Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe

Yejin Jung, Naotaka Noda, Junichiro Takaya, Masahiro Abo, Kohei Toh, Ken Tajiri, Changyi Cui, Lu Zhou, Shin-ichi Sato, and Motonari Uesugi

ACS Chemical Biology 2021

DOI: 10.1021/acschembio.1c00824

Covalent inhibitors of enzymes are increasingly appreciated as pharmaceutical seeds, yet discovering non-cysteine-targeting inhibitors remains challenging. Herein, we report an intriguing experience during our activity-based proteomic screening of 1601 reactive small molecules, in which we monitored the ability of library molecules to compete with a cysteine-reactive iodoacetamide probe. One epoxide molecule, F8, exhibited unexpected enhancement of the probe reactivity for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-limiting glycolysis enzyme. In-depth mechanistic analysis suggests that F8 forms a covalent adduct with an aspartic acid in the active site to displace NAD+, a cofactor of the enzyme, with concomitant enhancement of the probe reaction with the catalytic cysteine. The mechanistic underpinning permitted the identification of an optimized aspartate-reactive GAPDH inhibitor. Our findings exemplify that activity-based proteomic screening with a cysteine-reactive probe can be used for discovering covalent inhibitors that react with non-cysteine residues.

Lysine-Targeting Reversible Covalent Inhibitors with Long Residence Time

Rahi M. Reja, Wenjian Wang, Yuhan Lyu, Fredrik Haeffner, and Jianmin Gao

Journal of the American Chemical Society 2021

DOI: 10.1021/jacs.1c12702

We report a new reversible lysine conjugation that features a novel diazaborine product and much slowed dissociation kinetics in comparison to the previously known iminoboronate chemistry. Incorporating the diazaborine-forming warhead RMR1 to a peptide ligand gives potent and long-acting reversible covalent inhibitors of the staphylococcal sortase. The efficacy of sortase inhibition is demonstrated via biochemical and cell-based assays. A comparative study of RMR1 and an iminoboronate-forming warhead highlights the significance and potential of modulating bond dissociation kinetics in achieving long-acting reversible covalent inhibitors.

Structural insights into the potency and selectivity of covalent pan-FGFR inhibitors

Lingzhi Qu, Xiaojuan Chen, Hudie Wei, Ming Guo, Shuyan Dai, Longying Jiang, Jun Li, Sitong Yue, Zhuchu Chen & Yongheng Chen 

Commun Chem 5, 5 (2022). 

https://doi.org/10.1038/s42004-021-00623-x

FIIN-2, TAS-120 (Futibatinib) and PRN1371 are highly potent pan-FGFR covalent inhibitors targeting the p-loop cysteine of FGFR proteins, of which TAS-120 and PRN1371 are currently in clinical trials. It is critical to analyze their target selectivity and their abilities to overcome gatekeeper mutations. In this study, we demonstrate that FIIN-2 and TAS-120 form covalent adducts with SRC, while PRN1371 does not. FIIN-2 and TAS-120 inhibit SRC and YES activities, while PRN1371 does not. Moreover, FIIN-2, TAS-120 and PRN1371 exhibit different potencies against different FGFR gatekeeper mutants. In addition, the co-crystal structures of SRC/FIIN-2, SRC/TAS-120 and FGFR4/PRN1371 complexes reveal structural basis for kinase targeting and gatekeeper mutations. Taken together, our study not only provides insight into the potency and selectivity of covalent pan-FGFR inhibitors, but also sheds light on the development of next-generation FGFR covalent inhibitors with high potency, high selectivity, and stronger ability to overcome gatekeeper mutations.

A Series of Pyrazole Analogs Binding to KRASG12C as Potential Cancer Treatment

Gerard Rosse

ACS Med. Chem. Lett. 2022, 13, 1, 11–12

https://doi.org/10.1021/acsmedchemlett.1c00643

The compounds of this patent application represent a potential treatment for cancer characterized by KRAS, HRAS, and NRAS GC12 mutation. The compounds have the ability to inhibit the G12C mutant of KRAS, HRAS, and NRAS via the formation of an irreversible bond to cysteine 12. The irreversible binding locks KRAS in an inactivate state and disrupts downstream signaling.

Repurposing the Damage Repair Protein Methyl Guanine Methyl Transferase as a Ligand Inducible Fusion Degron

Gosia M. Murawska, Caspar Vogel, Max Jan, Xinyan Lu, Matthias Schild, Mikolaj Slabicki, Charles Zou, Saule Zhanybekova, Manisha Manojkumar, Georg Petzold, Peter Kaiser, Nicolas Thomä, Benjamin Ebert, and Dennis Gillingham

ACS Chemical Biology 2021

DOI: 10.1021/acschembio.1c00771

We successfully repurpose the DNA repair protein methylguanine methyltransferase (MGMT) as an inducible degron for protein fusions. MGMT is a suicide protein that removes alkyl groups from the O6 position of guanine (O6G) and is thereafter quickly degraded by the ubiquitin proteasome pathway (UPP). Starting with MGMT pseudosubstrates (benzylguanine and lomeguatrib), we first demonstrate that these lead to potent MGMT depletion while affecting little else in the proteome. We then show that fusion proteins of MGMT undergo rapid UPP-dependent degradation in response to pseudosubstrates. Mechanistic studies confirm the involvement of the UPP, while revealing that at least two E3 ligase classes can degrade MGMT depending on cell-line and expression type (native or ectopic). We also demonstrate the technique’s versatility with two clinically relevant examples: degradation of KRASG12C and a chimeric antigen receptor.