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.