Development of a covalent cereblon-based PROTAC employing a fluorosulfate warhead [@LynJonesChemBio, @RPNowak]

Radosław P. Nowak, Leah Ragosta, Fidel Huerta, Hu Liu, Scott B. Ficarro, Justin T. Cruite, Rebecca J. Metivier, Katherine A. Donovan, Jarrod A. Marto, Eric S. Fischer, Breanna L. Zerfas and Lyn H Jones

RSC Chem. Biol., 2023
https://doi.org/10.1039/D3CB00103B

Many cereblon (CRBN) ligands have been used to develop proteolysis targeting chimeras (PROTACs), but all are reversible binders of the E3 ubiquitin ligase. We recently described the use of sulfonyl exchange chemistry to design binders that covalently engage histidine 353 in CRBN for the first time. Here we show that covalent CRBN ligands can be used to develop efficient PROTAC degraders. We demonstrate that the fluorosulfate PROTAC FS-ARV-825 covalently labels CRBN in vitro, and in cells the BRD4 degrader is insensitive to wash-out and competition by potent reversible CRBN ligands, reflecting enhanced pharmacodynamics. We anticipate that covalent CRBN-based PROTACs will enhance degradation efficiencies, thus expanding the scope of addressable targets using the heterobifunctional degrader modality.

A covalent inhibitor of the YAP–TEAD transcriptional complex identified by high-throughput screening

Kayla Nutsch, Lirui Song, Emily Chen, Mitchell Hull, Arnab K. Chatterjee, Jian Jeffery Chen and Michael J. Bollong

RSC Chem. Biol., 2023

DOI: https://doi.org/10.1039/D3CB00044C

Yes-associated protein (YAP), the master transcriptional effector downstream of the Hippo pathway, regulates essential cell growth and regenerative processes in animals. However, the activation of YAP observed in cancers drives cellular proliferation, metastasis, chemoresistance, and immune suppression, making it of key interest in developing precision therapeutics for oncology. As such, pharmacological inhibition of YAP by targeting its essential co-regulators, TEA domain transcription factors (TEADs) would likely promote tumor clearance in sensitive tumor types. From a fluorescence polarization-based high throughput screen of over 800 000 diverse small molecules, here we report the identification of a pyrazolopyrimidine-based scaffold that inhibits association of YAP and TEADs. Medicinal chemistry-based optimization identified mCMY020, a potent, covalent inhibitor of TEAD transcriptional activity that occupies a conserved, central palmitoylation site on TEADs.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation [@MichaelBollong]

Lara Ibrahim, Caroline Stanton, Kayla Nutsch, Thu Nguyen, Chloris Li-Ma, Yeonjin Ko, Gabriel C. Lander, R. Luke Wiseman, Michael J. Bollong

Cell Chemical Biology, 2023

https://doi.org/10.1016/j.chembiol.2023.07.014 

Cross talk between metabolism and stress-responsive signaling is essential for maintaining cellular homeostasis. This cross talk is often achieved through covalent modification of proteins by endogenous, reactive metabolites that regulate key stress-responsive transcription factors like NRF2. Metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 repressor KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolism to NRF2 activation. Succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Multi-omic stratification of the missense variant cysteinome [@Keribackus]

Heta S Desai, Samuel Ofori, Lisa M Boatner,  Fengchao Yu, Miranda Villaneuva, Nicholas Ung, Alexey Nesvizhskii, Keriann M Backus

doi: https://doi.org/10.1101/2023.08.12.553095

Cancer genomes are rife with genetic variants; one key outcome of this variation is gain-of-cysteine, which is the most frequently acquired amino acid due to missense variants in COSMIC. Acquired cysteines are both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain uncharacterized. Here, we pair cysteine chemoproteomics, a technique that enables proteome-wide pinpointing of functional, redox sensitive, and potentially druggable residues, with genomics to reveal the hidden landscape of cysteine acquisition. For both cancer and healthy genomes, we find that cysteine acquisition is a ubiquitous consequence of genetic variation that is further elevated in the context of decreased DNA repair. Our chemoproteogenomics platform integrates chemoproteomic, whole exome, and RNA-seq data, with a customized 2-stage false discovery rate (FDR) error controlled proteomic search, further enhanced with a user-friendly FragPipe interface. Integration of CADD predictions of deleteriousness revealed marked enrichment for likely damaging variants that result in acquisition of cysteine. By deploying chemoproteogenomics across 11 cell lines, we identify 116 gain-of-cysteines, of which 10 were liganded by electrophilic druglike molecules. Reference cysteines proximal to missense variants were also found to be pervasive, 791 in total, supporting heretofore untapped opportunities for proteoform-specific chemical probe development campaigns. As chemoproteogenomics is further distinguished by sample-matched combinatorial variant databases and compatible with redox proteomics and small molecule screening, we expect widespread utility in guiding proteoform-specific biology and therapeutic discovery.

Simultaneous Covalent Modification of K-Ras(G12D) and K-Ras(G12C) with Tunable Oxirane Electrophiles

Zhongtang Yu, Xiaoqiang He, Ruiliu Wang, Xinxin Xu, Zhang Zhang, Ke Ding, Zhi-Min Zhang, Yi Tan, and Zhengqiu Li

Journal of the American Chemical Society 2023

DOI: 10.1021/jacs.3c05899

Owing to their remarkable pharmaceutical properties compared to those of noncovalent inhibitors, the development of targeted covalent inhibitors (TCIs) has emerged as a powerful method for cancer treatment. The K-Ras mutant, which is prevalent in multiple cancers, has been confirmed to be a crucial drug target in the treatment of various malignancies. However, although the K-Ras(G12D) mutation is present in up to 33% of K-Ras mutations, no covalent inhibitors targeting K-Ras(G12D) have been developed to date. The relatively weak nucleophilicity of the acquired aspartic acid (12D) residue in K-Ras may be the reason for this. Herein, we present the first compound capable of covalently engaging both K-Ras(G12D) and K-Ras(G12C) mutants. Proteome profiling revealed that this compound effectively conjugates with G12C and G12D residues, modulating the protein functions in situ. These findings offer a unique pathway for the development of novel dual covalent inhibitors.

Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility

Corteselli, E.M., Sharafi, M., Hondal, R. et al. 

Nat Commun 14, 4550 (2023). 

https://doi.org/10.1038/s41467-023-39664-2

Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX’s cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.