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Ligand-Based NMR Study of C-X-C Chemokine Receptor Type 4 (CXCR4)-Ligand Interactions on Living Cancer Cells Diego Brancaccio, Donatella Diana, Salvatore Di Maro, Francesco Saverio Di Leva, Stefano Tomassi, Roberto Fattorusso, Luigi Russo, Stefania Scala, Anna Maria Trotta, Luigi Portella, Ettore Novellino, Luciana Marinelli, and Alfonso Carotenuto J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01830 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 10, 2018

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Journal of Medicinal Chemistry

Ligand-Based NMR Study of C-X-C Chemokine Receptor Type 4 (CXCR4)-Ligand Interactions on Living Cancer Cells Diego Brancaccio,1,§ Donatella Diana,2,§ Salvatore Di Maro,3,§ Francesco Saverio Di Leva,1 Stefano Tomassi,3 Roberto Fattorusso,3 Luigi Russo,3 Stefania Scala,4 Anna Maria Trotta,4 Luigi Portella,4 Ettore Novellino,1 Luciana Marinelli,1,* and Alfonso Carotenuto1,* 1

Dipartimento di Farmacia, Università di Napoli Federico II, 80131, Naples, Italy.

Biostrutture e Bioimmagini, C.N.R., 80134 Naples, Italy.

3

2

Istituto di

Dipartimento di Scienze e Tecnologie

Ambientali, Biologiche e Farmaceutiche, Università degli Studi della Campania “Luigi Vanvitelli”, 81100 Caserta, Italy.

4

Molecular Immunology and Immunoregulation, Istituto Nazionale Tumori

"Fondazione G. Pascale", IRCCS-Napoli, 80131 Naples, Italy.

1 ACS Paragon Plus Environment

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KEYWORDS: Peptides-binding G protein-coupled receptors; Ligand-based NMR techniques;

trNOESY; Saturation transfer difference; WaterLOGSY; CXCR4 receptor.

Abstract Peptide-binding G protein-coupled receptors are key effectors in numerous pathological and physiological pathways. The assessment of the receptor-bound conformation of a peptidic ligand within a membrane receptor such as a GPCR is of great impact for a rational drug design of more potent analogues. In this work, we applied multiple ligand-based NMR methods to study the interaction of peptide heptamers, derived from the C-X-C Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) on membranes of human T-Leukemia cells (CCRFCEM cells). This study represents the first structural investigation reporting the receptor-bound conformation of a peptide to a GPCR directly on a living cell. The results obtained in the field of CXCL12/CXCR4 are proofs of concept, although important information for researchers dealing with the CXCR4 field arise. General application of the presented NMR methodologies is possible and surely may help to boost the development of new therapeutic agents targeting GPCRs.


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Introduction. G Protein-Coupled Receptors (GPCRs) family are well-established drug targets within pharmaceutical intervention, and to date about 50% of the marketed drugs exert their activity by modulating distinct members of this class of transmembrane signal pathway. Among GPCRs, peptides-binding receptors play a crucial role in many pathological and physiological pathways.1,2 However, the study of the binding mode between peptides and membrane receptor like GPCRs is far to be an easy task. In fact, most of peptide ligands, with the sole exception of highly constrained cyclic peptides, are endowed with very flexible structure in water solution, which limits the possibility to achieve information about the bioactive conformation. In this regard, Nuclear Magnetic Resonance (NMR) and other spectroscopic methodologies (i.e. Circular dichroism), have so far required the use of structuring solvents like fluoroalcohols,3 or membrane mimetic environment to solve the conformations of bioactive peptide.4,5 However, the above mentioned techniques still show some limitations, including the need to validate the putative peptide bioactive conformation, for example, by the synthesis of conformationally constrained analogues. On the other hand, investigations concerning GPCRs are further complicated by the need of a natural or artificial membrane environment to guarantee the correct structure and functionality of this class of trasducers.6

In the case of the membrane mimetic environments, the proper GPCRs folding,

especially in bicelles, depends on the combination of various factors, including the nature of the protein and the correct mixture of the longer chain and shorter chain lipids. Thus, a considerable work is preliminarily required to establish the most suitable membrane mimetic compositions for NMR studies on a GPCR.7 Regardless the mixture composition, the detergent micelles do not fully resemble the a real cell membrane, representing a poor mimics of the complex lipid environment in which these proteins function.8 On the other hand, the on-cell NMR allows to study the receptor in its native context, without any artificial modifications, in turn generating “physiological” structure-activity relationships which are crucial for the drug design process. Finally, the employment of computational approaches such as 3 ACS Paragon Plus Environment


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molecular docking of peptides to a GPCR, can be hampered not only by the low accuracy of the ligand conformation (see above) but also by the few experimental data available on peptidesbinding GPCRs. Indeed, a few structures of peptide-bound GPCR have been solved to date both by x-ray,9–11 and cryo-EM techniques.12,13 Thus, a direct observation of a peptide bound to its target GPCR exposed on a cell membrane could provide innovative information for accelerating the discovery of new therapeutic GPCR-modulating agents. The interaction between a small molecule or a peptide and a macromolecule or a macrosystem, like a membrane-bound receptor, can be studied by a few homonuclear NMR experiments, such as Saturation Transfer Difference (STD),14 Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY)15 and transferred NOESY (trNOESY)16. These techniques, also referred to as “ligand-based” techniques, can either detect binding events, providing important information on the bound conformation of the investigated ligand (trNOESY). One key advantage of the latter techniques is their ability to provide structural information without requiring any crystallization or isotopic labeling for relatively low affinity compounds endowed with KD values of 100 nM or higher and consequently in fast exchange between free and bound conformations.17 Since the macromolecule size is not a limiting factor in these experiments, ligand-based techniques can be even applied directly on living cells.6,18–20 Conversely high affinity ligands, with KD values lower than 1 nM and off rates in the range of 0.1–0.01 Hz, cannot be efficiently investigated by the saturation transfer. However, on cell NMR approaches have been already applied in aiding the drug design process.21,22 For instance, Madge at al.21 used on-cell STD NMR spectroscopy to investigate the binding epitope of Siglec-2 (CD22) ligands. The results facilitated the design of novel Siglec-2 ligands showing up to 88-fold higher affinity when compared to the starting ligands. Specifically, in this work we applied ligand-based NMR methods to study the interaction of heptapeptides, derived from the C-X-C Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) located on the cytosolic membrane of CCRF-CEM cells. This study represents the first structural investigation on the binding mode of a peptide to a GPCR directly on a 4 ACS Paragon Plus Environment

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living cell. As model for our investigation, we selected CXCR4 since it represents an important potential therapeutic target for various severe diseases involving immune system, including cancer.23 In particular, recent studies demonstrated that targeting CXCR4 in association with antiPDL1 antibody produces synergistic effect for restoring immune response to tumor.24 In this context, we have recently developed different peptides able to bind CXCR4 and to inhibit CXCL12dependent migration with variable potencies ranging from low micromolar25 to low nanomolar.26–28 Among these peptides, 1 (Figure 1) has been chosen as the starting point of this study since it has a suitable affinity (IC50 = 6.2 µM, Table 1) for ligand-based NMR studies. Due to the chemical instability of this compound in the cell environment used in our experiments, a novel series of potential CXCR4 antagonist was also developed (compounds 2-6, Figure 1) leading to the identification of a peptidomimetic analogue (3) endowed with similar affinity and higher serum stability than the lead 1 (Table 1). Moreover, as negative control for our experiments, a scrambled derivative (8) was synthesized operating a random permutation of the original sequence of 1. Finally, the more potent CXCR4 antagonist 7 (Figure 1), which is endowed with a low nM IC50 (IC50 = 0.053 µM, Table 1),26 and hence potentially out of the theoretical binding range for ligandbased NMR analyses, was also tentatively considered in our studies.



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Figure 1. Structures of the CXCR4 antagonists described in the study. Applied modifications are evidenced in red.


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Table 1. CXCR4 antagonists employed in this study.

X

IC50 (µM)a

Stabilityb

1

R

6.2 ± 1.3c

22 min

2

rd

> 10

NT

3.2 ±1.1

Unaltered up to 120 min

> 10

NT

5

> 10

NT

6

> 10

NT

0.053 ± 0.004c

Unaltered up to 120 minc

> 10

NT

0.006 ± 0.004c

NT

Entry

Sequence

3

4

7

XA[CRFFC]

XA[cdRFFC]

8 9

Ac-R

FR[CFARC]e

a

Half-maximal inhibitory concentration (IC50, µM, mean ± SD) of CXCR4 antagonist peptides necessary to reduce by 50% the binding to CXCR4 of CXCR4-specific mAb 12G5 in CCRF-CEM cells. bHuman plasma stability expressed as t1/2 (min). cAlready reported in reference 26. dSmall letters indicate amino acids with D-configuration. eFor the synthesis of 8 see the experimental section. Results and Discussion In our previous studies, we reported the identification and development of novel CXCL12-inspired CXCR4 antagonists, which could selectively bind the receptor in the micromolar (1)25 and 7 ACS Paragon Plus Environment


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nanomolar range (7) (Table 1).26 As starting point for our NMR interaction studies, we selected peptide 1, (Table 1) since it has an IC50 (low micromolar, Table 1) suitable for NMR analysis (see Introduction for details).17 We acquired both STD and WaterLOGSY spectra (Figures 2 and S1, Supporting Information) of 1 in a suspension of CCRF-CEM cells, a human T-Leukemia cell line which highly expresses CXCR4. STD spectrum of 1 in the presence of cell suspension shows positive signals (Figure 2b), while no signal is found in the STD spectra of either the peptide or cell suspension alone. This result is thus indicative of peptide-cells interaction. To assess that the latter occurs through the peptide binding to the CXCR4 receptor, a competition experiment was performed

using

plerixafor,

previously

known

as

AMD3100

(1,1′-[1,4-phenylenebis-

(methylene)]bis[1,4,8,11-tetraazacyclotetradecane]) (9, Figure 1), a potent and selective CXCR4 antagonist.29 After the addition of 9 to the 1/CCRF-CEM cells mixture, STD signals significantly decreased, demonstrating that 1 specifically binds CXCR4. Increased amounts of plerixafor from 0.010 to 0.100 mM were used for the STD competition experiments (Supporting Information, Figure S2). A consistent decrease of the S/N ratio was observed, which demonstrated that the effects observed were dose-dependent (Table 2). In the WaterLOGSY experiments, many signals of 1 exhibit positive phase after the addition of cell suspension to peptide NMR sample (Figure 2c), thus indicating that 1 interacts with cells. Again, 9 was then employed to perform competition experiments. As shown in Figure 2 (red line), many WaterLOGSY signals significantly decrease or invert after 9 addition, demonstrating that 1 specifically binds CXCR4. The effect is dose-dependent as it can be inferred from the S/N decrease at increasing concentration of 9 (Figure S2). Residual STD and WL signals are probably due to an incomplete displacement of the peptide from the receptor or unspecific peptide-cells interactions. In addition to the evaluation of ligand-receptor interaction, STD experiments can be also employed to derive ligand binding epitopes.30 In the STD spectrum of 1, aromatic signals of Phe5 and Phe6 are clearly visible with Hε’s of Phe6 being the highest signals (Table 3). Clearly, the relative intensities


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of STD signals (ηsat) reported in Table 3 indicate that the side chains of the triad Arg4, Phe5, Phe6 are the most involved in receptor interaction. Table 2. The S/N ratios in the STD spectra.a

a

Peptide

Cell Line

[9] mM

S/N

1

CCRF-CEM

-

13.3

1

CCRF-CEM

0.010

7.2

1

CCRF-CEM

0.020

6.3

1

CCRF-CEM

0.050

6.1

1

CCRF-CEM

0.100

5.9

3

CCRF-CEM

-

10.0

3

CHO+ b

-

35.9

3

CHO-

-

12.5

2

CHO+

-

9.6

2

CHO-

-

8.2

8

CHO+

-

10.2

8

CHO-

-

11.7

7

CCRF-CEM

-

6.7

The highest aromatic signal was considered. b Chinese hamster ovary.



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Figure 2. a) 1H NMR spectrum of 1 (0.500 mM) in the presence of a suspension of CCRF-CEM cells (20 x 106); b) STD and c) WaterLOGSY spectra in absence (black) and in presence (red) of plerixafor (9, AMD3100, 0.020 mM).

Unfortunately, we noticed from spectral modification that peptide 1 is relatively unstable and it is completely degraded within about 2 h during the acquisition of NMR spectra in the sample containing cell suspension. This is in accordance with the low peptide stability in human serum (t1/2 = 22’ in 90%).26 Henceforth, more time consuming NMR experiments such as 2D trNOESY could not be acquired for this peptide. To overcome this problem, a small library of analogues of 1 was designed and synthesized (Figure 1 and Table 1) with the aim of enhancing the peptide stability 10 ACS Paragon Plus Environment

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without affecting the affinity for the receptor. Considering that the Arg1-Ala2 dipeptide was the most engaged in the proteolytic degradation,26 the library was focused on the N-terminal modification of 1 leading to analogues 2-6 (Figure 1). First, we introduced D-Arg in place of L-Arg for the synthesis of peptide 2. In alternative, we synthesized 3-6 by replacing L-Arg1 with several previously reported arginine mimetics, including 3- and 4-guanidinobenzoic acids (3 and 4, respectively),31 1carbamimidoylpiperidine-4-carboxamide (5)32 and 4-guanidinobutyric acid (6).33 The design of these analogues, at least considering 3 and 4, was also aimed at increasing signals dispersion in the 1

H NMR spectrum of the peptides, particularly avoiding signals superposition of Arg1 and Arg4.

Scheme 1. Synthesis of compounds 2-6. numerals for clarity.

Intermediates of synthesis are reported as Roman



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As described in Scheme 1, the synthesis was performed on solid support by a classical Fmoc/tBu strategy. Specifically, the common sequence (I) (Ala-Cys-Arg-Phe-Phe-Cys) was assembled through reiterated coupling and deprotection cycles. At this step, the linear sequence of peptide 2 was completed with a residue of D-Arg, released from the resin; the resulting free thiols were oxidized by treatment with N-chlorosuccinimide. For compounds 3-6, the arginine mimetic moieties were synthesized directly on solid phase starting from the corresponding amino precursors (II-V), and conversion of the amino- into guanidine-groups was quantitatively monitored by analytical HPLC-UV-MS. At this stage, the linear peptidomimetics (II-IV) were released from the resin, and the disulfide bridge was formed as above described (3-6). By applying same strategy of 2, we synthesized 8 (see experimental section for details), a scrambled version of 1, which was employed as negative control in our experiments. All the compounds (2-6 and 8) were purified by preparative HPLC and finally characterized by analytical HPLC-UV-MS. All the novel compounds 2-6 and 8 were evaluated against CXCR4 measuring their capability to inhibit receptor binding of PE-conjugated-12G5 anti-CXCR4 antibodies in CEM-CCRF human T leukemia CXCR4 expressing cells (Table 1).34 The IC50 values of peptides 1 and 7, previously found by us,25,26 are also reported in Table 1 for comparison. As expected, 8 did not bind the receptor up to 10 µM concentration (maximum concentration exploited in the assay). Similarly, compound 2, obtained through the introduction of

D-Arg

1

in place of L-Arg1, turned out to be

inactive up to 10 µM concentration. Analogous results were observed for compounds 4-6, where LArg1 was replaced with 4-guanidinobenzoic acid (4), 1-carbamimidoylpiperidine-4-carboxamide (5) and 4-guanidinobutyric (6). Conversely, 3, resulting from the substitution of L-Arg1 with the 3guanidinobenzoic acid (GBA), showed affinity for the receptor comparable to 1 (IC50 = 3.2 µM). Thus, based on these results, 3 was the unique member of this small library to be considered as potential candidate for our investigation.


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As mentioned above, an ideal candidate for our cell based NMR binding experiments should possess a higher metabolic stability if compared to 1. Hence, the early metabolic stability of 3 was tested by incubating it and its parent peptide 1 in 90% human serum at 37 °C, following a previously described protocol.35 At several time intervals, aliquots of the mixture reaction were collected and the plasma proteins were precipitated with acetonitrile. The recovered supernatants were analyzed by ESI-RP-HPLC. As shown in Figure 3, 1 starts to be significantly degraded already after 15 min, and is fully converted into the corresponding pentapeptide metabolite H-[CysArg-Phe-Phe-Cys]-COOH within 30 min. Conversely, 3 remained unaltered up to 120 min, indicating that the 3-guanidino benzoic acid moiety at the N-terminus prevents the recognition by the proteases involved in the degradation process.

Figure 3. Human plasma stability profiles of 1 and 3 after different intervals of incubation with 90% human serum. Relative concentrations of peptides were determined by integration of the A230 peaks from analytical HPLC.

Encouraged by the good activity and the high plasma stability of 3, we performed a series of NMR experiments on this analogue. STD and WaterLOGSY spectra of 3 are reported in Figure 4 and the relevant ηsat% are reported in Table 3. As for the parent peptide 1, the residues mainly involved in



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the CXCR4 binding are Arg4, Phe5, Phe6 while saturation transfer on GBA signals, here clearly distinguishable from those of Arg1, is much less efficient compared to the other aromatics. Differently from what we experienced with 1, we managed to acquire the trNOESY spectrum of 3 in cell suspension. In fact, as well as in human serum (Figure 3), the stability of 3 was increased in this medium so that it remains unmodified after up to 6 hours (data not shown).


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Figure 4. a) 1H NMR, b) STD, and c) WaterLOGSY spectra of 3 (0.500 mM) in the presence of a suspension of CCRF-CEM cells (20 x 106).



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Table 3. The relative STD effect, ηsat%, for epitope mapping.a Residue

Atom

1

Arg1

Hγ

4

Arg1

Hδ

3

8b

6

Ala2

Hβ

8

15

6

Arg4

Hα

4

nd

6

Arg4

Hγ

10

9

8

Arg4

Hδ

5

17

8

Arg4

Hε

10

10

9

Phe5

Hδ

73

78

100

Phe6

Hε

100

100

91

3

7 24

a

Relative intensity of STD signal of the individual protons, ηsat%, normalized to that of the most intense not overlapped signal. bHighest aromatic signal of guanidinobenzoic acid residue. *Overlapped signals. nd: not determinable.

First, trNOESY confirms that 3 binds to the cell since we observe the appearence of negative NOE cross-peaks (Figure 5a) that are instead absent in the spectra acquired in buffer solution (data not shown). The exiguous number of cross-peaks present in the trNOESY spectrum prevented however a NOE-based structure calculation. In fact, almost all amide proton signals could not be detected due to their fast exchange rate in these sample conditions (pH 7.4). Nonetheless, a very informative group of NOEs, evidenced in Figure 5a, correlated the side chain signals of Arg4 with those of Phe5 and Phe5 with Phe6. These NOEs experimentally demonstrated the presence of a side chains cluster formed by residues Arg4, Phe5 and Phe6 in the peptide conformation when bound to the CXCR4 receptor on the cell surface. Interestingly, assigned NOEs are in accordance with the 3D structure of 3 obtained in sodium dodecyl sulfate (SDS) solution (Figure 5b, see Experimental Section and Supporting Information for details). Distances among Arg4, Phe5 and Phe6 centroids are highlighted


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and allow their use in any approach of rational drug design aimed at the finding of peptidic and non peptidic CXCR4 ligands. Herein as a control, a trNOESY spectrum of the peptide-cell suspension was acquired after the addition of plerixafor. In this case no cross peaks were observed indicating that observed NOEs come from the peptide/CXCR4 interaction (Supporting Information, Figure S3).

Figure 5. a) trNOESY spectrum of 3 in the presence of a suspension of CCRF-CEM cells (20 x 106). b) Lowest energy conformer of 3 (PDB ID code: 5OLF). Distances among the side chains (in Å) of the Arg4, Arg5, Phe6 cluster are reported as dotted lines. GBA: guanidinobenzoic acid residue.

To better characterize the interaction of the ligand with CXCR4, the same NMR experiments using CXCR4-null cell line (CHO-) and recombinant CXCR4-expressing cell line (CHO+) were performed as negative and positive controls, respectively. Figure 6 shows the STD and WaterLOGSY spectra of 3 in the presence of these two cell lines. It can be clearly inferred that the signal to noise (S/N) ratio in both these experiments is significantly higher for CHO+/3 solution (S/N = 35.9 vs 12.5, Table 2) in the same experimental conditions. To further support that the signals of active peptides were derived from the interactions with CXCR4, STD experiments using the inert peptides 2 and 8 (Table 1) in the presence of CHO+ and CHO- cells were also acquired as negative controls. Also in these cases (Supporting Information, Figure S4), the signal to noise ratio 17 ACS Paragon Plus Environment


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was relatively low compared to active peptide responses in the same conditions (S/N ~ 10 vs 36, Table 2). Hence, it can be safely stated that specific interactions of peptide with the CXCR4 receptor on cell surface is quantitatively discriminable from nonspecific cell-peptide interactions.

Figure 6. a) 1H NMR, b) STD, and c) WaterLOGSY spectra of 3 (0.500 mM) in the presence of a suspension of 20 x 106 CHO+ cells (black) or CHO- cells (red).


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In the attempt to compare the interaction mode of 3 and 7 with CXCR4, additional NMR experiments were then carried out on the latter peptide. Indeed, no correlation peaks could be observed in the trNOESY spectrum of 7 in the presence of CEM cells (data not shown) probably due to the high 7/CXCR4 binding constant (low dissociation rate constant, koff).17 In contrast, STD and WaterLOGSY spectra of peptide 7 (Figure 7) showed interpretable signals, while the STD spectrum has a lower signal-to-noise ratio compared to that obtained for 1 and 3 (S/N ~ 6.7 vs 13.3 for peptide 7 and 1, respectively, Table 2). Interestingly, STD spectrum of 7 provides very useful information. As for peptides 1 and 3, Arg4, Phe5, Phe6 protons are characterized by the most intense STD signals, indicating that the three ligands share the same binding epitope. However, an intriguing difference observed in the STD spectrum of 7 is the appearance/increased intensity of signals attributable to Arg1 side chain (Table 3). These signals demonstrate that the Arg1 residue of peptide 7 is involved in CXCR4 binding. This is an experimental confirmation of the differences found in the structural models built for both the 1/CXCR4 and 7/CXCR4 complexes and described by us in a recent paper.26 Indeed, in these models the Arg4 side chain of both the peptides makes tight salt bridge with the D972.63 carboxylate group of CXCR4, while Phe5 and Phe6 side chains are close and buried into aromatic pockets where they can establish favorable hydrophobic interactions with the receptor. However, according to the two models, the Arg1 side chain of 1 extends out of the binding site while Arg1 guanidinium group of 7 can form a stable salt bridge with the receptor D18745.51 carboxylate group, perfectly attuned with the present experimental results. Hence, the present case study demonstrates that ligand-based NMR experiments acquired in the presence of living cells can provide unique information about the nature and the arrangement of peptide binding epitopes, which might be used for ligand-receptor interaction mode analysis and rational drug design studies.



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Figure 7. a) 1H NMR, b) STD, and c) WaterLOGSY spectra of 7 (0.500 mM) in the presence of a suspension of CCRF-CEM cells (20 x 106).

In fact, when a cyclic peptide NMR structure is solved in SDS micelles and it is then submitted to docking simulations, the backbone is generally kept rigid while the rotameric states of the side chains are explicitly sampled. In the present case, however, NMR studies demonstrated that the ligand Arg4, Phe5 and Phe6 cluster is well structured also in the receptor-bound conformation. This 20 ACS Paragon Plus Environment

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allowed us to perform docking calculations where the conformational space of the side chains of the latter residues was limited so as to improve the efficiency and the reliability of docking results. Thus, herein, we benefited of on cell NMR data and carried out NMR-constrained docking calculations through the Glide-SP peptide mode (see the Methods section for details). Remarkably, such simulations converged toward only one highly populated binding mode (12 out of the 50 docking solutions; ≈ 24 %), whose stability was verified by molecular dynamics (MD) simulations (see the Experimental Section for technical details) (Supporting Information, Figure S5). In this pose (Figure 8a), the Arg4 side chain engages H-bonds with the H1133.29 and, more importantly, the D1714.60 side chains and cation-π interactions with H1133.29, Y1163.32, H2035.42, and W2526.48, while the Phe5 and Phe6 aromatic rings establish favorable contacts with the side chains of R18845.52, Y19045.54, V1965.35, H2035.42 and L2666.62. Besides the Arg4, Phe5 and Phe6 cluster, the C-ter carboxylate group of 3 can form a H-bond with the R30N-ter side chain, while the guandinium benzoic moiety extends out of the receptor binding cavity contacting the E32N-ter. Additional Hbonds are eventually formed by the Arg4 backbone CO and NH with the R18845.52 and E2887.39 side chains, respectively. Interestingly, this docking pose well overlaps with that of the co-crystallized ligand CVX15 (see Figure 8b) and is in agreement with the experimental data reporting the involvement of H1133.29, Y1163.32, H2035.42, Y2556.51 in peptide binding to CXCR4.36,37 On the other hand, missing contacts with the key residues D972.63 and D2626.58

would explain the

moderate CXCR4 affinity of 3 whose IC50 falls in the micromolar range.36,37 These results were then compared with those provided by standard docking calculations, where only the ligand cyclic backbone was considered rigid. As expected, these simulations displayed a lower convergence rate compared to the previous set of calculations. In detail, we found that in 20 out of 50 poses the Arg4, Phe5 and Phe6 could establish specific interactions with the CXCR4 receptor. Among these, 4 corresponded to the constrained-docking predicted binding mode (see above). The other 16 solutions could be grouped into three new clusters formed by 7, 7 and 2 members, respectively. Nevertheless, in none of the latter poses the NMR distances between the Arg4, Phe5 and Phe6 side 21 ACS Paragon Plus Environment


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chains were fully respected. Therefore, in absence of any experimental data about the peptide binding residues and their relative distances in the “bound” conformation, the choice of the “best” pose would have been surely challenging. Thus, these outcomes indicate that the employment of on cells NMR constraints can improve the efficiency of peptide docking calculations, by both increasing their convergence and reducing the numbers of false positives. Moreover, it can furnish precious help to the researcher for the choice of the correct pose. Finally, the use of on cell NMR data might save precious computational time and efforts as far as docking calculation converge toward a unique solution. In fact, more sophisticated computational methods, such as MD simulations, are mandatory when more than a docking conformation is obtained and the relative stability of such poses has to be evaluated.

Figure 8. a) Binding mode of 3 (green sticks) at the CXCR4 receptor9 (gray cartoons) as predicted by docking and MD simulations. Receptor amino acids important for ligand binding are shown as sticks. Hydrogen bonds are displayed as dashed black lines. Nonpolar hydrogens are omitted for clarity. b) Superposition between the predicted binding pose of 3 (green cartoon and sticks) and the co-crystallized ligand CVX159 (orange cartoons and sticks) at the CXCR4 receptor (gray cartoons). Residues 4, 6-13 and 15 in CVX15 as well as all nonpolar hydrogens are omitted for clarity.

Conclusions The experimental determination of the receptor-bound conformation of a peptide ligand to membrane receptors such as GPCRs is a precious ground for a rational drug design of more potent analogues. Here, we have shown for the first time that ligand-based NMR techniques like STD,


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WaterLOGSY and trNOESY are suitable tools for a direct observation of the binding of a peptide to a GPCR on living cells. A ligand-based NMR analysis was performed on CXCR4 peptide ligands in the presence of a suspension of CCRF-CEM cells, a human T-Leukemia cell line highly expressing this chemokine receptor, to identify the ligand interacting residues and their pharmacophoric arrangement. Experiments acquired on both known and ad hoc developed peptides, indicated that the Arg4, Phe5, Phe6 triad is mostly involved in receptor interaction. Moreover, trNOESY acquired on a novel stable derivative (3) furnished a very informative group of NOEs which is consistent with a cluster of the side chains of these residues in the ligand bound conformation. These NOEs thus demonstrated the presence of a well-defined side chains cluster involved in the binding to the CXCR4 receptor on the cell surface. The receptor-bound conformation of 3 furnishes precious details on CXCR4 binding epitopes, which can assist computational studies aimed at the definition of the ligand/receptor binding mode. In fact, we here showed that on-cell NMR driven docking calculations achieved a higher convergence compared to standard unrestrained simulations, which instead provided ambiguous results. Furthermore, such information can be used in any drug design approach aimed at finding novel, even non peptidic CXCR4 antagonists, such as pharmacophorebased virtual screening campaigns. Notably, the presented NMR protocol can also help to rationalize the ligand structure activity relationships as here demonstrated for 1, 3 and the more potent derivative 7. In fact, we found that besides the pharmacophoric residue cluster (Arg4, Phe5 and Phe6), a positively charged residue at the 1 position, if correctly spatially oriented, is important to achieve high CXCR4 binding potency by these analogues. The broad applicability of the presented NMR methodologies opens up new opportunities for medicinal chemists involved in design and discovery of new therapeutic agents targeting GPCRs.



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Experimental Section Chemistry Materials. Nα-Fmoc-protected amino acids, 2-Cl-trtCl resin, Fmoc-Rink Amide-Am resin, Obenzotriazole-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate

(HBTU),

N,N-

diisopropylethylamine (DIEA), triisopropylsilane (TIS), trifluoroacetic acid (TFA), piperidine and N-hydroxybenzotriazole (HOBt), from Iris-Biotech Gmbh (Marktredwitz, Germany). N,Ndimethylformamide (DMF), dichloromethane (DCM), Diethylether, N-Chlorosuccinimide, H2O, CH3CN for HPLC, N,N’-Di-Boc-Thiourea, HgCl2 and human serum were reagent grade and were acquired from commercial sources (Sigma-Aldrich, Milano, Italy) and used as received unless otherwise noted. Peptides were purified by preparative HPLC (Shimadzu HPLC system) equipped with a C18-bounded preparative RP-HPLC column (Phenomenex Kinetex 21.2 mm × 150 mm 5 µm). Peptides were analyzed by analytical HPLC (Shimadzu Prominance HPLC system) equipped with a C18-bounded analytical RP-HPLC column (Phenomenex Luna, 4.6 mm × 150 mm 5 µM) using a gradient elution (10−90% acetonitrile in water (0.1% TFA) over 20 min; flow rate = 1.0 mL/min; diode array UV-VIS detector). Molecular weights of compounds were confirmed by ESImass spectrometry using an Agilent 6110 quadrupole LC-MS system. General Procedure for the Synthesis of 2-6. 2-Cl-trtCl resin (65.5 mg, 1.60 mmol/g) was swollen in Dry DMF (2 ml) over 0.5 h, and a solution of Fmoc-L-Cys(trt)-OH (59.0 mg, 0.10 mmol, 1 equiv) and DIPEA (52 µl, 3eq) in DMF (2ml) was added. The mixture was stirred for 24 h. The residual chloride groups contained in the resin were capped by adding MeOH (200 µl) in DCM (2 ml) in presence of DIPEA (35 µl, 2eq) and stirring for 30 min to avoid eventually parallel synthesis of side products. Fmoc group removal was performed using 20 % piperidine in DMF (1 x 5 min and 1 x 25 min). The peptide resin was then washed with DCM (3 x 0.5 min) and DMF (3 x 0.5 min) and positive Kaiser ninhydrine38 and TNBS39 tests were observed. Fmoc-L-Phe-OH (155.0 mg, 0.4 mmol, 4 equiv), Fmoc-L-Arg(Pbf)-OH (259. 5 mg, 0.4mmol, 4 equiv), Fmoc-L-Cys(Trt)-OH (234.3 mg, 0.4 mmol, 4 equiv), Fmoc-L-Ala-OH (124.5 mg, 0.4 mmol, 4 equiv), Fmoc-D-Arg(Pbf)-OH 24 ACS Paragon Plus Environment

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(259. 5 mg, 0.4mmol, 4 equiv) or Fmoc-3-aminobenzoic acid (143.8 mg, 0.4 mmol, 4 equiv) or Fmoc-4-aminobenzoic acid (143.8 mg, 0.4 mmol, 4 equiv) or Fmoc-Isonipecotic acid (140.6 mg, 0.4 mmol, 4 equiv) or Fmoc-γ-aminobutyric acid (130.1 mg, 0.4 mmol, 4 equiv) were sequentially added to the resin bound H-L-Cys(Trt). Each coupling reaction was achieved using a 4-fold excess of amino acid with HBTU (151. 7 mg, 0.4 mmol, 4 equiv) and HOBt (61.2 mg, 0.4 mmol, 4 equiv) in the presence of DIPEA (140 µl, 0.8 mmol, 8 equiv) in DMF. Fmoc deprotections were accomplished with 20% piperidine in DMF solution (1 x 5 min, 1 x 25 min). Washings with DMF (3 x 0.5 min) and DCM (3 x 0.5 min) were performed through every coupling/deprotection step. Kaiser ninhydrine and TNBS tests were employed for monitoring the progress of peptide synthesis. General Procedure for the Synthesis of 8. 2-Cl-trtCl resin (65.5 mg, 1.60 mmol/g) was swollen in Dry DMF (2 ml) over 0.5 h, and a solution of Fmoc-L-Cys(trt)-OH (59.0 mg, 0.10 mmol, 1 equiv) and DIPEA (52 µl, 3eq) in DMF (2ml) was added. The mixture was stirred for 24 h. The residual chloride groups contained in the resin were capped by adding MeOH (200 µl) in DCM (2 ml) in presence of DIPEA (35 µl, 2eq) and stirring for 30 min to avoid eventually parallel synthesis of side products. Fmoc group removal was performed using 20 % piperidine in DMF (1 x 5 min and 1 x 25 min). The peptide resin was then washed with DCM (3 x 0.5 min) and DMF (3 x 0.5 min) and positive Kaiser ninhydrine38 and TNBS39 tests were observed. Fmoc-L-Arg(Pbf)-OH (259. 5 mg, 0.4mmol, 4 equiv), Fmoc-L-Ala-OH (124.5 mg, 0.4 mmol, 4 equiv), Fmoc-L-Phe-OH (155.0 mg, 0.4 mmol, 4 equiv), Fmoc-L-Cys(Trt)-OH (234.3 mg, 0.4 mmol, 4 equiv), Fmoc-L-Arg(Pbf)-OH (259. 5 mg, 0.4mmol, 4 equiv) and Fmoc-L-Phe-OH (155.0 mg, 0.4 mmol, 4 equiv) were sequentially added to the resin bound H-L-Cys(Trt). Each coupling reaction was achieved using a 4fold excess of amino acid with HBTU (151. 7 mg, 0.4 mmol, 4 equiv) and HOBt (61.2 mg, 0.4 mmol, 4 equiv) in the presence of DIPEA (140 µl, 0.8 mmol, 8 equiv) in DMF. Fmoc deprotections were accomplished with 20% piperidine in DMF solution (1 x 5 min, 1 x 25 min). Washings with DMF (3 x 0.5 min) and DCM (3 x 0.5 min) were performed through every coupling/deprotection



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step. Kaiser ninhydrine and TNBS tests were employed for monitoring the progress of peptide synthesis.

General Procedure for Guanidine formation. After removing the last Fmoc group, the resin bound peptide was treated with N,N’-Di-Boc-Thiourea (55.3 mg, 0.2 mmol, 2 equiv) and HgCl2 (54.3 mg, 0.2 mmol, 2 equiv) in DMF (2 ml) and the mixture was shaken for 3 h. The quantitative conversion of the amino- to guanidino-group was monitored by analytical HPLC-UV-MS.

General Procedure for Peptide Oxidation and Purification. The peptide was released from the solid support and all the protecting groups cleaved, treating the resin with TFA/DCM/TIS (80/15/5, v/v/v) (3 ml solvent/0.1 mmol) for 2 h. The resin was then filtered off and the crude linear peptide was recovered by precipitation with chilled ether to give a powder. The crude peptide (0.1 mmol) was dissolved in 45 ml of H2O and a solution of N-Chlorosuccinimmide (20 mg, 0.15 mmol, 1.5 eq) in H2O (5 ml) was added and the mixture was mechanical stirring for 30 min at room temperature. The solution mixture was finally purified by preparative RP-HPLC in 0.1% TFA with an ACN gradient (10−70% ACN in H2O over 20 min, flow rate of 15 mL/min) on a Phenomenex Kinetex C18 column (21.2 mm × 150 mm 5 µm). Analytical RP-HPLC were performed in 0.1% TFA with an ACN gradient (10−90% ACN in H2O over 20 min, flow rate of 1.0 mL/min) on a Phenomenex Luna C18 column (0.46 mm × 150 mm 5 µm). 2. Purity > 95%, tR 8.4 min; molecular formula: C39H57N13O8S2; calculated mass: 899.4; found: 900.3 (M+H+), 922.7 (M+Na+), 938.5 (M+K+). 3. Purity > 95%, tR 9.0 min; molecular formula: C41H52N12O8S2; calculated mass: 904.4; found: 905.6 (M+H+), 927.5 (M+Na+). 4. Purity > 95%, tR 9.5 min; molecular formula: C41H52N12O8S2; calculated mass: 904.4; found: 905.6 (M+H+), 927.5 (M+Na+), 949.5 (M+2Na+).


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5. Purity > 95%, tR 9.7 min; molecular formula: C40H56N12O10S2; calculated mass: 896.4; found: 897.5 (M+H+), 919.5 (M+Na+). 6. Purity > 95%, tR 9.2 min; molecular formula: C38H54N12O8S2; calculated mass: 870.4; found: 871.5 (M+H+). 8. Purity > 95%; tR 8.5 min; C39H57N13O8S2; calculated mass: 899.4; found: 900.2 (M+H+), 922.5 (M+Na+).

Human Serum Stability Serum stability was evaluated adopting a previously reported protocol.35 In detail, the reaction solution was prepared by mixing peptide in sterile water (1 mM) and human serum (at concentration 0.1 mM, 90% serum) and incubated at 37 °C. Aliquots were taken at different interval times (0, 15 min, 30 min, 60 min, 120 min), subjected to precipitation by addition of ACN/ 0.1% TFA solution, and then centrifuged (12 000 rpm, 15 min, 4 °C). The supernatant obtained was analysed by HPLC using a linear elution gradient from 10% to 90% ACN (0.1% TFA) in water (0.1% TFA) in 20 min. Binding Assay CXCR4 binding was evaluated as previously described.34 Briefly, 5 × 105 CCRF−CEM, HT29 cells were preincubated with increasing peptide concentrations (0.01 µM, 0.1 µM, 1 µM, 10 µM) in the binding buffer (PBS 1× plus 0.2% BSA and 0.1% NaN3) for 30 min at 37 °C, 5% CO2 and then labeled for 30 min using anti-CXCR4 PE-antibody (FAB170P, clone 12G5, R&D Systems, Minneapolis, MN, USA). Cell Culture and CXCR4 expression analysis Chinese hamster ovarian cells (CHO, indicated as CHO-) and CHO cells that were stably transfected with CXCR4 (CHO+) were a kind gift from Dr. David McDermott (NIAID, NIH,



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Bethesda, MD, USA). CCRF-CEM cells, human T-Leukemia cell line, CHO and CHO-CXCR4 were cultured in the recommended growth medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% L-glutamine , 1% penicillin/streptomycin and maintained in 95% air-5% CO2 at 37°C. For NMR analysis, cells were counted using Burker chamber and 20 x 106 cells were resuspended in 450 µl PBS. CXCR4 expression was assessed by flow cytometry using the mouse anti-human CXCR4 monoclonal antibody clone 12G5 (MAB170P, R&D Systems) as previously described.25,40 NMR Spectroscopy All NMR experiments were carried out at 298 K with an Inova 600 MHz spectrometer (Varian Inc., Palo Alto, CA, USA), equipped with a cryogenic probe that was optimized for 1H detection. For interaction studies of peptides with intact cells of CCRF-CEM, CHO-, and CHO+ cell lines, pellet of 20 x 106 cells was resuspended in 450 µL of PBS buffer (pH 7.4) and 50 µL of 2H2O. 1H and STD NMR spectra of peptides alone or cell suspension alone were acquired as reference. When reported, plerixafor (9) was added to the sample to the indicated concentration. STD spectra were acquired with 512 scans with on-resonance irradiation at -0.5 ppm for selective saturation of protein resonances and off-resonance irradiation at 30 ppm for reference spectra. A train of 40 Gaussian shaped pulses of 50 ms with 1ms delay between pulses were used, for a total saturation time of 2 s. STD spectra were obtained by internal subtraction of the saturated spectrum from the reference spectrum by phase cycling with a spectral width of 7191.66 Hz, relaxation delay 1.0 s, 8 k data points for acquisition, and 16 k for transformation. WaterLOGSY NMR experiments employed a 20 ms selective Gaussian 180° pulse at the water signal frequency and an NOE mixing time of 1 s. Both for STD and WaterLOGSY, the FIDs were multiplied by an exponential weighting (lb = 5 Hz) before Fourier transformation.


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2D [1H, 1H] and trNOESY spectra of 3 in the presence of CCRF-CEM cell were acquired with 32 or 64 scans per t1 increment, with a spectral width of 6712.0 Hz along both t1 and t2, 2048×256 data points in t2 and t1, respectively, and 1.0 s recycle delay. Water suppression was achieved by means of a double pulsed field gradient spin echo (DPFGSE) sequence.41 Reference trNOESY spectra of peptide 3 alone and 3 in the presence of CCRF-CEM and plerixafor (0.020 mM) were also acquired. All NMR data were processed with the software VNMRJ 1.1.D (Varian Inc.). 1D spectra were analyzed by using the software Bruker Topspin (www.bruker.com). 2D NOESY spectra were analyzed by using CARA (Computer Aided Resonance Assignment) software.42 For structure determination of 3, 2D DQF-COSY,43,44 TOCSY,45 and NOESY46 spectra of the peptide (2 mM) were recorded in 200 mM SDS-d25 micelle solution. Spectra were acquired in the phase-sensitive mode using the method from States.47 Data block sizes were 2048 addresses in t2 and 512 equidistant t1 values. Before Fourier transformation, the time domain data matrices were multiplied by shifted sin2 functions in both dimensions. A mixing time of 70 ms was used for the TOCSY experiments. NOESY experiments were run with mixing times in the range of 50-200 ms. The water signal was abolished by gradient echo.41 Proton assignments were obtained using the standard reported strategy (Table S1, Supporting Information).48

Structural Determinations of 3. The NOE-based distance restraints were obtained from NOESY spectra acquired with a mixing time of 100 ms. The NOE cross peaks were integrated with the XEASY49 program and were converted into upper distance bounds using the CALIBA program incorporated into the program package DYANA.50 Only NOE derived constraints (Supporting Information, Table S2) were considered in the annealing procedures. Non-standard GBA residue was added to DYANA residue library using the Biopolymer module of InsightII (Accelrys, San Diego, CA). An ensemble of 200 structures was generated with the simulated annealing of the program DYANA. From the produced 200 conformations, 50 structures were chosen, whose interproton distances best fitted NOE derived distances, and then refined through successive steps 29 ACS Paragon Plus Environment


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of restrained and unrestrained energy minimization calculations using the Discover algorithm (Accelrys, San Diego, CA) and the consistent valence force field (CVFF)51 An ensemble of the lowest 10 energy conformer of 3 is shown in Figure S6 (Supporting Information). Molecular graphics images were realized using the UCSF Chimera package.52

Molecular Docking Docking of the 3 NMR structure was performed in the crystal structure of CXCR4 in complex with CVX15 (PDB code: 3OE0).9 Both the ligand and the receptor were prepared using the Protein Preparation Wizard implemented in the Maestro Suite 2016.53 First, bond orders were assigned and missing hydrogen atoms added. A prediction of the side chains ionization and tautomeric states was then performed using Epik.54 Finally, an optimization of the receptor hydrogen-bonding network was performed, and the positions of the hydrogen atoms were minimized. Prior to docking, all the water molecules were deleted from the receptor structure. For the grid generation, a virtual box of 30 Å × 30 Å × 30 Å, surrounding the inner ligand binding cavity site, was created. Two independent sets of calculations, with the Arg4, Phe5 and Phe6 side chains alternatively constrained or unconstrained, were then performed in the Glide SP-peptide mode.55,56 During constrained docking calculations, the following ligand dihedral angles were kept fixed: N-Cα-Cβ-Cγ, Cα-Cβ-Cγ-Cδ and Cβ-Cγ-Cδ-Nε for Arg4; N-Cα-Cβ-Cγ for Phe5 and Phe6. Docking solutions were clustered on the basis of the ligand cyclic backbone rmsd (cutoff = 1.5 Å). Molecular Dynamics Prior to MD simulations, the receptor structure was refined as described in a previous publication.26 The

optimized

3/CXCR4

complex

was

then

embedded

in

a

1-Palmitoyl-2-

oleoylphosphatidylcholine (POPC) bilayer through the protocol applied in the same work.26 A 10 Å TIP3P water layer was then added on both the side of the POPC bilayer through the solvation 30 ACS Paragon Plus Environment

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module of VMD 1.9.3. The ff14SB57 and lipid1458 Amber force fields were used to parameterize the protein and the peptide, and the lipids, respectively. Missing parameters for the ligand GBA1 residue were generated according to a previously described procedure.26 Six Cl- counterions were added to ensure neutrality. The system was then submitted to over 100 ns MD simulations with NAMD 2.10.59 A 10 Å cutoff (switched at 8 Å) was used to calculate atom pair interactions. The long-range electrostatic interactions were computed by means of the particle mesh Ewald (PME) method using a 1.0 Å grid spacing in periodic boundary conditions. The RATTLE algorithm was applied to constrain bonds involving hydrogen atoms, thus allowing the use of a 2 fs integration time step interval. Each system was minimized and heated up to 300 K while applying harmonic constraints, which were gradually released along the equilibration process. To prevent any distortion in the receptor transmembrane helices their Cα carbons were constrained for further 10 ns. Production run was then performed in the NPT ensemble, at 1 atm and 300 K. CXCR4 residues were numbered according to both the wild-type primary sequence and the Ballesteros Weinstein scheme60 (as superscript). All of the pictures were rendered using PyMOL (www.pymol.org). ASSOCIATED CONTENT Supporting Information STD and WaterLOGSY NMR spectra of 1 alone and upon plerixafor titration; trNOESY spectrum of 3 in the presence of a suspension of CCRF-CEM cells and plerixafor; 1H and STD NMR spectra of 2 and 8 in the presence of a suspension of CHO+ cells; rmsd plot of the 3 Cα carbons along the MD production run; superposition of the ten lowest energy conformers of 3;

1

H NMR resonance

assignments of 3 in SDS solution; NOE derived upper limit constraints of 3; PDB coordinates of the predicted 3/CXCR4 complex. The Supporting Information is available free of charge on the ACS Publications website at DOI: …



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PDB ID Code NMR structure of 3 in SDS solution, 5OLF. Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION

Corresponding Author *Prof. Alfonso Carotenuto; e-mail: [email protected] *Prof. Luciana Marinelli; e-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. §These Authors contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This research was funded by Scientific Independence of Young Researchers (SIR) 2014 (Grant RBSI142AMA) to S.D.M, MIUR-PRIN 2015 (Grant FCHJ8E) to L.M., and Euro-Nanomed (Nano)systems with active targeting to sensitize colorectal cancer stem cells to antitumoral treatment (Target4Cancer) to S.S. We also acknowledge Prof. Vittorio Limongelli for granting access to the Swiss National Supercomputing Center HPC resources (CSCS, under project ID s712). ABBREVIATIONS ACN, Acetonitrile; CHO, chinese hamster ovary; CXCL12, C-X-C Motif Chemokine 12; CXCR4, C-X-C Chemokine Receptor Type 4; DIEA, N,N-Diisopropylethylamine; DQF-COSY, Cosy 32 ACS Paragon Plus Environment

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Double Quantum Filter; DPFGSE, Double Pulsed Field Gradient Spin Echo; FBS, Fetal Bovine Serum; GBA, guanidinobenzoic acid; HBTU, (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaﬂuorophosphate);

HOBT,

Hydroxybenzotriazole;

MD,

molecular

dynamics;

PDL1,

Programmed death-ligand 1; POPC, 1-Palmitoyl-2-oleoylphosphatidylcholine; RPMI, Roswell Park Memorial Institute; RP-HPLC, Reversed Phase High Performance Liquid Chromatography; SDS, sodium dodecyl sulfate; SP, standard precision; STD, Saturation Transfer Difference; TIS, Triisopropylsilane; TNBS,

2,4,6-Trinitrobenzenesulfonic acid; TOCSY, Total Correlation

Spettroscopy; trNOESY, Two-dimensional transferred Nuclear Overhauser Effect Spettroscopy; WaterLOGSY, Water Ligand Observed via Gradient Spectroscopy



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