Saturday, December 24, 2022

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.

Wednesday, December 21, 2022

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; 


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.

Tuesday, December 20, 2022

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; 


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 are readily extendable to reactive amino acids beyond cysteine, and related questions in chemical biology.

Wednesday, December 14, 2022

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; 


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.

Tuesday, December 13, 2022

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.

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.

Sunday, December 11, 2022

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.

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.

Monday, December 5, 2022

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,

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.

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...