Fragments in the clinic: AMG 510

Few cancer targets are as prominent as KRAS, a molecular switch that has been called “the beating heart of cancer.” Mutations that cause the switch to be stuck in the “on” state occur in roughly a quarter of human tumors. The protein’s role in driving cancer has been known for nearly forty years, but for most of that time it has been considered undruggable.

Until now.

When bound to GTP, KRAS is turned on, promoting cell proliferation until the GTP is hydrolyzed to GDP. Activating mutants impair this hydrolysis. An obvious approach to targeting KRAS would be to develop small molecules that bind in the nucleotide-binding pocket, as has been done successfully for kinases. Unfortunately, the extremely high affinity of KRAS for GTP, along with the high intracellular concentration of GTP, makes this impossible.

One activating mutant replaces a glycine with cysteine (G12C), providing a convenient handle for covalent inhibitors. In 2013, Kevan Shokat and colleagues at University of California San Francisco described in Nature how they used Tethering to identify fragments such as 6H05 that bound to this cysteine through a disulfide bond. Because disulfides are not stable in cells, they replaced this “warhead” with an acrylamide moiety and made a few other tweaks to arrive at compound 12. Extensive optimization described by Yi Liu and collaborators at Kura, Wellspring, and Janssen in a 2018 Cell paper led to molecules such as ARS-1620, which showed potent biochemical and cell-based activity and promising results in mouse xenograft models.

In 2014, Carmot Therapeutics began a collaboration with Amgen to discover covalent inhibitors of the G12C mutant of KRAS. Carmot’s technology, Chemotype Evolution, entails rapid synthesis and testing of large libraries around an existing molecule such as a fragment (see here for a nice animation). In this case, fragments chosen included simple acrylamides, the thought being that – unlike the disulfides in Tethering – these could be carried through into the final molecule.

Initial hit 2 was truncated and cyclized to compound 4, which had comparable activity but with fewer atoms and rotatable bonds. Additional iterations of Chemotype Evolution and medicinal chemistry ultimately led to compound 1, with nanomolar activity in cells containing KRAS^G12C. Crystallography revealed that the tetrahydroisoquinoline moiety bound in a previously cryptic pocket formed by movement of a histidine side chain. These interactions contributed to the high affinity of the molecule; more details are provided in a paper published last year in ACS Med. Chem. Lett. by Victor Cee, myself, and our collaborators at Amgen and Carmot.

Unfortunately, although compound 1 was more potent in cell assays than ARS-1620, it had low oral bioavailability and rapid clearance in mice and rats. However, as described in J. Med. Chem., Brian Lanman and colleagues at Amgen superimposed ARS-1620 with compound 1 and realized that it would be possible to access the cryptic pocket from the former compound. This strategy proved successful, ultimately leading to AMG510, which entered the clinic in August 2018. And although it is still early, a paper in Nature by Jude Canon, J. Russell Lipford, and collaborators at Amgen and elsewhere describes promising responses to the drug by a handful of patients with non-small-cell lung carcinoma.

Amgen is not the only company to have capitalized on the Tethering results: Mirati used the information to develop their clinical-phase MRTX849. And Janssen has also entered the clinic with JNJ-74699157.

There are multiple lessons here. First, as we’ve seen previously, a single fragment can lead to multiple clinical compounds. Second, progress often requires considerable changes to the initial fragment. This story is clearly a case of fragment-assisted drug discovery, and in the interest of space I’ve had to omit most of the lovely medicinal chemistry, not to mention biology and biophysics, detailed in these five papers.

Another lesson is that covalent fragments can enable lead discovery for targets not accessible through other means. But this enablement may require non-conventional molecules; the initial fragments violate the rule of three, and the disclosed clinical compounds have molecular weights in excess of 500 Da. Despite these challenges, remarkably rapid progress is possible: less than five years elapsed between the first Nature publication and the entry of AMG 510 into the clinic.

Most important, this research has led to possibly life-extending molecules – one of the responders described in the second Nature paper had been on drug for 42 weeks. Practical Fragments wishes her or him, and everyone else involved with the trials, the best of luck.