Wednesday, November 23, 2022

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

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

Monday, November 21, 2022

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

J. Med. Chem. 2022

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).


Friday, November 18, 2022

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


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


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.

Monday, November 14, 2022

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.

Thursday, November 10, 2022

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.

Friday, November 4, 2022

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;

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.

Thursday, November 3, 2022

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.

Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry

Roman C. Sarott, Inchul You, Yen-Der Li, Sean T. Toenjes, Katherine A. Donovan, Pooreum Seo, Martha Ordonez, Woong Sub Byun, Muhammad Murtaz...