Cryo-EM-guided subtractive optimization of a novel VCP/p97 inhibitor

We report the cryo-EM structure-guided discovery of GND-135, a novel small-molecule inhibitor of the VCP/p97 AAA ATPase that demonstrates efficient inhibition of VCP/p97 in biochemical, cellular, and pharmacokinetic assays and in a tumor efficacy mouse model of acute myeloid leukemia. Our approach overcomes the liability in the clinical-stage compound CB-5083 where Phase I studies showed off-target activity of CB-5083 for the enzyme PDE6. From the cryo-EM structural analysis of CB-5083 bound to PDE6 and VCP/p97, we identified critical ligand/protein interactions in both proteins and rationally designed a small molecule that retains key interactions necessary for VCP/p97 inhibition while eliminating PDE6 off-target activity. We refer to this approach as `subtractive optimization' because we are leveraging our ability to determine both on-target and off-target cryo-EM structures to guide the medicinal chemistry campaign to enable more targeted compound design. While this strategy is not possible in all cases, the use of cryo-EM to tune on-site binding while eliminating off-target binding could be a generally applicable strategy for informing molecular design and accelerating small-molecule drug discovery.