Small-Molecule Cyanamide Pan-TEAD·YAP1 Covalent Antagonists

Khuchtumur Bum-Erdene, I-Ju Yeh, Giovanni Gonzalez-Gutierrez, Mona K. Ghozayel, Karen Pollok, and Samy O. Meroueh

Journal of Medicinal Chemistry 2023

DOI: 10.1021/acs.jmedchem.2c01189

Transcriptional enhanced associate domains (TEADs) are transcription factors that bind to cotranscriptional activators like the yes-associated protein (YAP) or its paralog transcriptional coactivator with a PDZ-binding motif (TAZ). TEAD·YAP/TAZ target genes are involved in tissue and immune homeostasis, organ size control, tumor growth, and metastasis. Here, we report isoindoline and octahydroisoindole small molecules with a cyanamide electrophile that forms a covalent bond with a conserved cysteine in the TEAD palmitate-binding cavity. Time- and concentration-dependent studies against TEAD1-4 yielded second-order rate constants kinact/KI greater than 100 M–1 s–1. Compounds inhibited YAP1 binding to TEADs with submicromolar IC50 values. Cocrystal structures with TEAD2 enabled structure–activity relationship studies. In mammalian cells, compounds suppressed CTGF mRNA levels and inhibited TEAD1-4 transcriptional activity with submicromolar IC50 values. Inhibition of TEAD binding to YAP1 in mammalian cells was also observed. Several compounds inhibited the cell viability of sarcoma, hepatocellular carcinoma, glioblastoma, and breast cancer cells with single-digit micromolar IC50 values.

Targeted Protein Degradation through E2 Recruitment [@DanNomura]

Nafsika Forte, Dustin Dovala, Matthew J Hesse, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Daniel K Nomura

bioRxiv 2022.12.19.520812; 

doi: https://doi.org/10.1101/2022.12.19.520812

Targeted protein degradation (TPD) with Proteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds consisting of protein targeting ligands linked to recruiters of E3 ubiquitin ligases, has arisen as a powerful therapeutic modality to induce the proximity of target proteins with E3 ligases to ubiquitinate and degrade specific proteins in cells. Thus far, PROTACs have primarily exploited the recruitment of E3 ubiquitin ligases or their substrate adapter proteins but have not exploited the recruitment of more core components of the ubiquitin proteasome system (UPS). In this study, we used covalent chemoproteomic approaches to discover a covalent recruiter against the E2 ubiquitin conjugating enzyme UBE2D, EN67, that targets an allosteric cysteine, C111, without affecting the enzymatic activity of the protein. We demonstrated that this UBE2D recruiter could be used in heterobifunctional degraders to degrade neo-substrate targets in a UBE2D dependent manner, including BRD4 and the androgen receptor. Overall, our data highlight the potential for the recruitment of core components of the UPS machinery, such as E2 ubiquitin conjugating enzymes, for TPD, and underscore the utility of covalent chemoproteomic strategies for identifying novel recruiters for additional components of the UPS.

Proteome-wide structure-based accessibility analysis of ligandable and detectable cysteines in chemoproteomic datasets

Matthew E. H. White, Jesús Gil, Edward W. Tate

bioRxiv 2022.12.12.518491; 

doi: https://doi.org/10.1101/2022.12.12.518491

Covalent drug discovery, in particular targeting reactive cysteines, has undergone a resurgence over the past two decades, demonstrated by recent clinical successes of covalent inhibitors for high-priority cancer targets. Reactive cysteine profiling, first pioneered by the Cravatt lab, has emerged in parallel as a powerful approach for proteome-wide on- and off-target profiling. Thus far however, structural analysis of liganded cysteines has been restricted to experimentally determined protein structures. We combined AlphaFold-predicted amino acid side chain accessibilities for >95% of the human proteome with a meta-analysis of thirteen public cysteine profiling datasets, totalling 40,070 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further suggests that exposed cysteine residues are preferentially targeted by elaborated fragments and drug-like compounds. We finally propose a framework for benchmarking coverage of ligandable cysteines in future cysteine profiling approaches, considering both selectivity for high-priority residues and quantitative depth. All analysis and produced resources (freely available at www.github.com/TateLab) are readily extendable to reactive amino acids beyond cysteine, and related questions in chemical biology.

Proteomic discovery of chemical probes that perturb protein complexes in human cells [@michaellazear]

Michael Lazear, Jarrett Remsberg, Martin Jaeger, Katherine Rothamel, Hsuan-lin Her, Kristen DeMeester, Evert Njomen, Simon Hogg, Jahan Rahman, Landon Whitby, Sang Joon Won, Michael Schafroth, Daisuke Ogasawara, Minoru Yokoyama, Garrett Lindsey, Haoxin Li, Jason Germain, Sabrina Barbas, Joan Vaughan, Thomas Hanigan, Vincent Vartabedian, Christopher Reinhardt, Melissa Dix, Seong Joo Koo, Inha Heo, John Teijaro, Gabriel Simon, Brahma Ghosh, Omar Abdel-Wahab, Kay Ahn, Alan Saghatelian, Bruno Melillo, Stuart Schreiber, Gene Yeo, Benjamin Cravatt

bioRxiv 2022.12.12.520090; 

doi: https://doi.org/10.1101/2022.12.12.520090

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such binding-first assays affect protein function, nonetheless, often remains unclear. Here, we describe a function-first proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

Efficient Ligand Discovery Using Sulfur(VI) Fluoride Reactive Fragments [@arronaatkar]

Aatkar, A.; Vuorinen, A.; Longfield, O.; Gilbert, K.; Peltier-Heap, R.; Wagner, C.; Zappacosta, F.; Rittinger, K.; Chung, C.-wa; House, D.; Tomkinson, N.; Bush, J. ChemRxiv 2022.

https://doi.org/10.26434/chemrxiv-2022-6mt7k

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of 'beyond cysteine' covalent inhibitors, and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesised, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterised to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of ‘beyond cysteine’ covalent inhibitors.

Mapping the chemical space of active-site targeted covalent ligands for protein tyrosine phosphatases

Hong, S. ho; Xi, S. Y.; Johns, A. C.; Tang, L. C.; Li, A.; Jovanovic, M.; Shah, N. H. ChemRxiv 2022.

https://doi.org/10.26434/chemrxiv-2022-1rnqh

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required chemical parameters for covalent inhibition of tyrosine phosphatases. Our analysis juxtaposes the intrinsic electrophilicity of these compounds with their potency against several classical PTPs, revealing chemotypes that inhibit tyrosine phosphatases while minimizing excessive, potentially non-specific reactivity. We also assess sequence divergence at key residues in PTPs to explain their differential susceptibility to covalent inhibition. We anticipate that our study will inspire new strategies to develop covalent probes and inhibitors for tyrosine phosphatases.

A toolkit for covalent docking with GOLD: from automated ligand preparation with KNIME to bound protein–ligand complexes

Laurianne David, Anissa Mdahoma, Natesh Singh, Sébastien Buchoux, Emilie Pihan, Constantino Diaz, Obdulia Rabal

Bioinformatics Advances, Volume 2, Issue 1, 2022, vbac090, 

https://doi.org/10.1093/bioadv/vbac090

Current covalent docking tools have limitations that make them difficult to use for performing large-scale structure-based covalent virtual screening (VS). They require time-consuming tasks for the preparation of proteins and compounds (standardization, filtering according to the type of warheads), as well as for setting up covalent reactions. We have developed a toolkit to help accelerate drug discovery projects in the phases of hit identification by VS of ultra-large covalent libraries and hit expansion by exploration of the binding of known covalent compounds. With this application note, we offer the community a toolkit for performing automated covalent docking in a fast and efficient way.

The toolkit comprises a KNIME workflow for ligand preparation and a Python program to perform the covalent docking of ligands with the GOLD docking engine running in a parallelized fashion.

Engaging a Non-Catalytic Cysteine Residue Drives Unprecedented Selectivity of Caspase Inhibition

Van Horn, K.; Wang, D.; Medina-Cleghorn, D.; Lee, P.; Bryant, C.; Altobelli, C.; Jaishankar, P.; Leung, K.; Ng, R.; Ambrose, A.; Tang, Y.; Arkin, M.; Renslo, A. ChemRxiv 2022

https://doi.org/10.26434/chemrxiv-2022-5wfm6

The caspases are a family of cysteine dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human disease. Classical chemical tools to study caspase function lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we tar-geted a non-catalytic cysteine residue (C264) unique to Caspase-6, an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used structure-informed covalent ligand design to produce potent, irreversible inhibitors (e.g., 3a) and chemoproteomic probes (e.g., 13-t) of Caspase-6 that exhibit unprecedented se-lectivity over other caspase family members and high proteomic selectivity. This approach and the new tools described will enable rigorous interrogation of the role of Caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases

JDQ443, a Structurally Novel, Pyrazole-Based, Covalent Inhibitor of KRASG12C for the Treatment of Solid Tumors

J. Med. Chem. 2022

https://doi.org/10.1021/acs.jmedchem.2c01438

Rapid emergence of tumor resistance via RAS pathway reactivation has been reported from clinical studies of covalent KRASG12C inhibitors. Thus, inhibitors with broad potential for combination treatment and distinct binding modes to overcome resistance mutations may prove beneficial. JDQ443 is an investigational covalent KRASG12C inhibitor derived from structure-based drug design followed by extensive optimization of two dissimilar prototypes. JDQ443 is a stable atropisomer containing a unique 5-methylpyrazole core and a spiro-azetidine linker designed to position the electrophilic acrylamide for optimal engagement with KRASG12C C12. A substituted indazole at pyrazole position 3 results in novel interactions with the binding pocket that do not involve residue H95. JDQ443 showed PK/PD activity in vivo and dose-dependent antitumor activity in mouse xenograft models. JDQ443 is now in clinical development, with encouraging early phase data reported from an ongoing Phase Ib/II clinical trial (NCT04699188).

 

Assigning functionality to cysteines by base editing of cancer dependency genes

Haoxin Li, Jarrett Remsberg, Sang Joon Won, Kevin Zhao, Tony Huang, Bingwen Lu, Gabriel Simon, David Liu, Benjamin Cravatt

doi: https://doi.org/10.1101/2022.11.17.516964

Chemical probes are lacking for most human proteins. Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cell proliferation. We show that the resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, correctly predicts the essentiality of cysteines targeted by cancer therapeutics and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines on >110 cancer dependency proteins. We further demonstrate how measurements of reactivity in native versus denatured proteomes can discriminate essential cysteines amenable to chemical modification from those buried in protein structures, providing a valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

Structure-guided design and characterization of a clickable, covalent PARP16 inhibitor

Bejan, Daniel S. and Sundalam, Sunil and Jin, Haihong and Morgan, Rory K. and Kirby, Ilsa T. and Siordia, Ivan R. and Tivon, Barr and London, Nir and Cohen, Michael S.

Chemical Science, 2022

DOI https://doi.org/10.1039/D2SC04820E

PARP16—the sole ER-resident PARP family member—is gaining attention as a potential therapeutic target for cancer treatment. Nevertheless, the precise function of the catalytic activity of PARP16 is poorly understood. This is primarily due to the lack of inhibitors that are selective for PARP16 over other PARP family members. Herein, we describe a structure-guided strategy for generating a selective PARP16 inhibitor by incorporating two selectivity determinants into a phthalazinone pan-PARP inhibitor scaffold: (i) an acrylamide-based inhibitor (DB008) designed to covalently react with a non-conserved cysteine (Cys169, human numbering) in the NAD+ binding pocket of PARP16 and (ii) a dual-purpose ethynyl group designed to bind in a unique hydrophobic cavity adjacent to the NAD+ binding pocket as well as serve as a click handle. DB008 exhibits good selectivity for PARP16 versus other PARP family members. Copper-catalyzed azide–alkyne cycloaddition (CuAAC) confirmed that covalent labeling of PARP16 by DB008 in cells is dependent on Cys169. DB008 exhibits excellent proteome-wide selectivity at concentrations required to achieve saturable labeling of endogenous PARP16. In-cell competition labeling experiments using DB008 provided a facile strategy for evaluating putative PARP16 inhibitors. Lastly, we found that PARP16 is sequestered into a detergent-insoluble fraction under prolonged amino acid starvation, and surprisingly, treatment with PARP16 inhibitors prevented this effect. These results suggest that the catalytic activity of PARP16 regulates its solubility in response to nutrient stress.

A Peptide-Based Ligand-Directed Chemistry Enables Protein Functionalization

Yuena Wang, Rongtong Zhao, Chuan Wan, Xiaochun Guo, Fenfang Yang, Zhanfeng Hou, Rui Wang, Shuiming Li, Tiejian Feng, Feng Yin, and Zigang Li

Organic Letters 2022 24 (39), 7205-7209

DOI: 10.1021/acs.orglett.2c02974

The ligand-directed (LD) chemistry provides powerful tools for site-specific modification of proteins. We utilized a peptide with an appended methionine (Met) as a ligand; then, the Met thioether was modified into sulfonium which enabled a proximity induced group transfer onto protein cysteine in the vicinity upon peptide–target binding. The sulfonium warhead could be easily constructed with unprotected peptides, and the transferable group scope was conducted on model protein PDZ and its ligand peptides. In addition, a living cell labeling was successfully achieved.

Dual Inhibitors of Main Protease (MPro) and Cathepsin L as Potent Antivirals against SARS-CoV2

Santanu Mondal, Yongzhi Chen, Gordon J. Lockbaum, Sudeshna Sen, Sauradip Chaudhuri, Archie C. Reyes, Jeong Min Lee, Arshia N. Kaur, Nadia Sultana, Michael D. Cameron, Scott A. Shaffer, Celia A. Schiffer, Katherine A. Fitzgerald, and Paul R. Thompson

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.2c04626

Given the current impact of SARS-CoV2 and COVID-19 on human health and the global economy, the development of direct acting antivirals is of paramount importance. Main protease (MPro), a cysteine protease that cleaves the viral polyprotein, is essential for viral replication. Therefore, MPro is a novel therapeutic target. We identified two novel MPro inhibitors, D-FFRCMKyne and D-FFCitCMKyne, that covalently modify the active site cysteine (C145) and determined cocrystal structures. Medicinal chemistry efforts led to SM141 and SM142, which adopt a unique binding mode within the MPro active site. Notably, these inhibitors do not inhibit the other cysteine protease, papain-like protease (PLPro), involved in the life cycle of SARS-CoV2. SM141 and SM142 block SARS-CoV2 replication in hACE2 expressing A549 cells with IC50 values of 8.2 and 14.7 nM. Detailed studies indicate that these compounds also inhibit cathepsin L (CatL), which cleaves the viral S protein to promote viral entry into host cells. Detailed biochemical, proteomic, and knockdown studies indicate that the antiviral activity of SM141 and SM142 results from the dual inhibition of MPro and CatL. Notably, intranasal and intraperitoneal administration of SM141 and SM142 lead to reduced viral replication, viral loads in the lung, and enhanced survival in SARS-CoV2 infected K18-ACE2 transgenic mice. In total, these data indicate that SM141 and SM142 represent promising scaffolds on which to develop antiviral drugs against SARS-CoV2.

Rational Chemical Design of Molecular Glue Degraders

Ethan S Toriki, James W Papatzimas, Kaila Nishikawa, Dustin Dovala, Lynn M McGregor, Matthew J Hesse, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Daniel K. Nomura

ACS Cent. Sci. 2023, ;
doi: https://doi.org/10.1101/2022.11.04.512693

https://pubs.acs.org/doi/10.1021/acscentsci.2c01317

Targeted protein degradation with molecular glue degraders has arisen as powerful therapeutic modality for eliminating classically undruggable disease-causing proteins through proteasome-mediated degradation. However, we currently lack rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. To overcome this challenge, we sought to identify chemical handles that would convert protein-targeting ligands into molecular glue degraders of their targets. Using the CDK4/6 inhibitor Ribociclib as a testbed, we identified a covalent handle that, when appended to the exit vector of Ribociclib, induced the proteasome-mediated degradation of CDK4 in cancer cells. Covalent chemoproteomic profiling of this CDK4 degrader revealed covalent interactions with cysteine 32 of the RING family E3 ubiquitin ligase RNF126. Optimization of this covalent scaffold led to an improved CDK4 degrader with a methoxyphenyl fumarate handle that showed improved interactions with RNF126. We then identified the minimum covalent chemical handle required for interaction with RNF126. With this knowledge in hand, we transplanted this covalent fumarate handle onto chemically related and un-related protein-targeting ligands to induce the degradation of several proteins across diverse protein classes, including BRD4, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, and SMARCA2. Our study undercovers a potential chemical rational design strategy for converting protein-targeting ligands into covalent molecular glue degraders.

Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase

Mark S. Tichenor, John J. M. Wiener, Navin L. Rao, Genesis M. Bacani, Jianmei Wei, Charlotte Pooley Deckhut, J. Kent Barbay, Kevin D. Kreutter, Leon Chang, Kathleen W. Clancy, Heather E. Murrey, Weixue Wang, Kay Ahn, Michael Huber, Elizabeth Rex, Kevin J. Coe, Jiejun Wu, Haopeng Rui, Kia Sepassi, Marcello Gaudiano, Mariette Bekkers, Ivo Cornelissen, Kathryn Packman, Mark Seierstad, Christos Xiouras, Scott D. Bembenek, Richard Alexander, Cynthia Milligan, Sriram Balasubramanian, Alec D. Lebsack, Jennifer D. Venable, Ulrike Philippar, James P. Edwards, and Gavin Hirst

Journal of Medicinal Chemistry 2022

DOI: 10.1021/acs.jmedchem.2c01026

Bruton’s tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

Fragment Optimization of Reversible Binding to the Switch II Pocket on KRAS Leads to a Potent, In Vivo Active KRASG12C Inhibitor

Joachim Bröker, Alex G. Waterson, Chris Smethurst, Dirk Kessler, Jark Böttcher, Moriz Mayer, Gerhard Gmaschitz, Jason Phan, Andrew Little, Jason R. Abbott, Qi Sun, Michael Gmachl, Dorothea Rudolph, Heribert Arnhof, Klaus Rumpel, Fabio Savarese, Thomas Gerstberger, Nikolai Mischerikow, Matthias Treu, Lorenz Herdeis, Tobias Wunberg, Andreas Gollner, Harald Weinstabl, Andreas Mantoulidis, Oliver Krämer, Darryl B. McConnell, and Stephen W. Fesik

Journal of Medicinal Chemistry 2022

DOI: 10.1021/acs.jmedchem.2c01120

Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRASG12C inhibitors. To date, KRASG12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRASG12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRASG12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.

Aryl Fluorosulfate Based Inhibitors That Covalently Target the SIRT5 Lysine Deacylase

Julie E. Bolding, Pablo Martín-Gago, Nima Rajabi, Luke F. Gamon, Tobias N. Hansen, Christian R. O. Bartling, Kristian Strømgaard, Michael J. Davies, Christian A. Olsen

 Angew. Chem. Int. Ed. 2022, e202204565

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase

Battisti, U..M., Gao, C., Nilsson, O., Akladios, F., Lulla, A., Bogucka, A., Nain-Perez, A., Håversen, L., Kim, W., Boren, J., Hyvönen, M., Uhlen, M., Mardinoglu, A. and Grøtli, M. 

ChemBioChem, 2022 

https://doi.org/10.1002/cbic.202200339

Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 = 0.29 µM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.

Covalent Protein Inhibitors via Tyrosine Conjugation with Cyclic Imine Mannich Electrophiles

Wang, S.; Hadisurya, M.; Tao, W. A.; Dykhuizen, E.; Krusemark, C. ChemRxiv 2022. https://doi.org/10.26434/chemrxiv-2022-tvgn1

Targeted covalent inhibitors (TCIs) have increased in popularity among drug candidates and chemical probes. Among current TCIs, the chemistry employed is largely limited to labeling cysteine and lysine side chains. Tyrosine is an attractive residue for TCIs due to its enrichment at protein-protein interfaces. Here, we investigate the utility of cyclic imine Mannich electrophiles as covalent warheads to specifically target a pro-tein tyrosine adjacent to an inhibitor binding pocket. We characterized the intrinsic reaction rates of several cyclic imines to tyrosine and identified the iminolactone to be suitable for a covalent inhibitor (second order rate constant of 0.0029 M-1 s-1). We appended the cyclic imine warheads to a CBX8 chromodomain inhibitor to label a non-conserved tyrosine, which markedly improves both the potency and selectivity of the inhibitor for CBX8 in vitro and in cells. These results indicate that Mannich electrophiles are promising and robust chemical warheads for tyrosine bioconjugation and covalent inhibitors.

Recent advances in the development of covalent inhibitors

Hyunsoo Kim,   Yoon Soo Hwang,   Mingi Kima  and  Seung Bum Park

RSC Med. Chem., 2021, 12, 1037-1045

https://doi.org/10.1039/D1MD00068C

The use of covalent inhibitors in the field of drug discovery has attracted considerable attention in the 2000s. As a result, more than 50 covalent drugs are currently on the market, and numerous covalent drug candidates are now under development. Therefore, interest in covalent drugs is expected to continue in the future. The purpose of this focused review is to provide an understanding of the development of covalent inhibitors by describing their inherent characteristics, possibilities, and limitations based on their mechanistic differences from noncovalent inhibitors. We also introduce the latest covalent warheads that can be applied to the development of potential covalent inhibitors.

Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization

Yue Liu, Rick C. Betori, Joanna Pagacz, Grant B. Frost, Elena V. Efimova, Ding Wu, Donald J. Wolfgeher, Tracy M. Bryan, Scott B. Cohen, Karl A. Scheidt, Stephen J. Kron

Cell Chemical Biology, 2022

https://doi.org/10.1016/j.chembiol.2022.09.002

Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT’s non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT’s role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.

Genetically encoded chemical crosslinking of RNA in vivo

Wei Sun, Nanxi Wang, Hongjiang Liu, Bingchen Yu, Ling Jin, Xingjie Ren, Yin Shen & Lei Wang 

Nature Chemistry, 2022

https://doi.org/10.1038/s41557-022-01038-4

Protein–RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein–RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics.

Targeted Protein Degradation by Electrophilic PROTACs that Stereoselectively and Site-Specifically Engage DCAF1

Yongfeng Tao, David Remillard, Ekaterina V. Vinogradova, Minoru Yokoyama, Sofia Banchenko, David Schwefel, Bruno Melillo, Stuart L. Schreiber, Xiaoyu Zhang, and Benjamin F. Cravatt

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.2c08964

Targeted protein degradation induced by heterobifunctional compounds and molecular glues presents an exciting avenue for chemical probe and drug discovery. To date, small-molecule ligands have been discovered for only a limited number of E3 ligases, which is an important limiting factor for realizing the full potential of targeted protein degradation. We report herein the discovery by chemical proteomics of azetidine acrylamides that stereoselectively and site-specifically react with a cysteine (C1113) in the E3 ligase substrate receptor DCAF1. We demonstrate that the azetidine acrylamide ligands for DCAF1 can be developed into electrophilic proteolysis-targeting chimeras (PROTACs) that mediated targeted protein degradation in human cells. We show that this process is stereoselective and does not occur in cells expressing a C1113A mutant of DCAF1. Mechanistic studies indicate that only low fractional engagement of DCAF1 is required to support protein degradation by electrophilic PROTACs. These findings, taken together, demonstrate how the chemical proteomic analysis of stereochemically defined electrophilic compound sets can uncover ligandable sites on E3 ligases that support targeted protein degradation.

Covalent drugs targeting histidine – An unexploited opportunity? [@LynJonesChemBio]

Jianwei Che  and  Lyn H Jones  

RSC Med. Chem., 2022

https://pubs.rsc.org/en/Content/ArticleLanding/2022/MD/D2MD00258B

Covalent drugs and chemical probes often possess pharmacological advantages over reversible binding ligands, such as enhanced potency and pharmacodynamic duration. The highly nucleophilic cysteine thiol is commonly targeted using acrylamide electrophiles, but the amino acid is rarely present in protein binding sites. Sulfonyl exchange chemistry has expanded the covalent drug discovery toolkit by enabling the rational design of irreversible inhibitors targeting tyrosine, lysine, serine and threonine. Probes containing the sulfonyl fluoride warhead have also been shown to serendipitously label histidine residues in proteins. Histidine targeting is an attractive prospect because the residue is frequently proximal to protein small molecule ligands and the imidazole side chain possesses desirable nucleophilicity. We recently reported the design of cereblon molecular glues to site-selectively modify a histidine in the thalidomide binding site using sulfonyl exchange chemistry. We believe that histidine targeting holds great promise for future covalent drug development and this Opinion highlights these opportunities.

CysDB: A Human Cysteine Database based on Experimental Quantitative Chemoproteomics [@Keribackus]

Boatner, L.; Palafox, M.; Schweppe, D.; Backus, K. 

ChemRxiv 2022

https://chemrxiv.org/engage/chemrxiv/article-details/632e3d710e3c6a3b31266100

Cysteine chemoproteomics studies provide proteome-wide portraits of the ligandability or potential ‘druggability’ of thousands of cysteine residues. Consequently, these studies are enabling resources for closing the druggability gap, namely achieving pharmacological manipulation of ~99% of the human proteome that remains untargeted by FDA approved small molecules. Recent interactive dataset repositories, such as OxiMouse and SLCABPP, have enabled users to interface more readily with cysteine chemoproteomics studies1,2. However, these databases remain limited to single studies and therefore do not provide a mechanism to perform cross-study analyses. Here we report CysDB as a curated community-wide repository of cysteine chemoproteomics data that incorporates high coverage data derived from nine studies generated by the Backus, Cravatt, Gygi, Wang, and Yang research groups. CysDB is a SQL relational database that is publicly available at https://backuslab.shinyapps.io/cysdb/ and features chemoproteomic measures of identification, hyperreactivity, and ligandability for 62,888 cysteines (24% of all cysteines the human proteome). The CysDB web application also includes annotations of functionality (UniProtKB/Swiss-Prot, Pfam, Panther), known druggability (FDA approved targets, DrugBank, ChEMBL), disease-relevance and genetic variation (ClinVar, Cancer Gene Census, Online Mendelian Inheritance in Man), and structural features (Protein Data Bank). Showcasing the utility of CysDB, here we report the discovery and enrichment of ligandable cysteines in undruggable classes of proteins, the observation that a subset of cysteines showed marked preference for specific classes of electrophiles (chloroacetamide vs acrylamide), and that ligandable cysteines are present in numerous undrugged disease-relevant proteins. Most importantly, we have designed CysDB for the incorporation of new datasets and features to support the continued growth of the druggable cysteineome.

Aryl Fluorosulfate Based Inhibitors that Covalently Target the SIRT5 Lysine Deacylase

Bolding, J..E., Martin-Gago, P., Rajabi, N., Gamon, L..F., Hansen, T..N., Bartling, C..R.O., Strømgaard, K., Davies, M..J. and Olsen, C..A. 

Angew. Chem. Int. Ed. 2022

https://doi.org/10.1002/anie.202204565

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl  ε - N -carboxyacyllysine  posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates.  It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Cysteine-Assisted Click-Chemistry for Proximity-Driven, Site-Specific Acetylation of Histones

Afonso, C..F., Marques, M..C., António, J..M.P., Cordeiro, C., Gois, P..M.P., Cal, P..M.S.D. and Bernardes, G..J.L. 

Angew. Chem. Int. Ed. 2022

https://doi.org/10.1002/anie.202208543

Post-translational modifications of histones are essential in the regulation of chromatin structure and function. Among these modifications, lysine acetylation is one of the most established. Earlier studies relied on the use of chromatin containing heterogeneous mixtures of histones acetylated at multiple sites. Differentiating the individual contribution of single acetylation events towards chromatin regulation is thus of great relevance. However, it is difficult to access homogeneous samples of histones, with a single acetylation, in sufficient quantities for such studies. By engineering histone H3 with a cysteine in proximity of the lysine of interest, we demonstrate that conjugation with maleimide-DBCO followed by a strain-promoted azide-alkyne cycloaddition reaction results in the acetylation of a single lysine in a controlled, site-specific manner. The chemical precision offered by our click-to-acetylate approach will facilitate access to and the study of acetylated histones.

X-ray Screening of an Electrophilic Fragment Library and Application toward the Development of a Novel ERK 1/2 Covalent Inhibitor

Jeffrey D. St. Denis, Gianni Chessari, Anne Cleasby, Benjamin D. Cons, Suzanna Cowan, Samuel E. Dalton, Charlotte East, Christopher W. Murray, Marc O’Reilly, Torren Peakman, Magdalini Rapti, and Jessie L. Stow

Journal of Medicinal Chemistry 2022

DOI: 10.1021/acs.jmedchem.2c01044

Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 μM), was developed.

A covalent inhibitor of K-Ras(G12C) induces MHC class I presentation of haptenated peptide neoepitopes targetable by immunotherapy

Cancer Cell, Volume 40, Issue 9, 12 September 2022, Pages 1060-1069.e7

https://doi.org/10.1016/j.ccell.2022.07.005

Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance.

Discovery of a Covalent Inhibitor of KRASG12C (AMG 510) for the Treatment of Solid Tumors

Brian A. Lanman, Jennifer R. Allen, John G. Allen, Albert K. Amegadzie, Kate S. Ashton, Shon K. Booker, Jian Jeffrey Chen, Ning Chen, Michael J. Frohn, Guy Goodman, David J. Kopecky, Longbin Liu, Patricia Lopez, Jonathan D. Low, Vu Ma, Ana E. Minatti, Thomas T. Nguyen, Nobuko Nishimura, Alexander J. Pickrell, Anthony B. Reed, Youngsook Shin, Aaron C. Siegmund, Nuria A. Tamayo, Christopher M. Tegley, Mary C. Walton, Hui-Ling Wang, Ryan P. Wurz, May Xue, Kevin C. Yang, Pragathi Achanta, Michael D. Bartberger, Jude Canon, L. Steven Hollis, John D. McCarter, Christopher Mohr, Karen Rex, Anne Y. Saiki, Tisha San Miguel, Laurie P. Volak, Kevin H. Wang, Douglas A. Whittington, Stephan G. Zech, J. Russell Lipford, and Victor J. Cee

Journal of Medicinal Chemistry 2020 63 (1), 52-65

DOI: 10.1021/acs.jmedchem.9b01180

KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-“undruggable” target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).

A covalent inhibitor of K-Ras(G12C) induces MHC class I presentation of haptenated peptide neoepitopes targetable by immunotherapy

Zhang, Ziyang, Peter J. Rohweder, Chayanid Ongpipattanakul, Koli Basu, Markus-Frederik Bohn, Eli J. Dugan, Veronica Steri, Byron Hann, Kevan M. Shokat.

Cancer Cell 40, 1060-1069.e7. 

DOI https://doi.org/10.1016/j.ccell.2022.07.005

Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance.

Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine

Kavanagh, M.E., Horning, B.D., Khattri, R. et al. 

Nat Chem Biol 2022)

https://doi.org/10.1038/s41589-022-01098-0

The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as ‘silent’ ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.

Fast Cysteine Bioconjugation Chemistry

Chem. Eur. J. 2022, e202201843

https://doi.org/10.1002/chem.202201843

Cysteine bioconjugation serves as a powerful tool in biological research and has been widely used for the chemical modification of proteins, constructing antibody-drug conjugates, and enabling cell imaging studies. Cysteine conjugation reactions with fast kinetics and exquisite selectivity have been under heavy pursuit as they would allow clean protein modification with just stoichiometric amount of reagents, which minimizes side reaction, simplifies purification and broadens the functional group tolerance. In this concept, we summarize the recent advances of fast cysteine bioconjugation, and discuss the mechanism and chemical principles that underlie the high efficiencies of the newly developed cysteine reactive reagents.

Chemoselective Covalent Modification of K-Ras(G12R) with a Small Molecule Electrophile

Ziyang Zhang, Johannes Morstein, Andrew K. Ecker, Keelan Z. Guiley, and Kevan M. Shokat

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.2c05377

KRAS mutations are one of the most common oncogenic drivers in human cancer. While small molecule inhibitors for the G12C mutant have been successfully developed, allele-specific inhibition for other KRAS hotspot mutants remains challenging. Here we report the discovery of covalent chemical ligands for the common oncogenic mutant K-Ras(G12R). These ligands bind in the Switch II pocket and irreversibly react with the mutant arginine residue. An X-ray crystal structure reveals an imidazolium condensation product formed between the α,β-diketoamide ligand and the ε- and η-nitrogens of arginine 12. Our results show that arginine residues can be selectively targeted with small molecule electrophiles despite their weak nucleophilicity and provide the basis for the development of mutant-specific therapies for K-Ras(G12R)-driven cancer.

Advances in covalent drug discovery

Boike, L., Henning, N.J. & Nomura, D.K.  Nat Rev Drug Discov (2022). 

https://doi.org/10.1038/s41573-022-00542-z

Covalent drugs have been used to treat diseases for more than a century, but tools that facilitate the rational design of covalent drugs have emerged more recently. The purposeful addition of reactive functional groups to existing ligands can enable potent and selective inhibition of target proteins, as demonstrated by the covalent epidermal growth factor receptor (EGFR) and Bruton’s tyrosine kinase (BTK) inhibitors used to treat various cancers. Moreover, the identification of covalent ligands through ‘electrophile-first’ approaches has also led to the discovery of covalent drugs, such as covalent inhibitors for KRAS(G12C) and SARS-CoV-2 main protease. In particular, the discovery of KRAS(G12C) inhibitors validates the use of covalent screening technologies, which have become more powerful and widespread over the past decade. Chemoproteomics platforms have emerged to complement covalent ligand screening and assist in ligand discovery, selectivity profiling and target identification. This Review showcases covalent drug discovery milestones with emphasis on the lessons learned from these programmes and how an evolving toolbox of covalent drug discovery techniques facilitates success in this field.

Systematic exploration of privileged warheads for covalent kinase drug discovery

Zhao, Z.; Bourne, P. E. ChemRxiv 2022.

https://doi.org/10.26434/chemrxiv-2022-nlb0m

Kinase-targeted drug discovery for cancer therapy has advanced significantly in the last three decades. Currently, diverse kinase inhibitors or degraders have been reported, such as allosteric inhibitors, covalent inhibitors, macrocyclic inhibitors, and PROTAC degraders. Out of these, covalent kinase inhibitors (CKIs) have been attracting attention due to their enhanced selectivity and exceptionally strong affinity. Eight covalent kinase drugs have been FDA approved thus far. Here, we review current developments in CKIs. We explore the characteristics of the CKIs: the features of nucleophilic amino acids and the preferences of electrophilic warheads. We provide systematic insights into privileged warheads for repurposing to other kinase targets. Finally, we discuss trends in CKI development across the whole proteome. 

Lysine-Targeted Reversible Covalent Ligand Discovery for Proteins via Phage Display

Mengmeng Zheng, Fa-Jie Chen, Kaicheng Li, Rahi M. Reja, Fredrik Haeffner, and Jianmin Gao

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.2c07375

Binding via reversible covalent bond formation presents a novel and powerful mechanism to enhance the potency of synthetic inhibitors for therapeutically important proteins. Work on this front has yielded the anticancer drug bortezomib as well as the antisickling drug voxelotor. However, the rational design of reversible covalent inhibitors remains difficult even when noncovalent inhibitors are available as a scaffold. Herein, we report chemically modified phage libraries, both linear and cyclic, that incorporate 2-acetylphenylboronic acid (APBA) as a warhead to bind lysines via reversible iminoboronate formation. To demonstrate their utility, these APBA-presenting phage libraries were screened against sortase A of Staphylococcus aureus, as well as the spike protein of SARS-CoV-2. For both protein targets, peptide ligands were readily identified with single-digit micromolar potency and excellent specificity, enabling live-cell sortase inhibition and highly sensitive spike protein detection, respectively. Furthermore, our structure–activity studies unambiguously demonstrate the benefit of the APBA warhead for protein binding. Overall, this contribution shows for the first time that reversible covalent inhibitors can be developed via phage display for a protein of interest. The phage display platform should be widely applicable to proteins including those involved in protein–protein interactions.

Covalent Proteomimetic Inhibitor of the Bacterial FtsQB Divisome Complex

Felix M. Paulussen, Gina K. Schouten, Carolin Moertl, Jolanda Verheul, Irma Hoekstra, Gregory M. Koningstein, George H. Hutchins, Aslihan Alkir, Rosa A. Luirink, Daan P. Geerke, Peter van Ulsen, Tanneke den Blaauwen, Joen Luirink, and Tom N. Grossmann

Journal of the American Chemical Society 2022

DOI: 10.1021/jacs.2c06304

The use of antibiotics is threatened by the emergence and spread of multidrug-resistant strains of bacteria. Thus, there is a need to develop antibiotics that address new targets. In this respect, the bacterial divisome, a multi-protein complex central to cell division, represents a potentially attractive target. Of particular interest is the FtsQB subcomplex that plays a decisive role in divisome assembly and peptidoglycan biogenesis in E. coli. Here, we report the structure-based design of a macrocyclic covalent inhibitor derived from a periplasmic region of FtsB that mediates its binding to FtsQ. The bioactive conformation of this motif was stabilized by a customized cross-link resulting in a tertiary structure mimetic with increased affinity for FtsQ. To increase activity, a covalent handle was incorporated, providing an inhibitor that impedes the interaction between FtsQ and FtsB irreversibly. The covalent inhibitor reduced the growth of an outer membrane-permeable E. coli strain, concurrent with the expected loss of FtsB localization, and also affected the infection of zebrafish larvae by a clinical E. coli strain. This first-in-class inhibitor of a divisome protein–protein interaction highlights the potential of proteomimetic molecules as inhibitors of challenging targets. In particular, the covalent mode-of-action can serve as an inspiration for future antibiotics that target protein–protein interactions.

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.

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

https://doi.org/10.1038/s41589-022-01065-9

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.

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

doi: https://doi.org/10.1101/2022.05.10.491316

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.