Pharmacoperone Identification for Therapeutic Rescue of Misfolded Mutant Proteins

G protein-coupled receptors (GPCRs), which includes the gonadotropin-releasing hormone (GnRH) receptor (GnRHR), comprises the largest family of validated drug targets – more than half of all approved drugs derive their benefits by selective targeting of GPCRs. Most drugs in this class are either agonists or antagonists of GPCRs and high throughput screens have typically been designed and performed with a view toward identification of such compounds as lead drug candidates. This manuscript presents the case that valuable drugs which effect the trafficking of GPCRs may have been overlooked because pharmacoperones have been selected from existing screens that identify agonists and antagonists. A “gain of activity assay” is proposed; this assay relies on the expression of a mutant of the GnRHR that is known to be rescuable by pharmacoperone drugs, and which is restored to activity in their presence. Accordingly, “hits” are identified by the appearance of activity. The gene for the mutant is under control of tetracycline and may be prevented from being expressed. This is a valuable feature since it allows false positives to be identified. Such drugs will show apparent activity whether or not the mutant is expressed. This assay will enable identification of these drugs from chemical libraries and does not rely on their activity as agonists or antagonists

Gonadotropin and Gonadal Steroid Release in Response to a Gonadotropin-Releasing Hormone Agonist in G_q^ɑ and G_(11)^ɑ Knockout Mice

In this study, we used mice lacking the G_(11)^α[ G_(11) knockout (KO)] or G_q^α gene (G_q KO) to examine LH release in response to a metabolically stable GnRH agonist (Buserelin). Mice homozygous for the absence of G_(11)^α and G_q^α appear to breed normally. Treatment of (5 wk old) female KO mice with the GnRH agonist Buserelin (2 μg/100 μl, sc) resulted in a rapid increase of serum LH levels (reaching 328 ± 58 pg/25 μl for G_(11) KO; 739 ± 95 pg/25 μl for G_q KO) at 75 min. Similar treatment of the control strain, 129SvEvTacfBr for G_(11) KO or the heterozygous mice for G_q KO, resulted in an increase in serum LH levels (428 ± 57 pg/25 μl for G_(11) KO; 884 ± 31 pg/25 μl for G_q KO) at 75 min. Both G_(11) KO and G_q KO male mice released LH in response to Buserelin (2 μg/100 μl of vehicle; 363 ± 53 pg/25μ l and 749 ± 50 pg/25 μl 1 h after treatment, respectively). These values were not significantly different from the control strain. In a long-term experiment, Buserelin was administered every 12 h, and LH release was assayed 1 h later. In female G11 KO mice and control strain, serum LH levels reached approximately 500 pg/25 μl within the first hour, then subsided to a steady level (∼100 pg/25 μl) for 109 h. In male G_(11) KO mice and in control strain, elevated LH release lasted for 13 h; however, LH levels in the G_(11) KO male mice did not reach control levels for approximately 49 h. In a similar experimental protocol, the G_q KO male mice released less LH (531 ± 95 pg/25 μl) after 13 h from the start of treatment than the heterozygous male mice (865 ± 57 pg/25 μl), but the female KO mice released more LH (634 ± 56 pg/25 μl) after 1 h from the start of treatment than the heterozygous female mice (346 ± 63 pg/25 μl). However, after the initial LH flare, the LH levels in the heterozygous mice never reached the basal levels achieved by the KO mice. G_(11) KO mice were less sensitive to low doses (5 ng/per animal) of Buserelin than the respective control mice. Male G_(11) KO mice produced more testosterone than the control mice after 1 h of stimulation by 2 μg of Buserelin, whereas there was no significant difference in Buserelin stimulated testosterone levels between G_q KO and heterozygous control mice. There was no significant difference in Buserelin stimulated estradiol production in the female G_q KO mice compared with control groups of mice. However, female G_(11) KO mice produced less estradiol in response to Buserelin (2 μg) compared with control strain. Although there were differences in the dynamics of LH release and steroid production in response to Buserelin treatment compared with control groups of mice, the lack of complete abolition of these processes, such as stimulated LH release, and steroid production, suggests that these G proteins are either not absolutely required or are able to functionally compensate for each other

Receptor antagonism/agonism can be uncoupled from pharmacoperone activity

Pharmacoperones rescue misrouted mutants of the vasopressin receptor type 2 (V2R) and enable them to traffic to the correct biological locus where they function. Previously, a library of nearly 645,000 structures was interrogated with a high throughput screen; pharmacoperones were identified for V2R mutants with a view toward correcting the underlying mutational defects in nephrogenic diabetes insipidus. In the present study, an orthologous assay was used to evaluate hits from the earlier study. We found no consistent relation between antagonism or agonism and pharmacoperone activity. Active pharmacoperones were identified which had minimal antagonistic activity. This increases the therapeutic reach of these drugs, since virtually all pharmacoperone drugs reported to date were selected from peptidomimetic antagonists. Such mixed-activity drugs have a complex pharmacology limiting their therapeutic utility and requiring their removal prior to stimulation of the receptor with agonist

Gonadotropin and Gonadal Steroid Release in Response to a Gonadotropin-Releasing Hormone Agonist in G_q^ɑ and G_(11)^ɑ Knockout Mice

In this study, we used mice lacking the G_(11)^α[ G_(11) knockout (KO)] or G_q^α gene (G_q KO) to examine LH release in response to a metabolically stable GnRH agonist (Buserelin). Mice homozygous for the absence of G_(11)^α and G_q^α appear to breed normally. Treatment of (5 wk old) female KO mice with the GnRH agonist Buserelin (2 μg/100 μl, sc) resulted in a rapid increase of serum LH levels (reaching 328 ± 58 pg/25 μl for G_(11) KO; 739 ± 95 pg/25 μl for G_q KO) at 75 min. Similar treatment of the control strain, 129SvEvTacfBr for G_(11) KO or the heterozygous mice for G_q KO, resulted in an increase in serum LH levels (428 ± 57 pg/25 μl for G_(11) KO; 884 ± 31 pg/25 μl for G_q KO) at 75 min. Both G_(11) KO and G_q KO male mice released LH in response to Buserelin (2 μg/100 μl of vehicle; 363 ± 53 pg/25μ l and 749 ± 50 pg/25 μl 1 h after treatment, respectively). These values were not significantly different from the control strain. In a long-term experiment, Buserelin was administered every 12 h, and LH release was assayed 1 h later. In female G11 KO mice and control strain, serum LH levels reached approximately 500 pg/25 μl within the first hour, then subsided to a steady level (∼100 pg/25 μl) for 109 h. In male G_(11) KO mice and in control strain, elevated LH release lasted for 13 h; however, LH levels in the G_(11) KO male mice did not reach control levels for approximately 49 h. In a similar experimental protocol, the G_q KO male mice released less LH (531 ± 95 pg/25 μl) after 13 h from the start of treatment than the heterozygous male mice (865 ± 57 pg/25 μl), but the female KO mice released more LH (634 ± 56 pg/25 μl) after 1 h from the start of treatment than the heterozygous female mice (346 ± 63 pg/25 μl). However, after the initial LH flare, the LH levels in the heterozygous mice never reached the basal levels achieved by the KO mice. G_(11) KO mice were less sensitive to low doses (5 ng/per animal) of Buserelin than the respective control mice. Male G_(11) KO mice produced more testosterone than the control mice after 1 h of stimulation by 2 μg of Buserelin, whereas there was no significant difference in Buserelin stimulated testosterone levels between G_q KO and heterozygous control mice. There was no significant difference in Buserelin stimulated estradiol production in the female G_q KO mice compared with control groups of mice. However, female G_(11) KO mice produced less estradiol in response to Buserelin (2 μg) compared with control strain. Although there were differences in the dynamics of LH release and steroid production in response to Buserelin treatment compared with control groups of mice, the lack of complete abolition of these processes, such as stimulated LH release, and steroid production, suggests that these G proteins are either not absolutely required or are able to functionally compensate for each other

Therapeutic Rescue of Misfolded Mutants: Validation of Primary High Throughput Screens for Identification of Pharmacoperone Drugs

Functional rescue of misfolded mutant receptors by small non-peptide molecules has been demonstrated. These small, target-specific molecules (pharmacological chaperones or "pharmacoperones") serve as molecular templates, promote correct folding and allow otherwise misfolded mutants to pass the scrutiny of the cellular quality control system (QCS) and be expressed at the plasma membrane (PM) where they function similarly to wild type (WT) proteins. In the case of the gonadotropin releasing hormone receptor (GnRHR), drugs that rescue one mutant typically rescue many mutants, even if the mutations are located at distant sites (extracellular loops, intracellular loops, transmembrane helices). This increases the value of these drugs. These drugs are typically identified, post hoc, from "hits" in screens designed to detect antagonists or agonists. The therapeutic utility of pharmacoperones has been limited due to the absence of screens that enable identification of pharmacoperones per se.We describe a generalizable primary screening approach for pharmacoperone drugs based on measurement of gain of activity in stable HeLa cells stably expressing the mutants of two different model G-protein coupled receptors (GPCRs) (hGnRHR[E(90)K] or hV2R[L(83)Q]). These cells turn off expression of the receptor mutant gene of interest in the presence of tetracycline and its analogs, which provides a convenient means to identify false positives.The methods described and characterized here provide the basis of novel primary screens for pharmacoperones that detect drugs that rescue GPCR mutants of specific receptors. This approach will identify structures that would have been missed in screens that were designed to select only agonists or antagonists. Non-antagonistic pharmacoperones have a therapeutic advantage since they will not compete for endogenous agonists and may not have to be washed out once rescue has occurred and before activation by endogenous or exogenous agonists

Quality Control Autophagy Degrades Soluble ERAD-Resistant Conformers of the Misfolded Membrane Protein GnRHR

Molecular chaperones triage misfolded proteins via action as substrate selectors for quality control (QC) machines that fold or degrade clients. Herein, the endoplasmic reticulum (ER) associated Hsp40 JB12 is reported to participate in partitioning mutant conformers of GnRHR, a G-protein coupled receptor, between ER-associated degradation (ERAD) and a novel ERQC-autophagy pathway for membrane proteins. ERQC-autophagy degrades E90K-GnRHR because pools of its partially folded and detergent soluble degradation intermediates are resistant to ERAD. S168R-GnRHR is globally misfolded and disposed of via ERAD, but inhibition of p97, the protein retrotranslocation motor, shunts S168R-GnRHR from ERAD to ERQC autophagy. Partially folded and grossly misfolded forms of GnRHR associate with JB12 and Hsp70. Elevation of JB12 promotes ERAD of S168R-GnRHR, with E90K-GnRHR being resistant. E90K-GnRHR elicits association of the Vps34 autophagy initiation complex with JB12. Interaction between ERassociated Hsp40s and the Vps34 complex permits the selective degradation of ERAD-resistant membrane proteins via ERQC-autophagy

Calnexin regulated gonadotropin-releasing hormone receptor plasma membrane expression

A significant proportion of human gonadotropin-releasing hormone receptors (GnRHRs) are normally retained in the endoplasmic reticulum (ER); however, nearly all rat GnRHRs are routed to the plasma membrane. When mutations are introduced into either receptor, considerably more of the proteins are recognized by the quality control system (QCS) as misfolded and retained compared with wild-type (WT) receptor, resulting in decreased signaling in the presence of agonist. Calnexin, a component of the QCS, decreased plasma membrane expression of the GnRHRs, an effect that was mediated by a physical interaction between the receptor and the calnexin. Only the human receptor showed reduced signaling because it had fewer spare receptors compared with the rat GnRHR, allowing calnexin to affect signaling. Calnexin did not affect receptor signaling when K(191) was deleted from the human WT GnRHR. Removal of this amino acid decreases receptor misfolding and increases plasma membrane expression. K(191) is not present in the rat WT GnRHR. A pharmacological chaperone that corrects GnRHR misfolding, increased expression of the human WT GnRHR in the presence of calnexin. Calnexin apparently retains misfolded GnRHRs but routes correctly folded receptors to the plasma membrane. Mutation of a calnexin protein kinase C consensus phosphorylation site promoted increased retention of the human GnRHR, suggesting that calnexin phosphorylation controls the retention mechanism. We conclude that a proportion of the human and the rat WT GnRHR appears to be retained in the ER by calnexin, an effect that decreases GnRHR signaling capacity