Fragment Optimization of Reversible Binding to the Switch II Pocket on KRAS Leads to a Potent, In Vivo Active KRASG12C Inhibitor

Joachim Bröker, Alex G. Waterson, Chris Smethurst, Dirk Kessler, Jark Böttcher, Moriz Mayer, Gerhard Gmaschitz, Jason Phan, Andrew Little, Jason R. Abbott, Qi Sun, Michael Gmachl, Dorothea Rudolph, Heribert Arnhof, Klaus Rumpel, Fabio Savarese, Thomas Gerstberger, Nikolai Mischerikow, Matthias Treu, Lorenz Herdeis, Tobias Wunberg, Andreas Gollner, Harald Weinstabl, Andreas Mantoulidis, Oliver Krämer, Darryl B. McConnell, and Stephen W. Fesik

Journal of Medicinal Chemistry 2022

DOI: 10.1021/acs.jmedchem.2c01120

Activating mutations in KRAS are the most frequent oncogenic alterations in cancer. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRASG12C inhibitors. To date, KRASG12C inhibitors have been discovered by first covalently binding to the cysteine at position 12 and then optimizing pocket binding. We report on the discovery of the in vivo active KRASG12C inhibitor BI-0474 using a different approach, in which small molecules that bind reversibly to the switch II pocket were identified and then optimized for non-covalent binding using structure-based design. Finally, the Michael acceptor containing warhead was attached. Our approach offers not only an alternative approach to discovering KRASG12C inhibitors but also provides a starting point for the discovery of inhibitors against other oncogenic KRAS mutants.

Aryl Fluorosulfate Based Inhibitors That Covalently Target the SIRT5 Lysine Deacylase

Julie E. Bolding, Pablo Martín-Gago, Nima Rajabi, Luke F. Gamon, Tobias N. Hansen, Christian R. O. Bartling, Kristian Strømgaard, Michael J. Davies, Christian A. Olsen

 Angew. Chem. Int. Ed. 2022, e202204565

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ-N-carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate-based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne-tagged affinity-labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase

Battisti, U..M., Gao, C., Nilsson, O., Akladios, F., Lulla, A., Bogucka, A., Nain-Perez, A., Håversen, L., Kim, W., Boren, J., Hyvönen, M., Uhlen, M., Mardinoglu, A. and Grøtli, M. 

ChemBioChem, 2022 

https://doi.org/10.1002/cbic.202200339

Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 = 0.29 µM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.

Covalent Protein Inhibitors via Tyrosine Conjugation with Cyclic Imine Mannich Electrophiles

Wang, S.; Hadisurya, M.; Tao, W. A.; Dykhuizen, E.; Krusemark, C. ChemRxiv 2022. https://doi.org/10.26434/chemrxiv-2022-tvgn1

Targeted covalent inhibitors (TCIs) have increased in popularity among drug candidates and chemical probes. Among current TCIs, the chemistry employed is largely limited to labeling cysteine and lysine side chains. Tyrosine is an attractive residue for TCIs due to its enrichment at protein-protein interfaces. Here, we investigate the utility of cyclic imine Mannich electrophiles as covalent warheads to specifically target a pro-tein tyrosine adjacent to an inhibitor binding pocket. We characterized the intrinsic reaction rates of several cyclic imines to tyrosine and identified the iminolactone to be suitable for a covalent inhibitor (second order rate constant of 0.0029 M-1 s-1). We appended the cyclic imine warheads to a CBX8 chromodomain inhibitor to label a non-conserved tyrosine, which markedly improves both the potency and selectivity of the inhibitor for CBX8 in vitro and in cells. These results indicate that Mannich electrophiles are promising and robust chemical warheads for tyrosine bioconjugation and covalent inhibitors.

Recent advances in the development of covalent inhibitors

Hyunsoo Kim,   Yoon Soo Hwang,   Mingi Kima  and  Seung Bum Park

RSC Med. Chem., 2021, 12, 1037-1045

https://doi.org/10.1039/D1MD00068C

The use of covalent inhibitors in the field of drug discovery has attracted considerable attention in the 2000s. As a result, more than 50 covalent drugs are currently on the market, and numerous covalent drug candidates are now under development. Therefore, interest in covalent drugs is expected to continue in the future. The purpose of this focused review is to provide an understanding of the development of covalent inhibitors by describing their inherent characteristics, possibilities, and limitations based on their mechanistic differences from noncovalent inhibitors. We also introduce the latest covalent warheads that can be applied to the development of potential covalent inhibitors.

Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization

Yue Liu, Rick C. Betori, Joanna Pagacz, Grant B. Frost, Elena V. Efimova, Ding Wu, Donald J. Wolfgeher, Tracy M. Bryan, Scott B. Cohen, Karl A. Scheidt, Stephen J. Kron

Cell Chemical Biology, 2022

https://doi.org/10.1016/j.chembiol.2022.09.002

Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT’s non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT’s role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.

Genetically encoded chemical crosslinking of RNA in vivo

Wei Sun, Nanxi Wang, Hongjiang Liu, Bingchen Yu, Ling Jin, Xingjie Ren, Yin Shen & Lei Wang 

Nature Chemistry, 2022

https://doi.org/10.1038/s41557-022-01038-4

Protein–RNA interactions regulate RNA fate and function, and defects can lead to various disorders. Such interactions have mainly been studied by nucleoside-based UV crosslinking methods, which lack broad in vivo compatibility and the ability to resolve specific amino acids. In this study we genetically encoded latent bioreactive unnatural amino acids into proteins to react with bound RNA by proximity-enabled reactivity and demonstrated genetically encoded chemical crosslinking of proteins with target RNA (GECX-RNA) in vivo. Applying GECX-RNA to the RNA chaperone Hfq in Escherichia coli identified target RNAs with amino acid specificity. Combining GECX-RNA with immunoprecipitation and high-throughput sequencing of an N6-methyladenosine reader protein in mammalian cells allowed the in vivo identification of unknown N6-methyladenosine on RNA with single-nucleotide resolution throughout the transcriptome. GECX-RNA thus affords resolution at the nucleotide and amino acid level for interrogating protein–RNA interactions in vivo. It also enables the precise engineering of covalent linkages between a protein and RNA, which will inspire innovative solutions for RNA-related research and therapeutics.