A study headed by researchers at the Centre for Genomic Regulation (CRG) in Barcelona has provided for the first time evidence that a single drug, already licensed for medical use, can stabilize nearly all mutated versions of a human protein, regardless of where the mutation is in the sequence. The researchers, Taylor Mighell, PhD, a postdoctoral researcher at the Centre for Genomic Regulation (CRG) in Barcelona, and Ben Lehner, PhD, ICREA Research Professor, group leader at the Wellcome Sanger Institute and CRG, reported on their work in Nature Structural & Molecular Biology, in a paper titled “A small molecule stabilizer rescues the surface expression of nearly all missense variants in a GPCR.”
They say that their study results provide proof-of-principle that small molecule binding can rescue destabilizing variants throughout a protein’s structure, and that applying their principle to other proteins could enable the development of new treatments for different types of rare diseases.
Rare genetic diseases pose “a formidable challenge for global health,” Mighell and Lehner stated. A rare disease is any disease affecting fewer than one in 2,000 people. Though individual prevalence is low, there are thousands of different types, meaning around 300 million people worldwide live with a rare condition, the team suggested.
Most rare diseases are caused by mutations in DNA. The same gene can be mutated in many ways, so patients with “the same” rare disease can have different mutations driving the condition. As the investigators further pointed out, “Despite recent progress in computational methods, the identification of causal pathogenic variants and the determination of molecular mechanisms remains an arduous challenge.”
Because few individuals will have the same mutation, drug development is slow and commercially unattractive. Most treatments help manage symptoms rather than tackling the root cause of a rare disease. “… developing effective therapies for genetic diseases for which only a small number of patients carry each causal variant is extremely challenging,” the researchers pointed out.
V2R is a G-protein-coupled receptor (GPCR), one of the human body’s largest family of receptors. These roughly 800 genes are the targets of about a third of all approved drugs. Many rare and common diseases arise when GPCRs don’t fold or traffic correctly to the cell surface, even though their signaling parts are largely intact.
V2R is critical for normal kidney function, and mutations in V2R that impact on its function prevent kidney cells from responding to the hormone arginine vasopressin (AVP), resulting in an inability to concentrate urine, which causes excessive thirst and large volumes of dilute urine.
This ultimately leads to nephrogenic diabetes insipidus (NDI), also known as arginine vasopressin resistance, a rare disease affecting roughly one in 25,000 people. “Individuals with NDI experience chronic dehydration that can lead to severe clinical outcomes, and treatment options are available only to manage symptoms.”
The gene encoding V2R is known as AVPR2. “Hundreds of AVPR2 variants have been found in individuals with NDI, of which about half are missense variants; remainder are nonsense, small insertions or deletions or splice-site mutations,” Mighell and Lehner continued. But only a fraction of the missense mutations have been characterized experimentally.
The most frequent mechanism by which missense variants cause rare diseases is reduced protein abundance, they further commented. One promising therapeutic avenue for treating reduced abundance variants is the use of pharmacological chaperones (PCs; also known as correctors or stabilizers). These are small molecules that bind to and stabilize target proteins. “PCs have been approved as clinical treatments for specific variants, but protein energetics suggest their effects might be much more general,” the team noted.
For their reported study they engineered seven thousand versions of the vasopressin V2 receptor (V2R), creating all possible mutated variants in the lab. “First, we use a multiplexed assay to quantify the effects of all possible variants on the cell surface expression of V2R, revealing that more than half the known pathogenic variants strongly impair V2R expression, as do thousands of other missense variants throughout the protein.” Their data showed that more than half of NDI variants are poorly expressed, “… highlighting loss of stability as the major pathogenic mechanism.”
When the team carried out further experiments looking specifically at mutations observed in patients, they found that the oral medicine tolvaptan, which is clinically approved for other kidney conditions, restored receptor levels to near normal for 87% of destabilized mutations (60 out of 69 known disease-causing mutations, and 835 out of 965 predicted disease-causing mutations).
“Inside the cell, V2R travels through a tightly managed traffic system. Mutations cause a jam, so V2R never reaches the surface,” Mighell explained. “Tolvaptan steadies the receptor for long enough to allow the cell’s quality control system to wave it through.”
The research group has previously shown that most mutations affect a protein’s function by altering its stability, making the whole structure wobblier than normal. “Reduced expression due to impaired fold stability is the predominant mechanism by which missense variants cause disease, including in both soluble and membrane proteins,” the authors wrote.
They suggest that tolvaptan works regardless of where the mutation is because proteins switch between folded and unfolded forms. Most V2R mutations make the unfolded form more likely. When tolvaptan binds to V2R, it favors the folded form over the unfolded one.
The research is the first proof-of-principle study to demonstrate that a drug can act like a “nearly universal” pharmacological chaperone, meaning it can latch onto a protein and stabilize the structure regardless of where it’s mutated, in this case, in nearly nine out of ten cases. The findings could help tackle a longstanding challenge in rare disease medicine. “Previous experimental and computational approaches estimate that 40–60% of pathogenic variants are explained by loss of stability or abundance (which is in line with our findings here), suggesting a broad scope for PC therapy,” the investigators noted. “Such general PCs will not have to bind to specific sites in a protein, and they will not need to be tailored to each pathogenic variant.”
If future studies confirm the rescued receptors work normally, the study offers a new roadmap for rare-disease drug development. Rather than look for a drug that targets a single mutation, researchers could instead look for one that targets stabilizing an entire protein.
“If the behavior we found holds for other members of GPCR family, drug developers could swap spending years of hunting for bespoke therapeutic molecules and try looking for general or universal pharmacological chaperones instead, greatly accelerating the drug development pipeline for many genetic diseases,” concluded Lehner. “The variants that are not rescued by a PC can be rapidly identified by selection and sequencing experiments and excluded from clinical trials,” the authors further pointed out. “This approach to identifying changes in abundance that are not rescued by small-molecule binding is a potentially very general strategy to rapidly identify drug-binding sites in proteins.”
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