From noncovalent fragment to covalent KRASG12C inhibitor

Last week we highlighted Steve Fesik’s presentation at the Discovery on Target meeting in which he discussed the discovery of a covalent inhibitor of the oncology target KRAS^G12C. The paper describing this work, by Joachim Bröker, Alex Waterson, and collaborators at Boehringer Ingelheim and Vanderbilt University, has just appeared (open access) in J. Med. Chem.

 

A decade ago we described how the Fesik lab reported finding millimolar fragments that bind to the so-called switch I/II pocket on KRAS. In collaboration with researchers at Boehringer Ingelheim, these were optimized to sub-micromolar ligands that block nucleotide exchange (see here). However, these molecules hit all RAS isoforms and show only modest cell activity. In contrast, the approved drug sotorasib binds in a different pocket, called switch II, and forms a covalent bond with an oncogenic cysteine mutation, G12C.

 

To find molecules that would bind in the switch II pocket, the researchers first needed to block the switch I/II pocket, which seems to be a hot spot for fragment binding. They did so by introducing a cysteine mutation near the pocket and linking this via a disulfide to a small fragment. All this was done in the context of KRAS^G12V, a mutant that is more common in cancer than KRAS^G12C. The modified protein was then screened using two-dimensional protein-observed (HSQC) NMR against 13,000 fragments. This process identified 20 fragments that bind outside of the switch I/II pocket, including compound 1, which bound to the modified protein with mid-micromolar affinity.

 

 

A combination of SAR-by-catalog and synthesis suggested the importance of both the amino group and the nitrile, and these observations were confirmed by a crystal structure of compound 1 bound to the protein deep in the switch II pocket, as predicted from the NMR data. The crystal structure also revealed a vector to grow the molecule, leading to compound 12. This molecule had sufficiently high affinity to bind to KRAS^G12V without the introduction of the switch I/II blocking fragment. Further growing to compound 19 and addition of a phenyl group (compound 20b) led to low micromolar binders. Installation of an acrylamide warhead and further decoration led to BI-0474, which rapidly reacted with the mutant cysteine in KRAS^G12C. Interestingly, the initial fragment is carried through unchanged.

 

In addition to potent biochemical activity, BI-0474 showed low nanomolar cell activity. The “bioavailability was not yet optimized,” but intraperitoneal administration led to anti-turmor activity in mouse xenograft models. The paper also notes that “a more advanced orally available analogue from this series has recently entered phase I clinical trials.” As we noted earlier this year, this is BI 1823911.

 

It is worth contrasting this work with the discovery of sotorasib, which we discussed in 2020. Sotorasib traces its origins to covalent fragment screens, and an electrophile was maintained throughout the optimization process. In contrast, the new paper starts with a non-covalent fragment that was optimized before an electrophilic warhead was introduced. This is probably more typical of how covalent drugs are discovered, as exemplified last year for the BTK inhibitor TAK-020. However, it is not necessarily easy; Steve mentioned in his presentation earlier this month that achieving the optimal configuration of the warhead took some effort.

 

KRAS has become a poster child for the power of fragment-based approaches to deliver drugs against previously intractable targets. The fact that the new molecules have good non-covalent affinity broadens the range of ligandable oncogenic mutants beyond KRAS^G12C. Indeed, the researchers end by noting that their approach has led to “molecules that are highly attractive for further use in the discovery of inhibitors against other KRAS mutants.”

 

Let’s hope they – and others – succeed.