Physical and Functional Analysis of the Putative Rpn13 Inhibitor RA190

Paige Dickson, Daniel Abegg, Ekaterina Vinogradova, Junichiro Takaya, Hongchan An, Scott Simanski, Benjamin F. Cravatt, Alexander Adibekian, Thomas Kodadek

Cell Chem. Biol., 2020

DOI: https://doi.org/10.1016/j.chembiol.2020.08.007

Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13. We find no evidence that this is the case. In vitro, RA190 is has no measurable effect on any of the known interactions of Rpn13. In cellulo, we see no physical engagement of Rpn13 by RA190, either on C88 or any other residue. However, chemical proteomics experiments in two different cell lines reveal that dozens of other proteins are heavily engaged by RA190. Finally, increasing or reducing the level of Rpn13 in HeLa and melanoma cells had no effect on the sensitivity of HeLa or melanoma cells to RA190. We conclude that Rpn13 is not the physiologically relevant target of RA190.

Selective N-Terminal Cysteine Protein Modification with Cyclopropenones [@gbernardes_chem]

Istrate, A.; Navo, C. D.; Sousa, B. B.; Marques, M. C.; Deery, M.; Bond, A.; Corzana, F.; Jiménez-Osés, G.; Bernardes, G.

ChemRxiv. 2020
https://doi.org/10.26434/chemrxiv.12866873.v1

Protein conjugates are valuable tools to create therapeutics, such as antibody-drug conjugates, or to study biological processes. Despite a number of protein conjugation strategies having been developed over recent years, the ability to modify one specific amino acid on a protein in the presence of other side chains with similar reactivity remains a challenge. We used the reaction between a monosubstituted cyclopropenone (CPO) probe and the 1,2-aminothiol of an N-terminal cysteine to give a stable 1,4-thiazepa-5-none linkage under mild, biocompatible conditions. The method relies on the ability of cyclopropenones to ring-open after sequential sulfhydryl and α-amine conjugation to be truly specific for N-terminal cysteine. We show that our CPO probes selectively label N-terminal cysteine containing peptides and proteins even in the presence of internal, solvent-exposed cysteines, which can be subsequently modified by using conventional cysteine modification reagents. The ability to distinguish and specifically target N-terminal cysteine residues on a protein will facilitate the construction of elaborate multi-labelled bioconjugates.

An Irreversible Inhibitor to Probe the Role of Streptococcus pyogenes Cysteine Protease SpeB in Evasion of Host Complement Defenses

Jordan L. Woehl, Seiya Kitamura, Nicholas Dillon, Zhen Han, Landon J. Edgar, Victor Nizet, and Dennis W. Wolan

ACS Chemical Biology 2020 15 (8), 2060-2069

DOI: 10.1021/acschembio.0c00191

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, 2S-alkyne, based on X-ray crystallographic analysis and structure–activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.

The Missing Link between (Un)druggable and Degradable KRAS

Elena De Vita, Maria Maneiro, and Edward W. Tate

ACS Central Science Article ASAP

DOI: 10.1021/acscentsci.0c00920

More than four decades since their first identification, small monomeric guanosine triphosphatases (GTPases) remain among the most studied oncogenic proteins. Three GTPases in the rat sarcoma viral oncogene (RAS) family, KRAS, NRAS, and HRAS, are activated by “gain-of-function” mutations in up to 25% of all cancers, driving proliferative signaling to support tumor growth.(1) Their flat topology and lack of a clear ligand binding site (with the exception of the undruggable GTP pocket) has given rise to a wide range of indirect strategies to target RAS proteins with varying degrees of success (Figure 1A).(1) Mutant KRAS remains among the hottest targets in oncology, and concerted efforts to target oncogenic KRASG12C culminated in 2019–2020 with the discovery of allosteric covalent inhibitors that attack the nucleophilic cysteine residue, which is present uniquely in this specific KRAS mutant. The work of numerous academic and industry teams ultimately delivered four KRASG12C covalent inhibitors currently in clinical trials for cancer.(2) Among these, Mirati Therapeutics’ drug candidate MRTX849 (Figure 1A) has shown promising results and tolerability in patients affected by nonsmall cell lung cancer (NSCLC) and colorectal cancer (CRC) driven by KRASG12C mutant.(3) Nevertheless, the search for improved strategies continues, and in the current issue, the Crews laboratory has used MRTX849 as a warhead in the first cell-active PROteolysis TArgeting Chimera (PROTAC) against KRASG12C.(4) This work elegantly illustrates the differentiating opportunities and rational design challenges of covalent PROTACs, and delivers striking insights into the trade-offs of inhibition versus degradation.