Potent Negative Allosteric Modulators Research Articles

SAT182 Effects Of An Orally Bioavailable Nonpeptide Parathyroid Hormone Receptor Type 1 (PTH1R) Antagonist In Rodent Models Of Primary Hyperparathyroidism (PHPT)

Abstract Disclosure: A.S. Antwan: Employee; Self; Crinetics Pharmaceuticals. E. Rico-Bautista: Employee; Self; Crinetics Pharmaceuticals. J. Pontillo: Employee; Self; Crinetics Pharmaceuticals. S. Wang: Employee; Self; Crinetics Pharmaceuticals. M. Johns: Employee; Self; Crinetics Pharmaceuticals. B. Ramms: Employee; Self; Crinetics Pharmaceuticals. M.A. Fowler: Employee; Self; Crinetics Pharmaceuticals. J.B. Nguyen: Employee; Self; Crinetics. A.A. Castellanos Gonzalez: Employee; Self; Crinetics. B. Fleck: Employee; Self; Crinetics Pharmaceuticals. K. Retting: Employee; Self; Crinetics Pharmaceuticals. D. Dalvie: Employee; Self; Crinetics Pharmaceuticals. S.F. Betz: Employee; Self; Crinetics Pharmaceuticals. S. Markison: Employee; Self; Crinetics Pharmaceuticals. Primary hyperparathyroidism (PHPT) is a condition resulting from an over-secretion of parathyroid hormone (PTH) from one or more overactive parathyroid glands. PTH is a peptide hormone that regulates blood calcium concentrations through effects on bone, intestines, and kidney. PTH acts on parathyroid hormone receptor type 1 (PTH1R) in the kidney to increase calcium reabsorption and block phosphate reabsorption. The loss of phosphate ions then causes an increase in ionized calcium in the blood and cAMP in the urine. At the bone, continuous PTH infusion stimulates osteoclast activity while inhibiting osteoblast activity, leading to breakdown of bone and release of calcium into the extracellular fluid. Conditions that cause excess PTH, such as PHPT, result in elevated concentrations of plasma calcium, increased bone loss, increased fracture risk, and higher susceptibility to kidney stone development. PHPT affects approximately 100,000 patients per year, but many patients are asymptomatic and remain undiagnosed. While a partial or total parathyroidectomy may be suitable for patients diagnosed with severe cases of PHPT, there are limited treatment options for those who do not meet the criteria or do not wish to undergo surgery. The goals for medicinal treatment are to normalize blood calcium levels and urinary calcium excretion, as well as increase bone mineral density to reduce fracture risk. Blocking PTH action directly via a potent PTH1R antagonist may provide an important new therapeutic mechanism to treat patients with PHPT. Using iterative medicinal chemistry and pharmacology, Crinetics has identified several potent and orally bioavailable PTH1R antagonists with good drug-like properties. Lead molecules were evaluated in vivo in preclinical rodent models for their ability to suppress the effects of excess PTH on serum calcium, bone turnover, and cAMP levels in the kidney. One of these compounds, ANT-5, was characterized as a potent negative allosteric modulator of both human and rat PTH1R. In rats, oral administration of ANT-5 dose-dependently suppressed PTH-induced hypercalcemia. ANT-5 also suppressed PTH-stimulated urinary cAMP in rats, indicating its ability to inhibit the actions of PTH in the kidney. We further evaluated the effect of ANT-5 in a rodent model of continuous PTH infusion-induced bone turnover. Once-daily oral administration of ANT-5 mitigated PTH-induced bone resorption, suggesting that inhibiting PTH1R may have beneficial clinical effects on bone density. The culmination of these studies has led to a subset of candidate molecules that are being evaluated in safety studies to identify the compound(s) suitable for evaluation in human clinical trials. Support: Crinetics Pharmaceuticals. Presentation: Saturday, June 17, 2023

FRI538 Discovery And Characterization Of Orally Bioavailable Nonpeptide Thyroid Stimulating Hormone Receptor (TSHR) Antagonists For The Treatment Of Graves’ Disease

Abstract Disclosure: M.A. Fowler: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. C. Regan: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. J. Zhao: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. E. Coutinho: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. B. Fleck: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. A.A. Castellanos: Employee; Self; Crinetics Pharmaceuticals. E. Muller: Employee; Self; Crinetics Pharmaceuticals. M. Johns: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. Y. Tang: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. E. Sturchler: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. M. Chen: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. K. Retting: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. D. Dalvie: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. S.F. Betz: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. S. Markison: Employee; Self; Crinetics Pharmaceuticals. Stock Owner; Self; Crinetics Pharmaceuticals. Graves’ disease is an autoimmune condition that affects approximately 1 in 100 people in the United States and 2-3% of the population worldwide. It is characterized by the production of autoantibodies against TSHR, and the pathology of Graves’ disease is driven by TSHR stimulatory antibodies (TSAb) that result in heightened activation of TSHR. This overstimulation results in hyperthyroidism due to excessive production of thyroid hormones. Approximately 30% of Graves’ disease patients also develop thyroid eye disease (TED or Graves’ orbitopathy) due to overactivation of TSHR in orbital fibroblasts leading to excessive production of hyaluronic acid, adipogenesis, cytokine production, and fibrosis. This can cause a myriad of debilitating symptoms including pain, swelling, blurry vision, diplopia, and proptosis. Several treatments for Graves’ hyperthyroidism are available including anti-thyroid drugs, radioactive iodine (RAI), and surgery. RAI and surgery are definitive treatments for Graves’ hyperthyroidism, but often result in hypothyroidism. In addition, none of the current treatments for Graves’ hyperthyroidism are effective in treating TED and, in some cases, such as with RAI, worsen the condition. Blocking TSHR activation directly via a TSHR antagonist may provide an important new therapeutic mechanism to treat patients with Graves’ disease that would effectively treat both the hyperthyroidism and TED. We have identified several potent and orally bioavailable nonpeptide allosteric antagonists with acceptable drug-like properties. One analog, TSHRant-1, demonstrated potent negative allosteric modulator activity at both the human and rat TSHR. To evaluate the in vivo pharmacodynamics of TSHant-1, we developed a rat model of hyperthyroidism. In this model, subcutaneous administration of the TSAb, M22, to female rats resulted in a robust and long-lasting rise in levels of the thyroid hormone thyroxin (T4). Oral administration of TSHRant-1 dose-dependently suppressed M22-stimulated T4, providing evidence that a nonpeptide allosteric antagonist of TSHR may serve as an effective treatment for Graves’ disease and associated orbitopathy (TED). Presentation: Friday, June 16, 2023

Negative allosteric modulators of NMDA receptors with GluN2B subunit: Alanine-derived benzoxazolone bioisosteres of 2-methyl-3-benzazepine-1,7-diols.

Inspired by besonprodil, the phenol of potent negative allosteric modulators of GluN2B-N-methyl-d-aspartate (NMDA) receptors was replaced by a benzoxazolone system. To increase the similarity to the lead compounds, an additional methyl moiety was installed in the 8-position of tricyclic oxazolobenzazepines, resulting in compounds 6. The additional methyl moiety originates from alanine, which was introduced by a Mitsunobu reaction of benzoxazolylethanol 7 with N-triflyl-protected alanine methyl ester. A crucial feature of the synthesis was the protection of the oxazolone ring by an allyl moiety, which was cleaved off at the end of the synthesis by RhCl3 -catalyzed isomerization. Due to the additional methyl moiety, the intramolecular Friedel-Crafts acylation of acid 10 to afford ketone 11 required careful optimization to minimize the formation of the side product tetrahydroisoquinoline 16. Alkylation or reductive alkylation of secondary amine 13 led to diastereomeric oxazolobenzazepines cis-14 and trans-14, which were separated by flash chromatography. Phenylbutyl derivatives cis-6a and trans-6a revealed twofold higher GluN2B affinity than analog 5a without 8-CH3 group. The methylated oxazolobenzazepines 6 and 14 did not interact with the phencyclidine binding site of NMDA receptors and σ2 receptors. However, the σ1 receptor preferred cis-configured oxazolobenzazepines. The highest σ1 receptor affinities were obtained for cis-14a (Ki = 26 nM) and cis-6b (Ki = 30 nM).

Synthesis and Characterization of 5-(2-Fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7-carboxamide as a PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

Metabotropic glutamate receptor 2 (mGluR2) is a therapeutic target for several neuropsychiatric disorders. An mGluR2 function in etiology could be unveiled by positron emission tomography (PET). In this regard, 5-(2-fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7-carboxamide ([11C]13, [11C]mG2N001), a potent negative allosteric modulator (NAM), was developed to support this endeavor. [11C]13 was synthesized via the O-[11C]methylation of phenol 24 with a high molar activity of 212 ± 76 GBq/μmol (n = 5) and excellent radiochemical purity (>99%). PET imaging of [11C]13 in rats demonstrated its superior brain heterogeneity and reduced accumulation with pretreatment of mGluR2 NAMs, VU6001966 (9) and MNI-137 (26), the extent of which revealed a time-dependent drug effect of the blocking agents. In a nonhuman primate, [11C]13 selectively accumulated in mGluR2-rich regions and resulted in high-contrast brain images. Therefore, [11C]13 is a potential candidate for translational PET imaging of the mGluR2 function.

Synthesis of GluN2A-selective NMDA receptor antagonists with an electron-rich aromatic B-ring.

Glutamatergic N-Methyl-d-aspartate (NMDA) receptors are heterotetrameric ion channels that can be comprised of different subunits. GluN2A subunit-containing NMDA receptors are associated with diseases like anxiety, depression, and schizophrenia. However, the exact contribution of these NMDA receptor subtypes is still unclear. To understand better the role of the GluN2A-containing receptors, novel ligands were designed. In co-crystallization with the isolated binding site, TCN-201 (1) and analogs adopt a U-shape conformation with parallel orientation of rings A and B. In order to increase the π/π-interactions between these rings, ring B of TCN-201 was replaced bioisosterically by different electron-rich thiazole, oxazole, and isoxazole heterocycles. The inhibitory activity was measured by two-electrode voltage clamp experiments with Xenopus laevis oocytes expressing GluN2A-containing NMDA receptors. It was found that 21c, 31a, 37a, and 37b were able to inhibit the ion channel. The isoxazole derivative 37b was the most potent negative allosteric modulator displaying 40% of the TCN-201 activity at a concentration of 10μM.

Identification and Development of a New Positron Emission Tomography Ligand 4-(2-Fluoro-4-[11C]methoxyphenyl)-5-((1-methyl-1H-pyrazol-3-yl)methoxy)picolinamide for Imaging Metabotropic Glutamate Receptor Subtype 2 (mGlu2).

Metabotropic glutamate receptor 2 (mGlu2) is a known target for treating several central nervous system (CNS) disorders. To develop a viable positron emission tomography (PET) ligand for mGlu2, we identified new candidates 5a-i that are potent negative allosteric modulators (NAMs) of mGlu2. Among these candidates, 4-(2-fluoro-4-methoxyphenyl)-5-((1-methyl-1H-pyrazol-3-yl)methoxy)picolinamide (5i, also named as [11C]MG2-1812) exhibited high potency, high subtype selectivity, and favorable lipophilicity. Compound 5i was labeled with positron-emitting carbon-11 (11C) to obtain [11C]5i in high radiochemical yield and high molar activity by O-[11C]methylation of the phenol precursor 12 with [11C]CH3I. In vitro autoradiography with [11C]5i showed heterogeneous radioactive accumulation in the brain tissue sections, ranked in the order: cortex > striatum > hippocampus > cerebellum ≫ thalamus > pons. PET study of [11C]5i indicated in vivo specific binding of mGlu2 in the rat brain. Based on the [11C]5i scaffold, further optimization for new candidates is underway to identify a more suitable ligand for imaging mGlu2.

Atom-based 3D-QSAR, induced fit docking, and molecular dynamics simulations study of thieno[2,3-b]pyridines negative allosteric modulators of mGluR5

Atom-based three dimensional-quantitative structure–activity relationship (3D-QSAR) model was developed on the basis of 5-point pharmacophore hypothesis (AARRR) with two hydrogen bond acceptors (A) and three aromatic rings for the derivatives of thieno[2,3-b]pyridine, which modulates the activity to inhibit the mGluR5 receptor. Generation of a highly predictive 3D-QSAR model was performed using the alignment of predicted pharmacophore hypothesis for the training set (R2 = 0.84, SD = 0.26, F = 45.8, N = 29) and test set (Q2 = 0.74, RMSE = 0.235, Pearson-R = 0.94, N = 9). The best pharmacophore hypothesis AARRR was selected, and developed three dimensional-quantitative structure activity relationship (3D-QSAR) model also supported the outcome of this study by means of favorable and unfavorable electron withdrawing group and hydrophobic regions of most active compound 42d and least active compound 18b. Following, induced fit docking and binding free energy calculations reveals the reliable binding orientation of the compounds. Finally, molecular dynamics simulations for 100 ns were performed to depict the protein–ligand stability. We anticipate that the resulted outcome could be supportive to discover potent negative allosteric modulators for metabotropic glutamate receptor 5 (mGluR5).

1,3,5-Trisubstituted Pyrazoles as Potent Negative Allosteric Modulators of the mGlu2/3 Receptors.

The metabotropic glutamate subtype 2 (mGlu2 ) receptor is a presynaptic membrane receptor distributed widely in brain that provides feedback inhibitory control of glutamate release. Inhibition of the mGlu2 receptor function with a negative allosteric modulator (NAM) enhances activity-dependent glutamate release, which may be of therapeutic benefit for the treatment of neurological and psychiatric disorders. An attractive pyrazole hit was identified after a high-throughput screening (HTS) campaign. The evolution of this hit is described by structure-activity relationship (SAR) studies on specific parts of the molecule. From near micromolar potency we could obtain compounds with single-digit nanomolar activity in the mGlu2 NAM GTPγS assay. In addition to SAR on in vitro potency, a more detailed overview is given with a specific set of compounds on the excellent agreement between in vitro potency, free brain concentration, and ex vivo mGlu2 receptor occupancy. Finally, to obtain improved drug-like compounds, plans for future research are suggested toward increasing free brain concentration while maintaining high in vitro potency.

Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s).

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.

Synthesis and biological evaluation of negative allosteric modulators of the Kv11.1(hERG) channel

We synthesized and evaluated a series of compounds for their allosteric modulation at the Kv11.1 (hERG) channel. Most compounds were negative allosteric modulators of [3H]dofetilide binding to the channel, in particular 7f, 7h–j and 7p. Compounds 7f and 7p were the most potent negative allosteric modulators amongst all ligands, significantly increasing the dissociation rate of dofetilide in the radioligand kinetic binding assay, while remarkably reducing the affinities of dofetilide and astemizole in a competitive displacement assay. Additionally, both 7f and 7p displayed peculiar displacement characteristics with Hill coefficients significantly distinct from unity as shown by e.g., dofetilide, further indicative of their allosteric effects on dofetilide binding. Our findings in this investigation yielded several promising negative allosteric modulators for future functional and clinical research with respect to their antiarrhythmic propensities, either alone or in combination with known Kv11.1 blockers.

A double effect molecular switch leads to a novel potent negative allosteric modulator of metabotropic glutamate receptor 5

Compounds that modulate the function of G-protein-coupled receptors (GPCRs) by binding to their allosteric sites are of potential interest for the treatment of multiple CNS and non-CNS disorders. Allosteric ligands can act either as positive (PAM), negative (NAM), or silent (SAM) receptor modulators and have numerous advantages over classic orthosteric compounds, including improved GPCR-subtype selectivity; the capacity to adapt to physiological conditions; and better safety profiles. Despite these benefits, allosteric modulators are difficult to design and optimize and are often prone to “molecular switching”: a structural phenomenon by which very subtle chemical variations in the ligand result in unexpected changes in selectivity profiles or pharmacology, changing PAMs to NAMs or vice versa. Here, we report the discovery of a nanomolar and subtype selective NAM of metabotropic glutamate receptor 5 (mGlu5) through a targeted “double effect molecular switch” of a potent mGlu4 PAM, and suggests a promising approach towards the discovery of novel mGluR allosteric modulators.

Tanshinone II A sulfonate, but not tanshinone II A, acts as potent negative allosteric modulator of the human purinergic receptor P2X7.

Tanshinone II A sulfonate (TIIAS) was identified as a potent, selective blocker of purinergic receptor P2X7 in a compound library screen. In this study, a detailed characterization of the pharmacologic effects of TIIAS on P2X7 is provided. Because TIIAS is a derivative of tanshinone II A (TIIA) and both compounds have been used interchangeably, TIIA was included in some assays. Fluorometric and electrophysiologic assays were used to characterize effects of TIIAS and TIIA on recombinantly expressed human, rat, and mouse P2X7. Results were confirmed in human monocyte-derived macrophages expressing native P2X7. In all experiments, involvement of P2X7 was verified using established P2X7 antagonists. TIIAS, but not TIIA, reduces Ca(2+) influx via human P2X7 (hP2X7) with an IC50 of 4.3 µM. TIIAS was less potent at mouse P2X7 and poorly inhibited rat P2X7. Monitoring of YO-PRO-1 uptake confirmed these findings, indicating that formation of the hP2X7 pore is also suppressed by TIIAS. Electrophysiologic experiments revealed a noncompetitive mode of action. TIIAS time-dependently inhibits hP2X7 gating, possibly by binding to the intracellular domain of the receptor. Inhibition of native P2X7 in macrophages by TIIAS was confirmed by monitoring Ca(2+) influx, YO-PRO-1 uptake, and release of the proinflammatory cytokine interleukin-1β. Fluorometric experiments involving recombinantly expressed rat P2X2 and human P2X4 were conducted and verified the compound's selectivity. Our data suggest that hP2X7 is a molecular target of TIIAS, but not of TIIA, a compound with different pharmacologic properties.

Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mechanistic studies with new potent negative allosteric modulators

Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu₂) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu₂/₃ receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu₂/₃ NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu₂ is characterized. Site directed mutagenesis, functional measurements and β₂-adrenoceptor-based modelling of mGlu₂ were employed to identify important molecular determinants of two new potent mGlu₂/₃ NAMs. RO4988546 and RO5488608 affected both [³H]-LY354740 agonist binding at the orthosteric site and the binding of a tritiated positive allosteric modulator (³H-PAM), indicating that NAMs and PAMs could have overlapping binding sites in the mGlu₂ TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu₂ and directly interact with the NAMs: Arg³·²⁸, Arg³·²⁹, Phe³·³⁶, His(E2.52) , Leu⁵·⁴³, Trp⁶·⁴⁸, Phe⁶·⁵⁵ and Val⁷·⁴³. The mGlu₂ specific residue His(E2.52) is likely to be involved in selectivity and residues located in the outer part of the binding pocket are more important for [³H]-LY354740 agonist binding inhibition, which is independent of the highly conserved Trp⁶·⁴⁸ residue. This is the first complete molecular investigation of the allosteric binding pocket of mGlu₂ and Group II mGluRs and provides new information on what determines mGlu₂ NAMs selective interactions and effects.

A Novel Family of Potent Negative Allosteric Modulators of Group II Metabotropic Glutamate Receptors

Group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, play a number of important roles in mammalian brain and represent exciting new targets for certain central nervous system disorders. We now report synthesis and characterization of a novel family of derivatives of dihydrobenzo[1,4]diazepin-2-one that are selective negative allosteric modulators for group II mGluRs. These compounds inhibit both mGluR2 and mGluR3 but have no activity at group I and III mGluRs. The novel mGluR2/3 antagonists also potently block mGluR2/3-mediated inhibition of the field excitatory postsynaptic potentials at the perforant path synapse in hippocampal slices. These compounds induce a rightward shift and decrease the maximal response in the glutamate concentration-response relationship, consistent with a noncompetitive antagonist mechanism of action. Furthermore, radioligand binding studies revealed no effect on binding of the orthosteric antagonist [(3)H]LY341495 [2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid]. Site-directed mutagenesis revealed that a single point mutation in transmembrane V (N735D), previously shown to be an important residue for potentiation activity of the mGluR2 allosteric potentiator LY487379 [N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethylamine], is not critical for the inhibitory activity of negative allosteric modulators of group II mGluRs. However, this single mutation in human GluR2 almost completely blocked the enhancing activity of biphenyl-indanone A, a novel allosteric potentiator of mGluR2. Our data suggest that these two positive allosteric modulators of mGluR2 may share a common binding site and that this site may be distinct from the binding site for the new negative allosteric modulators of group II mGluRs.

Modeling and Mutagenesis of the Binding Site of Calhex 231, a Novel Negative Allosteric Modulator of the Extracellular Ca2+-sensing Receptor

A model of the Ca2+-sensing receptor (CaSR) seven transmembrane domains was constructed based on the crystal structure of bovine rhodopsin. This model was used for docking (1S,2S,1'R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane (Calhex 231), a novel potent negative allosteric modulator that blocks (IC50 = 0.39 microm) increases in [3H]inositol phosphates elicited by activating the human wild-type CaSR transiently expressed in HEK293 cells. In this model, Glu-8377.39 plays a pivotal role in anchoring the two nitrogen atoms of Calhex 231 and locating the aromatic moieties in two adjacent hydrophobic pockets delineated by transmembrane domains 3, 5, and 6 and transmembrane domains 1, 2, 3, and 7, respectively. To demonstrate its validity, we have mutated selected residues and analyzed the biochemical and pharmacological properties of the mutant receptors transfected in HEK293 cells. Two receptor mutations, F684A3.32 and E837A7.39, caused a loss of the ability of Calhex 231 to inhibit Ca2+-induced accumulation of [3H]inositol phosphates. Three other mutations, F688A3.36, W818A6.48, and I841A7.43, produced a marked increase in the IC50 of Calhex 231 for the Ca2+ response, whereas L776A5.42 and F821A6.51 led to a decrease in the IC50. Our data validate the proposed model for the allosteric interaction of Calhex 231 with the seven transmembrane domains of the CaSR. Interestingly, the residues at the same positions have been shown to delimit the antagonist-binding cavity of many diverse G-protein-coupled receptors. This study furthermore suggests that the crystal structure of bovine rhodopsin exhibits sufficient mimicry to the ground state of a very divergent class 3 receptor to predict the interaction of antagonists with the heptahelical bundle of diverse G-protein-coupled receptors.