Exploring Targeted Degradation Strategy for Oncogenic KRASG12C

Mei Zeng, Yuan Xiong, Nozhat Safaee, Radoslaw P. Nowak, Katherine A. Donovan, Christine J. Yuan, Behnam Nabet, Thomas W. Gero, Frederic Feru, Lianbo Li, Sudershan Gondi, Lincoln J. Ombelets, Chunshan Quan, Pasi A. Jänne, Milka Kostic, David A. Scott, Kenneth D. Westover, Eric S. Fischer Nathanael S. Gray

Cell Chemical Biology, 2019

DOI: https://doi.org/10.1016/j.chembiol.2019.12.006.

KRAS is the most frequently mutated oncogene found in pancreatic, colorectal, and lung cancers. Although it has been challenging to identify targeted therapies for cancers harboring KRAS mutations, KRAS G12C can be targeted by small-molecule inhibitors that form covalent bonds with cysteine 12 (C12). Here, we designed a library of C12-directed covalent degrader molecules (PROTACs) and subjected them to a rigorous evaluation process to rapidly identify a lead compound. Our lead degrader successfully engaged CRBN in cells, bound KRAS G12C in vitro, induced CRBN/KRAS G12C dimerization, and degraded GFP-KRAS G12C in reporter cells in a CRBN-dependent manner. However, it failed to degrade endogenous KRAS G12C in pancreatic and lung cancer cells. Our data suggest that inability of the lead degrader to effectively poly-ubiquitinate endogenous KRAS G12C underlies the lack of activity. We discuss challenges for achieving targeted KRAS G12C degradation and proposed several possible solutions which may lead to efficient degradation of endogenous KRAS G12C.

A chemical proteomic probe for the mitochondrial pyruvate carrier complex

Yamashita, Y., Vinogradova, E., Zhang, X., Suciu, R. and Cravatt, B.

Angew. Chem. Int. Ed.. 2019 
doi:10.1002/anie.201914391

Target engagement assays are crucial for establishing the mechanism‐of‐action of small molecules in living systems. Integral membrane transporters, due to their specialized biophysical properties and activity assays, can present a challenging protein class for assessing cellular engagement by small molecules. Here, we describe the chemical proteomic discovery of alpha‐chloroacetamide (aCA) compounds that covalently modify cysteine‐54 (C54) of the MPC2 subunit of the mitochondrial pyruvate carrier (MPC) complex. We leverage this finding to create an alkyne‐modified aCA, YY4‐yne, that serves as a versatile cellular target engagement probe for MPC2 in click chemistry‐enabled western blotting or global mass spectrometry‐based proteomic experiments. Using YY4‐yne, we demonstrate that UK‐5099, an alpha‐cyanocinnamate inhibitor of the MPC complex, first discovered more than 30 years ago, but still with a poorly defined mechanism‐of‐action, engages MPC2 with remarkable selectivity in human cells. These findings support a model where UK‐5099 inhibits the MPC complex by binding to C54 of MPC2 in a covalent reversible manner that can be quantified in cells using the YY4‐yne probe.

Light-Activatable, 2,5-Disubstituted Tetrazoles for the Proteome-Wide Profiling of Aspartates and Glutamates in Living Bacteria

Kathrin Bach Bert L. H. Beerkens Patrick R. A. Zanon Stephan M. Hacker
ChemRxiv, 2019
doi: 10.26434/chemrxiv.11352101.v1

Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, that do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids

Kinetic Optimization of Lysine-Targeting Covalent Inhibitors of HSP72

Jonathan Pettinger, Michael Carter, Keith Jones, and Matthew D. Cheeseman

Journal of Medicinal Chemistry 2019

DOI: 10.1021/acs.jmedchem.9b01709

The covalent inhibition mechanism of action, which overcomes competition with high-affinity, high-abundance substrates of challenging protein targets, can deliver effective chemical probes and drugs. The success of this strategy has centered on exposed cysteine residues as nucleophiles but the low abundance of cysteine in the proteome has limited its application. We have recently reported our discovery that lysine-56 in the difficult-to-drug target HSP72 could form a covalent bond with a small-molecule inhibitor. We now disclose the optimization of these targeted covalent inhibitors using rational design. Essential to our optimization was the development of a new covalent fluorescence polarization assay, which allows for the direct measurement of the key kinetic parameter in covalent inhibitor design, kinact/KI, extrapolation of the underlying parameters, kinact and Ki, and direct comparison to reversible analogues. Using our approach, we demonstrate a >100-fold enhancement in covalent efficiency and key learnings in lysine-selective electrophile optimization.

Discovery of a covalent inhibitor of KRASG12C (AMG 510) for the treatment of solid tumors

Brian A Lanman, Jennifer R. Allen, John G. Allen, Albert K Amegadzie, Kate S. Ashton, Shon K. Booker, Jian Jeffrey Chen, Ning Chen, Michael J Frohn, Guy Goodman, David J Kopecky, Longbin Liu, Patricia Lopez, Jonathan D Low, Vu Ma, Ana Elena Minatti, Thomas T Nguyen, Nobuko Nishimura, Alexander J. Pickrell, Anthony B. Reed, Youngsook Shin, Aaron Siegmund, Nuria A. Tamayo, Christopher M Tegley, Mary C Walton, Hui-Ling Wang, Ryan P. Wurz, May Xue, Kevin C Yang, Pragathi Achanta, Michael D. Bartberger, Jude Canon, L Steven Hollis, John D McCarter, Christopher Mohr, Karen Rex, Anne Y Saiki, Tisha San Miguel, Laurie Volak, Kevin H Wang, Douglas A. Whittington, Stephan G Zech, J. Russell Lipford, and Victor J. Cee

Journal of Medicinal Chemistry 2019

DOI: 10.1021/acs.jmedchem.9b01180

KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-“undruggable” target, however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in Phase I clinical trials (NCT03600883).