Vinyl sulfonamide synthesis for irreversible tethering via a novel α-selenoether protection strategy

Med. Chem. Commun., 2018 
DOI: 10.1039/C8MD00566D

Vinyl sulfonamides are valuable electrophiles for targeted protein modification and inhibition. We describe a novel approach to the synthesis of terminal vinyl sulfonamides which uses mild oxidative conditions to induce elimination of an α-selenoether masking group. The method complements traditional synthetic approaches and typically yields vinyl sulfonamides in high purity after aqueous work-up without requiring column chromatography of the final electrophilic product. The methodology is applied to the synthesis of covalent fragments for use in irreversible protein tethering and crucially enables the attachment of diverse fragments to the vinyl sulfonamide warhead via a chemical linker. Using thymidylate synthase as a model system, ethylene glycol is identified as a effective linker for irreversible protein tethering.

Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology

Matthias Gehringer and Stefan A. Laufer
Journal of Medicinal Chemistry 
DOI: 10.1021/acs.jmedchem.8b01153

Targeted covalent inhibitors (TCIs) are designed to bind poorly conserved amino acids by means of reactive groups, the so-called warheads. Currently, targeting non-catalytic cysteine residues with acrylamides and other α,β-unsaturated carbonyl compounds is the predominant chemical strategy in TCI development. The recent ascent of covalent drugs has stimulated considerable efforts to characterize alternative warheads for the covalent-reversible and irreversible engagement of non-catalytic cysteine residues as well as other amino acids. This Perspective article provides an overview of warheads beyond α,β-unsaturated amides that were recently used in the design of targeted covalent ligands. Promising reactive groups that have not yet demonstrated their utility in TCI development are also highlighted. Special emphasis is placed on the discussion of reactivity and case studies illustrating applications in medicinal chemistry and chemical biology.

A Chemoproteomic Strategy for Direct and Proteome-wide Covalent Inhibitor Target-site Identification

Christopher Michael Browne, Baishan Jiang, Scott B Ficarro, Zainab M Doctor, Jared Lee Johnson, Joseph D Card, Sindhu Carmen Sivakumaren, William M Alexander, Tomer Yaron, Charles Joseph Murphy, Nicholas P Kwiatkowski, Tinghu Zhang, Lewis C. Cantley, Nathanael S Gray, and Jarrod A. Marto

Journal of the American Chemical Society 2018

DOI: 10.1021/jacs.8b07911

Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically-addressable cysteine-thiols, and inform structure-based drug design programs.

Overview of Current Type I/II Kinase Inhibitors

Zheng Zhao, Philip E. Bourne

https://arxiv.org/abs/1811.09718

Research on kinase-targeting drugs has made great strides over the last 30 years and is attracting greater attention for the treatment of yet more kinase-related diseases. Currently, 42 kinase drugs have been approved by the FDA, most of which (39) are Type I/II inhibitors. Notwithstanding these advances, it is desirable to target additional kinases for drug development as more than 200 diseases, particularly cancers, are directly associated with aberrant kinase regulation and signaling. Here, we review the extant Type I/II drugs systematically to obtain insights into the binding pocket characteristics, the associated features of Type I/II drugs, and the mechanism of action to facilitate future kinase drug design and discovery. We conclude by summarizing the main successes and limitations of targeting kinase for the development of drugs.

Covalent Docking Identifies a Potent and Selective MKK7 Inhibitor [@london_lab]

Amit Shraga, Evgenia Olshvang, Natalia Davidzohn, Payam Khoshkenar, Nicolas Germain, Khriesto Shurrush, Silvia Carvalho, Liat Avram, Shira Albeck, Tamar Unger, Bruce Lefker, Chakrapani Subramanyam, Robert L. Hudkins, Amir Mitchell, Ziv Shulman, Takayoshi Kinoshita, Nir London,
Covalent Docking Identifies a Potent and Selective MKK7 Inhibitor,
Cell Chemical Biology, 2018

DOI: 10.1016/j.chembiol.2018.10.011

The c-Jun NH2-terminal kinase (JNK) signaling pathway is central to the cell response to stress, inflammatory signals, and toxins. While selective inhibitors are known for JNKs and for various upstream MAP3Ks, no selective inhibitor is reported for MKK7––one of two direct MAP2Ks that activate JNK. Here, using covalent virtual screening, we identify selective MKK7 covalent inhibitors. We optimized these compounds to low-micromolar inhibitors of JNK phosphorylation in cells. The crystal structure of a lead compound bound to MKK7 demonstrated that the binding mode was correctly predicted by docking. We asserted the selectivity of our inhibitors on a proteomic level and against a panel of 76 kinases, and validated an on-target effect using knockout cell lines. Lastly, we show that the inhibitors block activation of primary mouse B cells by lipopolysaccharide. These MKK7 tool compounds will enable better investigation of JNK signaling and may serve as starting points for therapeutics.

Balancing reactivity and antitumor activity: Heteroarylthio acetamide derivatives as potent and time-dependent inhibitors of EGFR

Riccardo Castelli, Nicole Bozza, Andrea Cavazzoni, Mara Bonelli, Federica Vacondio, Francesca Ferlenghi, Donatella Callegari, Claudia Silva, Silvia Rivara, Alessio Lodola, Graziana Digiacomo, Claudia Fumarola, Roberta Alfieri, Pier Giorgio Petronini, Marco Mor

European Journal of Medicinal Chemistry, 2018
DOI: 10.1016/j.ejmech.2018.11.029

Second- and third-generation inhibitors of EGFR possess an acrylamide group which alkylates Cys797, allowing to overcome resistance due to insurgence of T790M mutation. Less reactive warheads, yet capable to bind the target cysteine, may be useful to design newer and safer inhibitors. In the present work, we synthesized a 2-chloro-N-(4-(phenylamino)quinazolin-6-yl)acetamide (8) derivative as a prototype of EGFR inhibitor potentially able to react with Cys797 by nucleophilic substitution. We then tuned the reactivity of the acetamide fragment by replacing the chlorine leaving group with (hetero)-aromatic thiols or carboxylate esters. Among the synthesized derivatives, the 2-((1H-imidazol-2-yl)thio)acetamide 16, while showing negligible reactivity with cysteine in solution, caused long-lasting inhibition of wild-type EGFR autophosphorylation in A549 cells, resulted able to bind recombinant EGFR L858R/T790M in a time-dependent manner, and inhibited both EGFR autophosphorylation and proliferation in gefitinib-resistant H1975 (EGFR L858R/T790M) lung cancer cells at low micromolar concentration.

Neolymphostin A is a Covalent Phosphoinositide-3-kinase (PI3-K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor that Employs an Unusual Electrophilic Vinylogous Ester

Gabriel Castro-Falcón, Grant Seiler, Ozlem Demir, Manoj Rathinaswamy, David Hamelin, Reece M. Hoffmann, Stefanie Makowski, Anne-Catrin Letzel, Seth Field, John Burke, Rommie E. Amaro, and Chambers C. Hughes

Journal of Medicinal Chemistry 2018
DOI: 10.1021/acs.jmedchem.8b00975

Using a novel chemistry-based assay for identifying electrophilic natural products from unprocessed extracts, we identified the PI3-kinase/mTOR dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent on the neolymphostin core was the exact site for enzyme conjugation. Tandem MS/MS experiments on PI3Kα treated with the inhibitor revealed that neolymphostin covalently modified Lys802 with a shift in mass of +306 amu, corresponding to addition of the inhibitor and elimination of methanol. The binding pose of the inhibitor bound to PI3Kα was modelled, and hydrogen-deuterium exchange mass spectrometry experiments supported this model. Against a panel of kinases, neolymphostin showed good selectivity for PI3-kinase and mTOR. In addition, the natural product blocked AKT phosphorylation in live cells with an IC50 of ~3 nM. Taken together, neolymphostin is the first reported example of a covalent kinase inhibitor from the bacterial domain of life.

Structure-based engineering of irreversible inhibitors against histone lysine demethylase KDM5A

John R Horton, Clayton B Woodcock, Qin Chen, Xu Liu, Xing Zhang, John Shanks, Ganesha Rai, Bryan T Mott, Daniel J Jansen, Stephen C Kales, Mark J Henderson, Matthew Cyr, Katherine Pohida, Xin Hu, Pranav Shah, Xin Xu, Ajit Jadhav, David J. Maloney, Matthew D. Hall, Anton Simeonov, Haian Fu, Paula M. Vertino, and Xiaodong Cheng

J. Med. Chem., 2018
DOI: 10.1021/acs.jmedchem.8b01219

The active sites of hundreds of human α-ketoglutarate (αKG) and Fe(II)-dependent dioxygenases are exceedingly well preserved, which challenges the design of selective inhibitors. We identified a non-catalytic cysteine (Cys481 in KDM5A) near the active sites of KDM5 histone H3 lysine 4 demethylases – which is absent in other histone demethylase families - that could be explored for interaction with the cysteine-reactive electrophile acrylamide. We synthesized analogs of a thienopyridine-based inhibitor chemotype, namely 2-((3-aminophenyl)(2-(piperidin-1-yl)ethoxy)methyl)thieno[3,2-b]pyridine-7-carboxylic acid (N70) and a derivative containing a (dimethylamino)but-2-enamido)phenyl moiety (N71) designed to form a covalent interaction with Cys481. We characterized the inhibitory and binding activities against KDM5A and determined the co-crystal structures of the catalytic domain of KDM5A in complex with N70 and N71. Whereas the non-covalent inhibitor N70 displayed αKG-competitive inhibition that could be reversed after dialysis, inhibition by N71 was dependent on enzyme concentration and persisted even after dialysis, consistent with covalent modification.

Genetic Incorporation of Olefin Cross-Metathesis Reaction Tags for Protein Modification

Bhaskar Bhushan, Yuya A. Lin, Martin Bak, Anuchit Phanumartwiwath, Nan Yang, Matthew K. Bilyard, Tomonari Tanaka, Kieran L. Hudson, Lukas Lercher, Monika Stegmann, Shabaz Mohammed, and Benjamin G. Davis

J. Am. Chem. Soc., 2018
DOI: 10.1021/jacs.8b09433

Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has been reported to date. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encodable Met analogue that is not only processed by translational cellular machinery but also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)) as well as metabolic labeling of protein in human cells and was reactive toward CM in several representative proteins. This expands the use of CM in the toolkit for “tag-and-modify” functionalization of proteins.

Celastrol binds to its target protein via specific noncovalent interactions and reversible covalent bond

Duo Zhang, Ziwen Chen, Caocao Hu, Siwei Yan, Zhuoer Li, Baohuan Lian, Yang Xu, Ding Rong, Zhiping Zeng, Xiao-kun Zhang and Ying Su

Chem. Commun., 2018
DOI: 10.1039/C8CC06140H

Celastrol is one of the most studied natural products. Our studies show for the first time that celastrol can bind to its target protein via specific noncovalent interactions, that position celastrol next to the thiol group of the reactive cysteine for reversible covalent bond formation. Such specific noncovalent interactions confer celastrol’s binding specificity and demonstrate the feasibility of improving the efficacy and selectivity of celastrol for therapeutic application.

Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases

Jacob Lauwring Andersen, Borbala Gesser, Erik Daa Funder, Christine Juul Fælled Nielsen, Helle Gotfred-Rasmussen, Mads Kirchheiner Rasmussen, Rachel Toth, Kurt Vesterager Gothelf, J. Simon C. Arthur, Lars Iversen & Poul Nissen

Nature Communications 2018, 9, 4344

Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment.

A metabolite-derived protein modification integrates glycolysis with KEAP1–NRF2 signalling

Michael J. Bollong, Gihoon Lee, John S. Coukos, Hwayoung Yun, Claudio Zambaldo, Jae Won Chang, Emily N. Chin, Insha Ahmad, Arnab K. Chatterjee, Luke L. Lairson, Peter G. Schultz & Raymond E. Moellering 

Nature (2018) DOI: 10.1038/s41586-018-0622-0

 that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3,4,5 and transcription6. An important ‘sensor’ protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1–NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases.

Reversible Covalent Reaction of Levosimendan with Cardiac Troponin C in Vitro and in Situ

Brittney A. Klein, Béla Reiz, Ian M. Robertson, Malcolm Irving, Liang Li, Yin-Biao Sun, and Brian D. Sykes

Biochemistry, 2018, 57 (15), pp 2256–2265

DOI: 10.1021/acs.biochem.8b00109

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC–levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.

A protein functionalization platform based on selective reactions at methionine residues

Michael T. Taylor, Jennifer E. Nelson, Marcos G. Suero & Matthew J. Gaunt

Nature, 2018, DOI: 10.1038/s41586-018-0608-y

Nature has a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a marked increase in their functional diversity1. Inspired by this, chemical tools have been developed for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations that are suitable for effective protein functionalization is limited, because the stringent demands inherent to biological systems preclude the applicability of many potential processes2. These chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near-perfect conversion2,3,4,5,6,7. Although many bioconjugation methods exist at cysteine, lysine and tyrosine, a method targeting a less-explored amino acid would considerably expand the protein functionalization toolbox. Here we report the development of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the electrophilic reactivity of a bespoke hypervalent iodine reagent, the S-Me group in the side chain of methionine can be targeted. The bioconjugation reaction is fast, selective, operates at low-micromolar concentrations and is complementary to existing bioconjugation strategies. Moreover, it produces a protein conjugate that is itself a high-energy intermediate with reactive properties and can serve as a platform for the development of secondary, visible-light-mediated bioorthogonal protein functionalization processes. The merger of these approaches provides a versatile platform for the development of distinct transformations that deliver information-rich protein conjugates directly from the native biomacromolecules.

Type II Kinase Inhibitors Targeting Cys-Gatekeeper Kinases Display Orthogonality with Wild Type and Ala/Gly-Gatekeeper Kinas

Cory A. Ocasio, Alexander A. Warkentin, Patrick J. McIntyre, Krister J. Barkovich, Clare Vesely, John Spencer, Kevan M. Shokat, and Richard Bayliss

ACS Chemical Biology, 2018,  Article ASAP

DOI: 10.1021/acschembio.8b00592

Analogue-sensitive (AS) kinases contain large to small mutations in the gatekeeper position rendering them susceptible to inhibition with bulky analogues of pyrazolopyrimidine-based Src kinase inhibitors (e.g., PP1). This “bump-hole” method has been utilized for at least 85 of ∼520 kinases, but many kinases are intolerant to this approach. To expand the scope of AS kinase technology, we designed type II kinase inhibitors, ASDO2/6 (analogue-sensitive “DFG-out” kinase inhibitors 2 and 6), that target the “DFG-out” conformation of Cys-gatekeeper kinases with submicromolar potency. We validated this system in vitro against Greatwall kinase (GWL), Aurora-A kinase, and cyclin-dependent kinase-1 and in cells using M110C-GWL-expressing mouse embryonic fibroblasts. These Cys-gatekeeper kinases were sensitive to ASDO2/6 inhibition but not AS kinase inhibitor 3MB-PP1 and vice versa. These compounds, with AS kinase inhibitors, have the potential to inhibit multiple AS kinases independently with applications in systems level and translational kinase research as well as the rational design of type II kinase inhibitors targeting endogenous kinases.

A road map for prioritizing warheads for cysteine targeting covalent inhibitors

Pé. Ábrányi-Balogh, Láó. Petri, Tí. Imre, Pé. Szijj, A. Scarpino, M. Hrast, A. Mitrović, Urš.Peč. Fonovič, K. Németh, Héè. Barreteau, D.I. Roper, K. Horváti, Gyö.G. Ferenczy, J. Kos, J. Ilaš, S. Gobec, Gyö.M. Keserű, A road map for prioritizing warheads for cysteine targeting covalent inhibitors, European Journal of Medicinal Chemistry, 2018, doi: 10.1016/j.ejmech.2018.10.010

Covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors.

Elucidating the catalytic power of glutamate racemase by investigating a series of covalent inhibitors

Nicholas Robert Vance  Katie R. Witkin  Patrick W. Rooney  Yalan Li  Marshall Pope Michael Ashley Spies

ChemMedChem 2018, doi: 10.1002/cmdc.201800592

The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to reduce off‐target liabilities. Herein, we present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase, discovered though structure‐based virtual screening. A combination of enzyme kinetics, mass spectrometry, and surface‐plasmon resonance experiments details a highly specific 1,4‐conjugate addition of a small molecule inhibitor with a catalytic cysteine of glutamate racemase. Molecular dynamics simulations and quantum mechanics‐molecular mechanics geometry optimizations reveal the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency comparable to β‐lactam antibiotics, with significant activity against methicillin‐resistant S. aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action against a target in the cell wall biosynthesis pathway.

Covalent modification of biomolecules through maleimide-based labeling strategies

Cyrille Sabot, Pierre-Yves Renard, Kevin Renault, and Jean Wilfried Fredy

Bioconjugate Chem., 2018
DOI: 10.1021/acs.bioconjchem.8b00252

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site selective modification of proteins through thio-Michael addition of biothiols and, to a lesser extent, via Diels‒Alder (DA) reactions in combination with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody-drug conjugates (ADCs) such as Adcetris® used in cancer therapies. Among the keys to success is the availability of several maleimides N-functionalized by fluorophores, affinity tags, spin labels, pharmacophores, as well as their unique reactivity features in terms of selectivity or kinetics. However, maleimide conjugate reactions have long been thought to be irreversible, and it is only recently that systematic studies regarding their reversibility and stability towards hydrolysis have been reported. This review provides an overview of the diversity of applications of maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being circumvented by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged in the preparation of fluorogenic probes mainly involved in the specific detection of thiol analytes. A summary of reported structures, their photophysical features and relative efficiency are discussed in the last part of the review.

Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide

Tang Li, René Maltais, Donald Poirier, and Sheng-Xiang Lin


J. Phys. Chem. Lett., 2018, 9, 5275–5280

DOI: 10.1021/acs.jpclett.8b02225

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) plays a pivotal role in the progression of estrogen-related diseases because of its involvement in the biosynthesis of estradiol (E2), constituting a valuable therapeutic target for endocrine treatment. In the present study, we successfully cocrystallized the enzyme with the reversible inhibitor 2-methoxy-16β-(m-carbamoylbenzyl)-E2 (2-MeO-CC-156) as well as the enzyme with the irreversible inhibitor 3-(2-bromoethyl)-16β-(m-carbamoylbenzyl)-17β-hydroxy-1,3,5(10)-estratriene (PBRM). The structures of ternary complexes of 17β-HSD1–2-MeO-CC-156–NADP+ and 17β-HSD1–PBRM–NADP+ comparatively show the formation of a covalent bond between His221 and the bromoethyl side chain of the inhibitor in the PBRM structure. A dynamic process including beneficial molecular interactions that favor the specific binding of a low-reactivity inhibitor and subsequent N-alkylation event through the participation of His221 in the enzyme catalytic site clearly demonstrates the covalent bond formation. This finding opens the door to a new design of alkyl halide-based specific covalent inhibitors as potential therapeutic agents for different enzymes, contributing to the development of highly efficient inhibitors.

Discovery and Optimization of Inhibitors of the Parkinson’s Disease Associated Protein DJ-1

ACS Chem. Biol., 2018

DOI: 10.1021/acschembio.8b00701

DJ-1 is a Parkinson’s disease associated protein endowed with enzymatic, redox sensing, regulatory, chaperoning, and neuroprotective activities. Although DJ-1 has been vigorously studied for the past decade and a half, its exact role in the progression of the disease remains uncertain. In addition, little is known about the spatiotemporal regulation of DJ-1, or the biochemical basis explaining its numerous biological functions. Progress has been hampered by the lack of inhibitors with precisely known mechanisms of action. Herein, we have employed biophysical methodologies and X-ray crystallography to identify and to optimize a family of compounds inactivating the critical Cys106 residue of human DJ-1. We demonstrate these compounds are potent inhibitors of various activities of DJ-1 in vitro and in cell-based assays. This study reports a new family of DJ-1 inhibitors with a defined mechanism of action, and contributes toward the understanding of the biological function of DJ-1.

Design and Characterization of Novel Covalent Bromodomain and Extra-terminal Domain (BET) Inhibitors Targeting a Methionine

Olesya Kharenko, Reena G Patel, S. David Brown, Cyrus Calosing, Andre White, Damodharan Lakshminarasimhan, Robert K Suto, Bryan C Duffy, Douglas B. Kitchen, Kevin G McLure, Henrik C. Hansen, Edward H van der Horst, and Peter R. Young

J. Med. Chem., 2018
DOI: 10.1021/acs.jmedchem.8b00666

BET proteins are key epigenetic regulators that alter transcription by binding to acetylated lysine (AcLys) residues of histones and transcription factors through bromodomains (BDs). The disruption of this interaction with small molecule bromodomain inhibitors is a promising approach to treat various diseases including cancer, autoimmune and cardiovascular diseases. Covalent inhibitors can potentially offer a more durable target inhibition leading to improved in vivo pharmacology. Here we describe the design of covalent inhibitors of BRD4(BD1) that target a methionine in the binding pocket by attaching an epoxide warhead to a suitably oriented non-covalent inhibitor. Using thermal denaturation, MALDI-TOF mass spectrometry and an X-ray crystal structure, we demonstrate that these inhibitors selectively form a covalent bond with Met149 in BRD4(BD1) but not other bromodomains, and provide durable transcriptional and anti-proliferative activity in cell based assays. Covalent targeting of methionine offers a novel approach to drug discovery for BET proteins and other targets.

Covalent Modification of Biomolecules through Maleimide-Based Labeling Strategies

Kévin Renault, Jean Wilfried Fredy, Pierre-Yves Renard, and Cyrille Sabot

Bioconjugate Chem., 2018, 29 (8),  2497–2513
DOI: 10.1021/acs.bioconjchem.8b00252

Since their first use in bioconjugation more than 50 years ago, maleimides have become privileged chemical partners for the site-selective modification of proteins via thio-Michael addition of biothiols and, to a lesser extent, via Diels–Alder (DA) reactions with biocompatible dienes. Prominent examples include immunotoxins and marketed maleimide-based antibody–drug conjugates (ADCs) such as Adcetris, which are used in cancer therapies. Among the key factors in the success of these groups is the availability of several maleimides that can be N-functionalized by fluorophores, affinity tags, spin labels, and pharmacophores, as well as their unique reactivities in terms of selectivity and kinetics. However, maleimide conjugate reactions have long been considered irreversible, and only recently have systematic studies regarding their reversibility and stability toward hydrolysis been reported. This review provides an overview of the diverse applications for maleimides in bioconjugation, highlighting their strengths and weaknesses, which are being overcome by recent strategies. Finally, the fluorescence quenching ability of maleimides was leveraged for the preparation of fluorogenic probes, which are mainly used for the specific detection of thiol analytes. A summary of the reported structures, their photophysical features, and their relative efficiencies is discussed in the last part of the review.

How Reactive are Druggable Cysteines in Protein Kinases? [@RowleyGroup]

Ernest Awoonor-Williams and Christopher N. Rowley

J. Chem. Inf. Model., 2018
DOI: 10.1021/acs.jcim.8b00454

Targeted covalent inhibitors (TCIs) have been successfully developed as high-affinity and selective inhibitors of enzymes of the protein kinase family. These drugs typically act by undergoing an electrophilic addition with an active-site cysteine residue, so design of a TCI begins with the identification of a “druggable” cysteine. These electrophilic additions generally require deprotonation of the thiol to form a reactive anionic thiolate, so the acidity of the residue is a critical factor. Few experimental measurements of the pKa’s of druggable cysteines have been reported, so computational prediction could prove to be very important in selecting reactive cysteine targets. Here we report the computed pKa’s of druggable cysteines in selected protein kinases that are of clinical relevance for targeted therapies. The pKa’s of the cysteines were calculated using advanced computational methods based on all-atom replica-exchange thermodynamic integration molecular dynamics simulations in explicit solvent. We found that the acidities of druggable cysteines within protein kinases are diverse and elevated, indicating enormous differences in their reactivity. Constant-pH molecular dynamics simulations were also performed on selected protein kinases, and the results confirmed this varied range in the acidities of druggable cysteines. Many of these active-site cysteines have low exposure to solvent molecules, elevating their pKa values. Electrostatic interactions with nearby anionic residues also elevate the pKa’s of cysteine residues in the active site. The results suggest that some cysteine residues within kinase binding sites will be slow to react with a TCI because of their low acidity. Several oncogenic kinase mutations were also modeled and found to have pKa’s similar to that of the wild-type kinase.

Applications of Reactive Cysteine Profiling

Keriann M. Backus

Current Topics in Microbiology and Immunology book series, 2018
doi: 10.1007/82_2018_120

Cysteine thiols are involved in a diverse set of biological transformations, including nucleophilic and redox catalysis, metal coordination and formation of both dynamic and structural disulfides. Often posttranslationally modified, cysteines are also frequently alkylated by electrophilic compounds, including electrophilic metabolites, drugs, and natural products, and are attractive sites for covalent probe and drug development. Quantitative proteomics combined with activity-based protein profiling has been applied to annotate cysteine reactivity, susceptibility to posttranslational modifications, and accessibility to chemical probes, uncovering thousands of functional and small-molecule targetable cysteines across a diverse set of proteins, proteome-wide in an unbiased manner. Reactive cysteines have been targeted by high-throughput screening and fragment-based ligand discovery efforts. New cysteine-reactive electrophiles and compound libraries have been synthesized to enable inhibitor discovery broadly and to minimize nonspecific toxicity and off-target activity of compounds. With the recent blockbuster success of several covalent inhibitors, and the development of new chemical proteomic strategies to broadly identify reactive, ligandable and posttranslationally modified cysteines, cysteine profiling is poised to enable the development of new potent and selective chemical probes and even, in some cases, new drugs.

Rational Design of a Highly Reactive Dicysteine Peptide Tag For Fluorogenic Protein Labelling [@theKeillors]

Miroslava Strmiskova,  Kelvin Tsao  and  Jeffrey W Keillor
Org. Biomol. Chem., 2018
doi: 10.1039/C8OB01417E

Rationally designed libraries of a short helical peptide sequence containing two cysteine residues were screened kinetically for their reactivity towards complementary dimaleimide fluorogens. This screening revealed variant sequences whose reactivity has been increased by an order of magnitude relative to the original sequence. The most reactive engineered sequences feature mutant residues bearing positive charges, suggesting the pKa values of the adjacent thiol groups have been significantly lowered, through electrostatic stabilization of the thiolate ionization state. pH-rate profiles measured for several mutant sequences support this mechanism of rate enhancement. The practical utility of the enhanced reactivity of the final engineered dicysteine tag (‘dC10*’) was then demonstrated in the fluorogenic intracellular labelling of a specific protein in living cells.

Target Identification of Bioactive Covalently Acting Natural Products

Nomura D.K., Maimone T.J.

Current Topics in Microbiology and Immunology, 2018
doi: 10.1007/82_2018_121

There are countless natural products that have been isolated from microbes, plants, and other living organisms that have been shown to possess therapeutic activities such as antimicrobial, anticancer, or anti-inflammatory effects. However, developing these bioactive natural products into drugs has remained challenging in part because of their difficulty in isolation, synthesis, mechanistic understanding, and off-target effects. Among the large pool of bioactive natural products lies classes of compounds that contain potential reactive electrophilic centers that can covalently react with nucleophilic amino acid hotspots on proteins and other biological molecules to modulate their biological action. Covalently acting natural products are more amenable to rapid target identification and mapping of specific druggable hotspots within proteins using activity-based protein profiling (ABPP)-based chemoproteomic strategies. In addition, the granular biochemical insights afforded by knowing specific sites of protein modifications of covalently acting natural products enable the pharmacological interrogation of these sites with more synthetically tractable covalently acting small molecules whose structures are more easily tuned. Both discovering binding pockets and targets hit by natural products and exploiting druggable modalities targeted by natural products with simpler molecules may overcome some of the challenges faced with translating natural products into drugs.

A green BODIPY‐based, super‐fluorogenic, protein‐specific labelling agent

Yingche Chen, Kelvin Tsao, Sydney L. Acton, Jeffrey W. Keillor

Angewandte Chemie, 2018
doi: 10.1002/ange.201805482

We report the development of YC23, a novel green BODIPY‐based dimaleimide derivative that undergoes a Fluorogenic Addition Reaction (FlARe) with a genetically encodable peptide tag (dC10α) that can be fused to a protein of interest (POI). We also demonstrate the application of this reaction for the fluorogenic labelling of a specific POI in bacterial lysate and in living mammalian cells.

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315

Clarissa G. Jakob, Anup K. Upadhyay, Pamela L. Donner, Emily Nicholl, Sadiya N. Addo, Wei Qiu, Christopher Ling, Sujatha M. Gopalakrishnan, Maricel Torrent, Steven P. Cepa, Jason Shanley, Alexander R. Shoemaker, Chaohong C. Sun∥, Anil Vasudevan, Kevin R. Woller, J. Brad Shotwell, Bailin Shaw, Zhiguo Bian, and Jessica E. Hutti

J. Med. Chem, 2018 61 (15), 6647–6657

DOI: 10.1021/acs.jmedchem.8b00305

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.

Precedence and Promise of Covalent Inhibitors of EGFR and KRAS for Patients with Non-Small-Cell Lung Cancer

Hengmiao Cheng  and Simon Planken

ACS Med. Chem. Lett., 2018
DOI: 10.1021/acsmedchemlett.8b00311

Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogenic mutations are leading causes for lung cancer. Extensive drug discovery efforts targeting EGFR have led to the discovery and FDA approval of both reversible and covalent inhibitors. Second and third generation covalent inhibitors for EGFR have also been described, with the latter targeting specific emerging mutations. After decades of extensive effort, KRAS is widely regarded as an intractable therapeutic target; however, recent publications suggest covalent inhibition is a promising strategy to deliver inhibitors of the KRASG12C mutation.

C797S Resistance: The Undruggable EGFR Mutation in Non-Small Cell Lung Cancer?

Tobias Grabe, Jonas Lategahn, and Daniel Rauh

ACS Med. Chem. Lett., 2018
DOI: 10.1021/acsmedchemlett.8b00314

The first evidence of osimertinib resistance mediated by the epidermal growth factor receptor (EGFR) mutation C797S was reported three years ago. Since then, no major breakthroughs have been achieved to target the clinically relevant mutant variant that impedes covalent bond formation with irreversible EGFR inhibitors. Although several biochemically active compounds have been described, only a few inhibitors that potently act on the cellular level or in vivo have been introduced so far. Herein, we give an overview of current approaches in the field and highlight the challenges that need to be addressed in future research projects to overcome the C797S-mediated drug resistance.

Structural basis of substrate recognition and covalent inhibition of Cdu1 from Chlamydia trachomatis

Yesid Andres Ramirez  Thomas Adler  Eva Altmann  Christian Tiesmeyer  Theresa Klemm Florian Sauer  Stefan Kathman  Alexander Statsyuk  Christoph Sotriffer  Caroline Kisker

ChemMedChem, 2018

doi: 10.1002/cmdc.201800364

Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionary related protease adenain a target‐hopping approach screening on a focused set of adenain inhibitors has been pursued. The thereby identified cyano‐pyrimidine based inhibitors represent the first active‐site directed small molecule inhibitors for Cdu1. High‐resolution crystal structures of Cdu1 in complex with two covalently bound cyano‐pyrimidines as well as with its substrate ubiquitin have been obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into Cdu1s substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong foundation for future structure‐guided medicinal chemistry optimization of this cyano‐pyrimidine based scaffold towards more potent and specific Cdu1 inhibitors.

Mechanistic Insight through Irreversible Inhibition: DNA Polymerase θ Uses a Common Active Site for Polymerase and Lyase Activities

Daniel J. Laverty, Ifor P. Mortimer, and Marc M. Greenberg
J. Am. Chem. Soc. 2018 140 (29), 9034–9037
doi: 10.1021/jacs.8b04158

DNA polymerase θ (Pol θ) is a multifunctional enzyme. It is nonessential in normal cells, but its upregulation in cancer cells correlates with cellular resistance to oxidative damage and poor prognosis. Pol θ possesses polymerase activity and poorly characterized lyase activity. We examined the Pol θ lyase activity on various abasic sites and determined that the enzyme is inactivated upon attempted removal of the oxidized abasic site commonly associated with C4′-oxidation (pC4-AP). Covalent modification of Pol θ by the DNA lesion enabled determination of the primary nucleophile (Lys2383) responsible for Schiff base formation in the lyase reaction. Unlike some other base excision repair polymerases, Pol θ uses a single active site for polymerase and lyase activity. Mutation of Lys2383 significantly reduces both enzyme activities but not DNA binding. Demonstration that Lys2383 is required for polymerase and lyase activities indicates that this residue is an Achilles heel for Pol θ and suggests a path forward for designing inhibitors of this attractive anticancer target.

Arylfluorosulfate‐Based Warheads for Covalent Protein Labeling: A New Addition to the Arsenal

Pablo Martin-Gago, Christian Adam Olsen

Angewandte Chemie, 2018

doi: 10.1002/anie.201806037

Selective covalent modification of a targeted protein is a powerful tool in chemical biology and drug discovery, with applications ranging from identification and characterization of proteins and their functions to the development of targeted covalent inhibitors. Most covalent ligands contain an "affinity motif" and an electrophilic warhead that reacts with a nucleophilic residue of the targeted protein. Because the electrophilic warhead is prone to react and modify off‐target nucleophiles, its reactivity should be balanced carefully to maximize target selectivity. Arylfluorosulfates have recently emerged as latent electrophiles for selective labeling of context‐specific tyrosine and lysine residues in protein pockets. Here, we review the recent but intense introduction of arylfluorosulfates into the arsenal of available warheads for selective covalent modification of proteins. We highlight the untapped potential of this functional group for use in chemical biology and drug discovery.

The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1

Russell J.Pearson, David G. Blake, Mokdad Mezna, Peter M.Fischer, Nicholas J.Westwood, Campbell McInnes

Cell Chem. Biol. 2018

DOI: 10.1016/j.chembiol.2018.06.001

The polo kinase family are important oncology targets that act in regulating entry into and progression through mitosis. Structure-guided discovery of a new class of inhibitors of Polo-like kinase 1 (PLK1) catalytic activity that interact with Cys67 of the ATP binding site is described. Compounds containing the benzothiazole N-oxide scaffold not only bind covalently to this residue, but are reversible inhibitors through the formation of Meisenheimer complexes. This mechanism of kinase inhibition results in compounds that can target PLK1 with high selectivity, while avoiding issues with irreversible covalent binding and interaction with other thiol-containing molecules in the cell. Due to renewed interest in covalent drugs and the plethora of potential drug targets, these represent prototypes for the design of kinase inhibitory compounds that achieve high specificity through covalent interaction and yet still bind reversibly to the ATP cleft, a strategy that could be applied to avoid issues with conventional covalent binders.

Merits and Pitfalls in the Characterization of Covalent Inhibitors of Bruton’s Tyrosine Kinase

Christopher M. Harris, Sage E. Foley, Eric R. Goedken, Mark Michalak, Sara Murdock, and Noel S. Wilson

SLAS Discovery, 2018, 1–11

DOI: 10.1177/2472555218787445

In vitro analysis of covalent inhibitors requires special consideration, due to the time-dependent and typically irreversible nature of their target interaction. While many analyses are reported for the characterization of a final candidate, it is less clear which are most useful in the lead optimization phase of drug discovery. In the context of identifying covalent inhibitors of Bruton’s tyrosine kinase (BTK), we evaluated multiple techniques for characterizing covalent inhibitors. Several methods qualitatively support the covalent mechanism of action or support a particular aspect of interaction but were not otherwise informative to differentiate inhibitors. These include the time dependence of IC₅₀, substrate competition, mass spectrometry, and recovery of function after inhibitor removal at the biochemical and cellular level. A change in IC₅₀ upon mutation of the targeted BTK C481 nucleophile or upon removal of the electrophilic moiety of the inhibitor was not always a reliable indicator of covalent inhibition. Determination of k_inact and K_I provides a quantitative description of covalent interactions but was challenging at scale and frequently failed to provide more than the ratio of the two values, k_inact/K_I. Overall, a combination of approaches is required to assess time-dependent, covalent, and irreversible inhibitors in a manner suitable to reliably advance drug candidates.

Structure-Based Design, Synthesis, and Characterization of the First Irreversible Inhibitor of Focal Adhesion Kinase

Acebrón-Garcia-de-Eulate, Marta; Tomkiewicz-Raulet, Céline; Dawson, John; Lietha, Daniel; Frame, Margaret C.; Coumoul, Xavier; Garbay, Christiane; Etheve-Quelquejeu, Mélanie Chen, Huixiong

ACS Chem. Biol., Article ASAP

DOI: 10.1021/acschembio.8b00250

Focal Adhesion Kinase signaling pathway and its functions have been involved in the development and aggressiveness of tumor malignancy, it then presents a promising cancer therapeutic target. Several reversible FAK inhibitors have been developed and are being conducted in clinical trials. On the other hand, irreversible covalent inhibitors would bring many desirable pharmacological features including high potency and increased duration of action. Herein we report the structure-guided development of the first highly potent and irreversible inhibitor of the FAK kinase. This inhibitor showed a very potent decrease of autophosphorylation of FAK in squamous cell carcinoma. A cocrystal structure of the FAK kinase domain in complex with this compound revealed the inhibitor binding mode within the ATP binding site and confirmed the covalent linkage between the targeted Cys427 of the protein and the inhibitor.

Oridonin is a covalent NLRP3 inhibitor with strong anti-inflammasome activity

Hongbin He, Hua Jiang, Yun Chen, Jin Ye, Aoli Wang, Chao Wang, Qingsong Liu, Gaolin Liang, Xianming Deng, Wei Jiang & Rongbin Zhou
Nature Communications, 2018, 9, 2550, doi: 10.1038/s41467-018-04947-6

Oridonin (Ori) is the major active ingredient of the traditional Chinese medicinal herb Rabdosia rubescens and has anti-inflammatory activity, but the target of Ori remains unknown. NLRP3 is a central component of NLRP3 inflammasome and has been involved in a wide variety of chronic inflammation-driven human diseases. Here, we show that Ori is a specific and covalent inhibitor for NLRP3 inflammasome. Ori forms a covalent bond with the cysteine 279 of NLRP3 in NACHT domain to block the interaction between NLRP3 and NEK7, thereby inhibiting NLRP3 inflammasome assembly and activation. Importantly, Ori has both preventive or therapeutic effects on mouse models of peritonitis, gouty arthritis and type 2 diabetes, via inhibition of NLRP3 activation. Our results thus identify NLRP3 as the direct target of Ori for mediating Ori’s anti-inflammatory activity. Ori could serve as a lead for developing new therapeutics against NLRP3-driven diseases.

Identification of the histidine residue in vitamin D receptor that covalently binds to electrophilic ligands

Mami Yoshizawa, Toshimasa Itoh, Tatsuya Hori, Akira Kato, Yasuaki Anami, Nobuko Yoshimoto, and Keiko Yamamoto

J. Med. Chem., 2018

DOI: 10.1021/acs.jmedchem.8b00774

We designed and synthesized vitamin D analogues with an electrophile as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin D analogues 1-4 have an electrophilic enone group at the side chain for conjugate addition to His301 or His393 in the VDR. All compounds showed specific VDR-binding potency and agonistic activity. Covalent bond formations of 1-4 with the ligand-binding domain (LBD) of VDR was evaluated by electrospray ionization mass spectrometry. All compounds were shown to covalently bind to the VDR-LBD, and the abundance of VDR-LBD corresponding conjugate adducts of 1-4 increased with incubation time. Enone compounds 1 and 2 showed higher reactivity than the ene-ynone 3 and dienone 4 compounds. Furthermore, we successfully obtained co-crystals of VDR-LBD with analogues 1-4. X-ray crystallographic analysis showed a covalent bond with His301 in VDR-LBD. We successfully synthesized vitamin D analogues that form a covalent bond with VDR-LBD.

Beyond cysteine: recent developments in the area of targeted covalent inhibition

Herschel Mukherjee, Neil P Grimster

Current Opinion in Chemical Biology 2018, 44, 30–38
doi: 10.1016/j.cbpa.2018.05.011

Over the past decade targeted covalent inhibitors have undergone a renaissance due to the clinical validation and regulatory approval of several small molecule therapeutics that are designed to irreversibly modify their target protein. Invariably, these compounds rely on the serendipitous placement of a cysteine residue proximal to the small molecule binding site; while this strategy has afforded numerous successes, it necessarily limits the number of proteins that can be targeted by this approach. This drawback has led several research groups to develop novel methodologies that target non-cysteine residues for covalent modification. Herein, we survey the current literature of warheads that covalently modify non-cysteine amino acids in proteins.

The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition

ACS Chem. Biol., 2018
DOI: 10.1021/acschembio.8b00225

We report the unprecedented reaction between a nitroalkane and an active-site cysteine residue to yield a thiohydroximate adduct. Structural and kinetic evidence suggests the nitro group is activated by conversion to its nitronic acid tautomer within the active site. The nitro group, therefore, shows promise as a masked electrophile in the design of covalent inhibitors targeting binding pockets with appropriately placed cysteine and general acid residues

Selective Irreversible Inhibitors of the Wnt-Deacylating Enzyme NOTUM Developed by Activity-Based Protein Profiling

Radu M. Suciu, Armand B. Cognetta, III, Zachary E. Potter, and Benjamin F. Cravatt

ACS Med. Chem. Lett., 2018
DOI: 10.1021/acsmedchemlett.8b00191

Wnt proteins are secreted morphogens that play critical roles in embryonic development and tissue remodeling in adult organisms. Aberrant Wnt signaling contributes to diseases such as cancer. Wnts are modified by an unusual O-fatty acylation event (O-linked palmitoleoylation of a conserved serine) that is required for binding to Frizzled receptors. O-Palmitoleoylation of Wnts is introduced by the porcupine (PORCN) acyltransferase and removed by the serine hydrolase NOTUM. PORCN inhibitors are under development for oncology, while NOTUM inhibitors have potential for treating degenerative diseases. Here, we describe the use of activity-based protein profiling (ABPP) to discover and advance a class of N-hydroxyhydantoin (NHH) carbamates that potently and selectively inhibit NOTUM. An optimized NHH carbamate inhibitor, ABC99, preserves Wnt-mediated cell signaling in the presence of NOTUM and was also converted into an ABPP probe for visualizing NOTUM in native biological systems.

Organometallic Gold(III) Reagents for Cysteine Arylation

Marco S Messina, Julia M Stauber, Mary A Waddington, Arnold L. Rheingold, Heather D. Maynard, and Alexander M Spokoyny

J. Am. Chem. Soc., 2018

DOI: 10.1021/jacs.8b04115

An efficient method for chemoselective cysteine arylation of unprotected peptides and proteins using Au(III) organometallic complexes is reported. The bioconjugation reactions proceed rapidly (<5 min) at ambient temperature in various buffers and within a wide pH range (0.5-14). This approach provides access to a diverse array of S-aryl bioconjugates including fluorescent dye, complex drug molecule, affinity label, poly(ethylene glycol) tags and a stapled peptide. A library of Au(III) arylation reagents can be prepared as air-stable, crystalline solids in one step from commercial reagents. The selective and efficient arylation procedures presented in this work broaden the synthetic scope of cysteine bioconjugation and serve as promising routes for the modification of complex biomolecules.

Targeted Covalent Inhibition of Prolyl Oligopeptidase (POP): Discovery of Sulfonylfluoride Peptidomimetics

Salvador Guardiola, Roger Prades, Laura Mendieta, Arwin J. Brouwer, Jelle Streefkerk, Laura Nevola, Teresa Tarragó, Rob M.J. Liskamp, Ernest Giralt

Cell Chemical Biology, 2018

doi: 10.1016/j.chembiol.2018.04.013

Prolyl oligopeptidase (POP), a serine protease highly expressed in the brain, has recently emerged as an enticing therapeutic target for the treatment of cognitive and neurodegenerative disorders. However, most reported inhibitors suffer from short duration of action, poor protease selectivity, and low blood-brain barrier (BBB) permeability, which altogether limit their potential as drugs. Here, we describe the structure-based design of the first irreversible, selective, and brain-permeable POP inhibitors. At low-nanomolar concentrations, these covalent peptidomimetics produce a fast, specific, and sustained inactivation of POP, both in vitro and in human cells. More importantly, they are >1,000-fold selective against two family-related proteases (DPPIV and FAP) and display high BBB permeability, as shown in both lipid membranes and MDCK cells.

Rapid labelling and covalent inhibition of intracellular native proteins using ligand-directed N-acyl-N-alkyl sulfonamide

Tomonori Tamura, Tsuyoshi Ueda, Taiki Goto, Taku Tsukidate, Yonatan Shapira, Yuki Nishikawa, Alma Fujisawa & Itaru Hamachi

Nature Communications, 9, Article number: 1870, 2018,
doi:10.1038/s41467-018-04343-0

 Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M−1 s−1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.

Phage Display of Dynamic Covalent Binding Motifs Enables Facile Development of Targeted Antibiotics

Kelly A. McCarthy, Michael A. Kelly, Kaicheng Li, Samantha Cambray, Azade S. Hosseini , Tim van Opijnen, and Jianmin Gao

J. Am. Chem. Soc., 2018, 140 (19), 6137–6145 DOI: 10.1021/jacs.8b02461 

Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of our society and urgently calls for new strategies for infection diagnosis and antibiotic discovery. The antibiotic resistance problem at least partially arises from extensive use of broad-spectrum antibiotics. Ideally, for the treatment of infection, one would like to use a narrow-spectrum antibiotic that specifically targets and kills the disease-causing strain. This is particularly important considering the commensal bacterial species that are beneficial and sometimes even critical to the health of a human being. In this contribution, we describe a phage display platform that enables rapid identification of peptide probes for specific bacterial strains. The phage library described herein incorporates 2-acetylphenylboronic acid moieties to elicit dynamic covalent binding to the bacterial cell surface. Screening of the library against live bacterial cells yields submicromolar and highly specific binders for clinical strains of Staphylococcus aureus and Acinetobacter baumannii that display antibiotic resistance. We further show that the identified peptide probes can be readily converted to bactericidal agents that deliver generic toxins to kill the targeted bacterial strain with high specificity. The phage display platform described here is applicable to a wide array of bacterial strains, paving the way to facile diagnosis and development of strain-specific antibiotics.

Chemo- and Regioselective Lysine Modification on Native Proteins

Maria J. Matos, Bruno L. Oliveira, Nuria Martínez-Saez,  Ana Guerreiro, Pedro M. S. D. Cal,
Jean Bertoldo, María Maneiro, Elizabeth Perkins, Julie Howard, Michael J. Deery,
Justin M. Chalker, Francisco Corzana, Gonzalo Jimenez-Oses, and Goncalo J. L. Bernardes

J. Am. Chem. Soc., 2018, 140 (11), pp 4004–4017

DOI: 10.1021/jacs.7b12874

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.