Rescuing the Function of Missense-Mutated Tumor Suppressor VHL using Stabilizing Small Molecules

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed some of comments raised in the previous round of review and have opted to proceed to a Version of Record without additional review.]

Summary:

This is an excellent and strong paper. The authors not only show the mechanisms of action of destabilizing mutations in VHL, but notably, they also go on to computationally design and experimentally test an inhibitor that restores wild-type pVHL function, offering starting points for a new class of kidney cancer drugs. The approach that the authors take here can be used to target destabilizing mutations in repressor proteins, common in diseases, including cancer.

Strengths:

This paper is the culmination of an extraordinary amount of work, over years, including method development and testing by a broad range of tools and experiments. It is thorough and comprehensive. It is also well-written and easy to follow.

Reviewer #2 (Public review):

Summary:

Inactivating VHL mutations are common in clear cell renal cell carcinoma, and about half of those mutations unfold/destabilize the protein rather than directly interfering with critical protein-protein interactions. The authors identify a compound that can stabilize/refold mutant VHL and seemingly restore its ability to downregulate its major downstream targets.

Strengths:

The authors use a clever combination of virtual and cell-based screens, followed by suitable biophysical and cell-based validation assays, to arrive at a VHL refolder. This compound is suboptimal from an ADME point of view, but could be a starting point for further medicinal chemistry optimization. Success would have implications for other diseases linked to similar loss-of-function mutations.

Weaknesses:

In going from CP4 to CP4.29 the authors screened based on downregulation of HIF. This is logical but also introduces the danger of identifying chemicals that can downregulate HIF in an "off-target" manner i.e. non-specifically. It therefore essential to clearly show that CP4.29 downregulates steady-state levels of HIF and HIF target genes in cells with suitable (hydrophobic core) VHL mutants but not in isogenic cells lacking VHL.

Author response:

The following is the authors’ response to the original reviews.

We are most grateful to both reviewers for providing valuable feedback on our manuscript.

Reviewer 1 had solely favorable comments, with no suggestions for revision.

Reviewer 2 pointed out that experiment evaluating the effect of CP4 on pVHL half-life (originally included as Figure 3c) was difficult to evaluate because of CP4’s effect on pVHL abundance prior to cycloheximide treatment. We agree with this assessment, and we opted to remove this experiment from the revised manuscript since it was not central to our overarching conclusions.

Reviewer 2 also pointed out that experiment evaluating the effect of CP4.29 on HIF-2α half-life (originally included as Figure 4g) was not very compelling. We agree with this assessment, and we opted to remove this experiment from the revised manuscript since it was not central to our overarching conclusions.

We agree with Reviewer 2’s suggestion that additional experiments could further solidify that C4.29 downregulates HIF2 in a purely “on-target” manner, however we prefer to reserve such studies for the future.

Reviewer 2 also made several valuable suggestions for the text itself (awkward wordings / citations / clearer figure legends). We appreciate this feedback and have updated the text accordingly.