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.