Negative allosteric modulators of cannabinoid receptor 2: protein modeling,binding site identification and molecular dynamics simulations in the presence of an orthosteric agonist

Abstract

Selective activation of the cannabinoid receptor subtype 2 (CB2) shows promise for treating pain, inflammation, multiple sclerosis, cancer, ischemic/reperfusion injury and osteoporosis. Target selectivity and off-target side effects are two major limiting factors for orthosteric ligands, and therefore, the search for allosteric modulators (AMs) is a widely used drug discovery approach. To date, only a limited number of negative CB2 AMs have been identified, possessing only micromolar activity at best, and the CB2 receptor’s allosteric site(s) are not well characterized. Herein, we used computational approaches including receptor modeling, site mapping, docking, molecular dynamics (MD) simulations and binding free energy calculations to predict, characterize and validate allosteric sites within the complex of the CB2 receptor with bound orthosteric agonist CP55,940. After docking of known negative CB2 allosteric modulators (NAMs), dihydro-gambogic acid (DHGA) and trans-β-caryophyllene (TBC) (note that TBC also shows agonist activity), at the predicted allosteric sites, the best total complex with CB2, CP55,940 and NAM was embedded into a hydrated lipid bilayer and subjected to a 200 ns MD simulation. The presence of an AM affected the CB2–CP55,940 complex, altering the relative positioning of the toggle switch residues and promoting a strong π–π interaction between Phe117^3.36 and Trp258^6.48. Binding of either TBC or DHGA to a putative allosteric pocket directly adjacent to the orthosteric ligand reduced the binding free energy of CP55,940, which is consistent with the expected effect of a negative AM. The identified allosteric sites present immense scope for the discovery of novel classes of CB2 AMs.     

Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.     

Selective orthosteric free fatty acid receptor 2 (FFA2) agonists: identification of the structural and chemical requirements for selective activation of FFA2 versus FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [(35)S]guanosine 5'-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp(3)-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp(2)- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.     

Allosteric Hotspots in the Main Protease of SARS-CoV-2

Inhibiting the main protease of SARS-CoV-2 is of great interest in tackling the COVID-19 pandemic caused by the virus. Most efforts have been centred on inhibiting the binding site of the enzyme. However, considering allosteric sites, distant from the active or orthosteric site, broadens the search space for drug candidates and confers the advantages of allosteric drug targeting. Here, we report the allosteric communication pathways in the main protease dimer by using two novel fully atomistic graph-theoretical methods: Bond-to-bond propensity, which has been previously successful in identifying allosteric sites in extensive benchmark data sets without a priori knowledge, and Markov transient analysis, which has previously aided in finding novel drug targets in catalytic protein families. Using statistical bootstrapping, we score the highest ranking sites against random sites at similar distances, and we identify four statistically significant putative allosteric sites as good candidates for alternative drug targeting.     

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.     

Orthosteric and allosteric binding sites of P2X receptors

P2X receptors for ATP comprise a distinct family of ligand gated ion channels with a range of properties. They have been shown to be involved in a variety of physiological processes including blood clotting, sensory perception, pain sensation, bone formation as well as inflammation and may provide a number of novel drug targets. In addition to the orthosteric site for ATP binding it has been suggested that there may be additional allosteric sites that regulate agonist action at the receptor. There is currently no crystal structure available for P2X receptors and the lack of sequence similarity to other ATP binding proteins has meant that a mutagenesis-based approach has been used primarily to investigate receptor structure-function. This review aims to provide an overview of recent work that gives an insight into residues involved in ATP action and allosteric regulation.     

The Activity of GAT107, an Allosteric Activator and Positive Modulator of α7 Nicotinic Acetylcholine Receptors (nAChR), Is Regulated by Aromatic Amino Acids That Span the Subunit Interface

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.     

Benzodiazepine ligands can act as allosteric modulators of the Type 1 cholecystokinin receptor

The cholecystokinin (CCK₁) receptor is a G protein-coupled receptor important for nutrient homeostasis. The molecular basis of CCK-receptor binding has been debated, with one prominent model suggesting occupation of the same region of the intramembranous helical bundle as benzodiazepines. Here, we used a specific assay of allosteric ligand interaction to probe the mode of binding of devazepide, a prototypic benzodiazepine ligand. Devazepide elicited marked slowing of dissociation of pre-bound CCK, only possible through binding to a topographically distinct allosteric site. This effect was disrupted by chemical modification of a cysteine in the benzodiazepine-binding pocket. Application of an allosteric model to the equilibrium interaction between a series of benzodiazepine ligands and CCK yielded quantitative estimates of each modulator’s affinity for the allosteric site, as well as the degree of negative cooperativity for the interaction between occupied orthosteric and allosteric sites. The allosteric nature of benzodiazepine binding to the CCK₁ receptor provides new opportunities for small molecule drug development.     

Defining the Molecular Basis for the First Potent and Selective Orthosteric Agonists of the FFA2 Free Fatty Acid Receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective orthosteric FFA2 agonists. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons, and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 and the transmembrane domain regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in inhibition of lipolysis and glucagon-like peptide-1 secretion in murine-derived 3T3-L1 and STC-1 cell lines, respectively. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the binding site of FFA2 that will be invaluable in future ligand development at this receptor.     

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.     

Virtual screening of a natural compound library at orthosteric and allosteric binding sites of the neurotensin receptor

Abstract

Molecular dynamics (MD) simulation using the AMBER force field has been performed on the neurotensin (NT) receptor, a class A type G-protein-coupled receptor in its activated conformation co-crystallized with the non-peptide agonists. For structure-based hit molecule identification via natural chemical compound library, orthosteric sites on NT receptor have been mapped by docking using AutoDock4.0 and Vina with the known agonists and antagonists SR48692, SR142948, ML301 and ML314 of the receptor. Furthermore, clustering analysis on the MD trajectories by SIMULAID has been performed to filter receptor conformations for the allosteric binders from the Otava natural compound library. Comparative mappings of contrasting binding region patterns have been done between the crystal structure orthosteric sites as well as the binding regions in the SIMULAID-based cluster center conformations from MD trajectories with the FTmap server using the small organic molecule fragments as the probes. The distinct binding region in the cluster-based conformations in the extracellular region of the receptor has been identified for targeted docking by Otava natural chemical compound library using AutoDock4.0 and Vina docking suites to obtain putative allosteric binders. A group of compounds from the Otava library has been identified as showing high free energy in both AutoDock4.0 and Vina docking suites. Biophysical assessments on the natural compound computational hit molecules will be done to identify lead structures from the hit molecules.

Communicated by Ramaswamy H. Sarma     

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.     

The first synthetic agonists of FFA2: Discovery and SAR of phenylacetamides as allosteric modulators

Free fatty acid receptor 2 (FFA2) is a G-protein coupled receptor for which only short-chain fatty acids (SCFAs) have been reported as endogenous ligands. We describe the discovery and optimization of phenylacetamides as allosteric agonists of FFA2. These novel ligands can suppress adipocyte lipolysis in vitro and reduce plasma FFA levels in vivo, suggesting that these allosteric modulators can serve as pharmacological tools for exploring the potential function of FFA2 in various disease conditions.     

Novobiocin and peptide analogs of α-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p–[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.     

Data-driven analysis in drug discovery

In the process of drug discovery for new chemical entities, application of appropriate pharmacological models often is not possible because the molecular mechanism of the compound is not yet elucidated. Therefore, a data-driven approach using generic tools designed to quantify characteristic patterns of concentration-response curves is required. This article outlines the options available for quantifying agonist and antagonist activity. Specifically, for agonists, the use of the Operational model for the determination of functional effects (equimolar potency ratios for full agonists, calculation of relative efficacy) is described. For antagonists, the measurement of pKB (-log of the equilibrium dissociation constant of the antagonist-receptor complex) for orthosteric antagonists that do not alter basal response (simple competitive antagonists), increase basal response (partial agonists), and decrease basal response (in constitutively active systems; inverse agonists) is discussed. In addition, this article considers methods to discern orthosteric receptor antagonism from allosteric antagonism whereby the agonist and antagonist bind to separate sites and interact through a conformational change in the receptor. Methods for the measurement of the pKB for allosteric modulators as well as co-operativity constants for these modulators is described.     

Data-Driven Analysis in Drug Discovery

In the process of drug discovery for new chemical entities, application of appropriate pharmacological models often is not possible because the molecular mechanism of the compound is not yet elucidated. Therefore, a data-driven approach using generic tools designed to quantify characteristic patterns of concentration-response curves is required. This article outlines the options available for quantifying agonist and antagonist activity. Specifically, for agonists, the use of the Operational model for the determination of functional effects (equimolar potency ratios for full agonists, calculation of relative efficacy) is described. For antagonists, the measurement of pK_B (-log of the equilibrium dissociation constant of the antagonist-receptor complex) for orthosteric antagonists that do not alter basal response (simple competitive antagonists), increase basal response (partial agonists), and decrease basal response (in constitutively active systems; inverse agonists) is discussed. In addition, this article considers methods to discern orthosteric receptor antagonism from allosteric antagonism whereby the agonist and antagonist bind to separate sites and interact through a conformational change in the receptor. Methods for the measurement of the pK_B for allosteric modulators as well as co-operativity constants for these modulators is described.     

Identification of an allosteric binding site for Zn2+ on the beta2 adrenergic receptor

The activity of G protein-coupled receptors (GPCRs) can be modulated by a diverse spectrum of drugs ranging from full agonists to partial agonists, antagonists, and inverse agonists. The vast majority of these ligands compete with native ligands for binding to orthosteric binding sites. Allosteric ligands have also been described for a number of GPCRs. However, little is known about the mechanism by which these ligands modulate the affinity of receptors for orthosteric ligands. We have previously reported that Zn(II) acts as a positive allosteric modulator of the beta(2)-adrenergic receptor (beta(2)AR). To identify the Zn(2+) binding site responsible for the enhancement of agonist affinity in the beta(2)AR, we mutated histidines located in hydrophilic sequences bridging the seven transmembrane domains. Mutation of His-269 abolished the effect of Zn(2+) on agonist affinity. Mutations of other histidines had no effect on agonist affinity. Further mutagenesis of residues adjacent to His-269 demonstrated that Cys-265 and Glu-225 are also required to achieve the full allosteric effect of Zn(2+) on agonist binding. Our results suggest that bridging of the cytoplasmic extensions of TM5 and TM6 by Zn(2+) facilitates agonist binding. These results are in agreement with recent biophysical studies demonstrating that agonist binding leads to movement of TM6 relative to TM5.     

3- and 6-Substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as A1 adenosine receptor allosteric modulators and antagonists

A series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were prepared and evaluated as potential allosteric modulators at the A₁ adenosine receptor. The structure–activity relationships of the 3- and 6-positions of a series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were explored. Despite finding that 3- and 6-substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines possess the ability to recognize an allosteric site on the agonist-occupied A₁AR at relatively high concentrations, the structural modifications we have performed on this scaffold favor the expression of orthosteric antagonist properties over allosteric properties. This research has identified 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as novel class of orthosteric antagonist of the A₁AR and highlighted the close relationship between structural elements governing allosteric modulation and orthosteric antagonism of agonist function at the A₁AR.     

Regulation of signal transduction at M2 muscarinic receptor

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.