Phosphorus-Based Probes as Molecular Tools for Proteome Studies

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Phosphorus-based probes as molecular tools for proteome studies: recent advances in probe development and applications #ukasz Joachimiak, and Katarzyna M. Blazewska J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00249 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Journal of Medicinal Chemistry

Phosphorus-based probes as molecular tools for proteome studies: recent advances in probe development and applications Łukasz Joachimiak, Katarzyna M. Błażewska*

Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Żeromskiego Str. 116, 90-924 Łódź, Poland * Corresponding author Abstract Studies on the human proteome have engaged diverse techniques; however, none of them represent a predominant approach. Chemical biology has made a major contribution to our understanding of human biology, stimulating the generation of biological hypotheses. Tools such as functional probes have advanced studies on biological mechanisms and helped in elucidating off-target reactivity and potential toxicities of drugs and drug candidates. Here, we accentuate the recent developments in the design and applications of phosph(on)ate-containing probes. Phosphate esters and anhydrides are present in a number of vital cell constituents, and their significance can be reflected by a number of biological processes that involve phosphorus-bearing molecules. We discuss the use of phosph(on)ate-derived probes for 1) the identification of phosphate-requiring enzymes, their substrates, interacting partners; 2) developing screening assays; and 3) their potential as diagnostics. Limitations that as yet need to be overcome and possible measures to be undertaken will also be addressed.

1. Introduction

ACS Paragon Plus Environment

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Studies on the human proteome have engaged diverse techniques, none of which represent a predominant approach. Genetic manipulations, including targeted gene knockdown, knockout or overexpression techniques, may lead to phenotypes with altered protein function or protein-protein interactions. However, compensatory effects following a gene knockout can be developed as a consequence of the loss of protein function. Therefore, operating at the level of the gene or its transcribed product might not provide an adequate view of the temporal, spatial and dynamic properties of proteins in cells. The immunochemical methods that are often employed can be laborious and impractical for high-throughput studies. Additionally, such methods are not very sensitive to discriminating between interactions of a similar magnitude. The use of inhibitors that block catalytic activity may help to more adequately decipher the functions of proteins in the cell, while leaving doubt about potential side interactions and their influence on the observed phenotypic response. Over the past two decades, chemical biology has made a major contribution to our understanding of human biology, using chemical proteomics to stimulate the generation of biological hypotheses. Tools such as functional probes bearing an added functionality (a photoreactive group, an affinity tag, a fluorophore, or a bioorthogonal handle) are used for the capture, visualization, and identification of biomolecules.1, 2 They have advanced studies on many biological mechanisms, such as signaling pathways, cell cytoskeleton, mitotic spindle or proteasome function and have helped in elucidating off-target reactivity and potential toxicities.3 Highly sensitive, lowbackground fluorescent probes are used not only for in vitro bioassays but also enable the visualization of targeted proteins in living cells. Probes equipped with affinity tags are used for the selective labeling and isolation of target proteins, allowing their subsequent identification and analysis with quantitative MS. Probes equipped with a


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photoaffinity label are used to covalently crosslink their targets within the environment of the live cell, allowing for the detection even of non-covalent interactions. Tagging with an analytical label is also possible via a two-step approach through the use of a small size bioorthogonal handle. In such cases, the unwanted interactions that potentially affect the affinity of the probe with the target can be excluded, since a bulky residue is introduced only at the detection step.

Figure 1. Structures of selected physiologically relevant and inhibitor-based phosphorus-containing molecules that were used for the development of functional probes for profiling the interaction of proteins.

In this review, we report on the selected functional probes derived from phosphorusbearing inhibitors or natural biomolecules (Figure 1). Phosphate ester and anhydride functionalities are present in a number of vital cell constituents, including nucleotides, oligonucleotides, nucleic acids, dinucleoside polyphosphates, acetyl coenzyme A, isoprenoid

diphosphates,

phospholipids,

and

phosphorylated

proteins.

The

significance of such compounds can be reflected by a number of biological processes


3


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that involve phosph(on)ate-bearing molecules. For example, up to 30% of all human proteins are targeted by ~200 structurally and functionally diverse phosphatase domain-containing enzymes, while ~500 kinases are responsible for phosphate group attachment.4, 5 More than 2000 acetylated proteins and over 4000 lysine acetylation sites are identified in the human proteome, which require the activity of lysine acetyltransferases and acetyl coenzyme A as a substrate.4, 6 Serine hydrolases, one of the largest enzyme classes,7 constitute approximately 1% of all predicted human proteins, and their action can be modulated by fluorophosphonates. Phosphorus-containing probes have been used for studying the activity and identification of phosphate-requiring enzymes, their substrates, and their interacting partners. In this regard, we do not attempt to give a comprehensive overview on this matter; rather, we accentuate the recent developments (2010-2017) in the design and structural optimization of such probes, together with their applications. The synthetic procedures are not discussed here since they have been thoroughly reviewed previously.8 To improve the clarity of the tables, the structures of the reported probes are often minimized and instead abbreviated names of selected reporter tags are inserted, as defined in Figure 2.


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Figure 2. The structures of selected reporter tags used in the design of presented probes.

2. Nucleotide-derived probes A number of processes in the cell are mediated by nucleotides, with ATP being a predominant source of energy and phosphate groups. Such processes can be traced by the detection of a phosphate residue introduced onto a protein or by generating a signal upon cleavage of a phosphoric anhydride bond. For such applications, the nucleotide of interest needs to be appropriately labeled so that the modification does not render the molecule biologically inactive. Base-labeled nucleoside 5’


5


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triphosphates are present in commercial kits used for DNA sequencing, gene expression analysis and genotyping,9 while new polynucleotide sequencing strategies, which employ nucleotides modified at the terminal phosphate, have also been successfully developed.10 ATP analogs with an alkyne group on the γ-phosphate are often used for the design of easily available (via click chemistry) libraries of new inhibitors or chemical biology tools.11-13 Here, we focus on the application of nucleoside triphosphate-derived probes bearing an appropriate tag at the γ-phosphate, enabling the detection of nucleotide-dependent processes catalyzed by a number of enzymes, such as kinases, ATP-cleaving enzymes, diadenosine polyphosphate hydrolases, and decapping enzymes. While during the preparation of this manuscript, Hacker and co-workers partly covered this topic in an excellent review,10 we have included additional data on the structural optimization of the probes and have provided facile access to their applications in terms of applied techniques and biological models used, showing their universal character. Protein kinases constitute the major class of enzymes that utilize ATP as a phosphate group source. They modulate a wide variety of biological processes and are involved in cell migration, differentiation and growth. During phosphorylation, a γ-phosphate group is transferred from ATP (GTP and phosphoenol pyruvate can also serve as a phosphate donor) to an appropriate amino acid residue. The ATP binding pocket of kinases is highly conserved across ~500 human kinases and >1000 other nucleotidebinding proteins. Phosphate esters of serine, threonine, and tyrosine are predominantly formed, but the phosphorylation of histidine, lysine and arginine, generating high-energy phosphoramidate linkages, also occurs.14


6

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a) OO OO OO P P P N O O O H

O

Base

OR OR

OO OO P P -O O O

kinase

OO P N H

OO P N O H

+

protein identification kinase activity determination

-

kinase-protein complex identification

-

Base

OR OR

HO

= tag

b)

O

-

kinase

O N H

OO P N O H

OP

O

effector identification = tag: photoaffinity label (PAL)

Scheme 1. a) Incorporation of a reporter tag onto a protein through kinase-catalyzed phosphorylation; b) when a photoaffinity label is applied as a reporter tag, the previously phosphorylated protein can be further tethered with a processing enzyme or a neighboring protein upon photolysis. Red sphere: target protein; green spheres: neighboring proteins.

Among the different methods used for the detection of protein phosphorylation (immobilization through metal-affinity chromatography, 2D gel analysis, MS analysis, phosphate staining and phospho-specific antibodies15, most frequently used method involves

32

16

), historically, the

P radiolabeling with [γ-32P]ATP.17 In

addition to being lengthy (exposure times of weeks to months) and not very sensitive, the radioactive tracer does not allow isolation of the phosphorylated protein, which is one of the major limitations of this method. ATP-γ-S is an alternative phosphate mimetic, containing a small modification in the form of a sulfur atom in the γ-position and is accepted as a substrate by many kinases. After incorporation into the protein, it allows further functionalization to enable purification and visualization. However, a major limitation of this probe is the protein background in the cell, since thiol


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moieties of cysteines are characterized by reactivities similar to the thiophosphate group.18

19

An example of a strategy to differentiate between these two reactivities

was proposed by Garber and Carlson.20 After introduction of a thiophosphate moiety, the thiol groups of cysteines were blocked with a selective capping reagent in a radical-mediated, thiol-ene reaction and then a thiophosphoryl group is reacted with an electrophilic reagent, BODIPY-iodoacetamide, leading to fluorescently labeled protein. In this chapter, we will focus on ATP analogs labeled at the γ-phosphate with an appropriate visualizing handle in the form of fluorescent, affinity, photolabile, bioorthogonal or electrochemical functional tags. Pflum and co-workers21 studied how the γ-phosphate modification of ATP affects the efficiency of the conversion of probes 1-4 by kinases (Table 1, entry 1). It turned out that higher conversion was achieved for longer and more polar linkers, which probably assure sufficient distance from the kinase active site. Additionally, the structure of the functional tag also plays a role here, i.e., smaller functional tags can be attached via a shorter linker (e.g., an acetyl group combined with a two-carbon linker), and the higher polarity of a tag (e.g., sulfonamide in dansylated analog) may compensate for the hydrophobic character of the linker. Using the preferable polyethylene glycol linker, the biotin-labeled affinity probe 5 was developed and used as a substrate for various kinases, showing lower or comparable phosphorylation efficiency to ATP (53-81%16 and 44-96%22). Importantly, proteins phosphorylated with the biotin-tagged moiety were 17-28% less prone to phosphatase hydrolysis than natively phosphorylated biomolecules.16,

23

Thanks to the acidic lability of a phosphoramidate bond, upon protein isolation the


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biotin tag could be removed, giving a phosphorylated product identical to the native one and facilitating quantitative studies by HPLC and gel-based methods.24 The probe 5 was also used in the K-BILDS method (kinase-catalyzed biotinylation with inactivated lysates for discovery of substrates) that was used for the identification of kinase substrates.25 The lysate was first incubated with covalent inhibitor in order to inactivate all kinases. A kinase of interest was then added, catalyzing the phosphorylation of a particular substrate, which upon affinity purification could be analyzed by LC-MS/MS. The authors identified 279 candidate substrates of the studied kinase (PKA), including 56 previously known and 3 confirmed novel substrates. Although probe 5 is characterized by a high conversion and kinetic efficiency, it did not show cell permeation. To improve cell permeability of the ATP probe, Pflum and co-workers developed an analog 6.26 The improved cell permeability of this probe was ascribed to the partial neutralization of the triphosphate’s negative charge with a cationic polyamine linker, methylated spermine. The difference between the identity of proteins biotinylated in the cell vs. lysate demonstrates the significance of the environment on the phosphorylation process, indicating the influence of compartmentalization inside the cell.26

Table 1. Selected probes for protein kinase profiling.a Entry

Probe

Techniques used

Biological model


9


1

2

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Quantitative

proteomics

Isolated

PKA;

21

CK2; Abl

1) Quant. MS; 1D and 2D

1) pairs of PKA;

gels;

CK2; Abl and

sensitivity

phosphatases;

to

16

peptide

2) HPLC or SDS-PAGE; phospho-protein stain; 3)

K-BILDS;

PAGE;

22

SDS-

phosphoprotein

stain; LC-MS/MS

substrates; HeLa

lysate;

phosphatases: PP1,

CIP,

TCPTP;

25

2) 25 kinases; 3) FSBA-treated HeLa

lysates

incubated

with

kinase PKA

3

cell fixation; SDS-PAGE;

HeLa

cells;

visualization

HeLa

cell

streptavidin-Cy5)

(e.g. 26

lysate; PKA – MBP


10

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4

1) 7a-d: CV; SWV;

27

2) 7d, 8, 9: CV; SWV;

1) 28

3) 7b: Fc-Ab: in solution

plate;

fluorescence

imaging;

TOF-SIMS;

X-ray

its

peptide

2) Kinases: Src; Erk1; CDK2/cyclin A kinase;

photoelectron spectroscopy;

kinase Src and

substrate;

and on the surface; microaaray

isolated

3) Au-bound α-

29

4) 7b: Enzyme kinetics;

30

5) 7b: CV; SWV; EIS; Fc-Ab: in solution and on the surface; microaaray

plate;

fluorescence imaging

31

casein; caspase3; kinases: Src; CDK2;

CK2;

Erk1;

PKA;

HeLa lysate up to 30%; 4)

substrate:

Tau410, kinases:

GSK-

3β; Src; PKA; 10%

human

serum albumin; 5)

Bacterial

histidine kinases: PhoR; PhoQ; EnvZ;

5

Fc-Ab;

fluorescence

imaging; SECM


32

Src;

CDK2;

CK2α

11


6

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1) 11, 12: SDS-PAGE;

1) CK2 kinase;

WB; MALDI-TOF MS;

substrate:

docking;

33

casein; 2) CK2 kinase

2) 12: MS;34 3)

12:

strategy;

α-

K-CLASP streptavidin-

and

α-casein;

biotin-labeled protein;

Cy5; MS35

cell

lysate, homogenized tissue; 3) PKAα and CK2

plus

biotinylated peptide substrates, e.g., mutant kemptide; HeLa lysate

and

biotinylated kemptide

7

SDS-PAGE;

WB;

Fe3+-immobilized

MS;

affinity column

8

LC

36

H/19F NMR; irradiation fluorescence

detection; SDS-PAGE; ATP-γ-S studies

from

chicken

skeletal muscle

1

and

Actomyosin

ATP-binding isolated proteins: Hsp90;

ATP

or

GDH

competition

37


12

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9

in-gel

fluorescence

HEK 293T cell

studies; GTP and ATP

lysate (MudPIT

competition studies; LC-

experiments);

MS/MS

38

transfected cells overexpressing targets

a

In this table, the abbreviation ATP is used for the framed residue in entry 1; abbreviations:

PKA: cAMP-dependent protein kinase A; CK2: casein kinase 2; Abl: protein tyrosine kinase; K-BILDS: kinase-catalyzed biotinylation with inactivated lysates for discovery of substrates; PP1: protein phosphatase 1; CIP: calf intestinal phosphatase; TCPTP: tyrosine phosphatase Tcell protein; FSBA: covalent inhibitor, 5‘-(4-fluorosulfonylbenzoyl) adenosine hydrochloride; MBP: myelin basic protein (kinase substrate); CV: cyclic voltammetry; SWV: square-wave voltammetry; Src: sarcome-related kinase; Fc-Ab: polyclonal rabbit antiferrocene; EIS: electrochemical impedance spectroscopy; CDK2: cyclin-dependent kinase 2; GSK-3β: glycogen synthase kinase; PhoR, PhoQ, EnvZ: bacterial histidine kinases; SECM: scanning electrochemical microscopy; HSP 90: heat shock protein 90; GDH: L-glutamate dehydrogenase; HEK 293T: Human embryonic kidney cells 293; K-CLASP: kinase-catalyzed crosslinking and streptavidin purification.

Ferrocene-modified bioconjugates are popular redox reporters thanks to their electrochemical properties and stability in aqueous solution.32 Kraatz and co-workers have developed γ-ferrocene-modified ATP derivatives 7-10 for monitoring protein kinase activity and for inhibitor screening. The method is based on phosphorylation of peptides immobilized on a gold electrode with ferrocene-labeled phosphate, which leads to a quantifiable redox response directly related to the reactivity of the studied kinases.39 The authors observed that among the synthesized probes, the phosphoramide analogs were chemically more stable than their ester counterparts (Table 1, entries 4-5). Additionally, hydrophobic analogs 7 with a longer all-carbon linker (>=C6) were better co-substrates for protein kinases than analogs with a polar polyethylene glycol linker (8 and 9).28, 40 Such a trend may indicate that hydrophobic


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linkers of appropriate length provide for reduced steric crowding in the kinase binding site, while hydrophilic linkers may reduce the efficiency of phosphate transfer to peptides because of polar interactions.27 Interestingly, compound 10, bearing as a linker the lysine moiety functionalized through an amide bond with propargylamine, showed impaired interaction with some kinases, such as Src and CK2α, but not with other kinases (CDK2).32 This impaired interaction might result from the shortening of the linker and the introduction of an amide bond, which is capable of electrostatic interactions with the catalytic pocket and is more conformationally constrained. An alkyne tag present in probe 10 enabled introduction of a fluorescent dye via a click reaction, a feature that can be further utilized for developing a fluorescent assay, an alternative to immunodetection. The ferrocene-modified proteins could also be detected with polyclonal rabbit antiferrocene antibody, which was developed and successfully used in solution and on Au surfaces in an immunoarray format using 7b29 and 1032 as probes. No cross-reactivity was observed for phosphorylated and unphosphorylated protein samples, and multiple phosphorylation sites could be detected.29 Probe 7b was also an effective co-substrate for the autophosphorylation of histidine protein kinases in surface-based and antibody format.31 It was observed that the autophosphorylation efficiency depends on the concentration of the probe. This may be caused by the binding of Fe2+ or the ferrocenyl moiety to the sensory domain of the kinase. The interference of divalent cations (Mg2+) in autophosphorylation has been previously observed.31 Additionally, the presence of human serum albumin leads to a current density decrease (50-70%), but the signal remains sufficiently strong for quantification.30


14

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Another class of probes constitutes ATP analogs containing a photolabile group, which can be linked upon UV irradiation with the interacting protein. Such a probe enables identification of the phosphorylation site and the proteins interacting with kinases. Among the first probes of this class, an aryl-azide group was directly connected with γ-phosphate. Such analogs turned out to be fairly potent inhibitors of Src and Csk kinases.41 The ATP-derived probes 11 equipped with a benzophenone moiety,33 or an aryl-azide residue, as in 12,34 attached to γ-phosphate were used for kinase-substrate crosslinking. The benzophenone probe turned out to be a more efficient kinase co-substrate than the azide derivative (71% vs 51%) and produced more crosslinked complexes with CK2 and casein. Probe 12 was used in the K-CLASP approach (Kinase Catalyzed CrossLinking And Streptavidin Purification),35 in which a known kinase-substrate pair, with the latter modified with biotin, were studied. Upon generation of a covalent bond between the kinase (PKA) and substrate, the fluorescently labeled streptavidin-Cy5 was used for detection of such a conjugate. A total of 60% of the K-CLASP hits (out of 324 proteins identified via tandem MS) were accounted for in the PKA interactome and may result from direct or indirect (neighboring proteins) interactions. Although GTP-binding proteins are implicated in a plethora of cell processes, such as signaling, trafficking, and nucleotide metabolism, GTP-derived probes are much less explored compared with the ATP analogs. On the other hand, many strategies applied to the design of ATP probes could be extended to other nucleoside triphosphates. Hatanaka and co-workers36 used probes 13a-b for analyzing the interactions within the actomyosin complex, showing selective labeling of tropomyosin. The trifluoromethyl phenyldiazirine moiety and its biotinylated version were linked


15


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through a phosphoramide bond to an ATP moiety. Another concept was applied in the design of probe 14 for the identification of the ATP-binding proteins, Hsp90 and GDH.37 The linker between photolabile diazirine and ATP consisted of a nonfluorescent α-hydroxycinnamate moiety which, upon irradiation, was transferred into a fluorescent derivative of coumarin. Rosen and co-workers designed probe 15 equipped at the γ-phosphate residue within GTP with a photolabile group and a bioorthogonal alkyne tag.38 Blocking vicinal hydroxyl groups in sugar had minimal impact on affinity to the GTP-binding proteins. In addition to GTP-binding proteins, ATP-binding targets (26% among all hits), proteins binding purine nucleotides, and targets that are not annotated as GTP-binding proteins were identified. This cross-reactivity of 15 is greater than for the acyl-ATP probes (discussed below), which targeted only 1.5% of GTP-binding proteins.42 The acyl nucleotide derivatives 16-21 (Table 2) constitute a separate class of probes, in which a terminal phosphate in ATP (or GTP) or ADP is modified with an affinity tag linked via an acyl functional group. The first representatives were developed in 2007 as biotin-functionalized ATP analogs.42, 43 This strategy relies on the presence of conserved lysine residue(s) within the kinase active site that react with an acyl phosphate group forming an amide bond, with the simultaneous cleavage of the large ATP or ADP group (Scheme 2). The resulting conjugate is equipped with an uncharged affinity tag appropriate for the isolation and MS analysis of the conjugate. The dominant reactivity of acyl-ATP towards lysine residues may be responsible for the nonspecific labeling of proteins that do not bind ATP. In kinome analysis, this may not be a major issue since conserved lysine targets near ATP-binding sites are known, and through peptide sequence analysis other hits can be excluded. However, for the analysis of other ATP-binding proteins that do not have a conserved P-loop


16

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motif, a different approach is required, e.g., using ATP and ATP-probe competition to distinguish ATP-binding proteins from nonspecific labeling.44

Scheme 2. Mechanism of action of acyl-ATP as an activity-based probe for kinases.

Although biotin is a frequently used affinity tag for protein isolation, the high stability of its complex with streptavidin might impede the elution of the biotinylated molecules from resin. Desthiobiotin can come to the rescue here, since its lower affinity to avidin may enable a better recovery of peptides and improve detection sensitivity. However, comparison between biotin-labeled 16 and 17 and desthiobiotinlabeled probes 18 and 19 did not show a substantial difference.45 However, in some applications, desthiobiotin has shown higher stability during post-labeling procedures (e.g., in-gel digest and LC-MS/MS analysis), while biotin was possibly oxidized to the sulfoxide.46 The desthiobiotin ATP and ADP analogs 18a and 18b have been developed into commercial products used in a chemoproteomics platform (KiNativTM) to globally study potential kinase inhibitors in cell lysates. In KiNativ, IC50 values measure active site binding rather than enzymatic activity. Programming the instrument to collect MS/MS data on known kinase peptide ions at specified times improves the sensitivity and accuracy of quantification, overcoming the limitations of low kinase abundancy and the interference by highly abundant ATP binding proteins. Such a targeted MS


17


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approach can be assembled for any cell, tissue, or species type. Comparative studies between four MS platforms (DDA, DIA, MRM, and PRM, Table 2) have been conducted to evaluate their sensitivity, reproducibility of detection and quantification of low-abundance desthiobiotinylated proteins against a complex background of higher abundance labeled peptides from all ATP-binding proteins, showing the complementarity of all methods.47 This platform is complementary to another commercially available technology, kinobeads, based on kinase inhibitors immobilized on beads.48, 49

Table 2. Acyl-ATP probes for protein kinase profiling Ent

Probe structure

Techniques used

Biological model

1) 16, 17, 18a, 19: SDS-PAGE;

1) HL-60 lysate;

LC-MS/MS; 2D-LC separation;

2) HL60 and PC3 cell

SILAC;45

lines

2) 18a,b: LC-MS/MS;50

with inhibitors;

3) 18a,b: LC-MS/MS with DDA

3) H1993 lysate; kinase

and DIA; LC-MRM; LC-PRM;47

inhibitors

4) 16, 18a: in-gel digest; LC-

influence on down-stream

MS/MS; dependance of labeling

pathways;

on pH; salts; chelating agents; 46

4)

5)

(Arabidopsis); lysate from

ry 1

18a:

shotgun

proteomics

lysates

Plant

pretreated

and

their

proteome

analysis;51

leaf extract;

6) 18a, 18b: LC-MS/MS;

5) Profiling of pathogenic

comparison of kinobeads and

organism, Mycobacterium

active site labeling;48

tuberculosis

7) 18a, 18b: LC-MS/MS; WB52

6) Profiling Aurora kinase inhibitor, tozasertib; K562 cell lysate 7)


Inhibitor

Sensitive:

18

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COLO-205;

MCF-7;

inhibitor resistant: MDAMB-468;

BT

549;

xenograft tumor mouse model 2

3

Quantitative

affinity

profiling;

HeLa-S3; Jurkat-T cell

ICAP53, 54

lysates

LC-MS (control of reaction);

Tyrosine kinase c-SRC

WB; streptavidin purification55

and biotinylated quasisubstrates; HCT-15 cell lysate

SILAC: stable isotope labeling with amino acids in cell culture; DIA: data-independent acquisition; DDA: data-dependent acquisition; PRM: parallel reaction monitoring MS; MRM: multiple reaction monitoring MS; PC3: human prostate cancer cell line; ICAP: isotope-coded ATP-affinity probe.

Acyl nucleotide probes were used for profiling the activity and expression levels of ATP-binding proteins in cell lysates and tissues upon treatment with known kinase inhibitors. The biological models resembled natural conditions, which make it superior to assays based on isolated proteins. In addition to well-characterized ATP binding proteins, new hits were identified.50 The acyl-ATP strategy was extended to other NTP-binding proteins by exchanging adenosine for other nucleosides. Wang and co-workers identified unique nucleotidebinding motifs for kinases and GTP-binding proteins using the ATP- and GTPderived probes 16-19 (Table 2, Entry 1).45 The binding affinity ratio (RATP/GTP) differentiated specific from nonspecific interactions with lysine residues. The selected examples of the application of KiNativTM include studies on plant proteome (Arabidopsis),46 profiling the ATP-ome of Mycobacterium tuberculosis for normally growing and hypoxic-state bacteria,51 proteome-wide identification of ATP-


19


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binding proteins and kinase inhibitor target proteins,44 and profiling the kinase inhibitor in drug-sensitive and resistant cells.52 The last studies enabled the observation of the effect of kinase inhibitors on targeted proteins and indirect hits, the latter being responsive due to a secondary effect of inhibition of targeted kinases. KiNativTM also enabled the determination of the ability of the inhibitor to penetrate the cell.52 Wang and co-workers53 introduced an isotope-coded affinity probe 20 (ICAP), in which desthiobiotin was connected with ATP via a light (6 hydrogens) or heavy (6 deuterons) isotope-coded linker, enabling quantitative affinity profiling of ATPbinding proteins. This strategy guarantees the incorporation of an isotope label into the protein at the early stage of the experiment, minimizing potential errors during further processing. By using low and high concentrations of ICAP probes, nonspecific labeling of lysines not present in ATP-binding sites can be excluded. An interesting approach was proposed by Shokat and Riel-Mehan55 who used crosslinking probe 21 for the identification of kinase-substrate pairs. The lysine residue from kinase reacted with a phosphoanhydride group, becoming functionalized with a methylacrylate residue, which was further modified via the Michael addition of thiol from the substrate protein (Scheme 3).

Scheme 3. Protein-kinase complex formation via a two-step reaction, utilizing probe 21. Red sphere: target protein.


20

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Another class of probes comprises the ATP analogs containing a fluorescence donor and a fluorescence acceptor suitable for undergoing Förster resonance energy transfer (FRET). Upon disconnecting the fluorophores, the FRET signal is terminated. Alternatively, a combination of the fluorescence quencher and fluorophore gives a fluorogenic probe which, upon detachment of these two parts, becomes fluorescent. In both cases, the studied enzyme catalyzes cleavage of a phosphoanhydride bond, leading to the separation of the fluorophores and, as a consequence, decreases or increases, respectively, a fluorescence signal (Scheme 4).

Scheme 4. Application of a nucleotide-derived probe equipped with two fluorescent dyes for the determination of activity of an α,β-phosphoanhydride bond-cleaving enzyme (presented on the example of a triphosphate analog fluorescently labeled on the γ-phosphate and base residues).

In such analogs, one fluorescent dye is localized at a terminal phosphate and the second one on a heterobase (N6 or C2 position) or a sugar moiety (O2’ or O3’ position), depending on the preference of the studied enzyme towards a particular modification of a natural substrate. In the reported studies, both positions were modified with the six-carbon linker and functionalized with azide (for a terminal phosphate) or a trifluoroacetamide group (for a nucleoside). Such orthogonal groups were easily transformed into an amine group and selectively functionalized with


21


Page 22 of 110

different fluorophores. The linker was attached to a phosphate through an ester bond, based on the previous studies which have shown the higher stability of this connection over a wider pH range (2-12) compared with an amide-linked triphosphate.56 The authors synthesized and characterized a number of probes that were built either from a combination of different fluorophores, including sulfonated analogs with higher water solubility, or from less polar dyes with potentially higher cellular uptake. The probes showed up to a 140-fold increase in fluorescence upon the action of phosphodiesterase (Table 3, entry 1).57 Table 3. ATP-derived probes equipped with two fluorescent groups or two spin labels. Entry 1

Probe*

Techniques used 1)

22,

23a-e,

Biological model 24a:

fluorescence charecteristics;

from HPLC

analysis;57 2)

24b:

1) Phosphodiesterase I

(SVPD); 2)

HPLC;

C.adamanteus

Phosphodiesterase;

bacterium

HRMS;58

Desulfococcus biacutus;

3) 23a, 23f: confocal

cell-free extracts;

microscopy; FRET59

3)

cell

lysates;

recombinant

NudT2;

electroporation

for

delivery into HeLa cells 2

Fluorescence characteristics;

activity SDS-

of

UBA1;

APPBP1/UBA3;

PAGE; WB; TRASE;

SAE1/SAE2;

HTS assay60

Xenopus

laevis

egg

extract


22

Page 23 of 110 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


3

HPLC; HRMS; EPR61

SVPD used as a model

4

1) 27: development of

1)

HTS; fluorescent read-

cytotoxicity

out;

HEK293T; H1299 (Fhit

cytotoxicity

isolated

Fhit; in

studies; 62

negative) cell lines;

2) 28: development of

2) Ap4A phosphorylase:

HTS assay; docking of

Rv2612c

substrates63

NudT2: human diadenosine tetraphosphate hydrolase; TRASE: time-resolved ATPase sensor; UBA1: human E1 enzyme for ubiquitin; SVPD: snake venom phosphodiesterase, Fhit: fragile histidine triad protein; H1299 lung cancer: Fhit-deficient cancer cell line; HEK 293T: human embryonic kidney cells endogenously expressing Fhit; Rv2612c: Ap4A phosphorylase of Mycobacterium tuberculosis

The Marx group60 developed probe 25, which was used in the first direct assay, TRASE (Time-Resolved ATPase SEnsor), for monitoring ubiquitin-activating enzyme E1 (UBA1). UBA1 forms a thioester between ubiquitin and the E1 catalytic cysteine through the consumption of ATP. While modification at the N-6 position of ATP does not compromise UBA1 activity, the δ-modified tetraphosphate is a superior substrate for UBA1 compared with the γ-modified triphosphate analog. The assay was used in HTS format for the screening of 1279 compounds. It could also be applied to E1-related ubiquitin-like (Ubl) proteins pairs. Probes 23a and 23f

59

were used to study nucleotide hydrolysis in intact human cells

and lysates. In lysates, only the adenosine tetraphosphate Ap4 analog 23f was processed, while in intact HeLa cells, the effect (observed as a decrease of FRET efficiency) was observed for both analogs 23a and 23f. This indicated the difference


23


Page 24 of 110

between both of the environments due to a reduction in activity of some enzymes during lysate formation and/or as a result of the varying conditions in different cellular compartments. Acetone degradation by cell extracts of Desulfococcus biacutus bacteria was studied with probe 24b, which upon cleavage of a phosphoanhydride bond, restores the specific fluorescence of the donor.58 A similar approach was applied to studies of hydrolytic enzymes, but the fluorescence tags were exchanged with spin labels attached at different locations on the ATP analog 26.61 The effect of their dipole-dipole coupling was observed as an EPR signal, which vanishes upon cleavage by the respective enzymes. The advantages of this approach include the lack of paramagnetic centers in most biological systems, the use of weak radiation that is not damaging to biological samples, and the relatively small spin label size compared to the fluorescent labels. However, the experiment involves freezing the sample at -50 °C in 20% glycerol . The FRET approach was also applied for the development of a high-throughput screening assay for diadenosine triphosphate hydrolase (Fhit) and diadenosine tetraphosphate phosphorylase. The probes were based either on doubly labeled triphosphate 2762, 64 or tetraphosphate 2863 derivatives, in which the FRET pair was introduced on the terminal phosphate and an amine group of the nucleobase. Their fluorescent

characteristics

changed

upon

enzymatic

cleavage

by

Fhit

or

tetraphosphate phosphorylases. Libraries of 15136 and 42000 compounds were tested, identifying several inhibitors that were then subjected to more in-depth screening.

3. Acetyl-coenzyme A-derived probes for acetyltransferase studies


24

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Lysine acetylation is a dynamic, post-translational modification that is involved in pivotal cellular functions; there have been over ~4000 lysine acetylation sites identified in the humane proteome.6 An acetyl group is donated by the cofactor acetyl-coenzyme A (Ac-CoA) to an amine group of the lysine side chain in a process catalyzed by lysine acetyltransferase (KAT), also referred to as histone acetyltransferases (HAT). KATs play a key role in the regulation of gene expression, epigenetic programming, the cell cycle, apoptosis, metabolism, and signal transduction.65 Their altered expression and activity is observed in a number of pathological states.

Scheme 5. (a) Utilization of CoA-derived probes for the labeling of KAT substrate(s); b) Utilization of CoA-derived probes for the labeling of KAT. Red sphere: target protein; green spheres: neighboring proteins.

One of the main strategies developed for studies of KAT-mediated processes involves the use of functional probes derived from Ac-CoA, in which an acetyl group is exchanged for an acyl chain equipped with an analytical handle. Among such probes, two main classes can be distinguished (Scheme 5). One group bears an energy-rich thioester bond (as in Ac-CoA) between coenzyme A and the acyl group, which can be easily cleaved with the simultaneous acyl group transfer to the lysine side chain of the


25


Page 26 of 110

targeted protein (Scheme 5a). The second class of probes is derived from the “bisubstrate” inhibitor of KAT, which combines two substrates, lysine and the coenzyme A residue, through a stable, non-cleavable thioether bond (Scheme 5b). These probes are equipped with a photoaffinity handle in order to form covalent linkages with the interacting protein.66

Table 4. Acetyl coenzyme A-derived probes.1 Entry 1

Probe moiety

Techniques used SDS-PAGE, fluorescence;

Biological model in-gel

isolated p300 and

LC-

H3 peptides;

and

Human

KATs:

fluorescent assays for

GCN5,

MOF,

KATs activities;

p300

MS/MS67 2

Radioactive

SDS-PAGE,

3

4

in-gel

(WT

mutants);

and HEK

fluorescence65

293T cell lysate;

HPLC, LC/MS; assay

microsomal

for detection of 7-O-

fraction

acyl form of okadaic

digestive gland of

acid (DTX3) 68

bivalves;

kinetic parameters of

Polyhydroxyalkan

PhaCCc, PhaECav and

oate synthases:

selected CoA analogs;

wt-PhaCCs,

Enzyme activity assay69

A479S-PhaCCs;

of

the

PhaECav;


26

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5

37-40: SDS-PAGE,

pCAF HEK-293

photocrosslinking, in-

overexpression

gel fluorescence; WB;

extracts; HeLa

KAT inhibition assay;

cell lysates;

enrichment; LC-

recombinant

MS/MS70

p300, pCAF, Mof;

41: competitive chemical proteomics; LC-MS/MS; WB71 6

unbiased LC-MS/MS;

HeLa cell lysate;

competition with CoA; WB72

1

In this table, the Co-A abbreviation is used for the framed residue in entry 1.

In the first class of KAT probes, acyl residues of different lengths were attached to coenzyme A and were studied in diverse models. Two myristic acid-CoA analogs, modified with an alkynyl or azide terminal group, were used as substrate by Nmyristoyl transferase (NMT) for transfer to an N-terminal glycine residue.73 Hang and co-workers67 used alkynylated acyl-CoA analog 29 for the identification of acetylated proteins and to investigate KAT activity. Among the three analogs, compound 29b was the preferred acyl donor for the acylation of H3 histone protein, while the analog 29a with a shorter linker was not accepted as a substrate for the transcriptional coactivator p300, which could be the result of its relatively low stability. The


27


Page 28 of 110

bioorthogonally tagged analogs 30-3165 were used to identify other enzyme-cofactor pairs among the major families of human KATs. The shape of the binding pocket was adjusted by the introduction of up to two mutations. The three functional probes 32-34 were assessed as substrates for the in vitro acylation of okadaic acid (OA) by an enzyme identified in the digestive gland of shellfish. The alkynylated and azido analogs were superior to (E)-pentadeca-12,14dienoyl CoA, as they were more conveniently converted into fluorescent derivatives by bioorthogonal click reactions.68 For studies of the substrate specificity of polyhydroxyalkanoate synthases (PhaC, class I and class III), the Li group69 synthesized the two probes 35 and 36. The authors observed that probes with a shorter linker and an azide group were preferable substrates for PhaCs, while the introduction of a terminal triple bond led to a slight decrease or increase of the activity, the direction of the change being dependent on the synthase. The structurally simplest representatives of the second class of the bisubstrate-type of KAT probes, which contain a Lys-CoA scaffold, can be relatively promiscuous.72 The probe specificity can be increased by flanking the lysine residue with different peptide sequences, matching those from the natural substrate. Using the latter approach, Meier and co-workers70 developed a suite of probes for cofactor-based affinity profiling of representatives of three phylogenetically distinct families of KATs: p300, pCAF, and Mof. All of the probes were equipped with benzophenone as a photoaffinity group. The clickable probe 37 had a superior labeling efficiency of pCAF compared with the more bulky fluorescent probe 39. The probes were used to determine KAT’s activity in different settings; however, they cannot be used for studies in living cells since they are not cell-permeable. Chemoproteomic profiling


28

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with these probes has led to the identification of several previously unknown acyltransferase activities and the identification of two orphan KAT enzymes. This method requires further refinements in order to detect low-abundance KATs in nonoverexpressed native settings. The metabolic regulation of KATs was studied using biotin-labeled photoactive probe 41. The authors reported an inhibitory interaction between palmitoyl-CoA and the KAT enzymes,71 which may suggest that fatty acyl-CoAs are endogenous regulators of histone acetylation and are involved in the metabolic modulation of epigenetic signaling. In another study, Meier and co-workers72 developed a platform for establishing the potency and selectivity of reversible stimuli, such as the feedback metabolite CoA in complex proteomes. Three resin (sepharose)-linked capture probes 42-44 were synthesized, of which two are flanked by peptide sequences specific to different subfamilies of KATs. While the method worked for a number of KATs, it was unable to detect the activity of the p300/CBP family, probably due to difficulties in accommodating the bulky Sepharose-linked bisubstrates in the active site. On the other hand, these capture agents enrich several cofactor-dependent enzymes that do not harbor annotated acetyltransferase activity. 4. Isoprenoid diphosphate-derived probes for studies of post-translational prenylation GTPases are a class of proteins requiring post-translational modification for proper functional activity. In this process, the thioether bond between the C-terminal cysteine(s) of GTPase and the farnesyl or geranylgeranyl group is formed. The prenyl carbon chain is provided by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate

(GGPP).

This

post-translational


process

is

catalyzed

by

29


prenyltransferases,

such

as

protein

farnesyl

transferase

Page 30 of 110

(PFTase,

FTase),

geranylgeranyl transferase 1 (GGT-1) or geranylgeranyl transferase 2 (GGT-2, RabGGTase).74 Perturbation of this process has been implicated in several disease conditions, including numerous cancers, Parkinson’s disease, Alzheimer’s disease, and viral infections.74 Therefore, there is a need for the development of tools to identify the protein transferases and their substrates and to quantitatively detect the changes in the levels of prenylated proteins in certain diseases. One of the strategies utilizes probes derived from the natural substrates, prenyl diphosphates, equipped with a detection-enabling modification incorporated into the carbon chain (Scheme 6). Previously, most of these probes were radiolabeled (e.g., [3H]-mevalonate, [3H]-FPP or [3H]-GGPP).75 Currently, analytical handles, such as fluorophores, biotin or bioorthogonal groups, are broadly utilized. Since they constitute a bulkier modification compared with isotope labeling, they are incorporated at the expense of shortening the isoprenoid chain so that the overall length and steric volume resemble that of the prenyl moiety. Often, such modifications are introduced at the third isoprenoid unit proximal to C-12 of FPP.

76

In some cases, the modification

disqualifies the probe as a substrate for the target protein, but the affinity remains comparable to natural substrates. The probe, which is a substrate for all prenyltransferases, can be used for examining the entire prenylome. Nevertheless, selective probes that are a substrate for only one prenyl transferase are useful in examining the substrates of individual enzymes.


30

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Scheme 6. Labeling of prenyltransferases (PT) substrates with prenyl diphosphatederived probes. Red sphere: target protein; green sphere: neighboring protein.

The modified prenyl chain does not need to be combined with the pyrophosphate moiety to serve as a probe. Instead, bare prenyl alcohol analogs can be metabolically converted into the corresponding diphosphate and then can be built into the protein substrate. This strategy facilitates the synthesis of probes and possibly improves their permeability, which otherwise might be hindered by the ionic character of the diphosphate moiety.77-80 Another strategy introduces prenyl chains onto peptides by a chemical reaction in order to check how such a modification influences further processing of the peptide inside the cell. As was shown by the Distefano group,81 incorporation of modified prenyl moieties should not induce undesired physiological changes. Such was the case with a peptide-bearing prenyl chain that was modified with a diazirine group, which remained a substrate for its processing enzyme, to which it was efficiently crosslinked upon UV irradiation, showing higher efficiency compared with the benzophenone-modified analogs.82 A convenient route for the synthesis of differently modified prenyl diphosphate probes has been elaborated on by Alexandrov and co-workers.83 The biotin-tagged probe 45 (Table 5, entry 1), a substrate only for GGTase-II, was efficiently incorporated into the Rab GTPase structure.84 An analog with a shorter isoprenoid


31


Page 32 of 110

chain was shown to be a weaker substrate for GGTase-II. In the case of the BODIPYFL probes, 46a-b (Table 5, entry 1), both have shown affinity towards PFTase comparable to FPP, but only the probe with the shorter isoprenoid chain, 46a, was incorporated into the protein substrate by GGTase-II.83 Since bulky fluorophores or biotin moieties may adversely influence a probe’s affinity towards an enzyme, Tate and co-workers used the azide-modified probe 47 in tandem labeling of GGTase-I and GGTase-II substrates with a multifunctional agent, enabling both visualization and enrichment.85 With these probes, changes in prenylation in disease models can be detected.85 Using the previously developed probe 48,86 Distefano and co-workers found that the labeling pattern of proteins is affected by prenyl transferase inhibitors.87 Subsequently, Gibbs, Fierke and Distefano88 used the alkyne-modified FPP analogs 48-50 to detect endogenously farnesylated proteins using dansylated peptide substrates. While the linear analogs 48a-b showed a similar farnesylation pattern to FPP, the branched analogs 49 and 50 had the potential to catalyze prenylation of a wider variety of peptide substrates. The observed variability in reactivity and specificity of the probes implies that for thorough proteomic studies, the use of different analogs is recommended. Since FTase can process a number of functionalized probes that are characterized by a similar size and hydrophobicity to that of its natural substrate FPP, these probes can be used for site-specific modification of recombinant proteins that are equipped at the C-terminal with a CaaX sequence recognized by prenyl transferases (a: aliphatic amino acid, X: Ser, Met, Ala or Gln). Such modifications may improve the specificity, potency and pharmacokinetic profile of a protein.89 Selected applications of this strategy are listed below.


32

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Distefano and co-workers have used probes 51-52 to label GFP-CaaX and GIP-CaaX proteins with aldehyde groups. This modification was later used for protein isolation from a complex lysate mixture through immobilization and release from beads.89 Other studies from the same group90 demonstrated the simultaneous and selective labeling of proteins using two isoprenoid-derived probes, each specific for a different prenyl transferase, PFTase or GGT-1. Probe 55 was reported in that study for the first time. Thus, labeled proteins were later connected to form tail-to-tail dimers in a CuAAC reaction. In the future, this strategy may allow concurrent studies on farnesylated and geranylgeranylated proteins and the generation of large, multidomain fusion proteins that are not accessible by standard genetic methods. Trans-cyclooctene geranyl was site-selectively introduced onto peptides and proteins using probe 56, which allowed their further functionalization via tetrazine ligation without the use of toxic copper catalysts.91 Kim and co-workers used geranyl ketone pyrophosphate (GKPP) and FTase in the chemoenzymatic construction of a proteindrug conjugate. After being prenylated with ketone pyrophosphate, the labeled protein underwent oxime ligation with the cytotoxic drug.92 The labeling of protein with triorthogonal probe 58 enabled further modification of the protein with two different tags.93 The azide-functionalized probe 59 was used for enzymatic labeling of a GFP construct.94 The analog was site-specifically conjugated with an alkyne-modified DNA fragment and used in model studies of the influence of DNA-protein cross-links (DPCs), which are helix-distorting DNA lesions, on DNA processing in the organism. These studies indicated that polymerases cannot read through the larger DPC lesions.

Table 5. The isoprenoid pyrophosphate-derived probes. Entry

Probe

Techniques used


Biological model

33


1

Page 34 of 110

Microscope

isolated RGGT

fluorescence

and REP1;

imaging; flow

BHK cell line;

cytometry,

zebrafish

WB, kinetic

embryos

studies, SDSPAGE,83

2

SDS-PAGE, in-gel

Isolated GGT-1

fluorescence, WB85

and RGGT in

cell lysate; gm, Chm, C57BL/6J mice tissue models;

3

1) 48a: SDS

1) HeLa,

PAGE, in gel

MCF10A,

fluorescence 87

astrocytes

2) 48a: LC-

2) Plasmodium

MS/MS, in-gel

falciparum

fluorescence95

tissue culture;

3) 48-50: in vitro

isolated

continous

PfFPPS;

spectrofluorimetric

3) FTase and

assay88

dansylated peptides:


34

Page 35 of 110


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

4

MALDI; in vitro

Recombinant

fluorescence assay;

proteins with

immobilization/

CaaX box

release on agarose

(GIP-CVIM,

beads89

GFP-CVIA); E.coli extract with GIPCVIM

5

SDS PAGE, LC-

Rat GGT-1 and

MS; In vitro

rat PFTase;

labeling of

GFP-CVLL;

peptides catalyzed

RFP-CVIA;

by two prenyl

dansylated

transferases90

peptides with CaaX box

6

ESI-MS;

Isolated FTase,

Continuous

eGFP-CVIA,

fluorescence-based

Oregon Green-

assay, Size

CaaX; N-

exclusion

dansylates

purification;91

GCVIA


35


7

Page 36 of 110

Cytotoxicity;

EGFR-specific

protein-drug

repebody;

conjugate92

HCC827 and A431 cell lines

8

confocal

GFP-CVIA and

microscopy; flow

CNTF model

cytometry; LC MS;

proteins; HPB-

internalization

MLT cells

studies93 9

HPLC MS/MS;

6xHis-eGFP-

SDS-PAGE;

CVIA; DNA

polymerase

with C8-

assay94, 96

alkyne-dU

BHK: baby hamster kidney cells; gm: gunmetal; Chm: Choroideremia mouse - C57BL/6J mouse (wild type (wt) as control); MCF10A: non-tumorigenic epithelial cell line; CNTF: ciliary neurotrophic factor; EGFR: epidermal growth factor receptor; repebody: a non-antibody scaffold composed of leucine-rich repeat modules.

Non-covalent interactions of pyrophosphates and isoprenoid-interacting enzymes can be studied by utilizing the photoaffinity labeling method.93 In this approach, a natural prenyl diphosphate scaffold, constituting the recognition element, is equipped with a photoreactive group which, upon UV irradiation, generates highly reactive species that are susceptible to reaction with proximal amino acid residues. This topic has been recently reviewed by Distefano and co-workers.97 To the best of our knowledge, no studies on this matter have been reported since 2010; therefore, here we will only shortly illustrate this topic. The first isoprenoid analogs with azide moieties were reported in 1979.98 Later, the benzophenone (either in meta- or para-substituted form) was most commonly utilized


36

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for the modification of prenyl chains. Despite being the bulkiest among the known photolabile moieties, it bears a certain structural similarity to the isoprenoid chain. The most common linkage between a photoreactive group and a truncated prenyl chain is an ester, amide or flexible ether bond, with the latter either in the phenoxy or alkoxy form. The linking groups have their own limitations, such as low stability towards hydrolysis (for esters) and increased rigidity adversely affecting binding (for amides). The ionic end of the probe, a diphosphate residue, can be exchanged for a phosphonophosphate moiety making the compound more stable under acidic conditions but simultaneously eliminating it as an enzyme’s substrate. In addition to analogs functionalized only on one phosphorus group, as in the native prenyl pyrophosphates, there are examples of derivatives bearing two different moieties on both phosphorus atoms, e.g., a prenyl-resembling benzophenone residue on one phosphorus and a carbon chain functionalized with biotin or azide groups on the other,99,

100

or a farnesyl group on one phosphorus and benzophenone on the

other.101 However, none of these probes were substrates for the studied PFTases.

5. Phospholipid-derived probes for profiling lipid-protein interactions Biological membranes are barriers that separate the cell interior from the extracellular space. One of their main components are phospholipids which, apart from a structural role, participate in a wide range of cell signaling processes through interactions with membrane-associated proteins. They are differentiated as glycerophospholipids and sphingophospholipids, dependent on their core structure. Additional diversity results from the phosphate ester residue (e.g., choline, ethanolamine, serine, inositol polyphosphate) and the fatty acyl chains.


37


Page 38 of 110

The insertion of phospholipid-derived probes into biological systems might be difficult due to a negatively charged phosphate group that could introduce alterations within the structure. Even synthetic natural phospholipids can show different behaviors from those synthesized by the cellular machinery, since only the latter are delivered to targets through native pathways. The evaluation of a probe’s potency is often limited to artificial systems that do not reflect the native cell environment. An alternative is based on the utilization of labeled building blocks for their metabolic incorporation into phospholipid structures, as was recently done in studies of proteinphosphatidylcholine interactions.102 The position of the reporter tag in a phospholipid scaffold has a significant impact on the observed interaction profile. The physicochemical properties of lipids (hydrophobic tail and hydrophilic head) force an appropriate arrangement of the probe in the membrane, which may result in a diverse labeling of proteins on the surface and within the core of the membrane. Therefore, the utilization of probes with different locations of the reporter tag is recommended. The type of tag is also important, as has been shown for the phosphatidylcholine-derived bifunctional probes103 bearing either a benzophenone or a diazirine moiety. Studies with a model of the inner mitochondrial membrane from Saccharomyces cerevisiae have shown efficient labeling by both probes, but the observed interaction profiles were significantly different. The first class of probes described here consists of two modifications, a reporter tag and a photoaffinity label, the latter being responsible for linking the probe with the interacting protein (Scheme 7).


38

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Scheme 7. The mechanism of action of a probe derived from a phospholipid and modified in two positions, the sn-2 and head groups, with reporter and photoaffinity tags. PAL: photoaffinity label.

The Hazen group designed bifunctional phosphatidylcholine-mimicking probe 60, in which a diazirine moiety and an alkyne residue were incorporated into the choline and the fatty acid chain residue, respectively (Table 6, entry 1).104 This probe was used for labeling protein associated with high-density lipoprotein. Best and co-workers elaborated on methods for the modular synthesis of probes derived from phosphatidic acid105 and phosphatidylinositol polyphosphate (PIP).106 The latter approach was used for the synthesis of phosphatidylinositol 3,4,5trisphosphate (PI(3,4,5)P3) probes 61-63, which were utilized for the identification and characterization of protein binding partners from cancer cell extracts (Table 6, entry 2).107 The glycerol backbone was replaced by single or double aminohexyl chain(s) that served as a linker between an interacting phosphoinositol head group and a photoaffinity tag. The amine group was used as a coupling point for functionalization with appropriate handles. Probe 62 was found to be superior, and its shorter linker was beneficial for effective protein labeling. It could be surmised that while a longer linker might provide greater flexibility to the probe, it may also hinder benzophenone’s interaction with the protein, which is necessary for the formation of the covalent bond. LC MS/MS studies gave a number of hits, among which the


39


Page 40 of 110

previously known PI(3,4,5)P3 binding partners were found and new hits, potentially representing target effectors, were identified. On the other hand, a number of known PI(3,4,5)P3 binding proteins were not detected, which may be ascribed to their low abundance and low cross-linking efficiency or to the differentiation of the PIPnbinding domains depending on their affinity to different regions of membrane. Probe 62 corresponds to the soluble headgroup analog, which was confirmed by the identity of the labeled proteins. Bong

and

Bandyopadhyay

elaborated

a

method

for

the

synthesis

of

phosphatidylserine-derived probes 64-65, which are equipped with alkyne and benzophenone groups placed within the same (64) or different (65) fatty acyl chains in the sn-1/sn-2 positions (Table 6, entry 3). The probes were shown to label the isolated phosphatidylserine protein target prothrombin-1 but not its heat-denatured form.108 Phospholipid-derived probes have also been utilized as tools for the investigation of phospholipases. Phospholipases mediate cell signaling through the formation of fatty acids and lysophopholipids, which are generated upon their action. There are four classes of phospholipases (PLs): PLA, PLB, PLC and PLD, each of which catalyzes the cleavage of different bonds across the phospholipid scaffold (Scheme 8). One of the approaches for studying their activity is based on the utilization of doubly labeled fluorescent

substrate-based

probes,

where

the

fluorescence

changes

upon

phospholipase-mediated hydrolysis. Depending on the combination of fluorophores built into the probe, either a decrease in Förster resonance energy transfer (FRET) phenomenon or an increase in fluorescence is observed, thereby quantitatively reporting on the rate of substrate hydrolysis. Since there are four sites appropriate for cleavage upon PL activity, the selectivity of the probe can be achieved by placing the


40

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fluorescent groups at ends that are separated upon a particular enzyme’s action. When the activity of the sn-1 or sn-2 ester selective PLs (PLAs) is studied, the reporter groups must be located on fatty acyl chains. In the case of PLD or PLC activity, one of the reporter groups must be located on the head group of the phosphate ester.

Scheme 8. The mechanism of action of a phospholipid probe upon phospholipase PLA2-mediated cleavage. On the left: the general structure of a phospholipid with cleavage sites for different phospholipases marked with arrows (R: fatty acid chain).

Much work has been dedicated to the development of probes for phospholipase A, including a fluorogenic probe in which the ester in the sn-1 position was exchanged for a non-hydrolyzable ether109 or amide110 moiety. Potential permeability issues due to the ionic character of the phospholipid probe were addressed by the application of a prodrug strategy.109, 111 The Hajdu group110,

112

designed fluorogenic probes 66-68 that target secretory

phospholipase sPLA2. Analogs modified with amide or ester groups in the sn-1 position did not show a difference in hydrolysis rates. Lam and co-workers elaborated the selective probe 69, which is capable of reporting the activity of either the cPLA2 or sPLA2 forms depending on the experimental conditions, taking into consideration


41


Page 42 of 110

that a membrane-like environment is essential for cPLA2 activity (Table 6, entry 6).113 Brask designed the two fluorogenic probes 70-71, which were used for the development of a FRET-based assay for phospholipase activity.114 Analogs with one fluorescent tag that enables tracking of probe localization in cells using

fluorescence

microscopy

constitute

another

group

of

probes.

The

phosphatidylserine- or phosphatidylcholine-based probes 72-74 (Table 6, entry 8) were evaluated for their ability to penetrate selected eukaryotic cell lines. Among the six tested probes, the previously developed115 coumarin analog 73 can enter the cytosol

and

exhibit

desirable

fluorescent

and

photostability

properties.

Carboxyfluorescein probe 72 was not translocated, which might imply that the protein possibly responsible for its transport, aminophospholipid flippase, does not accept analogs with charged and highly polar groups attached to the 2-acyl chain.116 Another monofunctional probe, 75, was developed as a mimic of glycolipid, phosphatidyl inositol mannoside (PIM),117 which is a potential anti-inflammatory agent. It was previously discovered that exchanging the inositol moiety for piperidine does not affect the biological activity of the parent PIM.118 Here, a nitrogen atom in piperidine was used to equip the PIM scaffold with a biotin or a fluorescent label through an aminocaproyl spacer. While the rhodamine-labeled analog has shown significant toxicity, the non-toxic biotin derivative 75 was used in localization studies with a streptavidin-Alexa 532 conjugate. Phospholipases belonging to the serine hydrolase family were studied utilizing activity-based probes 76-77, in which the fluorophosphonate moiety, known for its selective binding of serine hydrolase (see section 7.1),119 was built into the phosphatidylcholine scaffold that contained a reporter handle. Depending on the


42

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position of the modification (sn-1 or sn-2), the probes showed selectivity towards the particular model lipases, PLA1 or PLA2, respectively. 120 A different group of analog probes were used, not for protein studies, but as artificial membrane fusion mediators that are potentially applicable as drug delivery systems. For

example,

the

phosphatidic

acid

scaffold

was

equipped

with

an

azadibenzocyclooctyne moiety enabling its functionalization through strain-promoted alkyne-azide cycloaddition (probes 78-79).121 The analog 80, bearing ferrocene, was also studied in this context.122 It was used for the formation of redox-active vesicles with encapsulated anticancer drugs.

Table 6. The phospholipid-derived probes. Probe

Techniques

Biological model

used

1

Affinity

site-specific

isolation LC-

mutagenesis

of

MS/MS104

Paraoxonase

1

(PON1)

2

62: LC-MS/MS;

purified protein

competition

Akt-PH domain;

studies with

cytosolic and

PI(3,4,5)P3; in-

membrane

gel fluorescence

fractions of:

detection107

MDA-MB-435, MDA-MB-231, MCF7, T47D; heat denatured proteomes


43


3

Page 44 of 110

SDS-PAGE,

64a, 64b: isolated

fluorescence

PT-1 and its heat

scanning108

denatured form

FRET-based

Bee-venom

enzyme activity

(PLA2)

4

measurement112

5

Enzymological

Bee-venom

characterization:

secretory

fatty-acid-

phospholipase A2

binding-protein

(sPLA2)

fluorescence assay and pHStat method110

6

Cytotoxicity

sPLA2,

with/without

cytotoxicity in

Triton X-100 or

BV-2

in liposomal

recombinant

solution113

cPLA2


cPLA2;

cells;

44

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7

FRET-based

Isolated

phospholipase

phospholipases:

assay;

PLA1,

PLA2,

PLC,

PLD,

kinetic

studies114

lipolase;

8

Fluorescence

Cell lines: V79,

microscopy for

CHO, A549;

localization studies: transport

into

cells116

9

cytokines

Bone

marrow-

TNFα, IL12p40

derived

release detected

macrophages;

by ELISA; Cytotoxicity; Inhibition of cytokine release;


45


Page 46 of 110

confocal microscopy117

1

SDS-PAGE, in-

Isolated

0

gel

phospholipases:

fluorescence;

DDHD1

inhibition

of

CDTA120

serine

and

DDHD2; mouse

brain,

testis proteome

1

membrane

1

fussion

Liposomes FRET

studies; STEM121

1

SECM, TEM,

HeLa and

2

DLS,

MRC-5

fluorescent imaging122 Akt: protein kinase B; PLA2: phospholipase A2; sPLA2: secretory Phospholipase A2; cPLA2: Ca2+dependent phospholipase A2; V79: Chinese hamster lung fibroblasts; CHO: Chinese hamster ovary cells; A549: human lung carcinoma cells; MDA-MB-435, MDA-MB-231, MCF7, T47D: cancer cell lines; BV-2: mouse microglial cells; MRC-5: normal lung fibroblast; CDTA: calcium-dependent transacylase; PT-1: human prothrombin-1; SECM: scanning electrochemical microscopy; TEM: transmission electron microscopy; STEM: scanning transmission electron microscopy; DLS: dynamic light scattering.

6. Phosphorus-containing probes as tools for the investigation of phosphatases


46

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Phosphatases belong to a wide group of enzymes responsible for the dephosphorylation of various substrates, such as proteins and nucleotides. Their participation in many intracellular signaling pathways is indispensable for proper cell functioning. The expression levels of acid and alkaline phosphatases has been routinely used for disease diagnosis and determination of the therapeutic efficiency of certain treatments.4 Determination of their activity can be helpful in distinguishing normal from abnormal cell behavior.4 The complexity of the dephosphorylation processes requires different approaches to elucidate the roles of phosphatases in many human diseases, one of which is the application of phosphorus-based functional probes.123 Much attention has been focused on the development of fluorogenic probes, which can be applied as screening tools for phosphatase inhibitors. On the other hand, activity-based probes (ABPs) which are capable of binding covalently to enzymes, may be used for determining phosphatase activity, abundance and localization. Phosphatase-targeting probes are often derived from enzyme-recognizable, phosphate ester-bearing mimics of native substrates, such as phosphorylated amino acids or peptides. Flanking of the phosphorylated amino acid residue with an appropriate protein sequence is often used to increase the specificity of the probe. The role of the phosphate moiety is often to retain the probe in the turn-off state, which upon enzyme-mediated cleavage changes into the spectroscopically detectable form (Scheme 9).


47


Page 48 of 110

Scheme 9. Fluorogenic moieties with a phosphate masking group release fluorescence through: a) restoration of conjugated π-electron system or aggregation– induced emission; b) cleavage of a phosphate bond, which triggers a quinone methide-based self-immolative linker expulsion.

Among the first fluorescent probes used were derivatives of coumarin and fluorescein which were efficiently hydrolyzed despite bearing only a minor resemblance to the natural substrates of phosphatase.4,

123-125

Further efforts in this area were directed

towards the elimination of disadvantages, such as low specificity and the diffusible nature of the probe.124, 126 In another approach, probes 81-82 were developed with a self-immolative spacer derived from 4-hydroxybenzyl alcohol (Table 7, entries 1 and 2). 127,

128

Upon

enzyme-induced dephosphorylation, the cascade reaction involving spacer expulsion and fluorescence release is triggered (Scheme 9b). This led to a significant improvement of the fluorogenic substrate specificity, as was shown in comparative studies.127 In the newer generation of probes, such as 83, fluorescent moieties characterized by longer excitation and emission wavelengths are applied (Table 7, entry 3). This feature eliminates the influence of background fluorescence during visualization of enzyme activity in living systems. Moreover, a positively charged fluorophore may play an additional role by neutralizing the negatively charged phosphate moiety, potentially improving the probe’s permeability.129 An alternative to conventional fluorescence imaging technique is two-photon (2P) fluorescence microscopy (TPFM). The TPFM technique is characterized by increased penetration depth, lower tissue autofluorescence and self-absorption, and reduced


48

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photodamage, which makes it suitable for imaging deep tissues and animals. Yao and co-workers designed the two-photon fluorogenic probe 84, in which the phosphataseresponsive warhead is equipped with a 2P fluorescence reporter and cell-penetrating peptides (CPP), the latter localizing the probe to a particular organelle (Table 7, entry 4).4 The phosphate was masked with a 2-nitrobenzyloxy group that was uncaged upon UV-irradiation. It protects the phosphate group from cleavage triggered by other proteins before entering the cell and potentially increases the probe’s permeability through the cell membrane. This strategy has allowed the spatiotemporal imaging of phosphatase activity only in predetermined intracellular locations. In another design, probe 85 was built from two partners, a 2P reporter (85b), having a π-conjugated fluorene moiety modified with two six-carbon chains for anchoring to the membrane, and a fluorescence quencher coupled with a photocaged phosphorylated phenolic group (85a) (Table 7, entry 5). The pairing partners come into contact due to electrostatic interactions localizing at the plasma membrane where phosphatase hydrolyzes the phosphate bond, leading to an increase of the fluorescence signal.130

Table 7. The probes for phosphatase profiling. Entr

Structure

Techniques

Biological

y

used

model

1

Fluorimetry127

ALP from bovine kidney


49


2

3

Page 50 of 110

Fluorescence

ALP;

characteristics

HeLa and

; confocal

HEK 293

microscopy128

cellsa

Near IR

Isolated

fluorescence

ALP; HEK

imaging129

293 and HeLaa; Kunming mice (ip)

TPFM4

4

Isolated phosphatase; HeLa and HepG2 cells; Drosophila brains

5

TPFM; FRET;

Eight normal

fluorescence

and cancer

characteristics

cell lines

; flow cytometry130


50

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6

7

Fluorescence

Isolated

characteristics

ALP; HeLa

; CLSM131

cells

Dual-mode

Isolated

fluorescence

ALP

„turn-on” biosensor: determination of ALP activity and presence of protamine132 8

Confocal

Isolated

microscopy;

ALP; Rat

fluorescence

BMSCs

characteristics ; flow cytometry133

9

confocal

Isolated

microscopy;

ALP; HeLa

co-staining

and HepG2;

with organelle

Saos-2 and

specific

U-2OS

trackers; nonand


51


Page 52 of 110

competitive ALP inhibitors134 10

Fluorescence

Isolated

characteristics

ALP; HeLa

; confocal

cells

microscopy135

11

in-gel

Purified

fluorescence

PTPs;

analysis;

Lysates:

WB136

HEK293T, NIH3T3

12

in-gel

Recombinan

fluorescence

t PTP1B; E.

analysis;

coli lysates

FACS137

overexpressing PTP1B

13

One- and two-

Isolated

photon

PTPs:

mictroscopy;

PTP1B (also

SDS-PAGE

denatured

138

and inhibited), TCPTP, PTPB, LMWPTP; Hela cell lysates


52

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ALP: alkaline phosphatase; aHeLa and HEK 293 cell lines used as models with high or low expression of ALP, respectively; CPP: cell-penetrating peptide; TPFM: two-photon fluorescence microscopy CLSM: confocal laser scanning microscopy; BMSCs: bone marrow mesenchymal stem cells; Saos-2 and U-2OSL osteosarcoma cell lines, with high and low ALP activity, respectively; PTP: protein tyrosine phosphatase; FACS: fluorescence-activated cell sorting.

The phenomenon of aggregation-induced emission (AIE) was also utilized for the design of alkaline phosphatase (ALP) probes. Unlike traditional fluorophores that undergo

fluorescence

quenching

upon

aggregation,

fluorogens

with

AIE

characteristics are non-emissive when dissolved and can only emit when aggregated (Scheme 9a). A typical AIE fluorogen has a propeller-shaped rotorlike structure, often built from phenyl rings, which undergoes low-frequency motions when dissolved, making the fluorogen non-emissive. In an aggregated state, these motions are restricted by intermolecular steric interactions, making the aggregates strongly fluorescent. One of the most popular AIE scaffolds is tetraphenylethylene (TPE). When this fluorogen is connected to a phosphate group, it does not aggregate, presumably as a result of its high solubility in water. Upon dephosphorylation (e.g., mediated by phosphatase), the solubility decreases leading to aggregation, which prevents the probe’s diffusion and allows the fluorescence readout at the site of action with improved resolution and contrast. A similar AIE fluorogen was used to design the dual-mode fluorescence “turn-on” biosensor 87, which was developed by Tang and co-workers.132 It enabled the observation of the response from two types of stimuli: electrostatic interactions of an anionic probe with a cationic protamine, which led to aggregation in the form of micelles; or the fluorescence turn-on due to aggregation upon cleavage of the phosphate residue (Table 7, entry 7). This system can be used for protamine and ALP


53


Page 54 of 110

detection. In other studies, probes with a multi-phosphate-substituted TPE core were evaluated (Table 7, entry 8).133 A probe with two phosphate moieties, 88, was found to be optimal, with a higher detection limit compared with a monophosphorylated analog and a less complex interaction scheme compared with a tetra-phosphatecontaining probe. In a recent report, the TPE scaffold was used for the development of probe 89 based on the excited-state intramolecular proton transfer (ESIPT) mechanism, a photochemical process occurring in the excited singlet state of a molecule with intramolecular hydrogen bonds. The spectral characteristic of the probe was improved and its fluorescence after phosphatase-catalyzed reaction is no longer blurred by autofluorescence from the living cell. Such a probe is suitable for imaging with confocal microscopy. High spatiotemporal resolution was achieved for probes based on TPE (89) (Table 7, entry 9)134 and the chalcone scaffold 90 (Table 7, entry 10).135 The potential of AIE probes in ALP activity monitoring, both in studies with isolated enzymes and living cells and in inhibitor screening, has been clearly demonstrated in various models.131-133, 139 As fluorescent probes cannot precisely identify which protein is responsible for changes in the fluorescence signal, activity-based probes (ABPs) have been developed to enable the proteomic analysis of phosphatases. Protein tyrosine phosphatases (PTP) belong to the most studied representatives of this class of enzymes. The nucleophilic cysteine in the active site can be utilized for covalent labeling with electrophilic probes. The common structural feature of such probes mimics the phosphotyrosine residue (Scheme 10), which is recognizable by PTPs. Such an approach was used by Zhang and co-workers in the design of non-hydrolyzable α-bromobenzylphosphonate


54

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inhibitor equipped with a biotin or rhodamine moiety.140,

141

Unfortunately, the

relatively unstable character of these probes has limited their applicability.142 Another approach relies on the utilization of a phosphotyrosine mimetic as a “latent trapping device”, which is activated after phosphatase-mediated dephosphorylation. The intermediate alcohol undergoes rearrangement to an electrophilic quinone methide which then undergoes a rapid reaction with a nearby nucleophile, labeling the target protein in activity-dependent manner (Scheme 10b).143

144

Although the

diffusible nature of quinone methide is sometimes limiting, its high cross-reactivity when applied to the crude proteome can also be advantageous, as is shown below.

Scheme 10. Phosphatase activity studies through quinone methide intermediate formation: a-b) upon dephosphorylation, a reactive electrophilic quinone methide is formed which is susceptible to reaction with a nearby nucleophile; in b) the dephosphorylation process requires prior decaging of a phosphate group. Upon cleavage of the P-O bond, a quencher is released leading to the generation of a fluorescent signal. Red sphere: target protein.

To increase the specificity of the probe, a latent quinone methide building block was incorporated into different peptide sequences (up to twelve amino acid residues),


55


Page 56 of 110

constituting an essential recognition element for native phosphatase (91, Table 7, entry 11), 136, 145 For tracking PTP activity in live cells in real time, another approach has been devised. Cell-penetrating peptides (CPP), each specific for a given subcellular organelle, were combined with a phosphorylated coumarin-derived amino acid residue. Additionally, the phosphate was masked with a photocleavable group, which upon photolysis is released and undergoes hydrolysis mediated by phosphatase. This triggers fluorescent coumarin formation, allowing its spatiotemporal observation (92, Table 7, entry 12).137 PTP activity can also be traced by the quenched activity-based probes 93-95, in which a recognition warhead and a fluorophore are combined with a fluorescent quencher, which is expelled from the probe upon PTP-mediated dephosphorylation and quinone methide formation (Table 7, entry 13, Scheme 7b). The authors took advantage of the diffusible nature of the quinone methide intermediate which, by binding to proteins in the vicinity of target enzyme, amplifies the fluorescence signal. Probe 95 was also equipped with a CPP residue in order to improve cell permeability. Additionally, the authors used a phosphate group in a caged form. Uncaging of the probe before incubation with HeLa cells resulted in membrane-localized fluorescence. Uncaging of the probe after incubation with HeLa cells indicated successful intracellular delivery of the probe because fluorescence was detected inside the cells.138

7. Probes derived from inhibitors bearing phosphorus In previous chapters, we have discussed the development of probes from naturally existing molecules. This chapter is devoted to probes derived from known inhibitors containing a phosphorus-based group responsible for the formation of a covalent bond


56

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with

the

targeted

protein.

We

focus

here

on

fluorophosphonates

and

diphenylphosphonates, which represent the first activity-based probes. These probes consist of a chemical group that covalently modifies the active site of an enzyme and an analytical handle that facilitates a simultaneous readout of enzyme activity. The chemical reactivity of fluorophosphonates and diphenylphosphonates originates from the phosphonate group, which reacts with the hydroxyl group in the active site, leading to the expulsion of a fluoride or phenoxy anion. While in previous chapters all probes were based on phosphate analogs, here all compounds bear one P-C bond. Since several distinct mechanisms of biological P-C bond cleavage have been reported,146-148 the widely held assumption that phosphonates are metabolically stable should be treated with caution. 7.1. Probes for serine hydrolases - fluorophosphonates Serine hydrolases (SHs) represent one of the largest and most diverse enzyme classes and play important roles in physiological and pathological processes.7 In viruses, bacteria and fungi, SHs are involved in vital functions, such as drug resistance or the pathogen life cycle. Therefore, a number of inhibitors and drugs targeting SHs have been developed. Among the many specific chemotypes, fluorophosphonates inactivate SHs by covalent modification of the conserved serine nucleophile while remaining virtually inert to other enzymes belonging to the hydrolase family (Scheme 11a). This unique property renders them exceptionally useful for the characterization of this class of enzymes and makes fluorophosphonates a useful tool for screening serine hydrolase inhibitors.149 7


57


Page 58 of 110

Scheme 11. a) The mechanism of action of a fluorophosphonate probe; b) schematic representation of inhibitor-based probes with an electrophilic site localized on phosphorus. In the third probe, upon cleavage of the P-O bond a quencher (dark gray rectangle) is released, leading to the generation of the fluorescent signal. Blue shape: serine hydrolase.

The first fluorophosphonate probes were introduced by Cravatt and co-workers,119 which

provided

a

foundation

for

further

development

in

the

field

of

fluorophosphonates and activity-based protein profiling. Fluorophosphonates helped to clarify the role of SHs in complex physiological and pathophysiological processes, revealing the relative activity state of SHs in their native environment and advancing evaluation of clinical inhibitor candidates. Although there is a limited diversity of fluorophosphonate probes, they also constitute a versatile and valuable tool for the identification of physiologically important proteins of this class, such as MAGLs (monoacylglycerol lipases), FAAHs (fatty acid amide hydrolases), and DAGLs (diacylglycerol lipases).149, 150 These probes were also used for profiling SH activity during HCV replication151 and in dysregulated cancer cells.152 Their applications and unique properties have recently been thoroughly reviewed;7, 149 therefore, here we will only report on selected recent examples of their applications.


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Probes 96 and 97 were used to track endocannabinoid hydrolase activity in different brain regions in mouse models, revealing differences between activity profiles.153

These probes were also used for the identification of off-targets of inhibitor BIA 102474, which were presumably responsible for the adverse effects of the inhibitor related to neurotoxicity that were revealed during phase I clinical trials. This is a prominent example of how universal these probes are and proves their utility for applied research.154 α,α-Difluoro benzyl phosphonate bioisosteres, which are phosphotyrosine mimics, show inhibitory activity against protein tyrosine phosphatases. Wagner and coworkers described mono-P-fluorophosphonates as potentially more cell permeable analogs. The authors developed fluorophosphonate probe 98, which enabled their studies on the mechanism of inhibition by this class of compounds, showing that it is not covalent in nature.155

Table 8. Fluorophosphonate probes. Entry

Structure of the probe

1


Techniques

Biological

used

model

1) SDS-

1) brains of

PAGE;

male

Avidine -

wild type

agarose

and CB1

beads; on

receptor

stage tip

knockout

dimethyl

mice

labeling;

2) human or

HPLC-Chip-

mouse brain

MS;153

proteome or

59


Page 60 of 110

2) SDS-

cell lysate

PAGE; in-gel fluorescence; SILAC; MS154 2

SDS-

Catalytic

PAGE155

domain of protein tyrosine phosphatase (PTP1B)

7.2. Diphenylphosphonate-derived probes Diphenyl phosphonates (DPPs) are an alternative to fluorophosphonates, with a reactivity restricted to serine proteases (Scheme 11). They covalently attach to a target serine protease through nucleophilic attack of the hydroxyl group of the serine residue on the electrophilic phosphonate moiety, with subsequent expulsion of a phenoxy group and the formation of a phosphonate ester that undergoes aging and loss of the second phenoxy group. Their development was pioneered by Powers and co-workers who synthesized and evaluated the first such biotinylated analog for studies of lymphocyte granzymes.156 Craik and Mahrus successfully used this type of probe to label granzymes A and B, which allowed the identification of granzyme B as the major effector of natural killer cell-mediated lysis.157 An even higher selectivity may be achieved by the introduction of a recognition element, an amino acid sequence corresponding to the native substrate, which is usually situated between a phosphonate warhead and a reporter handle. In certain


60

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examples, a benzguanidine moiety is essential for increasing the selectivity of the probe. Methods facilitating the synthesis of libraries of potentially selective DPP probes have been developed. Verhelst and co-workers established a method combining solution and solid phase synthesis to synthesize a library of ABPs (e.g., 99) with diverse recognition elements (Table 9, entry 1).158 It has been shown that even small changes within a probe’s structure may affect its selectivity; thus, this method may be helpful to fine tune the structure of ABPs. The role of optimizing the amino acid sequence in the structure to achieve the required selectivity has also been demonstrated by others.159

The majority of DPP probes contain a diphenylphosphonate moiety that is linked to the rest of the probe through a P-C bond. Verhelst and co-workers developed phosphoramidate probes 101, which retain activity toward serine protease. These compounds could be obtained using solid phase synthesis protocol which facilitates their preparation (Table 9, entry 3).160 Bunnet and co-workers developed fluorescent DPP probes 102-103 aiming at the detection of serine protease activity during inflammation processes (Table 9, entry 4).161 The inclusion of a fluorescent label in the probe enables direct read-out in gel without the need for carrying out a click reaction. Gütschow and co-workers developed coumarin-labeled activity-based probe 104 for the detection of the type II transmembrane serine protease, matriptase-2 (Table 9, entry 5).162 Drąg and co-workers optimized the structure of the probe for neutrophil serine protease by utilization of a combinatorial approach. This versatile method allows for the selection of a substrate peptide bearing an unnatural amino acid, which matches


61


Page 62 of 110

protease subsite preference. Thus, the identified sequence can be utilized as the core of diphenylphosphonate probes bearing a biotin tag for the imaging of neutrophiles.163, 164 (e.g., probe 105, Table 9, entry 6). Later, the same approach was applied for design of four fluorescent probes 106-109, each equipped with a different fluorophore of minimal wavelength overlap. Such a set of probes enables parallel imaging and localization studies of different neutrophil serine proteases.165 Taking advantage of the mechanism of action of diphenylphosphonate probes, Verhelst and co-workers designed quenched activity-based probes 110a-b (qABP) in the form of mixed alkyl aryl phosphonate esters (Table 9, entry 8).166 Upon covalent attachment of the probe to the reactive site, the quencher is expelled leading to the revival of the fluorescence signal.

Table 9. Diphenylphosphonate-derived activity-based probes. Entr

Structure of the probe

Techniques used

Biological model

SDS-PAGE158

purified proteases

y 1

(and in proteome background): bovine chymotrypsin, human cathepsin G, human neutrophil elastase, bovine trypsin SDS-PAGE; WB159

2

human neutrophil elastase; proteinase 3; Cathepsin G


62

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3

SDS-PAGE; in-gel

bovine trypsin;

fluorescence160

bovine chymotrypsin; and human neutrophil elastase

4

SDS-PAGE; in gel

recombinant

fluorescence;

proteases: elastase,

immunoprecipitiation

proteinase-3, trypsin,

161

chymotrypsin, thrombin, plasmin, and granzyme B

5

SDS-PAGE; in-gel

Transfected HEK

fluorescence162

cells expressing matriptase-2

6

SDS-PAGE; in-gel

NSP4

fluorescence164


63


7

8

Page 64 of 110

SDS-PAGE;

Isolated human

fluorescence

neutrophil serine

imaging; confocal

proteases; isolated

microscopy165

neutrophiles

SDS-PAGE; in-gel

isolated proteases:

fluorescence;

trypsin, elastase,

competition studies166

human urokinase plasminogen activator, cathepsin

G, bovine chymotrypsin;

NSP4: Human neutrophil serine protease 4.

8. Miscellaneous


64

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The Boons group167 developed SEEL (Selective ExoEnzymatic Labeling) to enrich and

identify

cell

surface

glycoproteins

with

the

use

of

recombinant

glycosyltransferases (sialyltransferase) and the exogenously administered biotinfunctionalized sugar nucleotides 111-112 (Table 10, Entry 1). This one-step technology improved cell surface labeling compared with the two-step approach,168 which required an additional step after the incorporation of the azide probe 113 to introduce biotin. The low efficiency of the two-step strategy might result from steric and electrostatic effects during the alkyne-azide reaction. The SEEL methodology has shown a high selectivity for cell surface proteins, since the employed reagents and enzyme do not cross the cell membrane. The authors expect that through the identification of altered cell surface-resident glycoproteins, molecular mechanisms of disease can be elucidated. Inositol hexakisphosphate (IP6) is an important signaling molecule, which shares a common structural pattern with the phosphatidylinositol probes described above (see chapter 5). IP6-based probes were used to identify their binding proteins. Affinity enrichment using compound 114 allowed for the identification of 77 IP6-binding proteins169, while compound 115 was shown to be selective for Ku protein, a factor required for DNA ligation by nonhomologous end joining.170 Jemielity and co-workers171 developed an acetylpyrene-labeled N7-methylguanine nucleotide as a fluorescent probe for monitoring the decapping scavenger pyrophosphatase, DcpS, which is a molecular target for spinal muscular atrophy. The enzymatically triggered cleavage of compound 118 results in the release of N7methylguanosine (m7GMP) and 5’-diphosphate, which induces a 2-fold increase in fluorescence.


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Kowalska and co-workers172 used the nucleoside 5’-fluorophosphate analogs 119-120 to develop a fluorescence screen for phosphohydrolase inhibitors. The assay is based on the enzyme-triggered cleavage of the P-F bond and uses the fluoride-sensitive fluorogenic probe bis-(tert-butyldimethylsilylfluorescein for the quantitative detection of the released fluoride anion. Autotaxin (ATX) is an enzyme with pyrophosphatase/phosphodiesterase activity. It resembles phospholipase D in terms of structure and catalytic activity and participates in many regulatory processes, including cancer cell invasion and metastasis. Therefore, this enzyme is considered to be a potential target of anticancer therapies and it may also be used as a biomarker for tracking tumor progression.173 Several probes based on natural substrate scaffolds have been made, some of which have become commercially available. Recently, a probe named TG-mTMP (119) with a nucleotide-resembling structure possessing high sensitivity and specificity was proposed as a tool for ATX inhibitor screening.174 TG-mTMP becomes strongly fluorescent upon autotaxin-mediated cleavage. The generated signal can be used to assess the inhibitory activity of the screened compounds. Another probe was designed for the specific labeling of autotaxin in an activitydependent manner.175 Probe 120 was equipped with a trapping device (a latent quinone methide) that was activated upon phosphate group cleavage. As the probe covalently binds to ATX, the quantity of this conjugate is related to the enzyme activity and can be used as an indication of inhibitor potency. NPP6 (choline-specific glycerophosphodiester phosphodiesterase) is representative of the nucleotide pyrophosphatase/phosphodiesterase family, with an activity profile similar to phospholipase C. Nagano and co-workers developed fluorogenic probe 121, which enables the distinction between activities related to enzymes with


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phospholipase C and D activity.176 It was shown that the increase of fluorescence was observed only in NNP6-rich cells, thereby confirming the utility of this probe in cellbased assays.

Table 10. Miscellaneous phosphorus-based probes. Structure

1

Techniques

Biological

used

model

LC-MS/MS;

recombinant

SEEL; confocal

ST6Gal1;

microscopy;

HeLa cells;

SDS-PAGE; WB; labeling efficiency167

2

WB;

Recombinant

colocalization

ST6Gal1

studies168

3

SDS−PAGE


169

HeLa cells

67


4

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SDS-PAGE; LC-MS/MS

170

cytosolic lysate of LIM1215; Ku protein

5

HPLC; UV-Vis

DcpS

spectroscopy; fluorescence assay171

6

HPLC;

DcpS and

fluorescence

PDE-1

characteristics; fluorescencebased HTS assay 172

7

fluorescence

Recombinant

characteristics

autotaxin

174


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8

SDS-PAGE;

Recombinant

fluorescence

autotaxin

imaging175

9

Fluorescence

Lysates of

imaging176

NIH3T3; HeLa cells: NPP6expressing or nonexpressing cells

SEEL: selective exoenzymatic labeling; ST6Gal1: alpha-(2,6)-sialyltransferase; PDE-1: snake-venom phosphodiesterase I; LIM1215: colonic carcinoma cell line; DcpS: a melal-independent mRNA decapping pyrophosphatase of the histidine triad family; PDE-I: a divalent cation-dependent nuclease.

Summary and future outlook In recent years, a number of probes containing phosph(on)ate functionality have been developed and evaluated in various biological models. In most cases, a phosph(on)ate played the role of a reacting center, while in other cases, it was used as a recognition site for a target protein. Therefore, a phosph(on)ate group is usually an indispensable part of such probes, and its depletion reduces or even abolishes their activity. On the other hand, one of the potential limitations of most phosph(on)ate-bearing probes is their reduced cell permeability, ascribed mainly to their negative charge. This may limit their application to in vitro analysis, although the possibility of active transport into the cell cannot be excluded at the moment. To overcome this problem, several strategies have been tried, such as the introduction of cell-penetrating peptides or a


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positively charged linker into probe’s structure or caging of a phosph(on)ate group, which is then programmed to be released inside the cell. The prodrug approach, successfully applied in phosphorus-containing drugs, has not yet been thoroughly studied in this respect. The activity of most reported probes was measured in biochemical assays using isolated proteins or in cell-based lysates. In vitro evaluation may be artificially affected by the unnatural concentration of reagents, the absence of regulatory parts of the protein sequence, and the missing biological context of the particular cell type. Therefore, profiling in a native cellular environment is superior to in vitro enzyme assays, and future directions should address extending the use of these probes to in vivo models. The phosph(on)ate probes were also used to quantify the level of proteins or the degree of their post-translational modification. Such studies reveal the potential of the probes as diagnostic tools. They can allow discrimination, for example, between normal and pathological cell phenotypes. Additionally, they can be used to determine which proteins are affected by the use of particular inhibitor. However, the change in the level of certain proteins upon the action of an inhibitor does not exclude the relevance of other undiscovered proteins to a particular process. Developing a method for the global characterization of the proteome remains a challenging task, and a substantial part of the proteome still awaits to be identified or functionally characterized. The low abundance of some enzymes, their limited stability, and high abundance of their natural substrates may impede the proper analysis of biological samples. The development of functional probes supported by new MS/MS platforms, which improve the sensitivity and accuracy of quantification, constitute one of the tools to overcome these limitations.


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As is typical for chemoproteomic studies, the pool of identified hits was usually larger than the group of targeted proteins. In addition to non-specific hits, which can be eliminated by the careful choice of control experiments and a thorough validation of the method, such results may indicate orphan activities of known proteins or lead to the identification of new proteins that may have been predicted earlier by bioinformatic tools. To deliver trustworthy data, high quality probes are indispensable. The rational design and synthesis of a variety of structurally diverse probes is recommended, since no single compound recapitulates the specificity and reactivity of a target protein. Therefore, the exploration stage of these probes cannot be undermined, and reasonable resources need to be applied here even if it involves laborious synthesis and HPLC purification. Systematic work in this respect should pay off with the discovery and validation of safer therapeutic targets and novel therapeutic agents.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Biographies Łukasz Joachimiak studied chemistry at the Lodz University of Technology, where he received his BSc in 2010 and MSc in 2012. He is currently finalizing his Ph.D. under supervision of Dr. Katarzyna Blażewska. His research interests concern development of phosphonocarboxylates as Rab GGTase (RGGT) inhibitors and utilization of prodrug strategy to improve therapeutic potential of biologically active compounds bearing phosphonic acid moiety.


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Katarzyna M. Błażewska received her Ph.D. in chemistry at the Technical University of Lodz (Poland). She was a postdoctoral fellow in the group of Prof. Charles McKenna at the University of Southern California (U.S.A.) and Fulbright Scholar with Prof. Eranthie Weerapana at Boston College (U.S.A.). She is currently an Assistant Professor at the Lodz University of Technology. From the beginning of her career, her research interests were focused on the synthesis and potential applications of phosphonates. Currently she concentrates on studies of Rab prenylation mediated by Rab GGTase, using medicinal chemistry and chemical biology tools. ACKNOWLEDGEMENTS This work was financially supported by the National Science Centre, Poland (2014/14/E/ST5/00491). We thank Dr. Edyta Gendaszewska-Darmach for careful reading of the manuscript and helpful discussion. We thank Ms. Julia Seferynowicz for creation of impactful graphical abstract. ABBREVIATIONS USED ALP, alkaline phosphatase; BHK, baby hamster kidney cells; BMSCs, bone marrow mesenchymal stem cells; cPLA2, Ca2+-dependent phospholipase A2; CDTA, calciumdependent transacylase; CIP, calf intestinal phosphatase; CK2, casein kinase 2; CPP, cell-penetrating peptide; CHO, Chinese hamster ovary cells; Chm, Choroideremia mouse; CNTF, ciliary neurotrophic factor; CLSM, confocal laser scanning microscopy; FSBA, covalent inhibitor, 5‘-(4-fluorosulfonylbenzoyl) adenosine hydrochloride; CDK2, cyclin-dependent kinase 2; DDA, data-dependent acquisition; DIA,

data-independent

acquisition;

DLS,

dynamic

light

scattering;

EIS,

electrochemical impedance spectroscopy; FACS, fluorescence-activated cell sorting; Fhit, fragile histidine triad protein; GSK-3β, glycogen synthase kinase; gm, gunmetal; HSP 90, heat shock protein 90; HEK 293T, human embryonic kidney cells 293;


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159. Grzywa, R.; Burchacka, E.; Lecka, M.; Winiarski, L.; Walczak, M.; Lupicka-Slowik, A.; Wysocka, M.; Burster, T.; Bobrek, K.; Csencsits-Smith, K.; Lesner, A.; Sienczyk, M. Synthesis of Novel Phosphonic-Type Activity-Based Probes for Neutrophil Serine Proteases and Their Application in Spleen Lysates of Different Organisms. ChemBioChem 2014, 15, 2605-2612. 160. Haedke, U. R.; Frommel, S. C.; Hansen, F.; Hahne, H.; Kuster, B.; Bogyo, M.; Verhelst, S. H. L. Phosphoramidates as Novel Activity-Based Probes for Serine Proteases. ChemBioChem 2014, 15, 1106-1110. 161. Edgington-Mitchell, L. E.; Barlow, N.; Aurelio, L.; Samha, A.; Szabo, M.; Graham, B.; Bunnett, N. Fluorescent Diphenylphosphonate-Based Probes for Detection of Serine Protease Activity during Inflammation. Bioorg. Med. Chem. Lett. 2017, 27, 254-260. 162. Haussler, D.; Mangold, M.; Furtmann, N.; Stirnberg, M.; Furtmann, N.; Bajorath, J.; Braune, A.; Blaut, M.; Guetschow, M. Phosphono Bisbenzguanidines as Irreversible Dipeptidomimetic Inhibitors and Activity-Based Probes of Matriptase-2. Chem. Eur. J. 2016, 22, 8525-8535. 163. Kasperkiewicz, P.; Poreba, M.; Snipas, S. J.; Parker, H.; Winterbourn, C. C.; Salvesen, G. S.; Drąg, M. Design of Ultrasensitive Probes for Human Neutrophil Elastase through Hybrid Combinatorial Substrate Library Profiling. Proc. Natl. Acad. Sci. 2014, 111, 2518-2523. 164. Kasperkiewicz, P.; Poreba, M.; Snipas, S. J.; Jack, L. S.; Kirchhofer, D.; Salvesen, G. S.; Drąg, M. Design of a Selective Substrate and Activity-Based Probe for Human Neutrophil Serine Protease 4. PLoS One 2015, 10, e0132818/1-e0132818/12. 165. Kasperkiewicz, P.; Altman, Y.; D'Angelo, M.; Salvesen, G. S.; Drąg, M. Toolbox of Fluorescent Probes for Parallel Imaging Reveals Uneven Location of Serine Proteases in Neutrophils. J. Am. Chem. Soc. 2017, 139, 10115-10125. 166. Serim, S.; Baer, P.; Verhelst, S. H. L. Mixed Alkyl Aryl Phosphonate Esters as Quenched Fluorescent Activity-Based Probes for Serine Proteases. Org. Biomol. Chem. 2015, 13, 2293-2299. 167. Sun, T.; Yu, S.-H.; Zhao, P.; Meng, L.; Moremen, K. W.; Wells, L.; Steet, R.; Boons, G.-J. One-Step Selective Exoenzymatic Labeling (SEEL) Strategy for the Biotinylation and Identification of Glycoproteins of Living Cells. J. Am. Chem. Soc. 2016, 138, 11575-11582. 168. Mbua, N. E.; Li, X.; Flanagan-Steet, H. R.; Meng, L.; Aoki, K.; Moremen, K. W.; Wolfert, M. A.; Steet, R.; Boons, G.-J. Selective Exo-Enzymatic Labeling of N-Glycans on the Surface of Living Cells by Recombinant ST6Gal I. Angew. Chem., Int. Ed. 2013, 52, 13012-13015. 169. Jiao, C.; Summerlin, M.; Bruzik, K. S.; Hanakahi, L. Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins. Biochemistry 2015, 54, 6312-6322. 170. Yin, M.-x.; Catimel, B.; Gregory, M.; Condron, M.; Kapp, E.; Holmes, A. B.; Burgess, A. W. Synthesis of an Inositol Hexakisphosphate (IP6) Affinity Probe to Study the Interactome from a Colon Cancer Cell Line. Integr. Biol. 2016, 8, 309-318. 171. Kasprzyk, R.; Kowalska, J.; Wieczorek, Z.; Szabelski, M.; Stolarski, R.; Jemielity, J. Acetylpyrene-Labelled 7-Methylguanine Nucleotides: Unusual Fluorescence Properties and Application to Decapping Scavenger Activity Monitoring. Org. Biomol. Chem. 2016, 14, 3863-3868. 172. Baranowski, M. R.; Nowicka, A.; Jemielity, J.; Kowalska, J. A Fluorescent HTS Assay for Phosphohydrolases Based on Nucleoside 5'-Fluorophosphates: its Application in Screening for Inhibitors of mRNA Decapping Scavenger and PDE-I. Org. Biomol. Chem. 2016, 14, 4595-4604. 173. Barbayianni, E.; Kaffe, E.; Aidinis, V.; Kokotos, G. Autotaxin, a Secreted Lysophospholipase D, as a Promising Therapeutic Target in Chronic Inflammation and Cancer. Prog. Lipid Res. 2015, 58, 76-96. 174. Kawaguchi, M.; Okabe, T.; Okudaira, S.; Nishimasu, H.; Ishitani, R.; Kojima, H.; Nureki, O.; Aoki, J.; Nagano, T. Screening and X-ray Crystal Structure-based Optimization of


82

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Autotaxin (ENPP2) Inhibitors, Using a Newly Developed Fluorescence Probe. ACS Chem. Biol. 2013, 8, 1713-1721. 175. Cavalli, S.; Houben, A. J. S.; Albers, H. M. H. G.; van Tilburg, E. W.; de Ru, A.; Aoki, J.; van Veelen, P.; Moolenaar, W. H.; Ovaa, H. Development of an Activity-Based Probe for Autotaxin. ChemBioChem 2010, 11, 2311-2317. 176. Kawaguchi, M.; Okabe, T.; Okudaira, S.; Hanaoka, K.; Fujikawa, Y.; Terai, T.; Komatsu, T.; Kojima, H.; Aoki, J.; Nagano, T. Fluorescence Probe for Lysophospholipase C/NPP6 Activity and a Potent NPP6 Inhibitor. J. Am. Chem. Soc. 2011, 133, 12021-12030. 177. Green, K. D.; Pflum, M. K. H. Kinase-catalyzed Biotinylation for Phosphoprotein Detection J. Am. Chem. Soc. 2007, 129, 10-11. 178. Arora, D. P.; Boon, E. M. Unexpected Biotinylation Using ATP-γ-biotin-LC-PEOamine as a Kinase Substrate Biochem. Biophys. Res. Commun. 2013, 432, 287-290.

Table of Contents Graphic


83

Journal of Medicinal Chemistry 1 Kinase 2 3 4 5 6 7

NH2 O O O P P O O O- O- n O

= tag, n = 1,2

O

Base ACS Paragon Plus Environment

OR OR

O Page 84 of 110 N H O O P P -O O O O- O- n

+ O

Base

OR OR

Page 85 of 110 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


O

O O O P P P O -O -O -O O O O

NH2

S O

Base

OR OR

SH O kinaseacrylamide

kinase


NH

O kinaseamide

NH


OO OO OO P P P O O O O

Page 86 of 110

-

O

Base

OO OO P P -+ O O O

enzyme

activity determination OR OR

OO P -O O

O

Base

OR OR

- fluorophores within FRET distance

- fluorophores after FRET is terminated


Page 87 of 110 1 2 3 4 5 6 7 8 9 10 11 12 a)13 14 15 16 17 18 19 20 21 22 23 b)24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


O

O

H N

S

O

O O O OP P O O O

OH

H N

+

KAT

HS

H N

OH OP(O)(OH) 2 H2N

OH

H N O

O O O OP P O O O

O

Base

O OH OP(O)(OH) 2

S O

O

Base

O

= tag

H N

N H

H N

OH

H N O

O O O OP P O O O

H N O

Base

KAT

O OH OP(O)(OH) 2

= photoaffinity label (PAL)


S O

H N

OH

H N O

O

O O O OP P O O O

O

Base

OH OP(O)(OH) 2


n

n = 1, 2

Page 88 of 110

PT

OPP

n

S

SH 1) Prenyl transferase substrates identification 2) modfiication of CaaX-containing proteins

= tag -OPP = pyrophosphate residue


PageO89 ofO 110 R 1 2 3 4 5

O O

= PAL

OO O P O

= tag

O 1.Incorporation into Journal of Medicinal Chemistry bilayer membrane 2. Photoaffinity labeling

O R

O

O O

OO P O

ACS Paragon Plus Environment Identification of membrane = membrane protein proteins



Page 90 of 110

a) Page 91 of 110 OO OH Phosphatase P 1 O 2 = non-fluorescent 3 reporter 4

b) Journal of Medicinal Chemistry OH

O ACS Paragon Plus Environment = fluorescent reporter

O

OOH Phosphatase P O

O + OH

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a) 20 F 21 O 22 Phosphatase 23 O P(OH)2 24 25 26 = tag 27 b) 28 29 30 O O O 31HN HN 32 O P(OR)2 UV 33 O O uncaging 34 O O 35 36 R = CH2C6H4(o-NO2) 37 38 39 quenched = = quencher 40 fluorophore 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 92 of 110

X

XH O

O O P(OH)2

OH

O Phosphatase

O

N H

XH

O = fluorophore


N H

X

OH

Page 93 of 110 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


b) a) OH

F

O P OEt

- FO

O P OEt

= tag

O P F OEt O recognition P site PhO OPh O recognition P site O OEt


Nucleoside triphosphate

Isoprenoid diphosphates Journal of Medicinal Chemistry

OO OO OO O- O- O O O P P 1 P Base P P O HO O O O 2 n O O 3 n = 3, 4 4 OH R 5 Base = adenine, guanine 6 R = H or OH NH2 Acetyl-CoA 7 8 N N OH 9 O O O O OH H N N P P 10 N N O O O O 11 S O O 12 13 OH (HO)2(O)PO 14 15 Diphenylphosphonates 16 Fluorophosphonates (sarin) 17 R 18 OPh F Paragon PeptidylACS HN P Plus Environment 19 OMe OPh P 20 O O R - side chain 21

Phospholipids

Page 94 of 110

O

O R1

O

O

R2

O

OO 3 P R O

R1, R2 = fatty acid chains R3 = choline, serine, ethanolamine, inositol Phosphotyrosine H2N

COOH O OH P O OH

Page 95 of 110 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


R1

O N N

O2N O

N+

Cl

Eclipse

R1 n

NH

O

5

N

N N

O2N

R2

N

Cl Me-Eclipse

n = 1, R1, R2 = H: Cy3; R1 = SO3H, R2 = Me: Sulfo-Cy3 n = 2, R1, R2 = H: Cy5; R1 = SO3H, R2 = Me: Sulfo-Cy5 n = 3, R1 = SO3H, R2 = Me: Sulfo-Cy7 O

CO2-

N

O +N

N+ O

N B F F

O

N N BODIPY FL

O

Cy7

CO2Na

TAMRA

O

O

O O

Me2N

N N

ONa FL

N

O

N

Et2N Dabcyl


NEt2 2-P dye

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

NH2 Page 96 of 110 N

Journal of Medicinal Chemistry N N O 2 N N OH 2 H O

XHN

O O P O OO CO2H OH

HO AcHN

X=biotin 111

O

N O OH OH

NH2 N

OH

O O P O OO CO2H OH

N

OH HO O

XHN

N H

2

N N N

2

O

X=biotin

O

N H

112

NH2 N N3 HO AcHN

OPO32-

O P O -O O OPO32-

OPO32-

OPO32-

OPO3

O OH OH

X=biotin 114

O

O O P O -O O OPO32-

OPO32-

O

NHX

OPO32-

2-

N

O O P O OO CO2H OH 113

OH

X

N H

X=Dynabead

OPO32OPO32-

115

O N+ N

O O O P O P P S -O -O O O O

O

O OH OH

116 O N+

NH

N

O P O F O

NH2

N

O OH OH 117

NH2 N

N

N

O P O F O

N

O OH OH 118

O O NH

O

O

O P O O O

O

O P HO O O

H N

O

O

O

O OH

119

F

N

NHX

O 120

HO

X = Sulfo-Cy5

O 5

5

O

O

O

O 121

O

O P N+ O - OACS Paragon Plus Environment O

NH N

NH2

O OH OH

O

NH H2N

Page 97 of 110

NH

Journal of Medicinal Chemistry O O

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

O

H N

H2N

N

N H

O 99

OPh P OPh O

N H

N N

OH

NH HN

O XHN

O

H N

N H

O

H N

N H

O

NH2

O N P H O O OH

O

100 X=biotin

S

S

R1 H2N

O

O

R2

H N

N H

N H

O or

R1 =

O OPh P OPh

NH-biotin 101

R2 = different residues

NH2

Sulfo-Cy5

O

102

H N

H2N

103

NH2+

N H

O

104

ClNH2

N H

NH

O

OPh P OPh O

N H

OPh P OPh

O

O

H N

NH2+

Cl-

Sulfo-Cy5

OPh N P H O OPh

N

O O O O

O

XHN

Ph

O O

H N

N H

4

O

O X=biotin

O

S

105

O

XHN

O H N

N H

Ph O

5

O

H N N

H N

N H

S

O

O XNH

O

O O

O

O

O OPh P OPh

H N

N

O

106 X=Cy5

Ph

O

Ph

O 4

O OPh P OPh

H N

N

OPh

O P

OPh

O O

O

107 X=BODIPY FL

O

HN

HN NH H2N

O

H N

XHN O

O OPh P OPh

H N

N

N H

4

O

O

NH 108 X=Cy7

H2N

Ph N

N

H N

O XHN 5

N H

H N

N

NH

OPh

O P

OPh Ph

O O

O 109 X=Cy3

N N N

R

O N H

O O P OEt

NH

O

HN

O O2S

N

ACS Paragon Plus Environment CO2-

N

O

N+

Ph N 110a: R = i-Pr 110b: R = p-[NH2-C(NH)-NH]-C6H4-

Ph O

N+

F EtO

O P

O O F Journal ofTAMRA Medicinal Chemistry Page 98 of 110 P N 7 N 7 H EtO H O 96

1 2 S N 3 FP-TAMRA H H 4 H NH 5 HN 97 6 O FP-Biotin 7 8 9 O 10 N 11 O N F H Plus Environment ACS Paragon 12 P N N NaO NHX 13 F F 98 14 X=FL

Page 99 of 110


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


O P O O-

O O

10

O O

6

HO HO HO

N N

Journal of Medicinal Chemistry C16H33

O

O

C16H33

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

O

H N

O

O

O P O O-

OPO32-

O

OH

O

H N

HN O O

H N

N H

O

O

O P O O-

OH OPO32OPO32OPO32-

HO

O

O O P O O-

O

H N

O

O

O O

O n

NH3+ -O

O

O

64a - n = 1 64b - n = 11

O

O

H N

O

O O

O

O P O O-

NH3+ -O

O

O

O

O O

N H N

O

9

O

S

O

O

9

O

O O P O O

O

N+

O OEt O

O O

N H HO

O

O

O

O

O

O

O

O

13

O O P O

N+

N O HO

H O N S O

O 9

O O P O O

H N

N O

N

N O S N O H

O O 9

O 9

O

O O P O O

H N

H O N S O N

O

N


NH O

O

O

OPO32-

S

HN

OH

HO

Page 100 of 110

O N H

O OH

F O P 8O H3C(CH2)15O

NHX

N H

O N

O HO HO HO

O

OH O

O O P O -O O

OO P O

OO P O

N+

N+

O O NH Hof Medicinal Chemistry Page 101 of 110 Journal HN S

H

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

OPP

N H

n

45a: n = 1 45b: n = 2 H N

N+ F BN F

OPP n

O 46a: n = 2 46a: n = 3

N3 OPP

3

47

O

OPP n

48a: n = 2 48b: n = 1

OPP 49

OPP 50

O

OPP 2

51

O O

OPP 2

O 52

O 48a

OPP

2

O O 2

O 53

N3 3

OPP

OPP

54

H2N

O

N

O

OPP 55

O

OPP

H

H

2

56

OPP O

2

57

O

OPP 2

58 O

N3 ACS Paragon Plus Environment OPP 2

59

NH2 N -Chemistry Journal ofHMedicinal OH O O O OH

O n

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

N

S

N

P

O

O

O

P

O

O

O

N

Co-A

n = 1, 2, 3

OH

(HO)2(O)PO n=1: 29a, n=3: 29b, n=3: 29c

O

H N

S

n

O

NH2

O O O OP P O O O

OH

H N

Page 102 of 110

N

N

N

N

N

O

N

O

n = 2,3,4

30

OH

(HO)2(O)PO

NH2 O N3 n

H N

S

OH

H N

O-

O

P

O

O

O OP O

O

N

N

N

O

N

O

n = 2,3

OH

(HO)2(O)PO

31

NH2 O 13

H N

S

OH

H N

O O

O

OP

N

O OP O

O

N

N

O

N

O 32

OH

(HO)2(O)PO

NH2 O N3 14

H N

S

O O O OP P O O O

OH

H N O

N O

N

N

N

O 33

OH

(HO)2(O)PO

NH2 O 9

H N

S

OH

H N O

O-

O O

P

N

O OP O

O

N

N

O

N

O 34

OH

(HO)2(O)PO

NH2 O HO

S

H N

O O O OP P O O O

OH

H N O

O

HO

S

H N

O O O OP P O O O

OH

(HO)2(O)PO

O

O O NH H N

GGTSKRATQ-Ahx H N HN

Co-A

O

N

N N

OH

O 3

NH TAG

NHBzPh

Co-A

O HN O APRKQLATK-CONH2

O

O

NH2

N

O

Ahx-BPyne

H2N

OH

N N N

H N

5

N N

O 36

HN

O

(HO)2(O)PO

H N O

O O

N

O 35

N3

N

Lys-CoA-BPyne, 37

TAG = TAMRA: H3K14-CoA-TAMRA, 39 TAG = biotin: H3K14CoA-biotin, 41 AhxBPyne AGGKGLGKGGKGRGS H N HN Co-A O HN O RHR-CONH2

Ahx-BPyne GGTSKRATQ H N HN Co-A O HN O APRKQLATK-CONH2

O

O

H4K16-CoA-BPYne, 40

H3K14-CoA-BPYne, 38

SEPHAROSE O O

NH 5

H N

HN H2N

O HN

GGTSKRATQ H N

Co-A O

O

Lys-CoA Sepharose, 42 O Co-A

AGGKGLGKGGKGRGS H N Co-A O HN O RHR-CONH2 HN


O HN O APRKQLATK-CONH2

H3K14-CoA-amine precursor to H3K14-CoA Sepharose 43

H4K16-CoA-amine precursor to H4K16-CoA Sepharose 44

Page 103 of 110

O R1

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

NH2

Journal of Medicinal Chemistry N H


4

O

22: R1 = Sulfo-Cy3; R2 = Sulfo-Cy5

N H

O N H

4

N

N

O

N

OH NH2

4

N


O R1

R2

N

O

n

N

N

N

n=1 OH O 23a: R1 = Sulfo-Cy3, R2 = Sulfo-Cy5 H N R2 23b: R1 = Sulfo-Cy5, R2 = Sulfo-Cy7 5 23c: R1 = Cy3, R2 = Cy5 O 23d: R1 = Sulfo-Cy3, R2 = Eclipse 23e: R1 = Cy3, R2 = Eclipse NH2 23f: n=2, R1 = Sulfo-Cy3, R2 = Sulfo-Cy5 N N O OO OO OO P P P N N R1 N O O O O O 4 H OH OH 24a: R1 = Sulfo-Cy3; R2 = Sulfo-Cy5 24b: R1 = Sulfo-Cy3; R2 = Me-Eclipse

R2

H N

O

NH

R2

R1

N

OO OO OO OO P P P P O O O O O

H N

O

25

O

N

N

N

O

n

R1 = Sulfo-Cy3 R2 = Sulfo-Cy5 n = 0, 1

OH OH O

N NH NH

O

O N H

O N

4

O

OP

O O

OP

O O

OP

O O

O-

N

P

N

O

O

26

O N N HN

n

N O

N OH OH N

ACSNH(Sulfo-Cy5) Paragon Plus Environment 27 - n=1 28 - n=2

N

NH N


N

OH OH

(Sulfo-Cy3)NH

OH OH

NH

O

N N

O

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

NH2

Journal of Medicinal Chemistry N O OO P O O

N H

5

O

H HN

N

OH OH NH

S

N

O

3

16

H

O N

O O OP O O

O N H

5

NH

N

OH OH NH

NH2

17

H

N

O O OP O O

HN

4

NH

N

n

4

NH O

N

OH OH O N

O O OP O O

HN

N

O

18a: n = 3 18b: n = 2

O

NH2

N

O

3

O

H HN S

Page 104 of 110

N

NH

N

N

O

3

NH2

OH OH

19

NH2 N

-

O HN NH

4

X N H X

X X

OO P O O

X X

3

O

OH OH

20, X=H, D

O

N O

NH2 O

N N O O O P P P N N O -O -O -O O OACSOParagon O Plus Environment 21

OH OH

N N

a) Page 105 of 110 1 2 3 4 5 6 7 8 9 10 b) 11 12 13 14 15 16 17 18 19


OO OO OO P P P N O O O H

O

Base

OR OR

OO OO P P -O O O

kinase

OO P N O H

+

protein identification kinase activity determination

-

kinase-protein complex identification

-

Base

OR OR

HO

= tag

OO P N H

O

-

-

kinase

OO P N O H ACS Paragon Plus Environment = tag: photoaffinity label (PAL)

OO P N O H effector identification



Page 106 of 110

Phosphorus-Based Probes as Molecular Tools for Proteome Studies

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