One Fluorine Atom Fixes Poor KRAS Inhibitors

KRAS is the most frequently mutated oncogene in human cancer. In the past few decades, the KRAS oncoprotein had always been deemed as an “undruggable target” due to lack of binding surface and tightly binding to its substrate GTP. In 2013, the Shokat group identified that the mutant cysteine KRAS^G12C creates a new allosteric pocket “switch-II pocket” which can be exploited to design covalent inhibitors. KRAS^G12C accounts for more than 50% of the incidences of KRAS mutations, involving in many cancers, such as non-small cell lung cancer (NSCLC), colorectal adenocarcinomas and pancreatic cancer. KARS^G12C has been an attractive target for drug discovery and development in both academia and industry.

MRTX849 is a potent, orally available covalent inhibitor of KRAS^G12C developed by Mirati Therapeutics and currently undergoing Phase I/II clinical trials. Recently, a paper published in Journal of Medicinal Chemistry reported the design and optimization of MRTX849. The medicinal chemistry optimization started with the previous reported covalent KRAS^G12C inhibitor compound 1, which showed potent inhibition in cells and desirable activity in vivo. However, it is not an ideal clinical candidate due to its poor pharmacokinetics. The metabolites of compound 1 in mouse hepatocytes indicated that the hydroxyl and acrylamide are the two major metabolic liabilities. Removal of the hydroxyl group in 1 led to compound 7 with lower clearance and higher bioavailability. Unfortunately, the potency of compound 7 in cells dropped significantly (IC₅₀ = 4400 nM) due to the loss of hydrogen bonding interactions.

To improve the potency, further modifications of compound 7 were performed to pursue additional interactions with the protein. A series of substituents were explored on the piperazine ring to displace the bound water molecule that interacts with Gly10 and Thr58. Compound 12a showed 400-fold more potency (IC₅₀ = 10 nM) compared with compound 7. Additionally, the negative inductive effect of the -CH₂CN group slightly increases the reactivity of the acrylamide warhead, a favorable feature for covalent inhibitors.

The co-crystal of compound 12 and KRAS^G12C further revealed a hydrophobic pocket towards the 8-position of the naphthyl group. Based on this observation, a series of small hydrophobic groups were explored to fill this cleft and an additional 10-fold potency improvement was achieved with compounds 18 and 19. However, both of them still have a very high clearance measured in dogs and predicted in humans, even higher than then liver blood flow, suggesting that the extrahepatic metabolism might be responsible for this high clearance. Subsequently, the researchers found out that GSH covalent conjugation to the acrylamide warhead, mediated by GST or its isoforms, is the major metabolic pathway for 18 and 19. To address this problem, MRTX849 containing a fluorine substituted acrylamide was finally developed, which can significantly reduce the GSH conjugation while maintain pretty good potency (IC₅₀ = 5-14 nM). It’s interesting that fluoro-substituted acrylamide shows less reactivity towards GSH.

MRTX849 also exhibited high selectivity towards KRAS^G12C in cells and moderate to high oral bioavailability of 26-63% across species. Last but not least, MRTX849 is capable of inducing durable and complete tumor regression after 16 days of treatment in a mouse model.

So far, the success of targeting KRAS^G12C with covalent inhibitor MRTX849 is remarkable and promising, which will prompt researchers to develop novel small molecules targeting not only KARS but also other “undruggable” targets.

Reference

1. Ostrem J. M., et.al. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature  2013, 503, 548–551.
2. Fell, J. B., et.al. Discovery of tetrahydropyridopyrimidines as irreversible covalent inhibitors of KRAS-G12C with in vivo activity. ACS Med. Chem. Lett. 2018, 9, 1230-1234.
3. Fell, J. B.,et.al. Identification of the Clinical Development Candidate MRTX849, a Covalent KRAS^G12CInhibitor for the Treatment of Cancer J. Med. Chem. 2020, 10.1021/acs.jmedchem.9b02052