Biophysical Requirements of Pharmacological Chaperones

The success of a pharmacological chaperone depends critically on biophysical properties that dictate affinity, specificity, and reversibility for its target, as well as cell permeability. With respect to permeability, it is imperative that the pharmacological chaperone penetrate both the plasma and ER membranes to bind and stabilize the newly synthesized protein during biosynthesis, thus facilitating ER export. With respect to affinity and specificity, the chaperone must bind the mutant form of the GPCR at concentrations that can be achieved in cells and tissues after administration of clinically relevant doses, as the magnitude of ligand-mediated rescue is correlated with binding affinity [22] . In addition, the concentration of pharmacological chaperone required to bind and stabilize the mutated GPCR must be relatively low in order to avoid offtarget interactions with other proteins. With respect to reversibility, pharmacological chaperones that are antagonists or partial agonists must be able to dissociate quickly from the receptor. In the case of molecules with high affinity and/or slow receptor off rates, there would be a risk that prolonged binding could interfere with the interaction of the rescued receptor with its endogenous hormone or neurotransmitter, thereby minimizing therapeutic benefit. Receptor agonists are also capable of promoting trafficking of mutant receptors to the cell surface. Indeed, for GPCRs such as the 8-opioid receptor, membrane- permeable agonists have been found to act as pharmacological chaperones [225]. However, functional rescue may be countered by their ability to activate the receptor and subsequently elicit internalization and desensitization, diminishing overall long--erm efficacy. Partial agonists may address the issues of rapid activation and subsequent desensitization but, like antagonists, could inhibit binding of the natural ligand. Hence, reversible, moderate -affinity competitive antagonists or partial agonists with a limited propensity to induce internalization and desensitization may provide the best opportunity for clinical efficacy. As discussed above, low-affinity pharmacological chaperones that target P23H opsin have been identified. These pharmacological chaperones can be displaced by 11-cis-retinal after introduction to the culture media, thus restoring mutant rhodopsin functionality -n vitro [137] - In the case of the NDI clinical trial, SR49059 was chosen as a tool for proof of concept because it has a 10-fold lower affinity for the V2R than the other V2R pharmacological chaperones that were tested -n vitro [62]. This lower affinity was seen as an advantage in the initial trial, and it remains to be seen if high-affinity V2R antagonists will be as efficacious as the lower affinity SR49059.

Lastly, noncompetitive allosteric modulators may offer an alternative to competitive ligands. As discussed above, prolonged incubation with the allosteric agonist NPS-568 increased the levels of cell surface wild-type and mutant CaR, most likely by favoring an active, more stable conformation, with reduced ubiquitination and premature degradation - 193] - These results support the hypothesis that activation of the CaR can facilitate trafficking through the secretory pathway, that is, receptors in an active conformation are more stable than those in an inactive conformation, allowing passage through the ER quality control system and avoidance of ERAD. As such, it could be envisioned that mutant forms of the CaR, with point mutations in the calcium-binding ECD, could be stabilized with allosteric agonists that favor an active conformation of the receptor in the calcium-rich environment of the ER. This strategy might also prove useful in the case of point mutations that affect the ability of the endogenous ligand to interact with the rescued cell surface receptor. Proof of concept for this approach with other mutant GPCRs with large ECDs remains to be established.

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