Sunday, August 7, 2022

Thiol Reactivity of N-Aryl α-Methylene-γ-lactams: Influence of the Guaianolide Structure [@KayBrummond]

 Daniel P. Dempe, Chong-Lei Ji, Peng Liu, and Kay M. Brummond

The Journal of Organic Chemistry, 2020

DOI: 10.1021/acs.joc.2c01530

The α-methylene-γ-lactam offers promise as a complementary warhead for the development of targeted covalent inhibitors. However, an understanding of the factors governing its electrophilic reactivity is needed to promote the development of lead compounds utilizing this motif. Herein we synthesize a series of N-aryl-substituted α-methylene-γ-lactams installed within the framework of a bioactive guaianolide analog. To determine the effects of the guaianolide structure on the electrophilic reactivity, these compounds were reacted with glutathione under biomimetic conditions, and the rate constants were measured. A linear free-energy relationship was observed with the Hammett parameter of the N-aryl group within the cis- or trans-annulated isomeric series of compounds. However, the trans-annulated compounds exhibited a ca. 10-fold increase in reactivity relative to both the cis-annulated compounds and the corresponding N-arylated 3-methylene-2-pyrrolidinones. Density functional theory calculations revealed that the reactivity of the trans-annulated stereoisomers is promoted by the partial release of the ring strain of the fused seven-membered ring in the thio-Michael addition transition state.

Thursday, July 21, 2022

Chemical acylation of an acquired serine suppresses oncogenic signaling of K-Ras(G12S) [@kevansf]

Zhang, Z., Guiley, K.Z. & Shokat, K.M. 

Nat Chem Biol, 2022

Drugs that directly impede the function of driver oncogenes offer exceptional efficacy and a therapeutic window. The recently approved mutant selective small-molecule cysteine-reactive covalent inhibitor of the G12C mutant of K-Ras, sotorasib, provides a case in point. KRAS is the most frequently mutated proto-oncogene in human cancer, yet despite success targeting the G12C allele, targeted therapy for other hotspot mutants of KRAS has not been described. Here we report the discovery of small molecules that covalently target a G12S somatic mutation in K-Ras and suppress its oncogenic signaling. We show that these molecules are active in cells expressing K-Ras(G12S) but spare the wild-type protein. Our results provide a path to targeting a second somatic mutation in the oncogene KRAS by overcoming the weak nucleophilicity of an acquired serine residue. The chemistry we describe may serve as a basis for the selective targeting of other unactivated serines.

Monday, July 11, 2022

Covalent Disruptor of YAP-TEAD Association Suppresses Defective Hippo Signaling

Mengyang Fan, Wenchao Lu, Jianwei Che, Nicholas Kwiatkowski, Yang Gao, Hyuk-Soo Seo, Scott B. Ficarro, Prafulla C. Gokhale, Yao Liu, Ezekiel A. Geffken, Jimit Lakhani, Kijun Song, Miljan Kuljanin, Wenzhi Ji, Jie Jiang, Zhixiang He, Jason Tse, Andrew S. Boghossian, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Joseph D. Mancias, Jarrod A. Marto, Sirano Dhe-Paganon, Tinghu Zhang, Nathanael S. Gray

bioRxiv 2022.05.10.491316


The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.

Wednesday, June 29, 2022

Oncogenic KRAS G12C: Kinetic and Redox Characterization of Covalent Inhibition

Minh V. Huynh, Derek Parsonage, Tom E. Forshaw, Venkat R. Chirasani, G. Aaron Hobbs, Hanzhi Wu, Jingyun Lee, Cristina M. Furdui, Leslie B. Poole, Sharon L. Campbell

Journal of Biological Chemistry, 2022

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 μM), and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, as well as identifying the occurrence of G12C oxidation with currently unknown biological ramifications.

Sunday, June 26, 2022

Discovery and Characterization of a Novel Series of Chloropyrimidines as Covalent Inhibitors of the Kinase MSK1

Adrian Hall, Jan Abendroth, Madison J. Bolejack, Tom Ceska, Sylvie Dell’Aiera, Victoria Ellis, David Fox, Cyril François, Muigai M. Muruthi, Camille Prével, Karine Poullennec, Sergei Romanov, Anne Valade, Alain Vanbellinghen, Jason Yano, and Martine Geraerts

ACS Medicinal Chemistry Letters 2022

DOI: 10.1021/acsmedchemlett.2c00134

We describe the identification and characterization of a series of covalent inhibitors of the C-terminal kinase domain (CTKD) of MSK1. The initial hit was identified via a high-throughput screening and represents a rare example of a covalent inhibitor which acts via an SNAr reaction of a 2,5-dichloropyrimidine with a cysteine residue (Cys440). The covalent mechanism of action was supported by in vitro biochemical experiments and was confirmed by mass spectrometry. Ultimately, the displacement of the 2-chloro moiety was confirmed by crystallization of an inhibitor with the CTKD. We also disclose the crystal structures of three compounds from this series bound to the CTKD of MSK1, in addition to the crystal structures of two unrelated RSK2 covalent inhibitors bound to the CTKD of MSK1.

Sunday, June 19, 2022

Nucleophilic covalent ligand discovery for the cysteine redoxome

Fu, L.; Jung, Y.; Tian, C.; Ferreira, R.; He, F.; Yang, J.; Carroll, K. ChemRxiv 2022.

The convergence of reactive cysteine-targeted electrophilic fragments and chemoproteomics have dramatically accelerated the discovery of ligandable sites in the proteome. Our genome encodes 214,000 cysteine residues, at least 20% of which are estimated to be redox-active. Oxidation blunts sulfur reactivity toward electrophiles but opens the door to a new class of nucleophilic covalent ligands that target cysteinyl sulfenic acids, which are widespread post-translational modifications. Here we report a quantitative analysis of nucleophilic fragments screened against the human sulfenome. Ligands were discovered for >500 sulfenated cysteines in >400 proteins, including sites not targeted by electrophiles with the same scaffold. Among these were compounds that preferentially react with hepatoma-derived growth factor (HDGF)-related proteins (HRPs) one of which was able to block nuclear transport of this oncoprotein. Nucleophilic fragments provide a rich resource for chemical biology and drug discovery, where ligandability in the human proteome extends beyond protein thiols.

Sunday, June 5, 2022

Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents

 Alena Istrate, Michael B. Geeson, Claudio D. Navo, Barbara B. Sousa, Marta C. Marques, Ross J. Taylor, Toby Journeaux, Sebastian R. Oehler, Michael R. Mortensen, Michael J. Deery, Andrew D. Bond, Francisco Corzana, Gonzalo Jiménez-Osés, and Gonçalo J. L. Bernardes

Journal of the American Chemical Society 2022
DOI: 10.1021/jacs.2c02185

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody–drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the β-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.

Friday, June 3, 2022

A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader

N. Connor Payne, Semer Maksoud, Bakhos A. Tannous, Ralph Mazitschek

Cell Chemical Biology, 2022


We describe a generalizable time-resolved Förster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras [PROTACs]) that is capable of both accurately quantifying protein levels in whole-cell lysates in less than 1 h and measuring small-molecule target engagement to endogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small-molecule inhibitors and PROTACs in a high-throughput format with unmodified cell lines. We furthermore validate our platform in the characterization of celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation.

Tuesday, May 31, 2022

Covalent labeling of a chromatin reader domain using proximity-reactive cyclic peptides

Meng Yao Zhang,   Hyunjun Yang, Gloria Ortiz,   Michael J. Trnka,   Nektaria Petronikolou,   Alma L. Burlingame,  William F. DeGrado  and  Danica Galonić Fujimori

Chemical Science, 2022

Chemical probes for chromatin reader proteins are valuable tools for investigating epigenetic regulatory mechanisms and evaluating whether the target of interest holds therapeutic potential. Developing potent inhibitors for the plant homeodomain (PHD) family of methylation readers remains a difficult task due to the charged, shallow and extended nature of the histone binding site that precludes effective engagement of conventional small molecules. Herein, we describe the development of novel proximity-reactive cyclopeptide inhibitors for PHD3—a trimethyllysine reader domain of histone demethylase KDM5A. Guided by the PHD3–histone co-crystal structure, we designed a sidechain-to-sidechain linking strategy to improve peptide proteolytic stability whilst maintaining binding affinity. We have developed an operationally simple solid-phase macrocyclization pathway, capitalizing on the inherent reactivity of the dimethyllysine ε-amino group to generate scaffolds bearing charged tetraalkylammonium functionalities that effectively engage the shallow aromatic ‘groove’ of PHD3. Leveraging a surface-exposed lysine residue on PHD3 adjacent to the ligand binding site, cyclic peptides were rendered covalent through installation of an arylsulfonyl fluoride warhead. The resulting lysine-reactive cyclic peptides demonstrated rapid and efficient labeling of the PHD3 domain in HEK293T lysates, showcasing the feasibility of employing proximity-induced reactivity for covalent labeling of this challenging family of reader domains.

Thursday, May 19, 2022

Reversible lysine-targeted probes reveal residence time-based kinase selectivity

Tangpo Yang, Adolfo Cuesta, Xiaobo Wan, Gregory B. Craven, Brad Hirakawa, Penney Khamphavong, Jeffrey R. May, John C. Kath, John D. Lapek Jr., Sherry Niessen, Alma L. Burlingame, Jordan D. Carelli & Jack Taunton 

Nat Chem Biol (2022).

The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe–kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.

Improved Electrophile Design for Exquisite Covalent Molecule Selectivity

José L. Montaño, Brian J. Wang, Regan F. Volk, Sara E. Warrington, Virginia G. Garda, Katherine L. Hofmann, Leo C. Chen, and Balyn W. Zaro

ACS Chemical Biology 2022

DOI: 10.1021/acschembio.1c00980

Covalent inhibitors are viable therapeutics. However, off-target reactivity challenges the field. Chemists have attempted to solve this issue by varying the reactivity attributes of electrophilic warheads. Here, we report the development of an approach to increase the selectivity of covalent molecules that is independent of warhead reactivity features and can be used in concert with existing methods. Using the scaffold of the Bruton’s tyrosine kinase (BTK) inhibitor Ibrutinib for our proof-of-concept, we reasoned that increasing the steric bulk of fumarate-based electrophiles on Ibrutinib should improve selectivity via the steric exclusion of off-targets but retain rates of cysteine reactivity comparable to that of an acrylamide. Using chemical proteomic techniques, we demonstrate that elaboration of the electrophile to a tert-butyl (t-Bu) fumarate ester decreases time-dependent off-target reactivity and abolishes time-independent off-target reactivity. While an alkyne-bearing probe analogue of Ibrutinib has 247 protein targets, our t-Bu fumarate probe analogue has only 7. Of these 7 targets, BTK is the only time-independent target. The t-Bu inhibitor itself is also more selective for BTK, reducing off-targets by 70%. We investigated the consequences of treatment with Ibrutinib and our t-Bu analogue and discovered that only 8 proteins are downregulated in response to treatment with the t-Bu analogue compared to 107 with Ibrutinib. Of these 8 proteins, 7 are also downregulated by Ibrutinib and a majority of these targets are associated with BTK biology. Taken together, these findings reveal an opportunity to increase cysteine-reactive covalent inhibitor selectivity through electrophilic structure optimization.

Covalent labeling of a chromatin reader domain using proximity-reactive cyclic peptides

Zhang, Meng Yao and Yang, Hyunjun and Ortiz, Gloria and Trnka, Michael J. and Petronikolou, Nektaria and Burlingame, Alma L. and DeGrado, William F. and Fujimori, Danica Galonić

Chem. Sci. 2022

Chemical probes for chromatin reader proteins are valuable tools for investigating epigenetic regulatory mechanisms and evaluating whether the target of interest holds therapeutic potential. Developing potent inhibitors for the plant homeodomain (PHD) family of methylation readers remains a difficult task due to the charged, shallow and extended nature of the histone binding site that precludes effective engagement of conventional small molecules. Herein, we describe the development of novel proximity-reactive cyclopeptide inhibitors for PHD3—a trimethyllysine reader domain of histone demethylase KDM5A. Guided by the PHD3–histone co-crystal structure, we designed a sidechain-to-sidechain linking strategy to improve peptide proteolytic stability whilst maintaining binding affinity. We have developed an operationally simple solid-phase macrocyclization pathway, capitalizing on the inherent reactivity of the dimethyllysine ε-amino group to generate scaffolds bearing charged tetraalkylammonium functionalities that effectively engage the shallow aromatic ‘groove’ of PHD3. Leveraging a surface-exposed lysine residue on PHD3 adjacent to the ligand binding site, cyclic peptides were rendered covalent through installation of an arylsulfonyl fluoride warhead. The resulting lysine-reactive cyclic peptides demonstrated rapid and efficient labeling of the PHD3 domain in HEK293T lysates, showcasing the feasibility of employing proximity-induced reactivity for covalent labeling of this challenging family of reader domains.

Chemoproteomics-Enabled Discovery of a Covalent Molecular Glue Degrader Targeting NF-κB

Elizabeth A King, Yoojin Cho, Dustin Dovala, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Daniel K Nomura

bioRxiv 2022.05.18.492542; 


Targeted protein degradation using heterobifunctional Proteolysis-Targeting Chimeras (PROTACs) or molecular glues has arisen as a powerful therapeutic modality for degrading disease targets. While PROTAC design is becoming more modular and straightforward, the discovery of novel molecular glue degraders has been more challenging. While several recent studies have showcased phenotypic screening and counter-screening approaches to discover new molecular glue degraders, mechanistically elucidating the ternary complex induced by the small molecule that led to the initial phenotype, i.e. identifying the degraded target and relevant components of the ubiquitin-proteasome system, has remained cumbersome and laborious. To overcome these obstacles, we have coupled the screening of a covalent ligand library for anti-proliferative effects in leukemia cells with quantitative proteomic and chemoproteomic approaches to rapidly discover both novel covalent molecular glue degraders and their associated ternary complex components and anti-proliferative mechanisms. We have identified a cysteine-reactive covalent ligand EN450 that impairs leukemia cell viability in a NEDDylation and proteasome-dependent manner. Chemoproteomic profiling revealed covalent interaction of EN450 with an allosteric C111 in the E2 ubiquitin ligase UBE2D. Follow-up quantitative proteomic profiling revealed the proteasome-mediated degradation of the oncogenic transcription factor NFKB1 as a putative degradation target. Subsequent validation studies demonstrated that EN450 induced the ternary complex formation between UBE2D and NFKB1 and that both UBE2D and NFKB1 were important for the anti-proliferative mechanisms of EN450. Our study thus puts forth the discovery of a novel molecular glue degrader that uniquely induced the proximity of an E2 ligase with a transcription factor to induce its degradation and anti-proliferative effects in cancer cells. Taken more broadly, our study showcases a rapid and modular approach for discovering novel covalent molecular glue degraders and their respective ternary complex components in an unbiased fashion.

Tuesday, May 10, 2022

Rapid covalent labeling of a GPCR on living cells using a nanobody-epitope tag pair to interrogate receptor pharmacology

Chino C CabaltejaRoss W Cheloha

Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications. To address these shortcomings, single domain antibodies (nanobodies, Nbs) that recognize short peptide epitopes have become increasingly prized. Here we characterize the interaction between a Nb (Nb6E) and a 14-mer peptide epitope. We identify residues in the peptide epitope essential for high affinity binding. Using this information in combination with computational modeling we propose a mode of interaction between Nb6E and this epitope. We apply this nanobody-epitope pair to augment the potency of a ligand at an engineered adenosine A2A receptor. This characterization of the nanobody-epitope pair opens the door to diverse applications including mechanistic studies of G protein-coupled receptor function.

Monday, May 2, 2022

In situ identification of cellular drug targets in mammalian tissue

 Zhengyuan Pang, Michael A. Schafroth, Daisuke Ogasawara, Yu Wang, Victoria Nudell, Neeraj K. Lal, Dong Yang, Kristina Wang, Dylan M. Herbst, Jacquelyn Ha, Carlos Guijas, Jacqueline L. Blankman, Benjamin F. Cravatt, Li Ye,

Cell 2022

The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.

Keywords: drug target engagement; drug distribution; molecular imaging; click chemistry; target identification; covalent inhibitor

Wednesday, April 27, 2022

Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease

Kneller, D.W., Li, H., Phillips, G. et al. 

Nat Commun 13, 2268 (2022).

Emerging SARS-CoV-2 variants continue to threaten the effectiveness of COVID-19 vaccines, and small-molecule antivirals can provide an important therapeutic treatment option. The viral main protease (Mpro) is critical for virus replication and thus is considered an attractive drug target. We performed the design and characterization of three covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2 created by splicing components of hepatitis C protease inhibitors boceprevir and narlaprevir, and known SARS-CoV-1 protease inhibitors. A joint X-ray/neutron structure of the Mpro/BBH-1 complex demonstrates that a Cys145 thiolate reaction with the inhibitor’s keto-warhead creates a negatively charged oxyanion. Protonation states of the ionizable residues in the Mpro active site adapt to the inhibitor, which appears to be an intrinsic property of Mpro. Structural comparisons of the hybrid inhibitors with PF-07321332 reveal unconventional F···O interactions of PF-07321332 with Mpro which may explain its more favorable enthalpy of binding. BBH-1, BBH-2 and NBH-2 exhibit comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.

Intracellular Formation of a DNA Damage-Induced, Histone Post-Translational Modification Following Bleomycin Treatment

Marco Paolo Jacinto, Stephen D. Fried, and Marc M. Greenberg
Journal of the American Chemical Society Article ASAP

DOI: 10.1021/jacs.2c02880

Evaluating the significance of various forms of DNA damage is complicated by discoveries that some lesions inactivate repair enzymes or produce more deleterious forms of damage. Histone lysines within nucleosomes react with the commonly produced C4′-oxidized abasic site (C4-AP) to concomitantly yield an electrophilic modification (KMP) on lysine and DNA strand scission. We developed a chemoproteomic approach to identify KMP in HeLa cells. More than 60 000 KMP-modified histones are produced per cell. Using LC-MS/MS, we detected KMP at 17 of the 57 lysine residues distributed throughout the four core histone proteins. Therefore, KMP constitutes a DNA damage-induced, nonenzymatic histone post-translational modification. KMP formation suggests that downstream processes resulting from DNA damage could have ramifications on cells.

Conditional covalent lethality driven by oncometabolite accumulation

Minervo Perez, Kellie D Nance, Daniel W Bak, Supuni Thalalla Gamage, Susana S Najera, Amy N Conte, W. Marston Linehan, Eranthie Weerapana, Jordan L Meier

bioRxiv 2022.04.26.489575; 


Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a cancer predisposition syndrome driven by mutation of the tumor suppressor fumarate hydratase (FH). Inactivation of FH causes accumulation of the electrophilic oncometabolite fumarate. In the absence of methods for reactivation, tumor suppressors can be targeted via identification of synthetic lethal interactions using genetic screens. Inspired by recent advances in chemoproteomic target identification, here we test the hypothesis that the electrophilicity of the HLRCC metabolome may produce unique susceptibilities to covalent small molecules, a phenomenon we term conditional covalent lethality. Screening a panel of chemically diverse electrophiles we identified a covalent ligand, MP-1, that exhibits FH-dependent cytotoxicity. Synthesis and structure-activity profiling identified key molecular determinants underlying the molecule's effects. Chemoproteomic profiling of cysteine reactivity together with clickable probes validated the ability of MP-1 to engage an array of functional cysteines, including one lying in the Zn-finger domain of the tRNA methyltransferase enzyme TRMT1. TRMT1 overexpression rescues tRNA methylation from inhibition by MP-1 and partially attenuates the covalent ligand's cytotoxicity. Our studies highlight the potential for covalent metabolites and small molecules to synergistically produce novel synthetic lethal interactions and raise the possibility of applying phenotypic screening with chemoproteomic target identification to identify new functional oncometabolite targets.

Thursday, April 21, 2022

Cell-Active, Reversible, and Irreversible Covalent Inhibitors that Selectively Target the Catalytic Lysine of BCR-ABL Kinase

Chen, P., Sun, J., Zhu, C., Tang, G., Wang, W., Xu, M., Xiang, M., Zhang, C., Zhang, Z., Gao, L. and Yao, S..Q. 

Angew. Chem. Int. Ed. 2022

Despite recent interests in developing lysine-targeting covalent inhibitors, no general approach is available to create such compounds. We report herein a general approach to develop cell-active covalent inhibitors of protein kinases by targeting the conserved catalytic lysine residue using key SuFEx and salicylaldehyde-based imine chemistries. We validated the strategy by successfully developing (irreversible and reversible) covalent inhibitors against BCR-ABL kinase. Our lead compounds showed high levels of selectivity in biochemical assays, exhibited nanomolar potency against endogenous ABL kinase in cellular assays, and were active against most drug-resistant ABL mutations. Among them, the salicylaldehyde-containing A5 is the first-ever reversible covalent ABL inhibitor that possessed time-dependent ABL inhibition with prolonged residence time and few cellular off-targets in K562 cells. Bioinformatics further suggested the generality of our strategy against the human kinome.

Friday, April 15, 2022

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent and Selective, Covalent Oral Inhibitor of KRASG12C

Andreas Weiss1*, Edwige Lorthiois1*, Louise Barys1, Kim S. Beyer1, Claudio Bomio-Confaglia1, Heather Burks2, Xueying Chen3, Xiaoming Cui3, Ruben de Kanter1, Lekshmi Dharmarajan1, Carmine Fedele2, Marc Gerspacher1, Daniel Alexander Guthy1, Victoria Head 1, Ashley Jaeger2, Eloísa Jiménez Núñez 1, Jeffrey D Kearns2, Catherine Leblanc1, Sauveur-Michel Maira1, Jason Murphy2, Helen Oakman2, Nils Ostermann1, Johannes Ottl1, Pascal Rigollier1, Danielle Roman1, Christian Schnell1, Richard Sedrani1, Toshio Shimizu4, Rowan Stringer1, Andrea Vaupel1, Hans Voshol1, Peter Wessels5, Toni Widmer5, Rainer Wilcken1, Kun Xu3, Frederic Zecri2, Anna F. Farago2#, Simona Cotesta1# and Saskia M. Brachmann

Cancer Discov

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRAS 46 G12C-mutated cancers, though resistance emerges and additional strategies are needed to improve 47 outcomes. JDQ443 is a structurally unique, covalent inhibitor of GDP-bound KRASG12C that forms 48 novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular 49 signaling and demonstrates selective antiproliferative activity in KRAS G12C-mutated cell lines, 50 including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven 51 antitumor efficacy in KRAS G12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor 52 xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with SHP2, 53 MEK or CDK4/6 inhibitors. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is 54 maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. 55 JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both 56 strategies showing antitumor activity in patients with KRAS G12C-mutated tumors.   

Monday, March 28, 2022

Discovery of SPH5030, a Selective, Potent, and Irreversible Tyrosine Kinase Inhibitor for HER2-Amplified and HER2-Mutant Cancer Treatment

Di Li, Yuanxiang Tu, Kaijun Jin, Lingjun Duan, Yuan Hong, Jia Xu, Na Chen, Zhihui Zhang, Hongjian Zuo, Wanchun Gong, Jing Zhang, Qian Wang, Hai Qian, Xuenan Wang, Ying Ke, and Guangxin Xia
Journal of Medicinal Chemistry 2022

DOI: 10.1021/acs.jmedchem.1c00710

Small-molecule irreversible tyrosine kinase inhibitors as high potent agents have led to improvements in disease-free and overall survival in patients with HER2-amplified cancer. The approved irreversible HER2 inhibitors, neratinib and pyrotinib, both lack HER2 selectivity, leading to off-target adverse events in patients. The development of HER2 mutation during treatment also hampers the progress of the treatment. We used a molecular hybridization strategy for structural optimizations, in conjunction with in vitro and in vivo drug-like property screening, to obtain a clinical candidate SPH5030. Overall, SPH5030 showed excellent activities against four frequent kinds of HER2 mutants and high relative HER2 selectivity compared with neratinib and pyrotinib, good pharmacokinetic characteristics with desirable bioavailabilities, and significant in vivo antitumor efficacy in xenograft mouse models, especially in a HER2 mutation A775_G776insYVMA xenograft mouse model with its potency much higher than those of neratinib and pyrotinib.

Friday, March 25, 2022

Chemical Proteomics Reveals Antibiotic Targets of Oxadiazolones in MRSA [@atbakker]

Bakker, A.; Kotsogianni, I.; Mirenda, L.; Straub, V.; Florea, B.; van den Berg, R.; Janssen, A.; Martin, N.; van der Stelt, M. ChemRxiv 2022


Phenotypic screening is a powerful approach to identify novel antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) infection, but elucidation of the targets responsible for antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode-of-action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones, identified in a phenotypic screen to have high antibacterial potency against multidrug resistant S. aureus. In situ competitive and comparative chemical proteomics with a tailor-made activity-based probe, in combination with transposon and resistance studies, revealed several cysteine and serine hydrolases as relevant targets. Our data showcase oxadiazolones as novel antibacterial chemotype with a polypharmacological mode-of-action, in which FabH, FphC and AdhE play a central role.

Monday, March 21, 2022

Structure-based design of a dual-warhead covalent inhibitor of FGFR4

  • Xiaojuan Chen, Huiliang Li, Qianmeng Lin, Shuyan Dai, Sitong Yue, Lingzhi Qu, Maoyu Li, Ming Guo, Hudie Wei, Jun Li, Longying Jiang, Guangyu Xu & Yongheng Chen 

  • Communications Chemistry volume 5,  36 (2022

  • DOI:

The fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR4) signaling pathways play critical roles in a variety of cancers, such as hepatocellular carcinoma (HCC). FGFR4 is recognized as a promising target to treat HCC. Currently, all FGFR covalent inhibitors target one of the two cysteines (Cys477 and Cys552). Here, we designed and synthesized a dual-warhead covalent FGFR4 inhibitor, CXF-009, targeting Cys477 and Cys552 of FGFR4. We report the cocrystal structure of FGFR4 with CXF-009, which exhibits a dual-warhead covalent binding mode. CXF-009 exhibited stronger selectivity for FGFR4 than FGFR1-3 and other kinases. CXF-009 can also potently inhibit the single cystine mutants, FGFR4(C477A) and FGFR4(C552A), of FGFR4. In summary, our study provides a dual-warhead covalent FGFR4 inhibitor that can covalently target two cysteines of FGFR4. CXF-009, to our knowledge, is the first reported inhibitor that forms dual-warhead covalent bonds with two cysteine residues in FGFR4. CXF-009 also has the potential to overcome drug induced resistant FGFR4 mutations and might serve as a lead compound for future anticancer drug discovery.


Wednesday, March 16, 2022

Single Mutation on Trastuzumab Modulates the Stability of Antibody–Drug Conjugates Built Using Acetal-Based Linkers and Thiol-Maleimide Chemistry

Xhenti Ferhati, Ester Jiménez-Moreno, Emily A. Hoyt, Giulia Salluce, Mar Cabeza-Cabrerizo, Claudio D. Navo, Ismael Compañón, Padma Akkapeddi, Maria J. Matos, Noelia Salaverri, Pablo Garrido, Alfredo Martínez, Víctor Laserna, Thomas V. Murray, Gonzalo Jiménez-Osés, Peter Ravn, Gonçalo J. L. Bernardes, and Francisco Corzana

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.1c07675

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.

Friday, March 11, 2022

Identification and In Silico Binding Study of a Highly Potent DENV NS2B-NS3 Covalent Inhibitor

Xincheng Lin, Jiawei Cheng, Yuming Wu, Yaoliang Zhang, Hailun Jiang, Jian Wang, Xuejun Wang, and Maosheng Cheng

ACS Medicinal Chemistry Letters  2022

DOI: 10.1021/acsmedchemlett.1c00653

Dengue virus (DENV), an arthropod-borne flavivirus, has developed rapidly in the past few decades and becoming the most widespread arbovirus in the world. The vital role of NS2B-NS3 in virus replication and maturation of viral proteins makes it the most promising target for anti-DENV drug discovery. In the current work, a potent NS2B-NS3 covalent inhibitor 23 (IC50 = 6.0 nM, kinac/Ki = 1581 M–1 s–1) was discovered through the chemical modification of a published covalent inhibitor 1 (IC50 = 500 nM, kinac/Ki = 156.1 M–1 s–1), followed by in vitro assay. Further comprehensive structure–activity relationship analysis through covalent docking and molecular dynamics simulation provides informative understanding of the binding modes of covalent inhibitors targeting NS2B-NS3.

Discovery of Potent and Selective Inhibitors against Protein-Derived Electrophilic Cofactors

Xie Wang, Zongtao Lin, Katelyn A. Bustin, Nate R. McKnight, William H. Parsons, and Megan L. Matthews

Journal of the American Chemical Society 2021
DOI: 10.1021/jacs.1c12748

Electrophilic cofactors are widely distributed in nature and play important roles in many physiological and disease processes, yet they have remained blind spots in traditional activity-based protein profiling (ABPP) approaches that target nucleophiles. More recently, reverse-polarity (RP)-ABPP using hydrazine probes identified an electrophilic N-terminal glyoxylyl (Glox) group for the first time in secernin-3 (SCRN3). The biological function(s) of both the protein and Glox as a cofactor has not yet been pharmacologically validated because of the lack of selective inhibitors that could disrupt and therefore identify its activity. Here, we present the first platform for analyzing the reactivity and selectivity of an expanded nucleophilic probe library toward main-chain carbonyl cofactors such as Glox and pyruvoyl (Pyvl) groups. We first applied the library proteome-wide to profile and confirm engagement with various electrophilic protein targets, including secernin-2 (SCRN2), shown here also to possess a Glox group. A broadly reactive indole ethylhydrazine probe was used for a competitive in vitro RP-ABPP assay to screen for selective inhibitors against such cofactors from a set of commercially available nucleophilic fragments. Using Glox-containing SCRN proteins as a case study, naphthyl hydrazine was identified as a potent and selective SCRN3 inhibitor, showing complete inhibition in cell lysates with no significant cross-reactivity detected for other enzymes. Moving forward, this platform provides the fundamental basis for the development of selective Glox inhibitors and represents a starting point to advance small molecules that modulate electrophile-dependent function.

Tuesday, March 8, 2022

Discovery of Potent PROTACs Targeting EGFR Mutants through the Optimization of Covalent EGFR Ligands

Hong-Yi Zhao, Hai-Peng Wang, Yu-Ze Mao, Hao Zhang, Minhang Xin, Xiao-Xiao Xi, Hao Lei, Shuai Mao, Dong-Hui Li, and San-Qi Zhang

Journal of Medicinal Chemistry 2022
DOI: 10.1021/acs.jmedchem.1c01827

Drug resistance caused by epidermal growth factor receptor (EGFR) mutation has largely limited the clinical use of EGFR tyrosine kinase inhibitors (EGFR-TKIs) for the treatment of non-small-cell lung cancer (NSCLC). Herein, to overcome the intractable problem of drug resistance, proteolysis targeting chimeras (PROTACs) targeting EGFR mutants were developed by optimizing covalent EGFR ligands. Covalent or reversible covalent pyrimidine- or purine-containing PROTACs were designed, synthesized, and evaluated. As a consequence, covalent PROTAC CP17, with a novel purine-containing EGFR ligand, was discovered as a highly potent degrader against EGFRL858R/T790M and EGFRdel19, reaching the lowest DC50 values among all reported EGFR-targeting PROTACs. Furthermore, CP17 exhibited excellent cellular activity against the H1975 and HCC827 cell lines with high selectivity. Mechanism investigation indicated that the lysosome was involved in the degradation process. Importantly, the covalent binding strategy was proven to be an effective approach for the design of PROTACs targeting EGFRL858R/T790M, which laid the practical foundation for further development of potent EGFR-targeting PROTACs.

Thiol Reactivity of N-Aryl α-Methylene-γ-lactams: Influence of the Guaianolide Structure [@KayBrummond]

 Daniel P. Dempe, Chong-Lei Ji, Peng Liu, and Kay M. Brummond The Journal of Organic Chemistry, 2020 DOI: 10.1021/acs.joc.2c01530 The α-meth...