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.
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