Rescuing the Function of Missense-Mutated Tumor Suppressor VHL using Stabilizing Small Molecules
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eLife Assessment
This study presents important findings by identifying small molecules that can stabilize and refold missense-mutated VHL tumor suppressor protein, offering a potential therapeutic approach for clear cell renal cell carcinoma. The computational design approach is well-executed, but the evidence is incomplete due to insufficient demonstration that HIF2 downregulation occurs through on-target VHL rescue rather than off-target effects. Additional experiments with appropriate controls are needed to establish the specificity of the mechanism.
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Abstract
Somatic mutations in the VHL gene, coupled with VHL loss of heterozygosity, drive sporadic clear cell renal cell carcinoma (ccRCC). From structural considerations, we hypothesized that certain mutations in the VHL gene thermodynamically destabilize the folded protein product; these mutations would increase the ratio of unfolded:folded pVHL, and thus cause loss of function. To test this, we used computational structure-based screening followed by biophysical characterization and cellular assays to identify small molecules that bind to the folded (native) conformation of pVHL and stabilize it. These studies led to creation of an agent, CP4.29, that stabilized the native folded structure of mutant pVHL, that restores wild-type pVHL activities to cells that harbor mutant VHL . These compounds may serve as starting points for further development into an unprecedented new class of kidney cancer drugs. The approach described herein may also serve as a blueprint for developing agents to correct destabilized mutations underlying other human diseases.
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eLife Assessment
This study presents important findings by identifying small molecules that can stabilize and refold missense-mutated VHL tumor suppressor protein, offering a potential therapeutic approach for clear cell renal cell carcinoma. The computational design approach is well-executed, but the evidence is incomplete due to insufficient demonstration that HIF2 downregulation occurs through on-target VHL rescue rather than off-target effects. Additional experiments with appropriate controls are needed to establish the specificity of the mechanism.
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Reviewer #1 (Public 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.
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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:
The authors need to …
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:
The authors need to tighten up the evidence that the VHL refolder is downregulating HIF2 in a strictly "on-target" manner.
(1) In Figure 3C, the increase in VHL stability looks very modest. Taking into account the increased abundance of the VHL protein at time 0 in the presence of CP4 compared to control, it is not so clear that VHL is decaying much more slowly in the presence of CP4. I understand that the fact that the signal is low in the absence of CP4 at time 1 hour makes it hard to quantify the half-life of p30 in the absence of the drug (is a longer exposure needed?). However, perhaps the authors could try to quantify the p19 half-life.
(2) 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 is 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. Another prediction is that these chemicals should be inert in cells with VHL mutations that directly abrogate HIF binding. So Figure 4E (HIF2 target genes) needs the use of the isogenic VHL-/- cells described later in the paper. And the steady-state levels of HIF2 should be measured in the isogenic cells (mutant VHL vs -/-) with or without CP4.29. Figure 4G, as it is done now, is too indirect and not very compelling. I don't understand why the "time 0" HIF2 levels aren't lower in the presence of CP4.29, and I think the half-life differences with treatment are very subtle to my eyeball densitometer (although I applaud the authors' attempt to quantify), with the exception of I180N. I agree that Figure 4F is encouraging, but hypoxia has many effects, and this experiment is not as "clean" as the VHL-/- experiments. The same applies to some of the pharmacologic agents in Figure 5.
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