtained by 19F NMR FAXS experiments for Neamine > Paromamine > Neomycin, known binders of 16S23 RNA, were in agreement with published data determined by other techniques. The two fluorinated spy molecules were also used to monitor RNA conformational changes upon ligand binding. Compound 49 binding does not alter the conformational equilibrium observed for the Neomycin aptamer target in the absence of Neomycin whereas compound 50 results in a shift toward the conformation observed in the presence of Neomycin. F HO HO H 2N

H 2N NH2 F rac-49: KD 16S23 RNA = 2 mM

O NH2

O HO HO

NH2

50: KD 16S23 RNA = 300 M

SH2 domain of v-Src. Inhibition of the function of the SH2 domain has been considered a possible tactic to modulate tyrosine kinase signaling. Fragments potentially binding at the phosphotyrosine binding site of this domain were selected by virtual screening and tested by 19F NMR FAXS in competition mode. This process resulted in the identification of two new ligands (51 and 52) whose binding was then characterized by ITC.165


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CF3

O

H N

O OH P O OH

O

Br Cl

51: KD SH2 = 177 M

O O P OH OH

52: KD SH2 = 104 M

Glucokinase (GK). This enzyme, also known as hexokinase D or hexokinase IV, catalyzes the phosphorylation of glucose to glucose 6-phosphate. Some research efforts aim at developing molecules that enhance GK activity as an innovative remedy for the treatment of type 2 diabetes. A suitable spy molecule was synthesized (compound 53), and used then to perform the FAXS experiment in competition mode on a series of potential binders. A close correlation between the 19F

signal intensity recovery of the spy molecule and the % of enzyme activation determined in an

enzymatic assay was established for the different binders.166 COOH

O O

O

N H

N

O F

53

Maltose-binding protein (MBP). This protein is a member of the periplasmic binding proteins family, which are implicated in active transport processes of small molecules into gram-negative bacteria. FAXS in competition mode with the 2-F-labeled maltose as a spy molecule was used to probe in an indirect mode protein–ligand or protein–protein interactions of proteins fused or tagged to the MBP. The 19F NMR signals of both gluco/manno-type-2-F-maltose-isomers (molecules 54 and 55) were observed: the α-gluco-type isomer binds to MBP while the nonbinding isomers (β-



gluco- and/or α/β-manno-type) act as internal references. This approach was also applied for studying the relative binding affinities of natural and artificial maltose derivatives to MBP.167

Glc

OH

Glc

O

O HO

F

OH

O HO

OH F O OH

Gluco-type isomer

Manno-type isomer

54

55

CONCLUSION In summary we have discussed the principles and reported several published applications of the 19F

ligand-based NMR screening method performed in direct and competition mode. The easy set-

up of the methodology along with its flexibility and high sensitivity to binding allows its application to a broad range of simple and complex biological and chemical systems. Many academic institutions and pharmaceutical companies have now invested in the methodology and apply it routinely in their projects. The novel NMR method for binding constant measurements reported in this work promises to be very useful in the rapid assess of the binding strength of the identified hits and their selected and synthesized close analogues. In the future there will be more successful stories originating from the 19F ligand-based NMR screening effort. It is envisaged that thanks to recent technological developments and commercially and in-house available fluorinated libraries the methodology will increasingly contribute to the hit identification and hit to lead optimization phases of drug discovery projects.


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AUTHORS INFORMATION Corresponding authors: 1C.

Dalvit, E-mail: [email protected];

2A.

Vulpetti, Phone 0041 793339044 E-mail:

[email protected]

Biography Claudio Dalvit studied biophysics at the University of Trento, Italy, and trained as postdoctoral researcher at the Carnegie-Mellon University, The Scripps Research Institute, and the University of Lausanne. He joined Sandoz (now Novartis) in 1989 as lab head prior to his position in 1999 as head of the biomolecular NMR group at Pharmacia & Upjohn and then senior scientist at the Italian Institute of Technology. From 2011 to 2015 he worked as senior NMR expert and associate professor at the University of Neuchâtel. His research interests are in NMR spectroscopy, from methodology development to its applications, enzymology and fragment-based drug discovery. In 2006 he was awarded with the gold medal of the Italian Society of Magnetic Resonance. Anna Vulpetti received her PhD degree in Chemistry from the University of Milan (Italy) in 1994 working on the development of a new synthetic route for Taxol side-chain synthesis. In 1995 she joined the Computer Aided Drug Design group of Pharmacia & Upjohn, and in 2006 she moved to Novartis, where, as Senior Investigator, she supports drug discovery projects in multiple disease areas and develops computational methods. Anna’s research interests include the development of novel methods for describing and comparing binding sites, fragment-based drug discovery, and the better understanding of the role of fluorine in drug design. In 2005 she was awarded with the Farmindustria Prize for Young Scientists excelling in Pharmaceutical Chemistry.



ABBREVIATIONS USED CSA, Chemical Shift Anisotropy; DOSY, Diffusion Ordered SpectroscopY; FAXS, Fluorine chemical shift Anisotropy and eXchange for Screening; FBDD, Fragment Based Drug Discovery; FP, Fluorescence Polarization; HB, Hydrogen Bond; HSA, Human Serum Albumin; HTS, High Throughput Screening; ITC, Isothermal Titration Calorimetry; LEF, Local Environment of Fluorine; NMR, Nuclear Magnetic Resonance; PDB, Protein Data Bank; PrOF, Protein Observed Fluorine; SAR, Structure-Activity Relationship; SPAM, Solubility Purity and Aggregation of the Molecule; SPR, Surface Plasmon Resonance; STD, Saturation Transfer Difference; WaterLOGSY, Water Ligand Observed by Gradient SpectroscopY.

ASSOCIATED CONTENT Supporting Information Figure S1

19F

NMR DQ screening in direct and competition format,

Figure S2 19F NMR DOSY screening, Annex S3 FAXS NMR theory, Figure S4 19F NMR pulse sequences for screening, Figure S5 and S6 NMR characterization of molecules undergoing transient intramolecular HB, Annex S7 derivation of equations (8-10).

REFERENCES

(1) O'Hagan, D. Fluorine in health care organofluorine containing blockbuster drugs. J. Fluorine Chem. 2010, 131, 1071-1081.


Page 72 of 93

Page 73 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(2) Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of fluorine in medicinal chemistry. J. Med. Chem. 2015, 58, 8315-8359. (3) Kirk, K. L. Fluorine in medicinal chemistry: recent therapeutic applications of fluorinated small molecules. J. Fluorine Chem. 2006, 127, 1013-1029. (4) Bohm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Müller, K.; Obst-Sander, U.; Stahl, M. Fluorine in medicinal chemistry. ChemBioChem 2004, 5, 637-643. (5) Dolbier, W. R. Fluorine chemistry at the millennium. J. Fluorine Chem. 2005, 126, 157-163. (6) Hagmann, W. K. The many roles for fluorine in medicinal chemistry. J. Med. Chem. 2008, 51, 4359-4369. (7) Huchet, Q. A.; Kuhn, B.; Wagner, B.; Kratochwil, N. A.; Fischer, H.; Kansy, M.; Zimmerli, D.; Carreira, E. M.; Müller, K. Fluorination patterning: a study of structural motifs that impact physicochemical properties of relevance to drug discovery. J. Med. Chem. 2015, 58, 9041-9060. (8) Isanbor, C.; O'Hagan, D. Fluorine in medicinal chemistry: a review of anti-cancer agents. J. Fluorine Chem. 2006, 127, 303-319. (9) Müller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: looking beyond intuition. Science 2007, 317, 1881-1886. (10) Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc. Rev. 2008, 37, 320-330. (11) Swallow, S. Fluorine in Medicinal Chemistry. In Progress in Medicinal Chemistry, Lawton, G.; Witty, D. R., Eds.; Elsevier: 2015; Vol. 54, pp 65-133. (12) Wang, J.; Sanchez-Rosello, M.; Acena, J. L.; del Pozo, C.; Sorochinsky, A. E.; Fustero, S.; Soloshonok, V. A.; Liu, H. Fluorine in pharmaceutical industry: fluorine-containing drugs introduced to the market in the last decade (2001-2011). Chem. Rev. 2014, 114, 2432-2506.



(13) Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Acena, J. L.; Soloshonok, V. A.; Izawa, K.; Liu, H. Next generation of fluorine-containing pharmaceuticals, compounds currently in phase IIIII clinical trials of major pharmaceutical companies: new structural trends and therapeutic areas. Chem. Rev. 2016, 116, 422-518. (14) Alonso, C.; Martinez de Marigorta, E.; Rubiales, G.; Palacios, F. Carbon trifluoromethylation reactions of hydrocarbon derivatives and heteroarenes. Chem. Rev. 2015, 115, 1847-1935. (15) Campbell, M. G.; Ritter, T. Modern carbon-fluorine bond forming reactions for aryl fluoride synthesis. Chem. Rev. 2015, 115, 612-633. (16) Champagne, P. A.; Desroches, J.; Hamel, J. D.; Vandamme, M.; Paquin, J. F. Monofluorination of organic compounds: 10 years of innovation. Chem. Rev. 2015, 115, 90739174. (17) Charpentier, J.; Fruh, N.; Togni, A. Electrophilic trifluoromethylation by use of hypervalent iodine reagents. Chem. Rev. 2015, 115, 650-682. (18) Fujiwara, Y.; Dixon, J. A.; Rodriguez, R. A.; Baxter, R. D.; Dixon, D. D.; Collins, M. R.; Blackmond, D. G.; Baran, P. S. A new reagent for direct difluoromethylation. J. Am. Chem. Soc. 2012, 134, 1494-1497. (19) Fustero, S.; Simon-Fuentes, A.; Barrio, P.; Haufe, G. Olefin metathesis reactions with fluorinated substrates, catalysts, and solvents. Chem. Rev. 2015, 115, 871-930. (20) Molnar, I. G.; Gilmour, R. Catalytic difluorination of olefins. J. Am. Chem. Soc. 2016, 138, 5004-5007. (21) Ni, C.; Hu, M.; Hu, J. Good partnership between sulfur and fluorine: sulfur-based fluorination and fluoroalkylation reagents for organic synthesis. Chem. Rev. 2015, 115, 765-825.


Page 74 of 93

Page 75 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(22) O'Hagan, D.; Deng, H. Enzymatic fluorination and biotechnological developments of the fluorinase. Chem. Rev. 2015, 115, 634-649. (23) Prakash, G. K.; Hu, J. Selective fluoroalkylations with fluorinated sulfones, sulfoxides, and sulfides. Acc. Chem. Res. 2007, 40, 921-930. (24) Artamonov, O. S.; Slobodyanyuk, E. Y.; Volochnyuk, D. M.; Komarov, I. V.; Tolmachev, A. A.; Mykhailiuk, P. K. Synthesis of trifluoromethyl-substituted 3-azabicyclo[n.1.0]alkanes: advanced building blocks for drug discovery. Eur. J. Org. Chem. 2014, 2014, 3592-3598. (25) Meanwell, N. A. Fluorine and fluorinated motifs in the design and application of bioisosteres for drug design. J. Med. Chem. 2018, 61, 5822-5880. (26) Dalvit, C. Ligand- and substrate-based

19F

NMR screening: principles and applications to

drug discovery. Prog. Nucl. Magn. Reson. Spectrosc. 2007, 51, 243-271. (27) Vulpetti, A.; Dalvit, C. Fluorine local environment: from screening to drug design. Drug Discovery Today 2012, 17, 890-897. (28) Chen, H.; Viel, S.; Ziarelli, F.; Peng, L. 19F NMR: a valuable tool for studying biological events. Chem. Soc. Rev. 2013, 42, 7971-7982. (29) Norton, R. S.; Leung, E. W.; Chandrashekaran, I. R.; MacRaild, C. A. Applications of 19FNMR in fragment-based drug discovery. Molecules 2016, 21, 860-873. (30) Sugiki, T.; Furuita, K.; Fujiwara, T.; Kojima, C. Current NMR techniques for structure-based drug discovery. Molecules DOI: 10.3390/molecules23010148. Published Online: 12 January 2018. (31) Campos-Olivas, R. NMR screening and hit validation in fragment based drug discovery. Curr. Top. Med. Chem. 2011, 11, 43-67.



(32) Canales, M. Á.; Espinosa, J. F. Ligand-detected NMR Methods in Drug Discovery. In Biophysical Techniques in Drug Discovery; Canales, A. Ed.; The Royal Society of Chemistry: 2018; pp 23-43. (33) Urick, A. K.; Hawk, L. M.; Cassel, M. K.; Mishra, N. K.; Liu, S.; Adhikari, N.; Zhang, W.; dos Santos, C. O.; Hall, J. L.; Pomerantz, W. C. Dual screening of BPTF and Brd4 using proteinobserved fluorine NMR uncovers new bromodomain probe molecules. ACS Chem. Biol. 2015, 10, 2246-2256. (34) Urick, A. K.; Calle, L. P.; Espinosa, J. F.; Hu, H.; Pomerantz, W. C. Protein-observed fluorine NMR is a complementary ligand discovery method to 1H CPMG ligand-observed NMR. ACS Chem. Biol. 2016, 11, 3154-3164. (35) Gee, C. T.; Arntson, K. E.; Urick, A. K.; Mishra, N. K.; Hawk, L. M. L.; Wisniewski, A. J.; Pomerantz, W. C. K. Protein-observed

19F-NMR

for fragment screening, affinity quantification

and druggability assessment. Nat. Protoc. 2016, 11, 1414. (36) Arntson, K. E.; Pomerantz, W. C. Protein-observed fluorine NMR: a bioorthogonal approach for small molecule discovery. J. Med. Chem. 2016, 59, 5158-5171. (37) Marsh, E. N.; Suzuki, Y. Using

19F

NMR to probe biological interactions of proteins and

peptides. ACS Chem. Biol. 2014, 9, 1242-1250. (38) Gerig, J. T. Fluorine NMR of proteins. Prog. Nucl. Magn. Reson. Spectrosc. 1994, 26, 293370. (39) Sharaf, N. G.; Gronenborn, A. M. 19F-Modified Proteins and 19F-containing Ligands as Tools in solution NMR Studies of Protein Interactions. In Methods in Enzymology, Kelman, Z., Ed.; Elsevier: 2015; Vol. 565, pp 67-95.


Page 76 of 93

Page 77 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(40) Kitevski-LeBlanc, J. L.; Prosser, R. S. Current applications of 19F NMR to studies of protein structure and dynamics. Prog. Nucl. Magn. Reson. Spectrosc. 2012, 62, 1-33. (41) Salwiczek, M.; Nyakatura, E. K.; Gerling, U. I.; Ye, S.; Koksch, B. Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions. Chem. Soc. Rev. 2012, 41, 2135-2171. (42) Merkel, L.; Budisa, N. Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes. Org. Biomol. Chem. 2012, 10, 7241-7261. (43) Luck, L. A.; Falke, J. J. Fluorine-19 NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change. Biochemistry 2002, 30, 4248-4256. (44) Leung, E. W.; Yagi, H.; Harjani, J. R.; Mulcair, M. D.; Scanlon, M. J.; Baell, J. B.; Norton, R. S.

19F

NMR as a probe of ligand interactions with the iNOS binding site of SPRY domain-

containing SOCS box protein 2. Chem. Biol. Drug Des. 2014, 84, 616-625. (45) Kitevski-Leblanc, J. L.; Evanics, F.; Scott Prosser, R. Approaches to the assignment of 19F resonances from 3-fluorophenylalanine labeled calmodulin using solution state NMR. J. Biomol. NMR 2010, 47, 113-123. (46) Fielding, L. NMR methods for the determination of protein-ligand dissociation constants. Prog. Nucl. Magn. Reson. Spectrosc. 2007, 51, 219-242. (47) Kreutz, C.; Kahlig, H.; Konrat, R.; Micura, R. A general approach for the identification of site-specific RNA binders by 19F NMR spectroscopy: proof of concept. Angew. Chem., Int. Ed. 2006, 45, 3450-3453. (48) Kreutz, C.; Micura, R. Investigations on Fluorine-labeled Ribonucleic Acids by

19F

NMR

Spectroscopy. In Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P., Ed.; Wiley-VCH: Weinheim, 2008; pp 3-28.



(49) Scott, L. G.; Hennig, M.

19F-site-specific-labeled

Nucleotides for Nucleic Acid Structural

Analysis by NMR. In Methods in Enzymology, Kelman, Z., Ed.; Elsevier: 2016; Vol. 566, pp 5987. (50) Sochor, F.; Silvers, R.; Muller, D.; Richter, C.; Furtig, B.; Schwalbe, H. 19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy. J. Biomol. NMR 2016, 64, 63-74. (51) Medvecky, M.; Istrate, A.; Leumann, C. J. Synthesis and properties of 6'-fluoro-tricycloDNA. J. Org. Chem. 2015, 80, 3556-3565. (52) Dalvit, C.; Ardini, E.; Flocco, M.; Fogliatto, G. P.; Mongelli, N.; Veronesi, M. A general NMR method for rapid, efficient, and reliable biochemical screening. J. Am. Chem. Soc. 2003, 125, 14620-14625. (53) Dalvit, C.; Ardini, E.; Fogliatto, G. P.; Mongelli, N.; Veronesi, M. Reliable high-throughput functional screening with 3-FABS. Drug Discovery Today 2004, 9, 595-602. (54) Frutos, S.; Tarrago, T.; Giralt, E. A fast and robust 19F NMR-based method for finding new HIV-1 protease inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 2677-2681. (55) Kichik, N.; Tarrago, T.; Giralt, E. Simultaneous 19F NMR screening of prolyl oligopeptidase and dipeptidyl peptidase IV inhibitors. ChemBioChem 2010, 11, 1115-1119. (56) Stockman, B. J. 2-fluoro-ATP as a versatile tool for 19F NMR-based activity screening. J. Am. Chem. Soc. 2008, 130, 5870-5871. (57) Forino, M.; Johnson, S.; Wong, T. Y.; Rozanov, D. V.; Savinov, A. Y.; Li, W.; Fattorusso, R.; Becattini, B.; Orry, A. J.; Jung, D.; Abagyan, R. A.; Smith, J. W.; Alibek, K.; Liddington, R. C.; Strongin, A. Y.; Pellecchia, M. Efficient synthetic inhibitors of anthrax lethal factor. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 9499-9504.


Page 78 of 93

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(58) Berkowitz, D. B.; Karukurichi, K. R.; de la Salud-Bea, R.; Nelson, D. L.; McCune, C. D. Use of fluorinated functionality in enzyme inhibitor development: mechanistic and analytical advantages. J. Fluorine Chem. 2008, 129, 731-742. (59) Rydzik, A. M.; Leung, I. K.; Thalhammer, A.; Kochan, G. T.; Claridge, T. D.; Schofield, C. J. Fluoromethylated derivatives of carnitine biosynthesis intermediates - synthesis and applications. Chem. Commun. 2014, 50, 1175-1177. (60) Cobb, S. L.; Murphy, C. D. 19F NMR applications in chemical biology. J. Fluorine Chem. 2009, 130, 132-143. (61) Seitz, J. D.; Vineberg, J. G.; Wei, L.; Khan, J. F.; Lichtenthal, B.; Lin, C. F.; Ojima, I. Design, synthesis and application of fluorine-labeled taxoids as 19F NMR probes for the metabolic stability assessment of tumor-targeted drug delivery systems. J. Fluorine Chem. 2015, 171, 148-161. (62) Ojima, I. Strategic incorporation of fluorine into taxoid anticancer agents for medicinal chemistry and chemical biology studies. J. Fluorine Chem. 2017, 198, 10-23. (63) Dalvit, C. Fluorine NMR Spectroscopy for Biochemical Screening in Drug Discovery. In NMR of Biomolecules: towards Mechanistic Systems Biology; Bertini, I., McGreevy, K. S., Parigi, G., Eds.; Wiley-Blackwell: 2012; pp 314-327. (64) Veronesi, M.; Giacomina, F.; Romeo, E.; Castellani, B.; Ottonello, G.; Lambruschini, C.; Garau, G.; Scarpelli, R.; Bandiera, T.; Piomelli, D.; Dalvit, C. Fluorine nuclear magnetic resonance-based assay in living mammalian cells. Anal. Biochem. 2016, 495, 52-59. (65) Dalvit, C.; Flocco, M.; Veronesi, M.; Stockman, B. J. Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures. Comb. Chem. High Throughput Screening 2002, 5, 605-611.



Page 80 of 93

(66) Dalvit, C.; Fagerness, P. E.; Hadden, D. T.; Sarver, R. W.; Stockman, B. J. Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practical considerations, and range of applicability. J. Am. Chem. Soc. 2003, 125, 7696-7703. (67) Tengel, T.; Fex, T.; Emtenas, H.; Almqvist, F.; Sethson, I.; Kihlberg, J. Use of

19F

NMR

spectroscopy to screen chemical libraries for ligands that bind to proteins. Org. Biomol. Chem. 2004, 2, 725-731. (68) Gerig, J. T. Fluorine Nuclear Magnetic Resonance of Fluorinated Ligands. Methods in Enzymology; Oppenheimer, N. J., James, T. L., Eds.; Elsevier: 1989; Vol. 177, pp 3-23. (69) Claridge, T. D. High-Resolution NMR Techniques in Organic Chemistry; Elsevier: 2016 (70) Dolbier, W. R., Jr. Guide to Fluorine NMR for Organic Chemists; John Wiley and Sons: Hoboken, 2016. (71) Peng, C.; Frommlet, A.; Perez, M.; Cobas, C.; Blechschmidt, A.; Dominguez, S.; Lingel, A. Fast and efficient fragment-based lead generation by fully automated processing and analysis of ligand-observed NMR binding data. J. Med. Chem. 2016, 59, 3303-3310. (72) Diercks, T.; Ribeiro, J. P.; Canada, F. J.; Andre, S.; Jimenez-Barbero, J.; Gabius, H. J. Fluorinated carbohydrates as lectin ligands: versatile sensors in

19F

detected saturation transfer

difference NMR spectroscopy. Chem. – Eur. J. 2009, 15, 5666-5668. (73) Ribeiro, J. P.; Diercks, T.; Jimenez-Barbero, J.; Andre, S.; Gabius, H. J.; Canada, F. J. Fluorinated carbohydrates as lectin ligands:

19F-based

direct STD monitoring for detection of

anomeric selectivity. Biomolecules 2015, 5, 3177-3192. (74) Dalvit, C.; Piotto, M. 19F NMR transverse and longitudinal relaxation filter experiments for screening: a theoretical and experimental analysis. Magn. Reson. Chem. 2017, 55, 106-114.


Page 81 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(75) Furihata, K.; Usui, M.; Tashiro, M. Application of NMR screening methods with

19F

detection to fluorinated compounds bound to proteins. Magnetochemistry 2018, 4(1), 3. (76) Vulpetti, A.; Dalvit, C. Design and generation of highly diverse fluorinated fragment libraries and their efficient screening with improved

19F

NMR methodology. ChemMedChem 2013, 8,

2057-2069. (77) Buratto, R.; Mammoli, D.; Canet, E.; Bodenhausen, G. Ligand-protein affinity studies using long-lived states of fluorine-19 nuclei. J. Med. Chem. 2016, 59, 1960-1966. (78) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Discovering high-affinity ligands for proteins: SAR by NMR. Science 1996, 274, 1531-1534. (79) Hajduk, P. J.; Greer, J. A decade of fragment-based drug design: strategic advances and lessons learned. Nat. Rev. Drug Discovery 2007, 6, 211-219. (80) Erlanson, D. A.; Fesik, S. W.; Hubbard, R. E.; Jahnke, W.; Jhoti, H. Twenty years on: the impact of fragments on drug discovery. Nat. Rev. Drug Discovery 2016, 15, 605-619. (81) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. Recent developments in fragmentbased drug discovery. J. Med. Chem. 2008, 51, 3661-3680. (82) Erlanson, D. A.; McDowell, R. S.; O'Brien, T. Fragment-based drug discovery. J. Med. Chem. 2004, 47, 3463-3482. (83) Renaud, J. P.; Chung, C. W.; Danielson, U. H.; Egner, U.; Hennig, M.; Hubbard, R. E.; Nar, H. Biophysics in drug discovery: impact, challenges and opportunities. Nat. Rev. Drug Discovery 2016, 15, 679-698. (84) Erlanson, D. A.; Jahnke, W.; Mannhold, R.; Kubinyi, H.; Folkers, G. Fragment-based Drug Discovery: Lessons and Outlook. Wiley-VCH: Weinheim, 2015.



Page 82 of 93

(85) Dalvit, C. NMR methods in fragment screening: theory and a comparison with other biophysical techniques. Drug Discovery Today 2009, 14, 1051-1057. (86) Kovacs, H.; Moskau, D.; Spraul, M. Cryogenically cooled probes - a leap in NMR technology. Prog. Nucl. Magn. Reson. Spectrosc. 2005, 46, 131-155. (87) Dalvit, C.; Mongelli, N.; Papeo, G.; Giordano, P.; Veronesi, M.; Moskau, D.; Kummerle, R. Sensitivity improvement in 19F NMR-based screening experiments: theoretical considerations and experimental applications. J. Am. Chem. Soc. 2005, 127, 13380-13385. (88) Vulpetti, A.; Hommel, U.; Landrum, G.; Lewis, R.; Dalvit, C. Design and NMR-based screening of LEF, a library of chemical fragments with different local environment of fluorine. J. Am. Chem. Soc. 2009, 131, 12949-12959. (89) Carr, H. Y.; Purcell, E. M. Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys. Rev. 1954, 94, 630-638. (90) Meiboom, S.; Gill, D. Modified spin-echo method for measuring nuclear relaxation times. Rev. Sci. Instrum. 1958, 29, 688-691. (91) Takegoshi, K.; Ogura, K.; Hikichi, K. A perfect spin echo in a weakly homonuclear J-coupled two spin-system. J. Magn. Reson. 1989, 84, 611-615. (92) Aguilar, J. A.; Nilsson, M.; Bodenhausen, G.; Morris, G. A. Spin echo NMR spectra without J modulation. Chem. Commun. 2012, 48, 811-813. (93) Calle, L. P.; Espinosa, J. F. An improved

19F-CPMG

scheme for detecting binding of

polyfluorinated molecules to biological receptors. Magn. Reson. Chem. 2017, 55, 355-358. (94) Bohlen, J. M.; Rey, M.; Bodenhausen, G. Refocusing with chirped pulses for broad-band excitation without phase dispersion. J. Magn. Reson. 1989, 84, 191-197.


Page 83 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(95) Bohlen, J. M.; Bodenhausen, G. Experimental aspects of chirp NMR-spectroscopy. J. Magn. Reson., Ser. A 1993, 102, 293-301. (96) Hwang, T. L.; van Zijl, P. C. M.; Garwood, M. Broadband adiabatic refocusing without phase distortion. J. Magn. Reson. 1997, 124, 250-254. (97) Murphy, C. D.; Schaffrath, C.; O'Hagan, D. Fluorinated natural products: the biosynthesis of fluoroacetate and 4-fluorothreonine in streptomyces cattleya. Chemosphere 2003, 52, 455-461. (98) Dalvit, C.; Vulpetti, A. Technical and practical aspects of 19F NMR-based screening: toward sensitive high-throughput screening with rapid deconvolution. Magn. Reson. Chem. 2012, 50, 592597. (99) Congreve, M.; Carr, R.; Murray, C.; Jhoti, H. A ‘rule of three’ for fragment-based lead discovery? Drug Discovery Today 2003, 8, 876-877. (100) Baell, J. B.; Holloway, G. A. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53, 2719-2740. (101) Baell, J. B.; Nissink, J. W. M. Seven Year Itch: Pan-assay interference compounds (PAINS) in 2017-utility and limitations. ACS Chem. Biol. 2018, 13, 36-44. (102) Brown, R. D.; Martin, Y. C. Use of structure−activity data to compare structure-based clustering methods and descriptors for use in compound selection. J. Chem. Inf. Comput. Sci. 1996, 36, 572-584. (103) Bender, A.; Jenkins, J. L.; Scheiber, J.; Sukuru, S. C.; Glick, M.; Davies, J. W. How similar are similarity searching methods? A principal component analysis of molecular descriptor space. J. Chem. Inf. Model. 2009, 49, 108-119.



Page 84 of 93

(104) Duan, J.; Dixon, S. L.; Lowrie, J. F.; Sherman, W. Analysis and comparison of 2D fingerprints: insights into database screening performance using eight fingerprint methods. J. Mol. Graphics Modell. 2010, 29, 157-170. (105) Sastry, M.; Lowrie, J. F.; Dixon, S. L.; Sherman, W. Large-scale systematic analysis of 2D fingerprint methods and parameters to improve virtual screening enrichments. J. Chem. Inf. Model. 2010, 50, 771-784. (106) Morley, A. D.; Pugliese, A.; Birchall, K.; Bower, J.; Brennan, P.; Brown, N.; Chapman, T.; Drysdale, M.; Gilbert, I. H.; Hoelder, S.; Jordan, A.; Ley, S. V.; Merritt, A.; Miller, D.; Swarbrick, M. E.; Wyatt, P. G. Fragment-based hit identification: thinking in 3D. Drug Discovery Today 2013, 18, 1221-1227. (107) Vulpetti, A.; Landrum, G.; Rüdisser, S.; Erbel, P.; Dalvit, C.

19F

NMR chemical shift

prediction with fluorine fingerprint descriptor. J. Fluorine Chem. 2010, 131, 570-577. (108) Nilakantan, R.; Bauman, N.; Dixon, J. S.; Venkataraghavan, R. Topological torsion - a new molecular descriptor for SAR applications. Comparison with other descriptors. J. Chem. Inf. Comput. Sci. 1987, 27, 82-85. (109) Dalvit, C.; Vulpetti, A. Fluorine-protein interactions and 19F NMR isotropic chemical shifts: an empirical correlation with implications for drug design. ChemMedChem 2011, 6, 104-114. (110) Dalvit, C.; Invernizzi, C.; Vulpetti, A. Fluorine as a hydrogen-bond acceptor: experimental evidence and computational calculations. Chem. – Eur. J. 2014, 20, 11058-11068. (111) Linclau, B.; Peron, F.; Bogdan, E.; Wells, N.; Wang, Z.; Compain, G.; Fontenelle, C. Q.; Galland, N.; Le Questel, J. Y.; Graton, J. Intramolecular OH--fluorine hydrogen bonding in saturated, acyclic fluorohydrins: the γ-fluoropropanol motif. Chem. – Eur. J. 2015, 21, 1780817816.


Page 85 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(112) Champagne, P. A.; Desroches, J.; Paquin, J. F. Organic fluorine as a hydrogen-bond acceptor: recent examples and applications. Synthesis 2015, 47, 306-322. (113) Dalvit, C.; Vulpetti, A. Weak intermolecular hydrogen bonds with fluorine: detection and implications for enzymatic/chemical reactions, chemical properties, and ligand/protein fluorine NMR screening. Chem. – Eur. J. 2016, 22, 7592-7601. (114) Tressler, C. M.; Zondlo, N. J. Perfluoro-tert-butyl homoserine is a helix-promoting, highly fluorinated, NMR-sensitive aliphatic amino acid: detection of the estrogen receptor.coactivator protein-protein interaction by 19F NMR. Biochemistry 2017, 56, 1062-1074. (115) Papeo, G.; Giordano, P.; Brasca, M. G.; Buzzo, F.; Caronni, D.; Ciprandi, F.; Mongelli, N.; Veronesi, M.; Vulpetti, A.; Dalvit, C. Polyfluorinated amino acids for sensitive 19F NMR-based screening and kinetic measurements. J. Am. Chem. Soc. 2007, 129, 5665-5672. (116) Tressler, C. M.; Zondlo, N. J. Synthesis of perfluoro-tert-butyl tyrosine, for application in 19F

NMR, via a diazonium-coupling reaction. Org. Lett. 2016, 18, 6240-6243.

(117) Tressler, C. M.; Zondlo, N. J. (2S,4R)- and (2S,4S)-perfluoro-tert-butyl 4-hydroxyproline: two conformationally distinct proline amino acids for sensitive application in 19F NMR. J. Org. Chem. 2014, 79, 5880-5886. (118) Dalvit, C.; Papeo, G.; Mongelli, N.; Giordano, P.; Saccardo, B.; Costa, A.; Veronesi, M.; Ko, S. Y. Rapid NMR-based functional screening and IC50 measurements performed at unprecedentedly low enzyme concentration. Drug Develop. Res. 2005, 64, 105-113. (119) Dalvit, C.; Caronni, D.; Mongelli, N.; Veronesi, M.; Vulpetti, A. NMR-based quality control approach for the identification of false positives and false negatives in high throughput screening. Curr. Drug Discov. Technol. 2006, 3, 115-124.



(120) Zega, A. NMR methods for identification of false positives in biochemical screens. J. Med. Chem. 2017, 60, 9437-9447. (121) Dalvit, C.; Pevarello, P.; Tato, M.; Veronesi, M.; Vulpetti, A.; Sundström, M. Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water. J. Biomol. NMR 2000, 18, 65-68. (122) Dalvit, C.; Santi, S.; Neier, R. A ligand-based NMR screening approach for the identification and characterization of inhibitors and promoters of amyloid peptide aggregation. ChemMedChem 2017, 12, 1458-1463. (123) Rosenau, C. P.; Jelier, B. J.; Gossert, A. D.; Togni, A. Exposing the origins of irreproducibility in fluorine NMR spectroscopy. Angew. Chem., Int. Ed. 2018, 57, 9528-9533. (124) Lee, Y.; Zeng, H.; Ruedisser, S.; Gossert, A. D.; Hilty, C. Nuclear magnetic resonance of hyperpolarized fluorine for characterization of protein-ligand interactions. J. Am. Chem. Soc. 2012, 134, 17448-17451. (125) Arroyo, X.; Goldflam, M.; Feliz, M.; Belda, I.; Giralt, E. Computer-aided design of fragment mixtures for NMR-based screening. PLoS One 2013, 8, e58571. (126) Stark, J. L.; Eghbalnia, H. R.; Lee, W.; Westler, W. M.; Markley, J. L. NMRmix: a tool for the optimization of compound mixtures in 1D 1H NMR ligand affinity screens. J. Proteome Res. 2016, 15, 1360-1368. (127) Moumne, R.; Pasco, M.; Prost, E.; Lecourt, T.; Micouin, L.; Tisne, C. Fluorinated diaminocyclopentanes as chiral sensitive NMR probes of RNA structure. J. Am. Chem. Soc. 2010, 132, 13111-13113.


Page 86 of 93

Page 87 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(128) Dalvit, C.; Knapp, S. 19F NMR isotropic chemical shift for efficient screening of fluorinated fragments which are racemates and/or display multiple conformers. Magn. Reson. Chem. 2017, 55, 1091-1095. (129) Picaud, S.; Leonards, K.; Lambert, J. P.; Dovey, O.; Wells, C.; Fedorov, O.; Monteiro, O.; Fujisawa, T.; Wang, C. Y.; Lingard, H.; Tallant, C.; Nikbin, N.; Guetzoyan, L.; Ingham, R.; Ley, S. V.; Brennan, P.; Muller, S.; Samsonova, A.; Gingras, A. C.; Schwaller, J.; Vassiliou, G.; Knapp, S.; Filippakopoulos, P. Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia. Sci. Adv. 2016, 2, e1600760. (130) Goudreau, N.; Coulombe, R.; Faucher, A. M.; Grand-Maitre, C.; Lacoste, J. E.; Lemke, C. T.; Malenfant, E.; Bousquet, Y.; Fader, L.; Simoneau, B.; Mercier, J. F.; Titolo, S.; Mason, S. W. Monitoring binding of HIV-1 capsid assembly inhibitors using 19F ligand-and 15N protein-based NMR and X-ray crystallography: early hit validation of a benzodiazepine series. ChemMedChem 2013, 8, 405-414. (131) Buratto, R.; Mammoli, D.; Chiarparin, E.; Williams, G.; Bodenhausen, G. Exploring weak ligand-protein interactions by long-lived NMR states: improved contrast in fragment-based drug screening. Angew. Chem., Int. Ed. 2014, 53, 11376-11380. (132) Yanamala, N.; Dutta, A.; Beck, B.; van Fleet, B.; Hay, K.; Yazbak, A.; Ishima, R.; Doemling, A.; Klein-Seetharaman, J. NMR-based screening of membrane protein ligands. Chem. Biol. Drug Des. 2010, 75, 237-256. (133) Morris, G. A. Diffusion-Ordered Spectroscopy. In Encyclopedia of Magnetic Resonance; Harris, R. K., Wasylishen, R.E., Eds.; Wiley: Chichester, U.K., 2009.



(134) Dal Poggetto, G.; Favaro, D. C.; Nilsson, M.; Morris, G. A.; Tormena, C. F.

Page 88 of 93

19F

DOSY

NMR analysis for spin systems with nJFF couplings. Magn. Reson. Chem. 2014, 52, 172-177. (135) Dal Poggetto, G.; Antunes, V. U.; Nilsson, M.; Morris, G. A.; Tormena, C. F. 19F NMR matrix-assisted DOSY: a versatile tool for differentiating fluorinated species in mixtures. Magn. Reson. Chem. 2017, 55, 323-328. (136) Macheteau, J. P.; Oulyadi, H.; van Hemelryck, B.; Bourdonneau, M.; Davoust, D. 2D experiments for the characterization of fluorinated polymers: pulsed-field gradients H-1-F-19 hetero-COSY and its selective version. J. Fluorine Chem. 2000, 104, 149-154. (137) Battiste, J.; Newmark, R. Applications of 19F multidimensional NMR. Prog. Nucl. Magn. Reson. Spectrosc. 2006, 48, 1-23. (138) Castanar, L.; Moutzouri, P.; Barbosa, T. M.; Tormena, C. F.; Rittner, R.; Phillips, A. R.; Coombes, S. R.; Nilsson, M.; Morris, G. A. FESTA: an efficient nuclear magnetic resonance approach for the structural analysis of mixtures containing fluorinated species. Anal. Chem. 2018, 90, 5445-5450. (139) Dalvit, C. Theoretical analysis of the competition ligand-based NMR experiments and selected applications to fragment screening and binding constant measurements. Concepts Magn. Reson., Part A 2008, 32a, 341-372. (140) Dalvit, C.; Gossert, A. D.; Coutant, J.; Piotto, M. Rapid acquisition of 1H and

19F

NMR

experiments for direct and competition ligand-based screening. Magn. Reson. Chem. 2011, 49, 199-202. (141) Kim, Y.; Hilty, C. Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands. Angew. Chem., Int. Ed. 2015, 54, 4941-4944.


Page 89 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(142) Doerr, A. J.; Case, M. A.; Pelczer, I.; McLendon, G. L. Design of a functional protein for molecular recognition: specificity of ligand binding in a metal-assembled protein cavity probed by 19F

NMR. J. Am. Chem. Soc. 2004, 126, 4192-4198.

(143) Copeland, R. A. Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem. Anal. 2005, 46, 1-265. (144) Jordan, J. B.; Poppe, L.; Xia, X.; Cheng, A. C.; Sun, Y.; Michelsen, K.; Eastwood, H.; Schnier, P. D.; Nixey, T.; Zhong, W. Fragment based drug discovery: practical implementation based on 19F NMR spectroscopy. J. Med. Chem. 2012, 55, 678-687. (145) Jordan, J. B.; Whittington, D. A.; Bartberger, M. D.; Sickmier, E. A.; Chen, K.; Cheng, Y.; Judd, T. Fragment-linking approach using

19F

NMR spectroscopy to obtain highly potent and

selective inhibitors of beta-secretase. J. Med. Chem. 2016, 59, 3732-3749. (146) Casale, E.; Amboldi, N.; Brasca, M. G.; Caronni, D.; Colombo, N.; Dalvit, C.; Felder, E. R.; Fogliatto, G.; Galvani, A.; Isacchi, A.; Polucci, P.; Riceputi, L.; Sola, F.; Visco, C.; Zuccotto, F.; Casuscelli, F. Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors. Bioorg. Med. Chem. 2014, 22, 4135-4150. (147) Brasca, M. G.; Mantegani, S.; Amboldi, N.; Bindi, S.; Caronni, D.; Casale, E.; Ceccarelli, W.; Colombo, N.; De Ponti, A.; Donati, D.; Ermoli, A.; Fachin, G.; Felder, E. R.; Ferguson, R. D.; Fiorelli, C.; Guanci, M.; Isacchi, A.; Pesenti, E.; Polucci, P.; Riceputi, L.; Sola, F.; Visco, C.; Zuccotto, F.; Fogliatto, G. Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90). Bioorg. Med. Chem. 2013, 21, 7047-7063. (148) Fogliatto, G.; Gianellini, L.; Brasca, M. G.; Casale, E.; Ballinari, D.; Ciomei, M.; Degrassi, A.; De Ponti, A.; Germani, M.; Guanci, M.; Paolucci, M.; Polucci, P.; Russo, M.; Sola, F.; Valsasina, B.; Visco, C.; Zuccotto, F.; Donati, D.; Felder, E.; Pesenti, E.; Galvani, A.; Mantegani,



S.; Isacchi, A. NMS-E973, a novel synthetic inhibitor of Hsp90 with activity against multiple models of drug resistance to targeted agents, including intracranial metastases. Clin. Cancer Res. 2013, 19, 3520-3532. (149) Garavis, M.; Lopez-Mendez, B.; Somoza, A.; Oyarzabal, J.; Dalvit, C.; Villasante, A.; Campos-Olivas, R.; Gonzalez, C. Discovery of selective ligands for telomeric RNA Gquadruplexes (TERRA) through 19F-NMR based fragment screening. ACS Chem. Biol. 2014, 9, 1559-1566. (150) Phan, A. T.; Patel, D. J. Two-repeat human telomeric d(TAGGGTTAGGGT) sequence forms interconverting parallel and antiparallel G-quadruplexes in solution: distinct topologies, thermodynamic properties, and folding/unfolding kinetics. J. Am. Chem. Soc. 2003, 125, 1502115027. (151) Vulpetti, A.; Randl, S.; Rudisser, S.; Ostermann, N.; Erbel, P.; Mac Sweeney, A.; Zoller, T.; Salem, B.; Gerhartz, B.; Cumin, F.; Hommel, U.; Dalvit, C.; Lorthiois, E.; Maibaum, J. Structure-based library design and fragment screening for the identification of reversible complement factor D protease inhibitors. J. Med. Chem. 2017, 60, 1946-1958. (152) Altmann, E.; Hommel, U.; Lorthiois, E. L.; Maibaum, J. K.; Ostermann, N.; Quancard, J.; Randl, S. A.; Vulpetti, A.; Rogel, O. Complement Pathway Modulators and Uses Thereof. WO2014/ 002051 A2, 2014. (153) Vulpetti, A.; Schiering, N.; Dalvit, C. Combined use of computational chemistry, NMR screening, and X-ray crystallography for identification and characterization of fluorophilic protein environments. Proteins: Struct., Funct., Genet. 2010, 78, 3281-3291. (154) Milletti, F.; Vulpetti, A. Tautomer preference in PDB complexes and its impact on structurebased drug discovery. J. Chem. Inf. Model. 2010, 50, 1062-1074.


Page 90 of 93

Page 91 of 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(155) Aretz, J.; Wamhoff, E. C.; Hanske, J.; Heymann, D.; Rademacher, C. Computational and experimental prediction of human C-type lectin receptor druggability. Front. Immunol. 2014, 5, 323. (156) Borrok, M. J.; Kiessling, L. L. Non-carbohydrate inhibitors of the lectin DC-SIGN. J. Am. Chem. Soc. 2007, 129, 12780-12785. (157) Wamhoff, E. C.; Hanske, J.; Schnirch, L.; Aretz, J.; Grube, M.; Varon Silva, D.; Rademacher, C. 19F NMR-guided design of glycomimetic langerin ligands. ACS Chem. Biol. 2016, 11, 2407-2413. (158) Aretz, J.; Wratil, P. R.; Wamhoff, E. C.; Nguyen, H. G.; Reutter, W.; Rademacher, C. Fragment screening of n-acetylmannosamine kinase reveals noncarbohydrate inhibitors. Can. J. Chem. 2016, 94, 920-926. (159) Poppe, L.; Harvey, T. S.; Mohr, C.; Zondlo, J.; Tegley, C. M.; Nuanmanee, O.; Cheetham, J. Discovery of ligands for Nurr1 by combined use of NMR screening with different isotopic and spin-labeling strategies. J. Biomol. Screening 2007, 12, 301-311. (160) Miller, J. R.; Thanabal, V.; Melnick, M. M.; Lall, M.; Donovan, C.; Sarver, R. W.; Lee, D. Y.; Ohren, J.; Emerson, D. The use of biochemical and biophysical tools for triage of highthroughput screening hits - a case study with escherichia coli phosphopantetheine adenylyltransferase. Chem. Biol. Drug Des. 2010, 75, 444-454. (161) Zech, S. G.; Kohlmann, A.; Zhou, T.; Li, F.; Squillace, R. M.; Parillon, L. E.; Greenfield, M. T.; Miller, D. P.; Qi, J.; Thomas, R. M.; Wang, Y.; Xu, Y.; Miret, J. J.; Shakespeare, W. C.; Zhu, X.; Dalgarno, D. C. Novel small molecule inhibitors of choline kinase identified by fragmentbased drug discovery. J. Med. Chem. 2016, 59, 671-686.



(162) Skora, L.; Jahnke, W.

19F-NMR-based

Page 92 of 93

dual-site reporter assay for the discovery and

distinction of catalytic and allosteric kinase inhibitors. ACS Med. Chem. Lett. 2017, 8, 632-635. (163) Nagatoishi, S.; Yamaguchi, S.; Katoh, E.; Kajita, K.; Yokotagawa, T.; Kanai, S.; Furuya, T.; Tsumoto, K. A combination of 19F NMR and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the ATP-binding site of a kinase. Bioorg. Med. Chem. 2018, 26, 1929-1938. (164) Lombes, T.; Moumne, R.; Larue, V.; Prost, E.; Catala, M.; Lecourt, T.; Dardel, F.; Micouin, L.; Tisne, C. Investigation of RNA-ligand interactions by 19F NMR spectroscopy using fluorinated probes. Angew. Chem., Int. Ed. 2012, 51, 9530-9534. (165) Taylor, J. D.; Gilbert, P. J.; Williams, M. A.; Pitt, W. R.; Ladbury, J. E. Identification of novel fragment compounds targeted against the pY pocket of v-Src SH2 by computational and NMR screening and thermodynamic evaluation. Proteins: Struct., Funct., Genet. 2007, 67, 981990. (166) Assemat, O.; Antoine, M.; Fourquez, J. M.; Wierzbicki, M.; Charton, Y.; Hennig, P.; Perron-Sierra, F.; Ferry, G.; Boutin, J. A.; Delsuc, M. A. F-19 nuclear magnetic resonance screening of glucokinase activators. Anal. Biochem. 2015, 477, 62-68. (167) Braitsch, M.; Kahlig, H.; Kontaxis, G.; Fischer, M.; Kawada, T.; Konrat, R.; Schmid, W. Synthesis of fluorinated maltose derivatives for monitoring protein interaction by Beilstein J. Org. Chem. 2012, 8, 448-455.


19F

NMR.

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Table of Contents Graphic

Ligand-based

19F

NMR Screening

Direct Format

Competition Format

(in house, commercial, DOS, DCC and peptide fluorinated libraries)

(fragment and lead optimization, HTS validation, KI measurement)

Fluorinated Fragments

*

Reporter

F F

F

F

- protein - protein

+ protein

* + protein +competitor

+ protein

δ

19F

δ


19F

Ligand-Based Fluorine NMR Screening: Principles and Applications in

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