Combinatorially Screened Peptide as Targeted Covalent Binder

May 24, 2018 - In contrast with conventional medicines, targeted covalent drugs ... (e.g., chemoproteomics-based one(14,15)), and alternative general ...
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Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Combinatorially Screened Peptide as Targeted Covalent Binder: Alteration of Bait-Conjugated Peptide to Reactive Modifier Shuta Uematsu,† Yudai Tabuchi,† Yuji Ito,‡ and Masumi Taki*,† †

Department of Engineering Science, Bioscience and Technology Program, The Graduate School of Informatics and Engineering, The University of Electro-Communications (UEC), 1-5-1 Chofugaoka, Chofu, Tokyo 182-8585, Japan ‡ Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima, Kagoshima 890-0065, Japan S Supporting Information *

ABSTRACT: A peptide-type covalent binder for a target protein was obtained by combinatorial screening of fluoroprobe-conjugated peptide libraries on bacteriophage T7. The solvatochromic fluoroprobe works as a bait during the affinity selection process of phage display. To obtain the targeted covalent binder, the bait in the selected consensus peptide was altered into a reactive warhead possessing a sulfonyl fluoride. The reaction efficiency and site/position specificity of the covalent conjugation between the binder and the target protein were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and rationalized by a protein−ligand docking simulation.



INTRODUCTION In contrast with conventional medicines, targeted covalent drugs can form permanent bonds to target proteins and eternally deactivate them.1−5 This prolonged duration of inhibition6 would reduce dose frequency of drugs and improve quality-of-life of patients.7,8 The covalent drugs should be required to possess high target selectivity and less off-target reactions,7,9 to reduce irreversible side effects (i.e., toxicity).8,10 To retain maximum target selectivity with minimal off-target reactivity,10 numerous efforts for rational design of novel covalent drugs have been paid by way of, for example, computational methods,3,11 fragment-based drug discovery,12 and covalent tethering.13 In many cases, optimization and chemical synthesis of such ideal covalent drugs require additional time and steps. Moreover, if three-dimensional structures of target proteins are not well-known, the structure-based drug development will be hindered. Currently, the development should be performed by limited numbers of sophisticated methodologies (e.g., chemoproteomics-based one14,15), and alternative general principles for obtaining target covalent binders are requested. Contrary to the rational design, expanded combinatorial screening16−19 of such targeted covalent binders would be an alternative way to solve these problems. Indeed, screening of the covalent binders from DNA-encoded chemical libraries has been successfully reported.20 For the screenings, displayed peptide libraries would also be attractive mainly because the library diversity is large (up to ∼1012 and ∼1014 for phage and mRNA display, respectively18) and preparation of the library is fairly easy,21 compared with the chemical ones. However, to the best of our knowledge, there are no reports to obtain targeted © XXXX American Chemical Society

covalent binders from the displayed randomized peptide, presumably because (1) the introduction method of the warhead (i.e., reactive group) into the displayed peptides with vast diversity is limited, and (2) exclusive enrichment of the targeted covalent binders from the vast library seems difficult; the warhead in target-unrelated peptides could unfavorably conjugate to the target protein during the biopanning process (Figure S1). To avoid the latter problem of unnecessary side reactions, here we take a detoured selection strategy to obtain a peptidetype covalent binder possessing target specificity (Figure 1). First, we introduce several solvatochromic bait fragments, instead of the warhead, to designated cysteine on T7 phagedisplayed library peptides22 via the gp10-based thioetherification (10BASEd-T).23,24 Second, we obtain targeted noncovalent binders from each bait-conjugated peptide library. Third, when a consensus peptide sequence around the designated cysteine appears, no matter what kind of bait fragment is used, we alter the bait in the consensus peptide into a differently structured warhead, to obtain the targeted covalent binder.



RESULTS AND DISCUSSION As a model target protein, we chose glutathione S-transferase (GST) because rationally designed covalent GST binders in which a warhead is conjugated with artificial reporter tags25 or a natural ligand (i.e., glutathione)26 have been reported; their Received: April 30, 2018 Revised: May 22, 2018 Published: May 24, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00301 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Targeted covalent binders selected by combinatorial screening of bait fragment-conjugated peptide library on bacteriophage T7, followed by an alteration to a reactive warhead possessing sulfonyl fluoride. The chemical structures of different solvatochromic bait fragments (i.e., Prodan, 4DMN, and DBD), as well as that of an altered warhead, are shown in dashed inset. (A) Specific introductions of different solvatochromic bait fragments into a designated cysteine on displaying library peptides on a capsid protein (gp10) of bacteriophage T7 were performed. This gp10-based thioetherification (10BASEd-T) was carried out without side reactions or loss of phage infectivity. (B) From each bait-conjugated peptide library, target (i.e., GST) binders were selected by biopanning. Then, the peptide sequences were analyzed via a next-generation sequencer (NGS), and consensus sequences for every-bait-conjugated peptide around the designated cysteine were determined. For each bait-conjugated peptide, solvatochromic fluorescence change upon GST-binding was also confirmed. (C) To obtain the targeted covalent binder, the bait fragments in the consensus peptide were altered in the warhead. The covalent binder was mixed with the target protein, and the modified position on the target was analyzed by LC-MS/MS.

structure information as well as conjugation efficiency upon GST binding could be easily compared with our combinatorially screened binder. For the bait fragments with different shapes, we have chosen several small or middle-sized solvatochromic fluorophores with neutral charge,22,27 so that we would sense when the bait could be buried deeply into a pocket of the target protein through hydrophobic interaction (Figure 1). These fragments were independently reacted with a designated cysteine on a T7-displayed randomized peptide library, and three rounds of biopanning were performed against biotinylated-GST.22 After the selection process, amino acid sequences of the polyclonal binders possessing each fragment were directly analyzed by a next generation sequencer (NGS). As shown in Table 1, common consensus sequences for all of these three bait fragments were obtained. In the common consensus peptides, structural flexibility of the fragment which is dangling on the designated cysteine seemed to be widely allowed. Among them, we chose ZC*DGZ sequence (highlighted in blue, Table 1) for further experiments, because it does not contain any wobble amino acids (i.e., X). Next, a representative common peptide whose sequence is LNYCDGW (the common consensus sequence is underlined) was synthesized, because it is one of the most abundant sequences obtained from six rounds of biopanning against GST using 4-DMN-conjugated randomized peptide library.22 Then, its sulfhydryl group was conjugated with all of the bait fragments independently. When each bait-conjugated peptide was mixed with GST, a remarkable solvatochromic fluorescence change was observed (Figure S2). This means that the microenvironment around the bait changed after the addition of GST, and most probably, all of the bait fragments were located inside the hydrophobic pocket of GST, no matter what kind of the chemical structures were used.

Table 1. GST-Specific Binders Selected after Three Rounds of Biopanninga (A) C*: DBD-Conjugated Cysteine consensus sequence

frequency

ZC*DGZ 20/100 ZZC*DGZ 12/100 C*XDGZ 10/100 (B) C*: 4-DMN-Conjugated Cysteine consensus sequence

frequency

ZC*DGZ 9/100 ZXYC*XDGZ 2/100 (C) C*: Prodan-Conjugated Cysteine consensus sequence

frequency

C*XXXDDGZ ZC*DDGZ ZC*DGZ C*XDDGZ C*XXXXDGZ C*XDGZ C*XXXDGZ

10/100 8/100 7/100 6/100 6/100 5/100 4/100

a

C*, Z, and X represent bait-fragment conjugated cysteine, hydrophobic amino acids, and any amino acids, respectively. The consensus sequences from each bait fragment were summarized by frequency of the top 100 occurrences obtained from NGS data analysis. The bold letter stands for common consensus sequence for all of the differentbait-conjugated peptides. The detailed sequences with the abundance ratio for each monoclone of ZC*DGZ were presented in Table S1.

Encouraged by the solvatochromic fluorescence, next we altered the bait structures to a reactive warhead, and targetprotein specific covalent binding of the altered peptide was evaluated. We chemically synthesized a targeted covalentB

DOI: 10.1021/acs.bioconjchem.8b00301 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 2. (A) Synthesis of a targeted covalent-binder peptide; Fam stands for a fluorophore at the N-terminus. (B) Specific conjugation between the covalent binder and GST, confirmed by 15% SDS-PAGE/fluorescence imaging. Whole proteins were visualized by CBB staining (left panel), and a protein conjugated with the covalent binder was visualized by fluorescence in the same gel (right). Blue arrow represents the band of the target protein (i.e., GST). At this stage, the cross-linking reaction yield was less than a few percent and the structural information could not be obtained by LC-MS/MS analysis.

MS, and MS/MS analyses (Figure S4), and favorably, the warhead did not react with its intrinsic tyrosine residue. Then, the reaction efficiency and site/position specificity of the covalent conjugation between the Fam-eliminated covalent binder and GST were evaluated as follows. First, the covalent binder and GST were reacted, and SDS-PAGE was performed. Then, the GST band in the gel was excised and digested with trypsin. The resulting peptide fragments were analyzed by LCMS/MS. An intense absorbance in the chromatogram was newly detected after the conjugation (Figure 3A), and it was identified as the GST-conjugated covalent binder by MS (Figure 3B) and tandem mass spectroscopy (Figure 3C). The cross-linking site was determined to be the glutathione binding pocket; the cross-linked amino acid was a tyrosine which was located at the 111th position from the N-terminus. This position was exactly the same when the rationally designed binders25,26 were covalently conjugated (also see Figure S5). From measurement of the abundance ratio of unreacted peptide fragment possessing the 111th tyrosine, the crosslinking reaction yield of our covalent binder was estimated to be 37%, which was far superior to that of the rationally designed one (Figure S6). Finally, the conjugation of the covalent binder was rationalized by a protein−ligand docking simulation using MolDesk Basic/myPresto.31 Thirty separate poses resulted in docking to the glutathione binding pocket of GST with free energy in the range −12.4 to −7.83 kcal/mol. As shown in Figure 4, the docking model of the lowest energy suggested that the warhead was buried deep inside the hydrophobic region of the glutathione-binding pocket and located very close to the

binder peptide whose sequence is Fam−GGLNYC*DGW (Figure 2A; Fam, GG, and C* represent carboxyfluorescein, glycylglycine spacer, and warhead-conjugated cysteine, respectively), and its appropriate covalent conjugation with GST was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Figure 2B). As the warhead, we chose sulfonyl fluoride (SO2F),25,26,28 because the reactive group possesses an exquisite balance of aqueous stability and moderate reactivity toward nucleophilic amino acids (i.e., serine, threonine, lysine, tyrosine, cysteine, and histidine residues in the target protein).29,30 When 4-(2-bromoacetyl)benzene-1-sulfonyl fluoride was reacted with the sulfhydryl group of the precursor peptide, complex reaction products were formed and we could not separate the expected covalent binder peptide. In contrast, when ethenesulfonyl fluoride26 was reacted, only a single component of the covalent binder peptide was successfully obtained (Figure 2A; for details, see Figure S3). The covalent-binder peptide was mixed with GST in the presence of serum proteins, and the reaction mixture was analyzed by SDS-PAGE followed by fluorescence imaging/ coomassie brilliant blue (CBB) staining. An intense fluorescent band could be seen at an appropriate molecular weight (ca. 29 kDa) of the peptide-fused GST (Figure 2B). This means that we could successfully confirm the covalent-binding property, as well as the GST specificity, of the peptide. To avoid unfavorable steric hindrance upon target binding and nonspecific hydrophobic interactions arising from the fluorophore, LNYC*DGW peptide without Fam−GG was once again synthesized. The chemical structure of the covalent binder was tentatively identified by combination of 1H NMR, C

DOI: 10.1021/acs.bioconjchem.8b00301 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 4. Molecular docking simulation of the covalent binder (shown as a stick) to GST (PDB ID: 1UA5) using sievgene of myPresto; the best docking model with a lowest binding energy of −12.4 kcal/mol was presented. Fluorine atom in the warhead and conjugated tyrosine in GST were colored in cyan and dark red, respectively. GST was shown as a cartoon with side chains as a line description.

fragment-conjugated peptide library on phage, followed by an alteration of the bait to a reactive warhead. Although the example shown here is not completely representative, the choice of GST enables us to rationalize the target binding retrospectively by using a computational model based on the crystal structure. Current limitation of this methodology seems that the bait-conjugated peptide tends to bind to a promiscuous large binding pocket. Nevertheless, we believe the nonreactive bait-alteration concept will be useful in general for the future, to discover covalent drugs made of peptide as antibody−drug substitutes, especially aimed for cell-surface receptors.



MATERIALS AND METHODS Biopanning against GST by Using Bait FragmentConjugated Peptide Library. Synthesis of each baitconjugated peptide library on phage and biopanning against GST was performed as described previously.22 In brief, approximately 1.0 × 1011 pfu of T7Select10 library (-S-G-GG-X3-C-X5−7-C-X3; X represents any randomized amino acid) was modified with bromoacetamide (BA)-conjugated bait fragment via the 10BASEd-T. After modification, the T7 phage library was dissolved in selection buffer (PBS supplemented with 0.1% v/v TritonX-100). To remove nonspecific binders (e.g., beads and streptavidin binders), the modified T7 phage library was preincubated with streptavidincoupled beads for 16 h at 4 °C, and then the supernatant was further incubated with the GST-immobilized beads for 15 h. The latter beads were washed three times for 14 min in each with 0.2 mL of the selection buffer. The entire binding and washing processes were performed using an automated machine (Target Angler 8, Tamagawa Seiki, Japan). GSTbound phage was directly infected and amplified with E. coli BLT5403 strain. Increasing stringent conditions such as shortening the binding time (e.g., 400 min at the final round) and increasing the washing frequency/time (up to 5 times/70 min in total at the final round), were applied stepwise to each round. After 3 rounds of biopanning, a mixture of T7 phage polyclones was subjected to a next generation sequencer (NGS), to obtain the common consensus sequence. Synthesis of the Bait-Conjugated Peptides and Confirmation of Their Solvatochromic Fluorescence-

Figure 3. Identification of fragments derived from the covalent-binderconjugated GST peptide by LC-MS/MS analysis. (A) LC profiles of trypsin-digested peptide fragments derived from modified and unmodified GST. The absorbance above 270 nm was measured. (B) MS and (C) MS/MS spectra of the newly appeared peak (indicated by arrow in A). All the detected fragments were consistent with theoretical m/z values of the represented structure. The peptide fragment of IAYSK was derived from a constituent of the glutathione binding pocket of GST protein. Y* means conjugated tyrosine.

conjugated tyrosine of the 111th position. The geometry of the altered warhead is in good agreement with the solvatochromic fluorescence change of the bait-conjugated peptide upon GST binding (Figure S2). We speculate that this proximity between the warhead and the 111th tyrosine caused the site- and position-specific efficient cross-linking. In contrast, the lowest docking model of the rationally designed one suggested that the warhead was located outside of the pocket (Figure S7), which might result in the insufficient conjugation with the 111th tyrosine.



CONCLUSIONS In conclusion, we have established a precise and efficient conjugation method between a hydrophobic pocket of a target protein and a targeted peptide. The peptide as a covalent binder has been obtained by combinatorial screening of a bait D

DOI: 10.1021/acs.bioconjchem.8b00301 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Change upon GST-Binding. Each bait-conjugated peptide possessing LNYCDGW sequence was synthesized by reaction of each BA-conjugated bait fragment independently, according to the reported procedure.27 Then, each bait-conjugated peptide (0.07 mM) was mixed with equal molar of GST or streptavidin in PBS. It was incubated for 10 min at room temperature, and fluorescence spectrum was measured (Figure S2). Conjugation between GST and Covalent Binders. Preliminary Covalent Modification at the GlutathioneBinding Pocket Using a Reported Covalent Binder. A rationally designed covalent binder (abbreviated as E′C*G), which is known to irreversibly bind to the glutathione-binding pocket of human GST, was synthesized and identified according to the reported procedure26 in combination with MS/MS analysis (Figure S5B). The binder (5.2 mM) was mixed with S. japonicum GST (2.7 mM) in 20 mM phosphate buffer (pH 7.5), and incubated for 21 h at 37 °C in the dark. It was mixed with 1 × sample buffer, denatured at 95 °C, separated by 15% SDS-PAGE, followed by trypsinization/LCMS/MS analysis. The reported covalent binder successfully conjugated to a tyrosine located at deep inside the glutathione binding pocket of S. japonicum GST (Figure S5C). Specific Conjugation between Combinatorially Screened Covalent Binder and GST Confirmed by SDS-PAGE/ Fluorescence Imaging. Fluorescent peptide (Fam− GGLNYCDGW, 0.01 M) was dissolved in 20 mM phosphate buffer (pH 7.4)/10% acetonitrile, and reacted with ethenesulfonyl fluoride (ESF, 0.1 M) or 4-(2-bromoacetyl)benzenesulfony fluoride (BBSF, 5 mM) in the presence of neutralized tris(2-carboxyethyl)phosphine (TCEP, 2 mM). The mixture was reacted for more than 4 h at room temperature in the dark with vigorous shaking, and the reaction was monitored by HPLC (Figure S3). Only the ESF-conjugated covalent binder could be purified by reverse-phase HPLC, and it was used for further experiments. The purified covalent binder (0.10 mM) was mixed with GST (0.31 mM) in phosphate buffer (pH 7.5) in the presence of 40% (v/v) human serum (Sigma, H4522), and incubated for 21 h at 37 °C in the dark. It was mixed with 1× sample buffer, denatured at 95 °C, and separated by 15% SDS-PAGE. The binder-conjugated proteins were detected by fluorescence imaging, and whole proteins were visualized by CBB staining (Figure 2B). Detailed Identification of the Conjugated Site/Position on GST after the Reaction with Combinatorially Screened Covalent Binder. Peptide (LNYCDGW, 0.24 M) was dissolved in 20 mM phosphate buffer (pH 7.4)/10% acetonitrile, and reacted with ESF (0.26 M) in the presence of neutralized TCEP (2 mM). The mixture was reacted for more than 4 h at room temperature in the dark with vigorous shaking, and purified by reverse-phase HPLC (yield 60%), and identified with 1H NMR, MS, and MS/MS analyses (Figure S4). Covalent conjugation reaction with GST, and SDS-PAGE followed by trypsinization/LC-MS/MS analysis were performed under the same conditions described previously in Preliminary Covalent Modification section, except using the combinatorially screened covalent binder (i.e., LNYC*DGW) instead of the glutathione derivative (i.e., E′C*G).

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00301.



Additional detailed materials and methods; supplemental figures and tables (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-42-443-5980. ORCID

Masumi Taki: 0000-0002-1811-8029 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by a JSPS KAKENHI grant (#17K05925) to M.T. We appreciate Dr. L. Nelson (MD Anderson), Prof. T. Yamashita (Keio Univ.), and Prof. M. Tanaka (UEC) for critical reading of this manuscript. We would like to acknowledge Ms. M. Yonekura and Mrs. Y. Kato as assistant researchers for performing NGS analysis and data mining in silico. We also thank Dr. H. Nakamura (Biomodeling Research Inc.) for fruitful discussions about molecular modeling. Finally, we greatly appreciate a (blinded) peer reviewer who read our old manuscript so carefully, and gave us a lot of fruitful questions and comments to let us be aware of deep understanding.



REFERENCES

(1) Baillie, T. A. (2016) Targeted Covalent Inhibitors for Drug Design. Angew. Chem., Int. Ed. 55, 13408−13421. (2) Gan, J. P., Zhang, H. Y., and Humphreys, W. G. (2016) DrugProtein Adducts: Chemistry, Mechanisms of Toxicity, and Methods of Characterization. Chem. Res. Toxicol. 29, 2040−2057. (3) Smith, A. J. T., Zhang, X. Y., Leach, A. G., and Houk, K. N. (2009) Beyond Picomolar Affinities: Quantitative Aspects of Noncovalent and Covalent Binding of Drugs to Proteins. J. Med. Chem. 52, 225−233. (4) Johnson, D. S., Weerapana, E., and Cravatt, B. F. (2010) Strategies for discovering and derisking covalent, irreversible enzyme inhibitors. Future Med. Chem. 2, 949−964. (5) Lagoutte, R., Patouret, R., and Winssinger, N. (2017) Covalent inhibitors: an opportunity for rational target selectivity. Curr. Opin. Chem. Biol. 39, 54−63. (6) Copeland, R. A., Pompliano, D. L., and Meek, T. D. (2006) Drug-target residence time and its implications for lead optimization. Nat. Rev. Drug Discovery 5, 730−739. (7) Singh, J., Petter, R. C., Baillie, T. A., and Whitty, A. (2011) The resurgence of covalent drugs. Nat. Rev. Drug Discovery 10, 307−317. (8) Bauer, R. A. (2015) Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies. Drug Discovery Today 20, 1061−1073. (9) Pichler, W. J., Naisbitt, D. J., and Park, B. K. (2011) Immune pathomechanism of drug hypersensitivity reactions. J. Allergy Clin. Immunol. 127, S74−81. (10) Yang, Y. O., Shu, Y. Z., and Humphreys, W. G. (2016) LabelFree Bottom-Up Proteomic Workflow for Simultaneously Assessing the Target Specificity of Covalent Drug Candidates and Their OffTarget Reactivity to Selected Proteins. Chem. Res. Toxicol. 29, 109− 116.

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Bioconjugate Chemistry (11) De Cesco, S., Kurian, J., Dufresne, C., Mittermaier, A. K., and Moitessier, N. (2017) Covalent inhibitors design and discovery. Eur. J. Med. Chem. 138, 96−114. (12) Kathman, S. G., Xu, Z. Y., and Statsyuk, A. V. (2014) A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases. J. Med. Chem. 57, 4969−4974. (13) Kathman, S. G., and Statsyuk, A. V. (2016) Covalent tethering of fragments for covalent probe discovery. MedChemComm 7, 576− 585. (14) Moellering, R. E., and Cravatt, B. F. (2012) How Chemoproteomics Can Enable Drug Discovery and Development. Chem. Biol. 19, 11−22. (15) Saghatelian, A. (2017) Novel Biology and Druggable Targets via Chemoproteomics. Biochemistry 56, 6515−6516. (16) Ng, S., Jafari, M. R., and Derda, R. (2012) Bacteriophages and viruses as a support for organic synthesis and combinatorial chemistry. ACS Chem. Biol. 7, 123−138. (17) Heinis, C., and Winter, G. (2015) Encoded libraries of chemically modified peptides. Curr. Opin. Chem. Biol. 26, 89−98. (18) Morioka, T., Loik, N. D., Hipolito, C. J., Goto, Y., and Suga, H. (2015) Selection-based discovery of macrocyclic peptides for the next generation therapeutics. Curr. Opin. Chem. Biol. 26, 34−41. (19) Kawakami, T., and Murakami, H. (2012) Genetically Encoded Libraries of Non-standard Peptides. J. Nucleic Acids 2012, 713510. (20) Zambaldo, C., Daguer, J. P., Saarbach, J., Barluenga, S., and Winssinger, N. (2016) Screening for covalent inhibitors using DNAdisplay of small molecule libraries functionalized with cysteine reactive moieties. MedChemComm 7, 1340−1351. (21) Fukunaga, K., and Taki, M. (2012) Practical Tips for Construction of Custom Peptide Libraries and Affinity Selection by Using Commercially Available Phage Display Cloning Systems. J. Nucleic Acids 2012, 295719. (22) Uematsu, S., Midorikawa, T., Ito, Y., and Taki, M. (2017) Selection of Turning-on Fluorogenic Probe as Protein-Specific Detector Obtained via the 10BASE(d)-T. AIP Conf. Proc. 1807, 020028. (23) Fukunaga, K., Hatanaka, T., Ito, Y., and Taki, M. (2013) Gp10 based-thioetherification (10BASE(d)-T) on a displaying library peptide of bacteriophage T7. Mol. BioSyst. 9, 2988−2991. (24) Fukunaga, K., Hatanaka, T., Ito, Y., Minami, M., and Taki, M. (2014) Construction of a crown ether-like supramolecular library by conjugation of genetically-encoded peptide linkers displayed on bacteriophage T7. Chem. Commun. 50, 3921−3923. (25) Gu, C., Shannon, D. A., Colby, T., Wang, Z. M., Shabab, M., Kumari, S., Villamor, J. G., McLaughlin, C. J., Weerapana, E., Kaiser, M., et al. (2013) Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases. Chem. Biol. 20, 541−548. (26) Shishido, Y., Tomoike, F., Kimura, Y., Kuwata, K., Yano, T., Fukui, K., Fujikawa, H., Sekido, Y., Murakami-Tonami, Y., Kameda, et al. (2017) A covalent G-site inhibitor for glutathione S-transferase Pi (GSTP(1−1)). Chem. Commun. 53, 11138−11141. (27) Taki, M., Inoue, H., Mochizuki, K., Yang, J., and Ito, Y. (2016) Selection of Color-Changing and Intensity-Increasing Fluorogenic Probe as Protein-Specific Indicator Obtained via the 10BASE(d)-T. Anal. Chem. 88, 1096−1099. (28) Dubiella, C., Cui, H., Gersch, M., Brouwer, A. J., Sieber, S. A., Kruger, A., Liskamp, R. M., and Groll, M. (2014) Selective inhibition of the immunoproteasome by ligand-induced crosslinking of the active site. Angew. Chem., Int. Ed. 53, 11969−11973. (29) Narayanan, A., and Jones, L. H. (2015) Sulfonyl fluorides as privileged warheads in chemical biology. Chem. Sci. 6, 2650−2659. (30) Mukherjee, H., Debreczeni, J., Breed, J., Tentarelli, S., Aquila, B., Dowling, J. E., Whitty, A., and Grimster, N. P. (2017) A study of the reactivity of S-(VI)-F containing warheads with nucleophilic aminoacid side chains under physiological conditions. Org. Biomol. Chem. 15, 9685−9695.

(31) Fukunishi, Y., Mikami, Y., and Nakamura, H. (2003) The filling potential method: A method for estimating the free energy surface for protein-ligand docking. J. Phys. Chem. B 107, 13201−13210.

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DOI: 10.1021/acs.bioconjchem.8b00301 Bioconjugate Chem. XXXX, XXX, XXX−XXX