Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

Roszko, Kelly L; Bi, Ruiye; Gorvin, Caroline M; Bräuner‐Osborne, Hans; Xiong, X.; Inoue, Asuka; Strømgaard, Kristian; Gardella, Thomas J.; Mannstadt, Michael

doi:10.1172/jci.insight.91079

Cited by 30 publications

(30 citation statements)

References 42 publications

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“…In functional studies, FR and YM were found to be similarly potent and selective Gα q/11 protein inhibitors. Both are exceedingly useful for studying G q protein signalling and for dissecting signalling pathways (Inamdar, Patel, Manne, Dangelmaier, & Kunapuli, 2015;Roszko et al, 2017;Schrage et al, 2015). However, more readily available inhibitors would be highly desirable.…”

Section: Introductionmentioning

confidence: 99%

Cell‐permeable high‐affinity tracers for G_q proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

Kuschak

Namasivayam

Rafehi

et al. 2020

British J Pharmacology

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Background and Purpose G proteins are intracellular switches that transduce and amplify extracellular signals from GPCRs. The Gq protein subtypes, which are coupled to PLC activation, can act as oncogenes, and their expression was reported to be up‐regulated in cancer and inflammatory diseases. Gq inhibition may be an efficient therapeutic strategy constituting a new level of intervention. However, diagnostic tools and therapeutic drugs for Gq proteins are lacking. Experimental Approach We have now developed Gq‐specific, cell‐permeable 3H‐labelled high‐affinity probes based on the macrocyclic depsipeptides FR900359 (FR) and YM‐254890 (YM). The tracers served to specifically label and quantify Gq proteins in their native conformation in cells and tissues with high accuracy. Key Results FR and YM displayed low nanomolar affinity for Gαq, Gα11 and Gα14 expressed in CRISPR/Cas9 Gαq‐knockout cells, but not for Gα15. The two structurally very similar tracers showed strikingly different dissociation kinetics, which is predicted to result in divergent biological effects. Computational studies suggested a “dowel” effect of the pseudoirreversibly binding FR. A high‐throughput binding assay led to the discovery of novel Gq inhibitors, which inhibited Gq signalling in recombinant cells and primary murine brown adipocytes, resulting in enhanced differentiation. Conclusions and Implications The Gq protein inhibitors YM and FR are pharmacologically different despite similar structures. The new versatile tools and powerful assays will contribute to the advancement of the rising field of G protein research.

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Section: Introductionmentioning

confidence: 99%

Cell‐permeable high‐affinity tracers for G_q proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

Kuschak

Namasivayam

Rafehi

et al. 2020

British J Pharmacology

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“…(15) Similarly, NPS2143 reduced mutant Val62 Gα11 signaling responses in vitro and led to an increase in plasma PTH and calcium concentrations in two animal models of ADH2. (16,17) The calcilytic JTT-305 (MK-5442) resulted in increased serum calcium and decreased urinary calcium by stimulating endogenous PTH secretion and prevented renal calcification in knock-in mice with human CAR (C129S) and CAR (A843E) mutations. (18) We thus hypothesized that the calcilytic NPSP795 may be effective in treating ADH1, and that by assessing the effect of NPSP795 on subject mutations in vitro and comparing the results to clinical findings we could gain insight into genotype/phenotype correlations as well as potentially predict how patients with specific mutations may respond to NPSP795.…”

Section: Introductionmentioning

confidence: 99%

Treatment of Autosomal Dominant Hypocalcemia Type 1 With the Calcilytic NPSP795 (SHP635)

Roberts

Gafni

Brillante

et al. 2019

Journal of Bone and Mineral Research

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Autosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism caused by heterozygous, gain‐of‐function mutations of the calcium‐sensing receptor gene (CAR). Individuals are hypocalcemic with inappropriately low parathyroid hormone (PTH) secretion and relative hypercalciuria. Calcilytics are negative allosteric modulators of the extracellular calcium receptor (CaR) and therefore may have therapeutic benefits in ADH1. Five adults with ADH1 due to four distinct CAR mutations received escalating doses of the calcilytic compound NPSP795 (SHP635) on 3 consecutive days. Pharmacokinetics, pharmacodynamics, efficacy, and safety were assessed. Parallel in vitro testing with subject CaR mutations assessed the effects of NPSP795 on cytoplasmic calcium concentrations (Ca2+i), and ERK and p38MAPK phosphorylation. These effects were correlated with clinical responses to administration of NPSP795. NPSP795 increased plasma PTH levels in a concentration‐dependent manner up to 129% above baseline (p = 0.013) at the highest exposure levels. Fractional excretion of calcium (FECa) trended down but not significantly so. Blood ionized calcium levels remained stable during NPSP795 infusion despite fasting, no calcitriol supplementation, and little calcium supplementation. NPSP795 was generally safe and well‐tolerated. There was significant variability in response clinically across genotypes. In vitro, all mutant CaRs were half‐maximally activated (EC50) at lower concentrations of extracellular calcium (Ca2+o) compared to wild‐type (WT) CaR; NPSP795 exposure increased the EC50 for all CaR activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose‐dependent manner, and thus, serves as proof‐of‐concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the CaR, in vitro observations were not predictive of the in vivo phenotype or the response to calcilytics, suggesting that other parameters impact the response to the drug. © 2019 American Society for Bone and Mineral Research.

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“…Pertussis toxin and cholera toxin enzymatically inhibit G i/o and activate G s signaling, respectively, but their application is restricted owing to their long incubation time and severe side effects . Recent studies have revealed that abnormal expression and activity of G proteins and GPCRs, especially the G q/11 proteins, are often associated with tumorigenesis, cardiovascular disease and asthma, suggesting that G proteins, together with GPCRs and their linked signaling circuitry, are promising therapeutic targets for a number of diseases …”

Section: Introductionmentioning

confidence: 99%

Structure–Activity Relationship Studies of the Natural Product G_q/11 Protein Inhibitor YM‐254890

et al. 2019

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G proteins act as molecular switches in G protein‐coupled receptor signaling pathways and are key mediators for numerous important physiological processes. The natural product, cyclic depsipeptide YM‐254890, together with the structurally similar FR900359, is the only known selective inhibitor of the Gq/11 subfamily of G proteins. We recently reported the first total synthesis of YM‐254890 and FR900359, followed by synthesizing analogues to perform structure–activity relationship studies. However, incomplete information about their structure–activity relationship prevents the further development of potent and structurally simplified analogues. Herein we report the first systematic structure–activity relationship study toward the N‐methyldehydroalanine moiety in YM‐254890, by designing and synthesizing seven new analogues. Pharmacological characterization of the seven compounds for Gq/11‐, Gi/o‐ and Gs‐mediated signaling showed that the simplified analogue YM‐19 is the most potent Gq/11inhibitor among the new analogues. This study provides information for the future design of potent and simplified YM‐254890 analogues.

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Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

Cited by 30 publications

References 42 publications

Cell‐permeable high‐affinity tracers for G_q proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

Cell‐permeable high‐affinity tracers for G_q proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

Treatment of Autosomal Dominant Hypocalcemia Type 1 With the Calcilytic NPSP795 (SHP635)

Structure–Activity Relationship Studies of the Natural Product G_q/11 Protein Inhibitor YM‐254890

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Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

Cited by 30 publications

References 42 publications

Cell‐permeable high‐affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

Cell‐permeable high‐affinity tracers for Gq proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

Treatment of Autosomal Dominant Hypocalcemia Type 1 With the Calcilytic NPSP795 (SHP635)

Structure–Activity Relationship Studies of the Natural Product Gq/11 Protein Inhibitor YM‐254890

Contact Info

Product

Resources

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Cell‐permeable high‐affinity tracers for G_q proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

Cell‐permeable high‐affinity tracers for G_q proteins provide structural insights, reveal distinct binding kinetics and identify small molecule inhibitors

Structure–Activity Relationship Studies of the Natural Product G_q/11 Protein Inhibitor YM‐254890