Receptor antagonism/agonism can be uncoupled from pharmacoperone activity

Spicer, Timothy; Smith, Emery; Bannister, Thomas D.; Kenakin, Terry; Scampavia, Louis; Conn, P. Michael

doi:10.1016/j.mce.2016.07.003

Cited by 11 publications

(7 citation statements)

References 36 publications

(48 reference statements)

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“…SR121463B is a V2R antagonist and known pharmacoperone that was used in the current study, after being generously provided by Dr. Claudine Serradeil at Sanofi-Aventis and used as received. Other pharmacoperones were identified by us by high throughput screening of a large chemical library [ 22 , 23 ]. Several reagents were used as obtained from indicated vendors: 3-Isobutyl-1-methylxanthine (IBMX, Sigma Aldrich, St. Louis, MO), vasopressin (Tocris Biosciences, Bristol, England UK), fetal calf serum (FCS, Hyclone, Logan, UT), Dulbecco’s MEM (DMEM), PBS (GIBCO, Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias

et al. 2017

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Pharmacoperones are small molecules that diffuse into cells and rescue misfolded, mistrafficked protein mutants, restoring their function. These substances act with high target specificity, serving as templates to fold (or refold) receptors, enzymes, ion channels or other proteins and enable them to pass the scrutiny of the cellular quality control system (“rescue”). In the present study we demonstrate that a rescued mutant (L83Q) of the vasopressin type 2 receptor (V2R), shows a strong bias for Gs coupling unlike the WT V2 receptor, which couples to both Gs and Gq/11. Failure of the mutant to couple to Gq/11 was not due to a limiting quantity of G-proteins since other Gq/11-coupled receptors (WT V2R, histamine receptor and muscarinic receptor) responded appropriately to their ligands. Transfection with DNA encoding Gq enabled the V2 receptor mutant to couple to this G protein, but only modestly compared with the WT receptor. Fourteen V2R mutant pharmacoperones, of multiple chemical classes, obtained from a high throughput screen of a 660,000 structure library, and one V2R peptidomimetic antagonist rescues L83Q. The rescued mutant shows similar bias with all pharmacoperones identified, suggesting that the bias is intrinsic to the mutant protein’s structure, rather than due to the chemical class of the pharmacoperone. In the case of V2R mutant Y128S, rescue with a pharmacoperone revealed constitutive activity, also with bias for Gs, although both IP and cAMP were produced in response to agonist. These results suggest that particular rescued receptor mutants show functional characteristics that differ from the WT receptor; a finding that may be important to consider as pharmacoperones are developed as therapeutic agents.

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Section: Methodsmentioning

confidence: 99%

Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias

et al. 2017

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“…When the affinity is too high, the displacement will be difficult, diminishing potential therapeutic utility. Pharmacoperones do not have to be antagonists; agonists and allosteric modulators can also serve as pharmacoperones (158,180,353).…”

Section: Chemical Chaperones As Tools and Potential Therapeutics For Correcting Misfolded Proteins Causing Protein Conformational Diseasementioning

confidence: 99%

“…In the case of screens for two GPCRs, GnRHR (74) and V2R (180,336), the screens employ stable HeLa cell lines expressing the misfolded mutants of these receptors, human GnRHR E 90 K or human V2R Y 128 S, respectively. These mutants are expressed under the control of the tet (tetracycline) (OFF) transactivator.…”

Section: A Design Of Screens For Pharmacoperones For Gpcrsmentioning

confidence: 99%

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Pharmacoperones as Novel Therapeutics for Diverse Protein Conformational Diseases

Tao

Conn

2018

Physiological Reviews

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After synthesis, proteins are folded into their native conformations aided by molecular chaperones. Dysfunction in folding caused by genetic mutations in numerous genes causes protein conformational diseases. Membrane proteins are more prone to misfolding due to their more intricate folding than soluble proteins. Misfolded proteins are detected by the cellular quality control systems, especially in the endoplasmic reticulum, and proteins may be retained there for eventual degradation by the ubiquitin-proteasome system or through autophagy. Some misfolded proteins aggregate, leading to pathologies in numerous neurological diseases. In vitro, modulating mutant protein folding by altering molecular chaperone expression can ameliorate some misfolding. Some small molecules known as chemical chaperones also correct mutant protein misfolding in vitro and in vivo. However, due to their lack of specificity, their potential as therapeutics is limited. Another class of compounds, known as pharmacological chaperones (pharmacoperones), binds with high specificity to misfolded proteins, either as enzyme substrates or receptor ligands, leading to decreased folding energy barriers and correction of the misfolding. Because many of the misfolded proteins are misrouted but do not have defects in function per se, pharmacoperones have promising potential in advancing to the clinic as therapeutics, since correcting routing may ameliorate the underlying mechanism of disease. This review will comprehensively summarize this exciting area of research, surveying the literature from in vitro studies in cell lines to transgenic animal models and clinical trials in several protein misfolding diseases.

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“…Pharmacological chaperones acting on V2R, which neither are peptidomimetics nor act as antagonists, have also been discovered. 25 Thus, there may be pharmacological chaperones yet to be discovered that rescue mutant GPCR function without disturbing natural ligand interactions.…”

Section: Introductionmentioning

confidence: 99%

Screening of Chemical Libraries Using a Yeast Model of Retinal Disease

et al. 2019

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Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9- cis retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z′ factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.

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Receptor antagonism/agonism can be uncoupled from pharmacoperone activity

Cited by 11 publications

References 36 publications

Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias

Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias

Pharmacoperones as Novel Therapeutics for Diverse Protein Conformational Diseases

Screening of Chemical Libraries Using a Yeast Model of Retinal Disease

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