Covalent fragments vs Rgl2

Just over a year ago the FDA granted accelerated approval to sotorasib, the first marketed inhibitor of KRAS and the first approved fragment-derived covalent drug. In a recent ChemMedChem paper, Samy Meroueh and colleagues at Indiana University School of Medicine describe their efforts against a protein in a related pathway.

 

KRAS is a GTPase which cycles between an “on” state, where GTP is bound, and an “off” state, where GTP is hydrolyzed to GDP. KRAS is just one member of a superfamily of GTPases. Two other members also associated with cancer include RalA and RalB. Sotorasib acts by binding to a mutant form of KRAS in which a glycine is replaced by a cysteine, but this mutation does not occur in RalA or RalB. An alternative approach to targeting GTPases is to prevent them from becoming activated by guanine exchange factors (GEFs), which help exchange GDP to GTP. We’ve previously written about how fragments have led to noncovalent inhibitors of the GEF SOS1, which activates RAS proteins.

 

To sum up, there’s more than one way to block GTPase activity: directly, or by preventing activation by an associated GEF. The new paper focuses on Rgl2, a GEF that serves RalA and RalB.

 

Rgl2 sports four surface-exposed cysteine residues, so the researchers screened the protein against a library of 1260 electrophilic fragments at 75 µM for 24 hours at 4 °C and then assessed whether it could still activate RalB. 50 fragments inhibited guanine nucleotide exchange by at least 30%, and a dozen were studied in detail. All were time-dependent inhibitors and had EC₅₀ values from 2.6 to 120 µM at 24 hours.

 

Next, the researchers mutated each of the four surface-exposed cysteine residues to serine. The twelve fragments still inhibited all the mutants except C284S. SOS1 does not contain a cysteine at the position corresponding to C284, and indeed none of the twelve fragments significantly inhibited SOS1 activation of KRAS. All this suggests the fragments act via modification of C284.

 

The easiest and most direct measurement of covalent binding is with intact protein mass spectrometry, and the researchers confirmed that 10 of the 12 fragments did in fact form adducts. Interestingly, Rgl2 was modified two or three times by each fragment, which is perhaps not surprising given that they had relatively reactive warheads (chloroacetamides or propiolamides). Mass-spec studies with the mutants revealed that most of the modifications were at C284 and C508.

 

Whether or not these fragments are advanceable, the discovery that modification of C284 inhibits Rgl2 is useful. Interestingly, C284 is near but not at the Ral binding interface, and the researchers suggest that their fragments block protein activity allosterically. I believe such allosteric sites are common throughout the proteome, and readily addressable using covalent approaches. Watch this space!