Pharmacological chaperone approaches for rescuing GPCR mutants: Current state, challenges, and screening strategies

Abstract

A substantial number of G-protein coupled receptors (GPCRs) genetic disorders are due to mutations that cause misfolding or dysfunction of the receptor product. Pharmacological chaperoning approaches can rescue such mutant receptors by stabilizing protein conformations that behave similar to the wild type protein. For example, this can be achieved by improving folding efficiency and/or interaction with chaperone proteins. Although efficacy of pharmacological chaperones has been demonstrated in vitro for a variety of GPCRs, translation to clinical use has been limited. In this paper we discuss the history of pharmacological chaperones of GPCR’s and other membrane proteins, the challenges in translation to the clinic, and the use of different assays for pharmacological chaperone discovery.

Introduction

Folding of newly translated polypeptides is a complex and inherently inefficient process, the failing of which results in protein aggregation or degradation instead of formation of functional proteins. Membrane proteins, including G protein-coupled receptors (GPCRs), appear to be particularly prone to misfolding, as a number of studies have demonstrated protein folding efficiencies of 50% or lower [1], [2], [3], [4]. The energetic balance between folding and misfolding can be delicate and easily perturbed due to the relatively small change in free energy between folded and unfolded states [5]. In fact, single nucleotide mutations leading to single amino acid changes can alter energy of folding enough to reduce folding efficiency by several-fold [6]. It is therefore not surprising that a large number of diseases can be traced back to single point mutations. Recently, a study of several thousand missense mutations associated with a spectrum of Mendelian genetic diseases estimated that 28% could be due to poor protein folding and stability [7].

Pathologies due to misfolding can occur in one of two ways: i) reduction of functional protein levels or ii) formation of toxic aggregates. To prevent the maturation of improperly folded receptors and potential aggregation, cells have stringent quality control mechanisms that block protein maturation unless it satisfies certain conditions. Membrane protein quality control is accomplished by a series of cytosolic, endoplasmic reticulum (ER), and Golgi-resident chaperone proteins. Chaperone proteins recognize folding states based on exposed hydrophobic regions, which are uncharacteristic of properly folded membrane proteins, exposure of retention or export motifs, and finally the addition of post-translational modifications such as glycosylation [8]. Proteins that do not properly fold are exported from the ER and degraded by the ER-associated degradation (ERAD) system [9]. Although ER-quality control is essential for maintaining proteostasis, the stringency of ER quality control can lead to ER-retention of misfolded mutants that may otherwise be partially or fully functional. This provides an opportunity for pharmacological intervention by targeting the folding and maturation process. A pharmacological chaperone (pharmacochaperone or pharmacoperone, PC) is a small molecule that selectively binds to a target protein and increases maturation efficiency by stabilizing a favorable conformation that can pass the cell's quality control system [10], [11], [12]. PCs have been demonstrated to rescue mutations in membrane proteins, and have been particularly successful for rescuing GPCRs. GPCRs are flexible proteins that explore a wide range of conformational spaces with multiple energetic minima [13]. Such flexibility makes receptors susceptible to conformational defects due to mutations, but also allows for ligand interactions that stabilize distinct conformations for the same receptor [14]. This is reflected in the wide variety of possible ligand-GPCR interactions, including biased agonism and allosteric modulation. The wealth of GPCR ligands identified through drug development efforts and screening programs provide a large pool of candidate compounds that could act as PCs. Drug screening has also resulted in the development of assays to measure GPCR activity, allowing straightforward screening for ligands that increase mutant receptor function. Another consideration is that GPCR mutations can have a dominant negative phenotype due to receptor dimerization. Improperly folded mutant GPCRs can dimerize with their wild-type (WT) counterparts and result in ER-retention of the dimer/oligomer, as has been shown for the vasopressin 2 receptor (V2R) and the follicle stimulating hormone receptor (FSHR) [15], [16]. In the cases of these dominant negative mutants, rescuing a fraction of misfolded mutant proteins can significantly increase the total amount of functional receptors at the cell surface, as the PC will augment the surface expression of both the mutant and retained wild type protein.

There have been numerous demonstrations of PC approaches in vitro with potential for therapeutic applications, including rescue of V2R for nephrogenic diabetes insipidus, and GnRHR for hypogonadism [1], [17]. However, clinical success of these approaches has thus far been limited (Table 1). In this review we discuss some of the PCs currently identified for GPCRs and other membrane proteins, the hurdles of translating in vitro results to clinical application, and screening approaches for discovering novel PCs.