Courtesy of Michael Conn
Above are the loci for all known naturally occurring mutants of the human gonadotropin releasing hormone receptor (GnRHR) identified from patients with hypogonadotropic hypogonadism (HH). The vast majority of point mutations can be functionally rescued through the use of a pharmacological chaperone. Some others had not been attempted. Twelve mutations identified in yellow letters result in modest charge changes. A smaller number result in other changes to the tertiary structure. Also shown for reference is Lys191 (red circle), an amino acid present in primates, but not in rodents.
Gene therapy has been a major focus for research into treating a number of diseases caused by receptor protein mutations. But a potentially more direct therapy is emerging with the discovery of small molecules that restore correct function to mutant receptor proteins, without attempting to change the primary sequence.
"This is going to be an...
RESTORING PROPER FOLDING
The therapeutic potential of such mutant-receptor rescue techniques has already been demonstrated with human cells in vitro and in animal models. Michael Conn and colleagues at the Oregon National Primate Research Center in Beaverton have shown how all but two of 15 known mutations causing HH in humans, plus other deliberately engineered mutations, could be rescued in vitro by a small nonpeptide molecule called IN3.1
HH delays sexual development and is characterized by abnormally low levels of sex steroids and gonadotropin hormone. The disease results from mutations to the gonadotropin-releasing receptor (GnRHR), causing it to be misrouted so that it can never perform its binding function. This leads to depressed hormone production.
By correcting the misfolding directly rather than changing the underlying sequence, normal routing of the protein can be restored, says Conn. "This molecule serves as a nucleus, promoting correct folding, and therefore the appropriate and stable positioning of otherwise defective mutants in the plasma membrane," he explains.
The stabilizing molecule IN3 was originally designed as a GnRH antagonist. "It was chosen based on hydrophobicity and size, allowing it to enter cells," says Conn. But an antagonist must be removed from a receptor in order for it then to perform, and this can be done in the case of IN3, Conn says. Furthermore, he adds, cell cultures have indicated a lack of toxicity, suggesting that IN3 use should not cause side effects.
That normal binding was restored through the action of IN3, even though the mutations were at loci widely scattered across the GnRHR protein's sequence, suggests that the receptors were still basically competent. Conn and his colleagues deduced that the action of the molecule is nonspecific to any particular mutation and, therefore, the approach could potentially be used to treat other diseases caused by misfolding proteins.
Allen Spiegel, director of the National Institute of Diabetes & Digestive & Kidney Diseases, agrees that specific molecules might be capable of curing more than one disease, suggesting as an example a treatment for cystic fibrosis. Curcumin, an antioxidant found in turmeric, alters the calcium influx to the endoplasmic reticulum (ER), thereby preventing calcium-dependent chaperone binding of the misfolded but still functional cystic fibrosis transmembrane conductance regulator. Recent work shows that curcumin allows misfolded proteins to be transported correctly to their destination in the case of cystic fibrosis.2 Spiegel points out that other diseases, caused by similar calcium-dependent retention of misfolded proteins in the ER, might be amenable to the same treatment.
Misfolded proteins cannot always be rescued, though. Conn and his team examined a spectrum of site-directed mutations in the human GnRHR and found that substitutions resulting in replacement of an amino acid by one whose electron cloud volume is significantly larger tended to be less capable of rescue. The more bulky amino acids presumably restricted the steric freedom of the protein molecule, inhibiting correct folding, says Conn. But even in these cases, increasing the concentration of the IN3 rescue agent could often restore correct folding.
The work already done on mutant receptor rescue suggests that such research may lead to powerful generic therapies for diseases considered largely untreatable. Jeff Kelly, professor of chemistry at Scripps Research Institute, La Jolla, Calif., agrees: "I think that's quite possible."
Philip J. Hunter
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