Fragments vs RhoDGI2: Towards a chemical probe

Many readers of this blog will be familiar with KRAS, a mutant form of which was successfully targeted a few years ago by a covalent, fragment-derived drug, sotorasib. KRAS is just one member of a large family of molecular switches which are on when bound to GTP and off when bound to GDP. This exchange is facilitated or inhibited by other proteins, including guanine nucleotide dissociation inhibitors (GDIs). GDIs bind to the GDP-form of RAS proteins, keeping them in the off state, but they can also stabilize Ras proteins against proteasomal degradation, keeping them around longer.

 

RhoGDI2 is a GDI that regulates Rho GTPases, which are involved in multiple cell pathways. The biology is complicated though, and RhoGDI2 has been implicated as both a cancer driver and inhibitor. Clearly a chemical probe would be useful. In a new ACS Chem. Biol. paper, Wei He and collaborators at Tsinghua University and University of Science and Technology of China Hefei report the first steps.

 

The story begins with a 2017 paper in Biochim. Biophys. Acta. Gen. Subj. by Ke Ruan (one of the authors of the new paper) and colleagues. A ligand-detected NMR screen of just under 1000 fragments yielded 14 hits, three of which were confirmed by two-dimensional protein-observed NMR. Further experiments suggested these bound in the hydrophobic pocket that binds gerarnylgeranylated Rho GTPases. Compounds 1 and 2, though weak, became the starting points for fragment growing.

 

Borrowing from compounds 1 and 2 and adding a phenyl moiety led to compound 2102, which was crystallographically confirmed to bind in the substrate binding pocket. Further fragment-growing, guided by structure-based design, ultimately led to HR3119, with low micromolar affinity for RhoGDI2 as assessed by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). HR3119 has four diastereomers, and those with an R-configuration at the benzylic position (6R) were almost 100-more potent than 6S.

 

HR3119 blocked the interaction of RhoGDI2 with the Rho GTPase Rac1 in cell lysates. (6R)-HR3119 stabilized RhoGDI2 in a cellular thermal shift assay, while (6S)-HR3119 did not. (6R)-HR3119 also decreased migratory activity of a cancer cell line, consistent with the role of RhoGDI2 in actin dynamics. However, (6S)-HR3119 also showed activity in this assay, albeit at a higher concentration, suggesting off-target effects.

 

The biochemical and cell activity are still too weak to nominate (6R)-HR3119 as a chemical probe against RhoGDI2; ideally biochemical activity should be better than 100 nM and cell activity should be better than 1 µM. Nonetheless, this is a good starting point for further optimization, and a nice example of fragment-based lead discovery in academia.