Chaperones to the Rescue

Image by Joel Ito and P. Michael Conn The first clinical trials to test protein misfolding therapies are so new that researchers haven't yet agreed on a collective name for the compounds being administered. Variously dubbed chemical chaperones, pharmacological chaperones, and pharmacoperones, these small molecules correct the misfolding of proteins that recent research has implicated in a host of diseases, both rare and prevalent. In such "conformational" diseases, misfolded proteins may lose

Steve Bunk
Nov 10, 2002
Image by Joel Ito and P. Michael Conn

The first clinical trials to test protein misfolding therapies are so new that researchers haven't yet agreed on a collective name for the compounds being administered. Variously dubbed chemical chaperones, pharmacological chaperones, and pharmacoperones, these small molecules correct the misfolding of proteins that recent research has implicated in a host of diseases, both rare and prevalent.

In such "conformational" diseases, misfolded proteins may lose their function and prematurely degrade. Or they can aggregate, resulting in a toxic gain of function that characterizes neurodegenerative conditions. So far, the best therapeutic progress with pharmacological chaperones has been in loss-of-function diseases. Promising results have been achieved in a small clinical trial to treat nephrogenic diabetes insipidus, and recruitment is under way of patients with emphysema and chronic liver disease, conditions that can derive from the same misfolded protein. Encouraging in vitro results have been reported for cystic fibrosis, Fabry disease, hypercholesterolemia, and the aggregation of prions in spongiform encephalopathy. In mice, the mutant p53 tumor-suppressor protein has been successfully treated. Potential also exists to correct misfolding in retinitis pigmentosa, sickle cell disease, thalassemia, cataracts, and hypertrophic cardiomyopathy.1 Researchers express hope that the approach will one day offer an alternative to antibody treatments and gene therapy.

The latest published evidence that the technique works in vitro comes from research by Oregon Health and Science University physiology and pharmacology professor P. Michael Conn and colleagues. They rescued 11 of 14 receptor mutations known to cause hypogonadotropic hypogonadism, characterized by deficiencies in sexual development.1,2 These mutations are widely dispersed throughout the receptor protein for gonadotropin-releasing hormone (GnRH), which is central to regulation of reproductive function. Of the 14 receptor mutations, 13 have a single substitution in the normal 328 amino acid configuration. The researchers coaxed most of the mildly mutated proteins to fold and function properly by treating them with a synthetic antagonist to GnRH.

Conn's work shows that the antagonist can be removed after the correctly folded protein reaches the cell surface and the receptor will function normally, as measured by its participation in activating the production of inositol phosphate and release of intracellular calcium. This suggests that the drug need not interact at the same site as the native ligand; it can stabilize the protein allosterically. He therefore thinks of the pharmacoperone as a scaffolding or template for folding rather than as a competitive antagonist.

CELLULAR OSMOLYTES The use of antagonists to help receptors fold correctly was preceded by ground-clearing work from William J. Welch, a professor of medicine at the University of California, San Francisco. In the mid-1990s, his lab demonstrated that low molecular weight compounds called cellular osmolytes can correct receptor protein misfolding caused by a single amino acid deletion in the cystic fibrosis transmembrane conductance regulatory (CFTR) gene, the disease's most common mutation.3 Cellular osmolytes, which include sugars and polyols, can accumulate and somehow reverse the effects of mutations when cells are subjected to osmotic shock. Welch and colleagues prompted a fold similar enough to that of the native state to prevent rejection by quality-control apparatus in the endoplasmic reticulum (ER), thereby permitting the protein's movement from the ER to the cell surface.

He explains that the chemical chaperone diffuses into the cell and is thought to bind to the mutant receptor while the protein is folding, driving the energy slope toward the native state. For example, a hydrophobic region might be unable to interact with a hydrophilic mutant. The antagonist can create a thermodynamic barrier that allows proper enfolding of hydrophobic regions within the protein, while water-loving regions are exposed. The chaperone also might block incorrect folding, which it apparently senses. The protein then may have a chance to refold correctly (although this has never been demonstrated in vivo) or will be targeted for degradation.

Welch identifies a challenge to therapeutic rescue of receptor proteins. "There's a lot of excitement, but how important are receptor numbers, even in the wild-type situation?" He explains that in such diseases, the percentage of normal receptor proteins that mature can be relatively low. For instance, three-quarters of normal CFTR protein undergoes proteolysis before reaching the cell surface. This has been ascribed to misfolding but might also be due to regulatory effects on CFTR protein, which functions as a chloride channel. The cell may produce more channel than it needs to stave off death in the event of an osmotic challenge. Disruption of such regulation through pharmacological rescue could be toxic. Welch's group is now testing the hypothesis that, in response to extracellular chloride, wild-type CFTR protein maturation will increase.

Unfortunately, the demonstrated corrective effects of cellular osmolytes on misfolding are nonspecific, and the concentrations necessary to affect folding in vivo would be unacceptably high. A response to this problem came in 2000 from University of Montreal biochemist Michel Bouvier. His lab used a synthetic antagonist to rescue eight receptor protein misfoldings in nephrogenic diabetes insipidus, in which improper reabsorption of water in the kidneys leads to various metabolic disorders.4 Now, he and nephrologist Daniel G. Bichet at Sacre-Coeur Hospital in Montreal have conducted the first clinical trial of a pharmacological chaperone to treat a conformational disease. Results were good, and the paper is being considered for publication in a prestigious journal.

Image by Joel Ito and P. Michael Conn

PBA TRIAL UPCOMING The potential of chemical chaperones to treat chronic liver disease and emphysema has been established by David H. Perlmutter, pediatrics chairman at Children's Hospital of Pittsburgh.5 Both diseases can be caused by misfolding of the alpha-1-antitrypsin (alpha-1-AT) inhibitor. When the mutant protein is retained in the ER of liver cells rather than secreted into the blood and body fluids, it is thought to become toxic to the liver. Its depletion in the lung is thought to cause emphysema via a failure to block an enzyme that hydrolyzes the connective tissue elastin.

Recruitment is now under way by University of Florida professor of medicine Mark L. Brantly and colleagues to clinically test 4-phenylbutyric acid (PBA), a drug that Perlmutter showed was effective on mice transgenic for the human alpha-1-AT gene. PBA has been safely administered to children with disorders of the urea cycle, and therefore can bypass early phases of the drug approval process. The drawback is that PBA is nonspecific.

"I don't feel that we have a great idea of what the mechanism is," Brantly acknowledges. "It may be promoting binding of chaperones to the misfolded proteins rather than binding itself." Others, he points out, have found that PBA upregulates CFTR expression. Brantly hopes that the trial of about 15 patients, representing both diseases, will help to resolve the questions about mechanism.

Liver and lung injuries also pose measurement problems for drug developers. Perlmutter notes that patients with liver disease vary enormously in the rate of progress and severity of the illness, and emphysema can take three decades to develop. "Clinical effectiveness testing will be a complex problem," he warns.

In misfolding diseases that involve receptor proteins, antagonists are not the only likely pharmacological chaperones. Early this year, Bouvier and colleagues showed that both antagonists and agonists of opioid receptors can increase their trafficking from the ER to the plasma membrane, which has implications for pain therapy.6 He says the only stipulation is that the ligand must be lipophilic to penetrate the plasma and ER membranes. The disadvantage in using a high-affinity antagonist is that it will remain bound to the receptor for some time after it reaches the cell surface, complicating the pharmacokinetics. The trouble with an agonist is it can undergo rapid desensitization, curtailing its effectiveness. Bouvier thinks that compounds with partial agonistic activity could be "magic bullets" for misfolding diseases.

In any case, he notes that pharmaceutical giants aren't likely to undertake the effort and expense involved in screening for new compounds to treat diseases that are rare or occur primarily among the poor. Indeed, the drug in his trial came from a pharmaceutical company that had used it for another application. As Welch puts it, "The clever groups will set up assays that are user-friendly to screen already-FDA-approved compounds." Conn is now exploring opportunities to screen such libraries. "You can bet that pharmaceutical archives are absolutely full of molecules that have utility but have been passed over," he enthuses. Perlmutter adds, "My thinking is that companies will go for the overall strategy of developing chemical chaperones."

Image by Joel Ito and P. Michael Conn

For treatment of misfolding that causes aggregation--as in Alzheimer, Parkinson, and Creutzfeldt-Jakob diseases--the first challenge will be to design effective assays. For example, the most advantageous point to intervene with a compound in such diseases is uncertain. Researchers still aren't sure which is more toxic, the initial aggregation of misfolded proteins or the fibrils that form later.

Stephen M. Massa, a neurologist at the Veteran's Affairs Medical Center in San Francisco and UC-San Francisco, thinks that the chaperone approach to treating neurodegenerative diseases is, in principle, "not too much of a stretch." But he cautions, "There are no great ways to modulate the level of stress response proteins without causing a stress, which may have some untoward consequences for the tissue." There's no evidence yet, he adds, that such treatment would work on neurodegeneration over the long term.

Bouvier is optimistic about the future of pharmacological chaperones. He thinks that once proof of principle is firmly established, "There certainly will be therapeutics." But he reiterates, "We'll have to go with drugs that already exist."

Steve Bunk ( is a contributing editor.

1. P.M. Conn et al., "Protein origami: Therapeutic rescue of misfolding gene products," Molecular Interventions, 2:308-16, Sept. 1, 2002.

2. A. Leanos-Miranda et al., "Receptor-misrouting: an unexpectedly prevalent and rescuable etiology in gonadotropin-releasing hormone receptor-mediated hypogonadotropic hypogonadism," Journal of Clinical Endocrinology and Metabolism, 87:4825-8, October 2002.

3. C.R. Brown et al., "Chemical chaperones correct the mutant phenotype of the delta F508 cystic fibrosis transmembrane conductance regulator protein," Cell Stress Chaperones, 1:117-25, 1996.

4. J.-P. Morello et al., "Pharmacological chaperones rescue cell-surface expression and function of misfolded VA vasopressin receptor mutants," Journal of Clinical Investigation, 105:887-95, 2000.

5. J.A. Burrows et al., "Chemical chaperones mediate increased secretion of mutant alpha 1-antitrypsin (alpha 1-AT) Z: a potential pharmacological strategy for prevention of liver injury and emphysema in alpha 1-AT deficiency," Proceedings of the National Academy of Sciences, 97:1796-801, 2000.

6. U.E. Petaja-Repo et al., "Ligands act as pharmacological chaperones and increase the efficiency of delta opioid receptor mutations," EMBO Journal, 21:1628-37, 2002.