Discovery of a DCAF11-dependent cyanoacrylamide-containing covalent degrader of BET-proteins

Gary Tin, Marko Cigler, Matthias Hinterndorfer, Kevin D. Dong, Hana Imrichova, Steven P. Gygi, Georg E. Winter,

Bioorganic & Medicinal Chemistry Letters, 2024

https://doi.org/10.1016/j.bmcl.2024.129779

Targeted protein degradation is mediated by small molecules that induce or stabilize protein–protein interactions between targets and the ubiquitin–proteasome machinery. Currently, there remains a need to expand the repertoire of viable E3 ligases available for hijacking. Notably, covalent chemistry has been employed to engage a handful of E3 ligases, including DCAF11. Here, we disclose a covalent PROTAC that enables DCAF11-dependent degradation, featuring a cyanoacrylamide warhead. Our findings underscore DCAF11 as an interesting candidate with a capacity to accommodate diverse electrophilic chemistries compatible with targeted protein degradation.

Inclusive Pattern Generation Protocols to Decode Thiol-Mediated Uptake

Saidbakhrom Saidjalolov, Filipe Coelho, Vincent Mercier, Dimitri Moreau, and Stefan Matile

ACS Central Science 2024 10 (5), 1033-1043

DOI: 10.1021/acscentsci.3c01601

Thiol-mediated uptake (TMU) is an intriguing enigma in current chemistry and biology. While the appearance of cell-penetrating activity upon attachment of cascade exchangers (CAXs) has been observed by many and is increasingly being used in practice, the molecular basis of TMU is essentially unknown. The objective of this study was to develop a general protocol to decode the dynamic covalent networks that presumably account for TMU. Uptake inhibition patterns obtained from the removal of exchange partners by either protein knockdown or alternative inhibitors are aligned with original patterns generated by CAX transporters and inhibitors and patterns from alternative functions (here cell motility). These inclusive TMU patterns reveal that the four most significant CAXs known today enter cells along three almost orthogonal pathways. Epidithiodiketopiperazines (ETP) exchange preferably with integrins and protein disulfide isomerases (PDIs), benzopolysulfanes (BPS) with different PDIs, presumably PDIA3, and asparagusic acid (AspA), and antisense oligonucleotide phosphorothioates (OPS) exchange with the transferrin receptor and can be activated by the removal of PDIs with their respective inhibitors. These findings provide a solid basis to understand and use TMU to enable and prevent entry into cells.

Targeted anticancer pre-vinylsulfone covalent inhibitors of carbonic anhydrase IX

Aivaras Vaškevičius, Denis Baronas, Janis Leitans, Agnė Kvietkauskaitė, Audronė Rukšėnaitė, Elena Manakova, Zigmantas Toleikis, Algirdas Kaupinis, Andris Kazaks, Marius Gedgaudas, Aurelija Mickevičiūtė, Vaida Juozapaitienė, Helgi B Schiöth, Kristaps Jaudzems, Mindaugas Valius, Kaspars Tars, Saulius Gražulis, Franz-Josef Meyer-Almes, Jurgita Matulienė, Asta Zubrienė, Virginija Dudutienė, Daumantas Matulis

bioRxiv 2024

doi: https://doi.org/10.1101/2024.05.20.594908

The CA IX is a transmembrane protein, highly overexpressed in hypoxic solid tumors, important for cancer cell survival and proliferation because it acidifies tumor microenvironment helping invasion and metastases processes. We designed novel compounds that have several functionalities: 1) primary sulfonamide group recognizing carbonic anhydrases (CA), 2) high-affinity moieties specifically recognizing CA IX among all CA isozymes, and 3) forming a covalent bond with the His64 residue. Such targeted covalent compounds possess both high affinity and selectivity for the disease target protein followed by complete irreversible inactivation of the protein via covalent modification. Our designed prodrug candidates bearing moderately active pre-vinyl sulfone esters or weakly active carbamates optimized for mild covalent modification activity to avoid toxic non-specific modifications and selectively target CA IX. The lead inhibitors reached 2 pM affinity, highest among known CA IX inhibitors. The strategy could be used for any disease drug target protein bearing a His residue in the vicinity of the active site.

Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors

 José O. Castellón, Samuel Ofori, Nikolas R. Burton, Ashley R. Julio, Alexandra C. Turmon, Ernest Armenta, Carina Sandoval, Lisa M. Boatner, Evan E. Takayoshi, Marina Faragalla, Cameron Taylor, Ann L. Zhou, Ky Tran, Jeremy Shek, Tianyang Yan, Heta S. Desai, Oliver I. Fregoso, Robert Damoiseaux, and Keriann M. Backus

Journal of the American Chemical Society 2024

DOI: 10.1021/jacs.3c12240

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.

Unanticipated mechanisms of covalent inhibitor and synthetic ligand cobinding to PPARγ

Jinsai Shang, Douglas J. Kojetin

bioRxiv 2024

 doi: https://doi.org/10.1101/2024.05.15.594037

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous lipids and synthetic ligands, including covalent antagonist inhibitors such as GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent antagonist inhibitors weaken—but do not prevent—the binding of other synthetic ligands via an allosteric mechanism rather than direct ligand clashing. The covalent ligands shift the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with and reduces the orthosteric binding affinity of non-covalent synthetic ligands. Crystal structures reveal different non-covalent synthetic ligand-specific cobinding mechanisms ranging from alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent ligand binding pose. Our findings not only highlight the significant flexibility of the PPARγ orthosteric pocket and its ability to accommodate multiple ligands simultaneously, but also demonstrate that GW9662 and T0070907 should not be used as reliable chemical tools to inhibit the binding of other ligands to PPARγ.

Targeting PfCLK3 with Covalent Inhibitors: A Novel Strategy for Malaria Treatmen

Jamieson, A.; Brettell, S.; Jahna, O.; Begen, A.; Cann, G.; Olaniyan, N.; Sharma, S.; Yelland, T.; Hole, A.; Alam, B.; Gillespie, R.; Capper, M.; Milligan, G.; Clarke, D.; Tobin, A. 

ChemRxiv 2024

https://doi.org/10.26434/chemrxiv-2024-d62kp

Malaria continues to pose a significant global health threat, with the number of deaths exceeding 600,000 annually. Acquired resistance to frontline drugs by the most deadly parasite, Plasmodium falciparum, means this number is increasing each year. There is therefore an urgent unmet need for new medicines with novel mechanisms of action. In this work, we solved the co-crystal structure of the essential malarial kinase PfCLK3 with the reversible inhibitor TCMDC-135051 1. This facilitated the rational design of covalent inhibitors of this validated drug target. An allosteric cysteine residue (Cys368) that is poorly conserved in the human kinome was targeted to improve the selectivity of hit molecules. Structure-based drug design yielded chloroacetamide 4, which displays low nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum killing assays. Efficacy in parasites was maintained when 4 was washed out 6 hours after exposure. Compound 4 showed significantly improved kinase selectivity relative to TCMDC-135051 1, and cell viability experiments in HepG2 cultures also demonstrated an over 500-fold selectivity index relative to P. falciparum parasites. To our knowledge, compound 4 represents the first covalent inhibitor of a malarial kinase. Its covalency, efficacy and selectivity for PfCLK3 makes it a promising lead in the search for a single-dose cure for malaria.

DCAF16-Based Covalent Handle for the Rational Design of Monovalent Degraders

Melissa Lim, Thang Do Cong, Lauren M. Orr, Ethan S. Toriki, Andrew C. Kile, James W. Papatzimas, Elijah Lee, Yihan Lin, and Daniel K. Nomura

ACS Cent. Sci. 2024, 

https://doi.org/10.1021/acscentsci.4c00286

Targeted protein degradation with monovalent molecular glue degraders is a powerful therapeutic modality for eliminating disease causing proteins. However, rational design of molecular glue degraders remains challenging. In this study, we sought to identify a transplantable and linker-less covalent handle that could be appended onto the exit vector of various protein-targeting ligands to induce the degradation of their respective targets. Using the BET family inhibitor JQ1 as a testbed, we synthesized and screened a series of covalent JQ1 analogs and identified a vinylsulfonyl piperazine handle that led to the potent and selective degradation of BRD4 in cells. Through chemoproteomic profiling, we identified DCAF16 as the E3 ligase responsible for BRD4 degradation─an E3 ligase substrate receptor that has been previously covalently targeted for molecular glue-based degradation of BRD4. Interestingly, we demonstrated that this covalent handle can be transplanted across a diverse array of protein-targeting ligands spanning many different protein classes to induce the degradation of CDK4, the androgen receptor, BTK, SMARCA2/4, and BCR-ABL/c-ABL. Our study reveals a DCAF16-based covalent degradative and linker-less chemical handle that can be attached to protein-targeting ligands to induce the degradation of several different classes of protein targets.

Identification and Evaluation of Reversible Covalent Binders to Cys55 of Bfl-1 from a DNA-Encoded Chemical Library Screen

Simon C. C. Lucas, J. Henry Blackwell, Ulf Börjesson, David Hargreaves, Alexander G. Milbradt, Samiyah Ahmed, Mark J. Bostock, Carine Guerot, Andrea Gohlke, Olaf Kinzel, Michelle L. Lamb, Nidhal Selmi, Christopher J. Stubbs, Nancy Su, Qibin Su, Haiou Luo, Ting Xiong, Xiaoqian Zuo, Sana Bazzaz, Corey Bienstock, Paolo A. Centrella, Kyle E. Denton, Diana Gikunju, Marie-Aude Guié, John P. Guilinger, Christopher Hupp, Anthony D. Keefe, Takashi Satoh, Ying Zhang, and Emma L. Rivers

ACS Medicinal Chemistry Letters 2024

DOI: 10.1021/acsmedchemlett.4c00113

Bfl-1 is overexpressed in both hematological and solid tumors; therefore, inhibitors of Bfl-1 are highly desirable. A DNA-encoded chemical library (DEL) screen against Bfl-1 identified the first known reversible covalent small-molecule ligand for Bfl-1. The binding was validated through biophysical and biochemical techniques, which confirmed the reversible covalent mechanism of action and pointed to binding through Cys55. This represented the first identification of a cyano-acrylamide reversible covalent compound from a DEL screen and highlights further opportunities for covalent drug discovery through DEL screening. A 10-fold improvement in potency was achieved through a systematic SAR exploration of the hit. The more potent analogue compound 13 was successfully cocrystallized in Bfl-1, revealing the binding mode and providing further evidence of a covalent interaction with Cys55.

Ophiobolin A Covalently Targets Mitochondrial Complex IV Leading to Metabolic Collapse in Cancer Cells

 Flor A. Gowans, Danny Q. Thach, Zhouyang Zhu, Yangzhi Wang, Belen E. Altamirano Poblano, Dustin Dovala, John A. Tallarico, Jeffrey M. McKenna, Markus Schirle, Thomas J. Maimone, and Daniel K. Nomura

ACS Chemical Biology 2024

DOI: 10.1021/acschembio.4c00064

Ophiobolin A (OPA) is a sesterterpenoid fungal natural product with broad anticancer activity. While OPA possesses multiple electrophilic moieties that can covalently react with nucleophilic amino acids on proteins, the proteome-wide targets and mechanism of OPA remain poorly understood in many contexts. In this study, we used covalent chemoproteomic platforms to map the proteome-wide reactivity of the OPA in a highly sensitive lung cancer cell line. Among several proteins that OPA engaged, we focused on two targets: lysine-72 of cytochrome c oxidase subunit 5A (COX5A) and cysteine-53 of mitochondrial hypoxia induced gene 1 domain family member 2A (HIGD2A). These two subunit proteins are part of complex IV (cytochrome C oxidase) within the electron transport chain and contributed significantly to the antiproliferative activity of OPA. OPA activated mitochondrial respiration in a COX5A- and HIGD2A-dependent manner, leading to an initial spike in mitochondrial ATP and heightened mitochondrial oxidative stress. OPA compromised mitochondrial membrane potential, ultimately leading to ATP depletion. We have used chemoproteomic strategies to discover a unique anticancer mechanism of OPA through activation of complex IV leading to compromised mitochondrial energetics and rapid cell death.

Discovery and Preclinical Characterization of BIIB129, a Covalent, Selective, and Brain-Penetrant BTK Inhibitor for the Treatment of Multiple Sclerosis

Martin K. Himmelbauer, Bekim Bajrami, Rebecca Basile, Andrew Capacci, TeYu Chen, Colin K. Choi, Rab Gilfillan, Felix Gonzalez-Lopez de Turiso, Chungang Gu, Marc Hoemberger, Douglas S. Johnson, J. Howard Jones, Ekta Kadakia, Melissa Kirkland, Edward Y. Lin, Ying Liu, Bin Ma, Tom Magee, Srinivasa Mantena, Isaac E. Marx, Claire M. Metrick, Michael Mingueneau, Paramasivam Murugan, Cathy A. Muste, Prasad Nadella, Marta Nevalainen, Chelsea R. Parker Harp, Vatee Pattaropong, Alicia Pietrasiewicz, Robin J. Prince, Thomas J. Purgett, Joseph C. Santoro, Jurgen Schulz, Simone Sciabola, Hao Tang, H. George Vandeveer, Ti Wang, Zain Yousaf, Christopher J. Helal, and Brian T. Hopkins

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c00220

Multiple sclerosis (MS) is a chronic disease with an underlying pathology characterized by inflammation-driven neuronal loss, axonal injury, and demyelination. Bruton’s tyrosine kinase (BTK), a nonreceptor tyrosine kinase and member of the TEC family of kinases, is involved in the regulation, migration, and functional activation of B cells and myeloid cells in the periphery and the central nervous system (CNS), cell types which are deemed central to the pathology contributing to disease progression in MS patients. Herein, we describe the discovery of BIIB129 (25), a structurally distinct and brain-penetrant targeted covalent inhibitor (TCI) of BTK with an unprecedented binding mode responsible for its high kinome selectivity. BIIB129 (25) demonstrated efficacy in disease-relevant preclinical in vivo models of B cell proliferation in the CNS, exhibits a favorable safety profile suitable for clinical development as an immunomodulating therapy for MS, and has a low projected total human daily dose.

Histidine-Covalent Stapled Alpha-Helical Peptides Targeting hMcl-1

Giulia Alboreggia, Parima Udompholkul, Carlo Baggio, Kendall Muzzarelli, Zahra Assar, and Maurizio Pellecchia

Journal of Medicinal Chemistry 2024

DOI: 10.1021/acs.jmedchem.4c00277

Several novel and effective cysteine targeting (Cys) covalent drugs are in clinical use. However, the target area containing a druggable Cys residue is limited. Therefore, methods for creating covalent drugs that target different residues are being looked for; examples of such ligands include those that target the residues lysine (Lys) and tyrosine (Tyr). Though the histidine (His) side chain is more frequently found in protein binding locations and has higher desirable nucleophilicity, surprisingly limited research has been done to specifically target this residue, and there are not many examples of His-targeting ligands that have been rationally designed. In the current work, we created novel stapled peptides that are intended to target hMcl-1 His 252 covalently. We describe the in vitro (biochemical, NMR, and X-ray) and cellular design and characterization of such agents. Our findings further suggest that the use of electrophiles to specifically target His residues is warranted.