Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges

Communications Biology, Journal Year: 2024, Volume and Issue: 7(1)

Published: April 25, 2024

Abstract G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C that typically sense amino-acids their derivatives, calcium sensing (CaSR) tightly controls blood levels. Its has not yet been studied. Here, we demonstrate importance of inter-subunit disulfide bridges maintaining state CaSR, undetectable activity, unlike other receptors. Deletion these results strong is abolished by mutations preventing amino acid binding. It shows this link necessary to limit agonist effect acids on CaSR. Furthermore, human genetic deleting associated hypocalcemia result elevated CaSR activity. These highlight fine tuning

Language: Английский

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Silicon-Rhodamine Functionalized Evocalcet Probes Potently and Selectively Label Calcium Sensing Receptors In Vitro, In Vivo, and Ex Vivo

ACS Pharmacology & Translational Science, Journal Year: 2024, Volume and Issue: 7(5), P. 1557 - 1570

Published: April 25, 2024

The calcium sensing receptor (CaSR) is a ubiquitously expressed G-protein coupled (GPCR) that regulates extracellular signals via the parathyroid glands. CaSR has recently also been implicated in noncalcitropic pathophysiologies like asthma, gut inflammation, and cancer. To date, molecular tools enable bioimaging of tissues are lacking. Based on silico analyses available structure–activity relationship data ligands, we designed prepared silicon-rhodamine (SiR) conjugates clinically approved drug evocalcet. new probes EvoSiR4 EvoSiR6, with differing linker lengths at evocalcet carboxyl end, both showed 6-fold 3-fold increase potency toward (EC50 < 45 nM) compared to evocalcet-linker conjugate, respectively, an FLIPR-based cellular functional assay. specificity EvoSiR binding impact albumin was evaluated live cell experiments. Both strong binding, which facilitated clearance nonspecific interactions. Accordingly, zebrafish embryos, specifically labeled high expressing neuromasts lateral line vivo. assessed human ex vivo, showing specific absolute CaSR-associated fluorescence autofluorescence. In summary, functionalization by SiR led preparation potent fluorescent probes. versatile small-molecular probe can be employed CaSR-related biomedical where antibodies not applicable.

Language: Английский

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Glutathione-Based Photoaffinity Probe Identifies Caffeine as a Positive Allosteric Modulator of the Calcium-Sensing Receptor

ACS Chemical Biology, Journal Year: 2024, Volume and Issue: 19(7), P. 1661 - 1670

Published: July 8, 2024

The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, bone remodeling, other organs tissues. diverse functions of are mediated by many endogenous exogenous ligands, calcium, amino acids, glutathione, cinacalcet, etelcalcetide, that have distinct binding sites CaSR. However, strategies to evaluate ligand interactions with remain limited. Here, we developed glutathione-based photoaffinity probe, DAZ-G, analyzes We showed DAZ-G binds acid site acts as positive allosteric modulator Oxidized reduced glutathione phenylalanine effectively compete conjugation CaSR, while etelcalcetide cooperative effects. An unexpected finding was caffeine competes DAZ-G's effective concentration for activation (<10 μM) is easily attainable plasma ordinary consumption. Our report demonstrates utility new chemical probe discovers protein target caffeine, suggesting consumption can modulate

Language: Английский

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Discovery and pharmacological characterization of nanobodies acting as potent positive allosteric modulators of the calcium-sensing receptor

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 8, 2024

Abstract The calcium-sensing receptor (CaSR) is responsible for sustaining a stable blood calcium concentration. Consequently, genetic and acquired changes in this G protein-coupled can give rise to various homeostasis disorders. Synthetic positive allosteric modulators targeting CaSR are currently used treat hypercalcemia, but their usage highly limited due the high risk of severe hypocalcemia gastrointestinal intolerance. In study, we aimed generate pharmacologically active CaSR-specific nanobodies that could be employed as new generation pharmacological tools investigate function potentially serve drug modality effective treatment CaSR-related Nanobodies were generated by immunization llama with CHO cells recombinantly overexpressing myc-epitope-tagged human CaSR. Following construction phage display library representing repertoire nanobody genes, binding isolated FACS whole HEK293 HA-epitope-tagged Based on sequence comparison, 37 from 25 different families purified subsequent characterized vitro modulation signaling. screened agonist, well negative activity cellular assays downstream activation. We identified eight acting divided into five main based identity. most potent (Nb4) extracellular domain was slightly more than reference small molecule PAM NPS R-568. This study describes discovery characterization modulators. These class research CaSR, which developed therapeutics

Language: Английский

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The impact of nanobodies on GPCR structural biology and their potential as therapeutic agents

Molecular Pharmacology, Journal Year: 2024, Volume and Issue: 106(4), P. 155 - 163

Published: Aug. 6, 2024

The family of human G protein-coupled receptors (GPCRs) is comprised about 800 different members, with 35% current pharmaceutical drugs targeting GPCRs. However, GPCR structural biology, necessary for structure-guided drug design, has lagged behind that other membrane proteins, and it was not until the year 2000 when first crystal structure a (rhodopsin) solved. Starting in 2007, determination additional structures facilitated by protein engineering, new crystallization techniques, complexation antibody fragments, strategies. More recently, use camelid heavy-chain-only fragments (nanobodies) as crystallographic chaperones revolutionized field aiding more than 340 to date. In most cases, solved complexes nanobodies (Nbs) have revealed binding mode cognate or non-natural ligands; few same Nb acted an orthosteric allosteric modulator signaling. this review we summarize multiple ingenious strategies been conceived implemented last decade capitalize on discovery study GPCRs from perspective. Significance Statement are major pharmacological targets, their at high resolution essential design insights functions. Single domain antibodies greatly GPCRs, forming directly indirectly through partners.

Language: Английский

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