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Recent Advances in Selective and Irreversible Covalent Ligand Development and Validation

2019; Elsevier BV; Volume: 26; Issue: 11 Linguagem: Inglês

10.1016/j.chembiol.2019.09.012

2451-9456

Tinghu Zhang, John M. Hatcher, Mingxing Teng, Nathanael S. Gray, Milka Kostić,

Computational Drug Discovery Methods

Some of the most widely used drugs, such as aspirin and penicillin, are covalent drugs. Covalent binding can improve potency, selectivity, and duration of the effects, but the intrinsic reactivity represents a potential liability and may result in idiosyncratic toxicity. For decades, the cons were believed to outweigh the pros, and covalent targeting was deprioritized in drug discovery. Recently, several covalent inhibitors have been approved for cancer treatment, thus rebooting the field. In this review, we briefly reflect on the history of selective covalent targeting, and provide a comprehensive overview of emerging developments from a chemical biology stand-point. Our discussion will reflect on efforts to validate irreversible covalent ligands, expand the scope of targets, and discover new ligands and warheads. We conclude with a brief commentary of remaining limitations and emerging opportunities in selective covalent targeting. Some of the most widely used drugs, such as aspirin and penicillin, are covalent drugs. Covalent binding can improve potency, selectivity, and duration of the effects, but the intrinsic reactivity represents a potential liability and may result in idiosyncratic toxicity. For decades, the cons were believed to outweigh the pros, and covalent targeting was deprioritized in drug discovery. Recently, several covalent inhibitors have been approved for cancer treatment, thus rebooting the field. In this review, we briefly reflect on the history of selective covalent targeting, and provide a comprehensive overview of emerging developments from a chemical biology stand-point. Our discussion will reflect on efforts to validate irreversible covalent ligands, expand the scope of targets, and discover new ligands and warheads. We conclude with a brief commentary of remaining limitations and emerging opportunities in selective covalent targeting. Deep down, under all those western blots and microscopy images, many chemical biologists are lovers and practitioners of chemistry, a scientific discipline that is centrally interested in reactivity. Thus, many in the field have been exploiting chemical reactivity between small molecules and biomolecules to create tools for biological research and agents for disease treatment. This second area of interest has, in part, been inspired by examples of approved drugs that, although not developed as covalent, have since been shown to exert their therapeutic effects by covalently binding their targets. Most notable examples of these are aspirin and penicillin, which target cyclooxygenases and bacterial DD-transpeptidase, respectively (Singh et al., 2011Singh J. Petter R.C. Baillie T.A. Whitty A. The resurgence of covalent drugs.Nat. Rev. Drug Discov. 2011; 10: 307-317Crossref PubMed Scopus (1189) Google Scholar). More recently, a range of rationally designed covalent inhibitors has received US Food and Drug Administration (FDA) approval, causing a resurgence of interest in this field (Byrd et al., 2016Byrd J.C. Harrington B. O’Brien S. Jones J.A. Schuh A. Devereux S. Chaves J. Wierda W.G. Awan F.T. Brown J.R. et al.Acalabrutinib (ACP-196) in relapsed chronic lymphocytic leukemia.N. Engl. J. Med. 2016; 374: 323-332Crossref PubMed Scopus (671) Google Scholar) (Kisselev et al., 2012Kisselev A.F. van der Linden W.A. Overkleeft H.S. Proteasome inhibitors: an expanding army attacking a unique target.Chem. Biol. 2012; 19: 99-115Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, Kwong et al., 2011Kwong A.D. Kauffman R.S. Hurter P. Mueller P. Discovery and development of telaprevir: an NS3-4A protease inhibitor for treating genotype 1 chronic hepatitis C virus.Nat. Biotechnol. 2011; 29: 993-1003Crossref PubMed Scopus (218) Google Scholar, Rotella, 2013Rotella D.P. The discovery and development of boceprevir.Expert Opin. Drug Discov. 2013; 8: 1439-1447Crossref PubMed Scopus (18) Google Scholar, Li et al., 2008Li D. Ambrogio L. Shimamura T. Kubo S. Takahashi M. Chirieac L.R. Padera R.F. Shapiro G.I. Baum A. Himmelsbach F. et al.BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models.Oncogene. 2008; 27: 4702-4711Crossref PubMed Scopus (1162) Google Scholar, Yver, 2016Yver A. Osimertinib (AZD9291)—a science-driven, collaborative approach to rapid drug design and development.Ann. Oncol. 2016; 27: 1165-1170Crossref PubMed Scopus (62) Google Scholar). The idea that selective covalent inhibitors could be valuable is not a new one. As a review from the 1960s illustrates, reactions between nucleophilic side chains of proteinogenic amino acids and electrophilic warheads of small-molecule inhibitors were already considered decades ago (Baker, 1964Baker B.R. Factors in the design of active-site directed irreversible inhibitors.J. Pharm. Sci. 1964; 53: 347-364Abstract Full Text PDF PubMed Scopus (25) Google Scholar). The advantages of irreversible inhibition that this review noted remain relevant today and include: (1) improved effectiveness of irreversible versus reversible compounds; and (2) the potential for higher specificity over reversible compounds given that irreversible ligands form a covalent bond with a relatively unique nucleophile on the target. On the other hand, the noted challenges we still consider relevant are: (1) achieving target selectivity given the use of reactive warheads; (2) ensuring that reactivity of the irreversible inhibitors does not interfere with tissue distribution and/or intracellular delivery; and (3) community skepticism surrounding the idea of selective covalent targeting. The recent drug approvals may have minimized some of the community skepticism; however, further efforts are needed to address issues surrounding development of selective covalent ligands. Here, we will discuss the importance of validation, and comment on the standards that need to be satisfied before using these compounds as chemical probes. We will then comment on emerging opportunities in selective irreversible covalent targeting and conclude by reflecting on some of the limitations and current challenges. An important aspect of this topic that will not be covered here is the target selection process and how to optimize it to achieve maximum potency and selectivity by taking into account both the nature of the available reactive sites and the target half-life. We feel that this issue deserves to be covered separately and hope to see it written about in the near future. We would also like to note that many excellent reviews on different aspects of covalent targeting have recently been published (Jackson et al., 2017Jackson P.A. Widen J.C. Harki D.A. Brummond K.M. Covalent modifiers: a chemical perspective on the reactivity of α,β-unsaturated carbonyls with thiols via hetero-Michael addition reactions.J. Med. Chem. 2017; 60: 839-885Crossref PubMed Scopus (292) Google Scholar, Bandyopadhyay and Gao, 2016Bandyopadhyay A. Gao J. Targeting biomolecules with reversible covalent chemistry.Curr. Opin. Chem. Biol. 2016; 34: 110-116Crossref PubMed Scopus (71) Google Scholar, De Cesco et al., 2017De Cesco S. Kurian J. Dufresne C. Mittermaier A.K. Moitessier N. Covalent inhibitors design and discovery.Eur. J. Med. Chem. 2017; 138: 96-114Crossref PubMed Scopus (167) Google Scholar, Lagoutte et al., 2017Lagoutte R. Patouret R. Winssinger N. Covalent inhibitors: an opportunity for rational target selectivity.Curr. Opin. Chem. Biol. 2017; 39: 54-63Crossref PubMed Scopus (78) Google Scholar, Mukherjee and Grimster, 2018Mukherjee H. Grimster N.P. Beyond cysteine: recent developments in the area of targeted covalent inhibition.Curr. Opin. Chem. Biol. 2018; 44: 30-38Crossref PubMed Scopus (39) Google Scholar, Shannon and Weerapana, 2015Shannon D.A. Weerapana E. Covalent protein modification: the current landscape of residue-specific electrophiles.Curr. Opin. Chem. Biol. 2015; 24: 18-26Crossref PubMed Scopus (154) Google Scholar, Pettinger et al., 2017Pettinger J. Jones K. Cheeseman M.D. Lysine-targeting covalent inhibitors.Angew. Chem. Int. Ed. 2017; 56: 15200-15209Crossref PubMed Scopus (100) Google Scholar, Lonsdale and Ward, 2018Lonsdale R. Ward R.A. Structure-based design of targeted covalent inhibitors.Chem. Soc. Rev. 2018; 47: 3816-3830Crossref PubMed Google Scholar, Chaikuad et al., 2018Chaikuad A. Koch P. Laufer S.A. Knapp S. The cysteinome of protein kinases as a target in drug development.Angew. Chem. Int. Ed. 2018; 57: 4372-4385Crossref PubMed Scopus (126) Google Scholar, Hallenbeck et al., 2017Hallenbeck K.K. Turner D.M. Renslo A.R. Arkin M.R. Targeting non-catalytic cysteine residues through structure-guided drug discovery.Curr. Top. Med. Chem. 2017; 17: 4-15Crossref PubMed Scopus (46) Google Scholar, Zhao and Bourne, 2018Zhao Z. Bourne P.E. Progress with covalent small-molecule kinase inhibitors.Drug Discov. Today. 2018; 23: 727-735Crossref PubMed Scopus (120) Google Scholar, Cuesta and Taunton, 2019Cuesta A. Taunton J. Lysine-targeted inhibitors and chemoproteomic probes.Annu. Rev. Biochem. 2019; 88: 365-381Crossref PubMed Scopus (45) Google Scholar). Our main goal here is to provide a chemical biology perspective on this topic, as a complementary viewpoint to primarily drug development and medicinal chemistry discussions present in the current literature. Over the last decade, the chemical biology community has developed a set of guidelines for chemical probes, also known as tool compounds (Arrowsmith et al., 2015Arrowsmith C.H. Audia J.E. Austin C. Baell J. Bennett J. Blagg J. Bountra C. Brennan P.E. Brown P.J. Bunnage M.E. et al.The promise and peril of chemical probes.Nat. Chem. Biol. 2015; 11: 536-541Crossref PubMed Scopus (541) Google Scholar) (here, we will use the term tool compounds to avoid confusion with agents referred to as “covalent probes,” which are used for activity-based protein profiling [ABPP]). Although these guidelines have been defined for noncovalent ligands, they do provide a basic framework that can be applied to covalent tool compounds. In this section, we will comment on how to expand the existing guidelines, and discuss strategies to characterize and validate selective covalent ligands. Key questions that a validation process for a tool compound has to address are: (1) How potent is the compound in biochemical and cellular assays? (2) What is the mode of binding? (3) Are suitable negative control compounds available and is the compound chemically stable under given conditions? (4) Is the probe engaging the correct target in cells and are observed biological effects due to the on-target engagement? (5) How selective is the compound? and (6) For tool compounds to be used in living organisms, what are the pharmacokinetic/pharmacodynamic (PK/PD) properties (Müller et al., 2018Müller S. Ackloo S. Arrowsmith C.H. Bauser M. Baryza J.L. Blagg J. Böttcher J. Bountra C. Brown P.J. Bunnage M.E. et al.Donated chemical probes for open science.Elife. 2018; 7https://doi.org/10.7554/eLife.34311Crossref Scopus (9) Google Scholar)? In addition, three central questions for validating irreversible covalent ligands are: (1) Is the ligand engaging covalently and irreversibly with the target? (2) Are any biological effects due to noncovalent interactions with the target and/or off-targets? (3) Are any biological effects of an irreversible covalent ligand due to off-target reactivity? The validation process we developed to help answer these questions is shown in Figure 1, and we will use an example from our laboratory to illustrate the type of experiments that we commonly employ. Most efforts to develop selective irreversible covalent ligands have been designed to take advantage of the intrinsic nucleophilic nature of proteinogenic amino acid side chains, most notably the thiol group (-SH) of cysteines (Cys). Along these lines, our illustrative example THZ531 was developed as an irreversible covalent inhibitor for cyclin-dependent kinases 12 and 13 (CDK12/13) (Figure 2A) (Zhang et al., 2016Zhang T. Kwiatkowski N. Olson C.M. Dixon-Clarke S.E. Abraham B.J. Greifenberg A.K. Ficarro S.B. Elkins J.M. Liang Y. Hannett N.M. et al.Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.Nat. Chem. Biol. 2016; 12: 876-884Crossref PubMed Scopus (186) Google Scholar). In addition to THZ531, we also synthesized THZ531R, a compound in which α,β-unsaturated carbonyl was reduced thus eliminating the Cys-reactive Michael acceptor, and THZ532, an inactive enantiomer (Figure 2A). We used both THZ531R and THZ532 as negative controls throughout our validation process, thus fulfilling recommended step 1 in our covalent inhibitor validation process (Figure 1). In general, covalent inhibitors display concentration-dependent and incubation time-dependent activity in in vitro enzymatic assays (Strelow, 2017Strelow J.M. A perspective on the kinetics of covalent and irreversible inhibition.SLAS Discov. 2017; 22: 3-20Crossref PubMed Scopus (146) Google Scholar). Therefore, one of the steps in our validation process is measuring loss of activity as a function of preincubation times. In this experiment, we varied the length of incubation time with THZ531, and quantified kinase activity using a radiometric assay that measures the ability of recombinant CDK12 to phosphorylate a Pol II CTD-peptide substrate in the presence of its cofactor, cyclin K, normalized to the relative [32P] transfer under DMSO control (Figure 1, recommended step 2; Figure 2B). Although incubation time dependence of activity can be due to factors other than covalent target binding, we use these results as indicators of covalent inhibition. In addition, it is recommended that potency of irreversible inhibitors is expressed as kinact/KI, where kinact is the maximal rate of inactivation and KI is the reversible binding constant (Strelow, 2017Strelow J.M. A perspective on the kinetics of covalent and irreversible inhibition.SLAS Discov. 2017; 22: 3-20Crossref PubMed Scopus (146) Google Scholar). Contributions of affinity and reactivity to the overall potency need to be considered separately, and an in vitro method for characterizing these two components has been described (Schwartz et al., 2014Schwartz P.A. Kuzmic P. Solowiej J. Bergqvist S. Bolanos B. Almaden C. Nagata A. Ryan K. Feng J. Dalvie D. et al.Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance.Proc. Natl. Acad. Sci. U S A. 2014; 111: 173-178Crossref PubMed Scopus (180) Google Scholar). However, it is worth pointing out that in vitro characterization may not translate to in vivo conditions. Washout experiments, in which cells are first exposed to the inhibitor, then washed out and allowed to grow in inhibitor-free media, are also an important step in the validation process. Here, the growth rates with washout are compared with “no washout” conditions. The sustained effect of covalent inhibitors subjected to the washout experiments is attributed to the irreversible nature of their target engagement. For THZ531 validation we used Jurkat T cell acute lymphoblastic leukemia cells and demonstrated that THZ531 maintained the effects 72 h post-washout, whereas negative controls, including the reversible compound THZ531R, had no effect (Figure 1, recommended step 3; Figure 2C). Moreover, we also used a biotinylated analog of THZ531 (bioTHZ531) to treat Jurkat cell lysates and identify cellular targets that affinity purify with bioTHZ531 after extensive washouts to remove nonspecific and noncovalent targets. These pull-down experiments provided evidence that CDK12-cyclin K and CDK13-cyclin K are the main covalent targets. A potential caveat when using tagged analogs, such as bioTHZ531, is that the tag itself may introduce nonspecific interactions, and these effects need to be taken into account. Overall, experiments such as measuring incubation time dependence of activity, loss of activity upon removal of the reactive warhead, and cellular washout experiments provide multiple lines of supporting evidence of irreversible mechanism of action. More direct methods for confirming and visualizing covalent binding in vitro are mass spectrometry (MS) and X-ray crystallography. We employed both of these strategies to validate that THZ531 covalently binds CDK12 and CDK13 in vitro. MS experiments showed the formation of the covalent adduct (+558 Da corresponding to the addition of THZ531; Figure 1, recommended step 4; Figure 2D), and, upon proteolysis, identified a peptide fragment containing the exact site (Cys1039 on CDK12) of modification. A 2.7-Å crystal structure of CDK12-cyclin K bound to THZ531 confirmed these findings (Figure 1, recommended step 5). To further establish activity and selectivity in vivo, an essential step in validating covalent ligands are cell-based experiments that use resistance mutations. For example, cysteines are commonly mutated to a serine or an alanine, and the presumed target protein harboring the point mutation is introduced to cells either exogenously or using CRISPR/Cas9 knock-in technology. Our cellular work using a Cys1039Ser CDK12 mutant created using CRISPR/Cas9 demonstrated that this mutation was sufficient to make CDK12 refractory to covalent affinity pull-down and led to partial restoration of cellular proliferation and reduced apoptosis (Figure 1, recommended step 6; Figure 2F). Overall, cell-based experiments using resistance mutations provide essential evidence that a given phenotype induced by a covalent inhibitor is on-target and on-mechanism, something that neither biochemical experiments described above nor chemoproteomics experiments we describe below can address. There are several methods that can be used to map selectivity of covalent ligands. For example, THZ531 was profiled using kinase panel assays Ambit for in vitro characterization and KiNativ for cell-based profiling (Du et al., 2009Du J. Bernasconi P. Clauser K.R. Mani D.R. Finn S.P. Beroukhim R. Burns M. Julian B. Peng X.P. Hieronymus H. et al.Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy.Nat. Biotechnol. 2009; 27: 77-83Crossref PubMed Scopus (198) Google Scholar). Both sets of data suggested that CDK12 and 13 are the primary, but not the only, targets of THZ531. The major limitation of a profiling strategies like these is that they include a limited number of targets in the profiling panel. To address this issue, a range of chemoproteomic approaches has been developed to allow interrogations of a broader target space. For example, ABPP in combination with MS-based proteomics, such as stable isotope labeling with amino acids in cell culture or tandem mass tagging (TMT) click chemistry pull-down experiments, can be used to map selectivity (Drewes and Knapp, 2018Drewes G. Knapp S. Chemoproteomics and chemical probes for target discovery.Trends Biotechnol. 2018; 36: 1275-1286Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We recently contributed to the development of a chemoproteomic method called CITe-Id (for covalent inhibitor target-site identification) (Browne et al., 2019Browne C.M. Jiang B. Ficarro S.B. Doctor Z.M. Johnson J.L. Card J.D. Sivakumaren S.C. Alexander W.M. Yaron T.M. Murphy C.J. et al.A chemoproteomic strategy for direct and proteome-wide covalent inhibitor target-site identification.J. Am. Chem. Soc. 2019; 141: 191-203Crossref PubMed Scopus (45) Google Scholar), which is able to capture, identify, and quantify dose-dependent covalently bound Cys sites in cell lysates. Using CITe-Id we were able to demonstrate that a covalent kinase inhibitor THZ1, originally developed to target CDK7, binds covalently to additional proteins, including non-kinase targets. Although useful, chemoproteomic strategies have a number of limitations, including incomplete coverage of the proteome, and can lead to both false positives, by identifying proteins as targets when they are not, and false negatives, by not detecting the binding event. Therefore, as mentioned above, any comprehensive validation process must include cell-based target confirmation experiments, such as the use of knockdowns, target overexpression, and evolution of resistant mutants. The range of proteins targeted through a Cys residue now extends into the area of “undruggable” targets such as KRASG12C, a common oncogenic mutant of the small GTPase KRAS (Patricelli et al., 2016Patricelli M.P. Janes M.R. Li L.-S. Hansen R. Peters U. Kessler L.V. Chen Y. Kucharski J.M. Feng J. Ely T. et al.Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state.Cancer Discov. 2016; 6: 316-329Crossref PubMed Scopus (446) Google Scholar, Janes et al., 2018Janes M.R. Zhang J. Li L.-S. Hansen R. Peters U. Guo X. Chen Y. Babbar A. Firdaus S.J. Darjania L. et al.Targeting KRAS mutant cancers with a covalent G12C-specific inhibitor.Cell. 2018; 172: 578-589.e17Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). These two reports describe development and validation of covalent agents ARS-853 and ARS-1620, respectively, both inspired by previous work that demonstrated that covalent inhibitors targeting Cys12 in KRASG12C were feasible (Ostrem et al., 2013Ostrem J.M. Peters U. Sos M.L. Wells J.A. Shokat K.M. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions.Nature. 2013; 503: 548-551Crossref PubMed Scopus (1272) Google Scholar). Whereas ARS-853 was shown to be a cell-based KRASG12C inhibitor, ARS-1620 is active in vivo. Both compounds exhibit a strict requirement for Cys12 for their activity, because wild-type (WT) and mutant cell lines where residue 12 is not Cys (such as KRASG12S, KRASG12V, or KRASG12D) were insensitive. In addition, both compounds inhibit the guanosine diphosphate-bound form of KRASG12C and display a narrow selectivity window, with ARS-853 binding RTN4 and FAM213A in addition to KRASG12C, and ARS-1620 additionally hitting FAM213A and AHR. Selectivity profiles were measured by cysteine reactivity profiling, a chemoproteomic competition-based method, which profiled 2,740 surface-exposed Cys belonging to 1,584 annotated proteins for ARS-853, and 8,501 Cys residues belonging to 3,012 annotated proteins for ARS-1620. Finally, ARS-1620 analysis included the use of an inactive control, an atropisomer. Together, these studies produced quality tool compounds and demonstrated the value of covalent targeting as a way to discriminate between WT and disease-associated mutant proteins. Targeting serines (Ser) and threonines (Thr) has also been successfully exploited for tool compound and drug development. Although hydroxy group (-OH) of serines and threonines are relatively inert under physiological conditions, activated Ser and Thr residues are found in active sites of many enzymes. In fact, ABPP was originally developed for profiling serine hydrolases, a large family of diverse enzymes that use catalytic (activated) Ser to hydrolyze an amide, an ester, or a thioester bond (Bachovchin and Cravatt, 2012Bachovchin D.A. Cravatt B.F. The pharmacological landscape and therapeutic potential of serine hydrolases.Nat. Rev. Drug Discov. 2012; 11: 52-68Crossref PubMed Scopus (205) Google Scholar). In addition, a number of approved covalent drugs target Ser and Thr residues, including: aspirin (cyclooxygenase inhibitor), penicillin (bacterial DD-transpeptidase inhibitor), telaprevir and boceprevir (hepatitis C virus protease inhibitors), avibactam (β-lactamase inhibitor), carfilzomib, bortezomib, and ixazomib (proteasome inhibitors), rivastigmine (acetylcholinesterase inhibitor), and saxagliptin (dipeptidyl peptidase-4 inhibitor). Selective covalent targeting of activated Ser and Thr residues will not be further discussed here as this topic was covered in more detail in recent reviews (Mukherjee and Grimster, 2018Mukherjee H. Grimster N.P. Beyond cysteine: recent developments in the area of targeted covalent inhibition.Curr. Opin. Chem. Biol. 2018; 44: 30-38Crossref PubMed Scopus (39) Google Scholar, Shannon and Weerapana, 2015Shannon D.A. Weerapana E. Covalent protein modification: the current landscape of residue-specific electrophiles.Curr. Opin. Chem. Biol. 2015; 24: 18-26Crossref PubMed Scopus (154) Google Scholar). In the following sections we discuss emerging strategies for targeting lysines, tyrosines, histidines, and methionines, and comment on opportunities and limitations. This topic of targeting sites beyond cysteines has been a subject of several recent reviews (Mukherjee and Grimster, 2018Mukherjee H. Grimster N.P. Beyond cysteine: recent developments in the area of targeted covalent inhibition.Curr. Opin. Chem. Biol. 2018; 44: 30-38Crossref PubMed Scopus (39) Google Scholar, Pettinger et al., 2017Pettinger J. Jones K. Cheeseman M.D. Lysine-targeting covalent inhibitors.Angew. Chem. Int. Ed. 2017; 56: 15200-15209Crossref PubMed Scopus (100) Google Scholar, Jones, 2018Jones L.H. Reactive chemical probes: beyond the kinase cysteinome.Angew. Chem. Int. Ed. 2018; 57: 9220-9223Crossref PubMed Scopus (24) Google Scholar). We refer those interested in covalent lysine targeting, which includes discussion of aldehyde-containing warheads and reversible covalent inhibition, to a very recent review by Cuesta and Taunton, 2019Cuesta A. Taunton J. Lysine-targeted inhibitors and chemoproteomic probes.Annu. Rev. Biochem. 2019; 88: 365-381Crossref PubMed Scopus (45) Google Scholar, as those topics will not be discussed here. Lysine (Lys) side chains are sites of numerous post-translational modifications, such as methylation, ubiquitination, SUMOylation, and a range of acylations, with acetylation being the most well established (Jones, 2018Jones L.H. Reactive chemical probes: beyond the kinase cysteinome.Angew. Chem. Int. Ed. 2018; 57: 9220-9223Crossref PubMed Scopus (24) Google Scholar). However, targeting Lys ϵ-amino group with irreversible electrophilic ligands has been challenging due to its high pKa (∼10) resulting in full protonation under physiological pH (7.4). In addition, most lysine-targeting agents display low compatibility with in vivo applications, therefore limiting their use as pharmacological tools. Recent chemoproteomic profiling of lysines reactivity suggests that human proteome may contain a number of lysine residues that could be targeted using covalent strategy (Hacker et al., 2017Hacker S.M. Backus K.M. Lazear M.R. Forli S. Correia B.E. Cravatt B.F. Global profiling of lysine reactivity and ligandability in the human proteome.Nat. Chem. 2017; 9: 1181-1190Crossref PubMed Scopus (220) Google Scholar). Importantly, many of the lysine sites documented in that study were found in proteins for which no small-molecule ligands are currently available, therefore suggesting a potential opportunity to expand the ligandable proteome. Below we describe several recently described Lys-directed covalent ligands, focusing on literature examples that include validation processes that are similar to the one we propose in Figure 1. In an example from the kinase field, Anscombe et al., 2015Anscombe E. Meschini E. Mora-Vidal R. Martin M.P. Staunton D. Geitmann M. Danielson U.H. Stanley W.A. Wang L.Z. Reuillon T. et al.Identification and characterization of an irreversible inhibitor of CDK2.Chem. Biol. 2015; 22: 1159-1164Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar used vinyl sulfone as a warhead and coupled it to a reversible CDK2 inhibitor with a purine scaffold, to achieve covalent inhibition of CDK2, a cyclin-dependent kinase that belongs to a large family of closely related kinases. The covalent inhibitor, NU6300 (Figure 3A), was found to bind Lys89, one of the two lysine residues located in a solvent-exposed region in the vicinity of the ATP binding pocket, as confirmed by mutagenesis combined with MS-based analysis, and high-resolution crystal structure. In addition to preparing NU6300, the authors also synthesized NU6310, where the vinyl group of NU6300 was replaced by an ethyl group thus resulting in a noncovalent ATP-competitive inhibitor. To distinguish between covalent and noncovalent inhibition, the authors incubated CDK2/cyclin A (cyclin A is a cofactor required for CDK2 activity) overnight with either NU6300 or NU6310, then dialyzed to remove unbound inhibitor, and checked for activity using a peptide derived from a known cellular substrate of CDK2, retinoblastoma protein RB. Whereas NU6310 was fully washed out by this treatment, NU6300 remained bound and therefore inhibited CDK2 activity after dialysis. Furthermore, in a complementary in vitro experiment NU6300 activity varied with the preincubation time, as expected from a covalent inhibitor. In cells, treatment with NU6300 inhibited phosphorylation of RB protein and maintained the inhibition after a washout experiment, whereas another noncovalent inhibitor NU6302 showed diminished activity upon washout, strongly supporting the irreversible covalent mode of inhibition. In terms of selectivity, NU6300 was accessed across a limited kinase panel (131 kinases out of about 518 human kinases [Manning et al., 2002Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6217) Google Scholar]) under conditions that do not distinguish noncovalent from covalent inhibition, and subsequently tested under preincubation conditions. The limited selectivity profiling suggested that Aurora A, STK3/MST2, and MAP4K3 may represent potential off-targets. However, since no broader selectivity profiling was conducted, additional off-targets may exist. Finally, cell-based validation did not include experiments to demonstrate target engagement and on-target mechanism, which are caveats that follow-up work will need to address. A more recent example in this