Identification of acidic residues in the extracellular loops of the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+ and a positive allosteric modulator

We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca(2+) receptor (hCaR) in receptor activation by Ca(2+) and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca(2+), and on NPS R-568 augmentation of sensitivity to Ca(2+). Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp(758), Glu(759), and Glu(767), increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca(2+). Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca(2+). Mutation of Glu(837) in exo-loop 3 to alanine did not alter Ca(2+) sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu(837) drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu(837) at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR.     

Sodium functions as a negative allosteric modulator of the oxytocin receptor

The oxytocin receptor, a class A G protein coupled receptor (GPCR), is essentially involved in the physiology of reproduction. Two parameters are crucially important to support high-affinity agonist binding of the receptor: Mg²⁺ and cholesterol, both acting as positive modulators. Using displacement assays with a high-affinity fluorescent antagonist (OTAN-A647), we now show that sodium functions as a negative allosteric modulator of the oxytocin receptor. In membranes from HEK293 cells stably expressing the oxytocin receptor, oxytocin binding occurred with about 15-fold lower affinity when sodium chloride was increased from 0 to 300 mM, whereas antagonist binding remained largely unchanged. The effect was concentration-dependent, sodium-specific, and it was also observed for oxytocin receptors endogenously expressed in Hs578T breast cancer cells. A conserved Asp (Asp 85) is known to stabilize the sodium binding site in other GCPRs. Mutations of this residue into Ala or Asn are known to yield non-functional oxytocin receptors. When Asp 85 was exchanged for Glu, most of the oxytocin receptors were localized in intracellular structures, but a faint plasma membrane labeling with OTAN-A647 and the appearance of oxytocin-induced calcium responses indicated that these receptors were functional. However, a sodium effect was not detectable for the mutant D85E oxytocin receptors. Thus, the oxytocin receptor is allosterically controlled by sodium similar to other GPCRs, but it behaves differently concerning the involvement of the conserved Asp 85. In case of the oxytocin receptor, Asp 85 is obviously essential for proper localization in the plasma membrane.     

Screening for positive allosteric modulators: assessment of modulator concentration-response curves as a screening paradigm

Targeting allosteric binding sites represents a powerful mechanism for selectively modulating receptor function. The advent of functional assays as the screening method of choice is leading to an increase in the number of allosteric modulators identified. These include positive allosteric modulators that can increase the affinity of the orthosteric agonist and potentiate the evoked response. A common method for screening for positive allosteric modulators is to examine a concentration-response (C/R) curve to the putative modulator in the presence of a single, low concentration of agonist. The study reported here has used data simulations for positive allosteric modulators according to the allosteric ternary complex model to generate modulator C/R curves. The results are then compared to the mechanistic parameters used to simulate the data. It is clear from the simulations that the potency of a positive modulator C/R curve in a screening assay is the product of both its affinity and positive cooperativity. However, it is often difficult to tell which parameter dominates the response; not knowing the actual affinity or cooperativity of a ligand may have consequences for receptor selectivity. Further modeling demonstrates that the use and choice of single agonist concentration, as well as changes in the agonist curve Hill slope, can have significant effects on the modulator C/R curve. Finally, the quantitative relationship between modulator C/R curves and the allosteric ternary complex model is explored. These simulations emphasize the importance of careful interpretation of screening data and of conducting full mechanism of action studies for positive allosteric modulators.     

Calindol, a positive allosteric modulator of the human Ca(2+) receptor, activates an extracellular ligand-binding domain-deleted rhodopsin-like seven-transmembrane structure in the absence of Ca(2+)

The extracellular calcium-sensing human Ca(2+) receptor (hCaR),2 a member of the family-3 G-protein-coupled receptors (GPCR) possesses a large amino-terminal extracellular ligand-binding domain (ECD) in addition to a seven-transmembrane helical domain (7TMD) characteristic of all GPCRs. Two calcimimetic allosteric modulators, NPS R-568 and Calindol ((R)-2-{1-(1-naphthyl)ethyl-aminom-ethyl}indole), that bind the 7TMD of the hCaR have been reported to potentiate Ca(2+) activation without independently activating the wild type receptor. Because agonists activate rhodopsin-like family-1 GPCRs by binding within the 7TMD, we examined the ability of Calindol, a novel chemically distinct calcimimetic, to activate a Ca(2+) receptor construct (T903-Rhoc) in which the ECD and carboxyl-terminal tail have been deleted to produce a rhodopsin-like 7TMD. Here we report that although Calindol has little or no agonist activity in the absence of extracellular Ca(2+) for the ECD-containing wild type or carboxyl-terminal deleted receptors, it acts as a strong agonist of the T903-Rhoc. In addition, Ca(2+) alone displays little or no agonist activity for the hCaR 7TMD, but potentiates the activation by Calindol. We confirm that activation of Ca(2+) T903-Rhoc by Calindol truly the is independent using in vitro reconstitution with purified G(q). These findings demonstrate distinct allosteric linkages between Ca(2+) site(s) in the ECD and 7TMD and the 7TMD site(s) for calcimimetics.     

A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand

Many G protein-coupled receptors (GPCRs) possess allosteric binding sites distinct from the orthosteric site utilized by their cognate ligands, but most GPCR allosteric modulators reported to date lack signaling efficacy in their own right. McN-A-343 (4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride) is a functionally selective muscarinic acetylcholine receptor (mAChR) partial agonist that can also interact allosterically at the M(2) mAChR. We hypothesized that this molecule simultaneously utilizes both an allosteric and the orthosteric site on the M(2) mAChR to mediate these effects. By synthesizing progressively truncated McN-A-343 derivatives, we identified two, which minimally contain 3-chlorophenylcarbamate, as pure allosteric modulators. These compounds were positive modulators of the orthosteric antagonist N-[(3)H]methylscopolamine, but in functional assays of M(2) mAChR-mediated ERK1/2 phosphorylation and guanosine 5'-3-O-([(35)S]thio)triphosphate binding, they were negative modulators of agonist efficacy. This negative allosteric effect was diminished upon mutation of Y177A in the second extracellular (E2) loop of the M(2) mAChR that is known to reduce prototypical allosteric modulator potency. Our results are consistent with McN-A-343 being a bitopic orthosteric/allosteric ligand with the allosteric moiety engendering partial agonism and functional selectivity. This finding suggests a novel and largely unappreciated mechanism of "directed efficacy" whereby functional selectivity may be engendered in a GPCR by utilizing an allosteric ligand to direct the signaling of an orthosteric ligand encoded within the same molecule.     

A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.     

On mechanism of allosteric modulation of NMDA receptor via amino-terminal domains

A possible mechanism of action of the allosteric modulators of NMDA (N-methyl-d-aspartate) receptors is proposed that involves the stabilization of the twisted closed-clamshell configuration of the amino-terminal domains of GluN1 and GluN2B subunits by negative modulators while positive modulators stabilize a roughly parallel tight arrangement of these domains. These respective motions may play an important role in the transition between the open-channel and closed-channel states of the receptor. In addition, some features of the negative modulator binding site found by means of the molecular dynamics study and pocket analysis can be used in the rational design of the allosteric NMDA receptor modulators.     

A Monod-Wyman-Changeux mechanism can explain G protein-coupled receptor (GPCR) allosteric modulation

The Monod-Wyman-Changeux (MWC) model was initially proposed to describe the allosteric properties of regulatory enzymes and subsequently extended to receptors. Yet despite GPCRs representing the largest family of receptors and drug targets, no study has systematically evaluated the MWC mechanism as it applies to GPCR allosteric ligands. We reveal how the recently described allosteric modulator, benzyl quinolone carboxylic acid (BQCA), behaves according to a strict, two-state MWC mechanism at the M1 muscarinic acetylcholine receptor (mAChR). Despite having a low affinity for the M1 mAChR, BQCA demonstrated state dependence, exhibiting high positive cooperativity with orthosteric agonists in a manner that correlated with efficacy but negative cooperativity with inverse agonists. The activity of BQCA was significantly increased at a constitutively active M1 mAChR but abolished at an inactive mutant. Interestingly, BQCA possessed intrinsic signaling efficacy, ranging from near-quiescence to full agonism depending on the coupling efficiency of the chosen intracellular pathway. This latter cellular property also determined the difference in magnitude of positive cooperativity between BQCA and the orthosteric agonist, carbachol, across pathways. The lack of additional, pathway-biased, allosteric modulation by BQCA was confirmed in genetically engineered yeast strains expressing different chimeras between the endogenous yeast G(pa1) protein and human Gα subunits. These findings define a chemical biological framework that can be applied to the study and classification of allosteric modulators across different GPCR families.     

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.     

A hotspot for enhancing insulin receptor activation revealed by a conformation-specific allosteric aptamer

Aptamers are single-stranded oligonucleotides that bind to a specific target with high affinity, and are widely applied in biomedical diagnostics and drug development. However, the use of aptamers has largely been limited to simple binders or inhibitors that interfere with the function of a target protein. Here, we show that an aptamer can also act as a positive allosteric modulator that enhances the activation of a receptor by stabilizing the binding of a ligand to that receptor. We developed an aptamer, named IR-A43, which binds to the insulin receptor, and confirmed that IR-A43 and insulin bind to the insulin receptor with mutual positive cooperativity. IR-A43 alone is inactive, but, in the presence of insulin, it potentiates autophosphorylation and downstream signaling of the insulin receptor. By using the species-specific activity of IR-A43 at the human insulin receptor, we demonstrate that residue Q272 in the cysteine-rich domain is directly involved in the insulin-enhancing activity of IR-A43. Therefore, we propose that the region containing residue Q272 is a hotspot that can be used to enhance insulin receptor activation. Moreover, our study implies that aptamers are promising reagents for the development of allosteric modulators that discriminate a specific conformation of a target receptor.     

The allosteric site regulates the voltage sensitivity of muscarinic receptors

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein–coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M₁-Rs and M₃-Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G_q protein cycle. In the presence of ACh, M₁-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M₃-R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed “allosteric site” M₃/M₁-R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M₃-Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects.     

Modeling and mutagenesis of the binding site of Calhex 231, a novel negative allosteric modulator of the extracellular Ca(2+)-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.     

Homology modeling of the transmembrane domain of the human calcium sensing receptor and localization of an allosteric binding site

A homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca2+]i responses. The calcimimetic NPS R-568 increases the potency of Ca2+ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca2+ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function.     

"Molecular switches" on mGluR allosteric ligands that modulate modes of pharmacology

G-protein-coupled receptors (GPCRs) represent the largest class of drug targets, accounting for more than 40% of marketed drugs; however, discovery efforts for many GPCRs have failed to provide viable drug candidates. Historically, drug discovery efforts have focused on developing ligands that act at the orthosteric site of the endogenous agonist. Recently, efforts have focused on functional assay paradigms and the discovery of ligands that act at allosteric sites to modulate receptor function in either a positive, negative, or neutral manner. Allosteric modulators have numerous advantages over orthosteric ligands, including high subtype selectivity; the ability to mimic physiological conditions; the lack of densensitization, downregulation, and internalization; and reduced side effects. Despite these virtues, challenging issues have now arisen for allosteric modulators of metabotropic glutamate receptors (mGluRs): shallow SAR, ligand-directed trafficking, and the identification of subtle "molecular switches" that modulate the modes of pharmacology. Here, we will discuss the impact of modest structural changes to multiple mGluR allosteric ligands scaffolds that unexpectedly modulate pharmacology and raise concerns over metabolism and the pharmacology of metabolites.     

Evidence for distinct cation and calcimimetic compound (NPS 568) recognition domains in the transmembrane regions of the human Ca2+ receptor

The Ca(2+) receptor, a member of the family 3 of G protein-coupled receptors (GPCR), responds not only to its primary physiological ligand Ca(2+) but also to other di- and trivalent metals (Mg(2+), Gd(3+)) and the organic polycations spermine and poly-l-Arginine. As has been found for other family 3 GPCRs, the large amino-terminal extracellular domain (ECD) of the Ca(2+) receptor is the primary Ca(2+) binding domain. To examine how the signal is propagated from the ECD to the seven-transmembrane core domain (7TM) we constructed a Ca(2+) receptor mutant (T903-Rhoc) lacking the entire ECD but containing the 7TM. We have found that this structure initiates signaling in human embryonic kidney (HEK) 293 cells stably expressing the construct. One or more cation recognition sites are also located within the 7TM. Not only Ca(2+), but also several other Ca(2+) receptor-specific agonists, Mg(2+), Gd(3+), spermine, and poly-l-Arginine, can activate T903-Rhoc truncated receptor-initiated phosphoinositide hydrolysis in HEK 293 cells. The phenylalkylamine compound, NPS 568, identified as a positive allosteric modulator of the Ca(2+) receptor can selectively potentiate the actions of Ca(2+) and other polycationic agonists on the T903-Rhoc receptor. Similarly, organic polycations synergistically activate T903-Rhoc with di- and trivalent metals. Alanine substitution of all the acidic residues in the second extracellular loop of the T903-Rhoc receptor significantly impairs activation by metal ions and organic polycations in the presence of NPS 568 but not the synergistic activation of Ca(2+) with poly-l-Arginine. These data indicate that although the ECD has been thought to be the main determinant for Ca(2+) recognition, the 7TM core of the Ca(2+) receptor contains activating site(s) recognizing Ca(2+) and Gd(3+) as well as the allosteric modulators NPS 568 and organic polycations that may play important roles in the regulation of receptor activation.     

Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has a Kd of 5.6?μM (ΔH=-4.9 kcal/mol, -TΔS=-2.3 kcal/mol; where 1?kcal≈4.187?kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46?mM (ΔH=-1.2 kcal/mol, -TΔS=-3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders.     

Critical role for the second extracellular loop in the binding of both orthosteric and allosteric G protein-coupled receptor ligands

The second extracellular (E2) loop of G protein-coupled receptors (GPCRs) plays an essential but poorly understood role in the binding of non-peptidic small molecules. We have utilized both orthosteric ligands and allosteric modulators of the M2 muscarinic acetylcholine receptor, a prototypical Family A GPCR, to probe possible E2 loop binding dynamics. We developed a homology model based on the crystal structure of bovine rhodopsin and predicted novel cysteine substitutions that should dramatically reduce E2 loop flexibility via disulfide bond formation and significantly inhibit the binding of both types of ligands. This prediction was validated experimentally using radioligand binding, dissociation kinetics, and cell-based functional assays. The results argue for a flexible "gatekeeper" role of the E2 loop in the binding of both allosteric and orthosteric GPCR ligands.     

The impact of cryo-EM on determining allosteric modulator-bound structures of G protein-coupled receptors

G-protein coupled receptors (GPCRs) are important therapeutic targets for the treatment of human disease. Although GPCRs are highly successful drug targets, there are many challenges associated with the discovery and translation of small molecule ligands that target the endogenous ligand-binding site for GPCRs. Allosteric modulators are a class of ligands that target alternative binding sites known as allosteric sites and offer fresh opportunities for the development of new therapeutics. However, only a few allosteric modulators have been approved as drugs. Advances in GPCR structural biology enabled by the cryogenic electron microscopy (cryo-EM) revolution have provided new insights into the molecular mechanism and binding location of small molecule allosteric modulators. This review highlights the latest findings from allosteric modulator-bound structures of Class A, B, and C GPCRs with a focus on small molecule ligands. Emerging methods that will facilitate cryo-EM structures of more difficult ligand-bound GPCR complexes are also discussed. The results of these studies are anticipated to aid future structure-based drug discovery efforts across many different GPCRs.     

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA(A) receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA(A) receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.