Allosteric modulation of kinases and GPCRs: design principles and structural diversity

Allosteric binding sites, as opposed to traditional orthosteric binding sites, offer unparalleled opportunities for drug discovery by providing high levels of selectivity, mimicking physiological conditions, affording fewer side effects because of desensitization/downregulation, and engendering ligands with chemotypes divergent from orthosteric ligands. For kinases, allosteric mechanisms described to date include alteration of protein kinase conformation blocking productive ATP binding which appear 'ATP competitive' or blocking kinase activation by conformational changes that are 'ATP non-competitive'. For GPCRs, allosteric mechanisms impart multiple modes of target modulation (positive allosteric modulation (PAM), negative allosteric modulation (NAM), neutral cooperativity, partial antagonism (PA), allosteric agonism and allosteric antagonism). Here, we review recent developments in the design principles and structural diversity of allosteric ligands for kinases and GPCRs.     

Challenges in the development of mGluR5 positive allosteric modulators: the discovery of CPPHA

This Letter describes, for the first time, the synthesis and SAR, developed through an iterative analog library approach, that led to the discovery of the positive allosteric modulator (PAM) of the metabotropic glutamate receptor mGluR5 CPPHA. Binding to a unique allosteric binding site distinct from other mGluR5 PAMs, CPPHA has been the focus of numerous pharmacology studies by several laboratories.     

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.     

N-Aryl pyrrolidinonyl oxadiazoles as potent mGluR5 positive allosteric modulators

A novel series of N-aryl pyrrolidinonyl oxadiazoles were identified as mGluR5 positive allosteric modulators (PAMs). Optimization of the initial lead compound 6a led to the identification of the 12c (-) enantiomer as a potent compound with acceptable in vitro clearance, CYP, hERG and PK properties. Para substituted N-aryl pyrrolidinonyl oxadiazoles are mGluR5 PAMs while the meta and ortho substituted N-aryl pyrrolidinonyl oxadiazoles are negative allosteric modulators (NAMs). Para fluoro substitution on the N-aryl group and meta chloro or methyl substituents on the aryl oxadiazole moiety are optimal for mGluR5 PAM efficacy. The existence of an exquisitely sensitive 'PAM to NAM switch' within this chemotype making it challenging for simultaneous optimization of potency and drug-like properties.     

Glutamate Acts as a Partial Inverse Agonist to Metabotropic Glutamate Receptor with a Single Amino Acid Mutation in the Transmembrane Domain

Metabotropic glutamate receptor (mGluR), a prototypical family 3 G protein-coupled receptor (GPCR), has served as a model for studying GPCR dimerization, and growing evidence has revealed that a glutamate-induced dimeric rearrangement promotes activation of the receptor. However, structural information of the seven-transmembrane domain is severely limited, in contrast to the well studied family 1 GPCRs including rhodopsins and adrenergic receptors. Homology modeling of mGluR8 transmembrane domain with rhodopsin as a template suggested the presence of a conserved water-mediated hydrogen-bonding network between helices VI and VII, which presumably constrains the receptor in an inactive conformation. We therefore conducted a mutational analysis to assess structural similarities between mGluR and family 1 GPCRs. Mutational experiments confirmed that the disruption of the hydrogen-bonding network by T789Y^6.43 mutation induced high constitutive activity. Unexpectedly, this high constitutive activity was suppressed by glutamate, the natural agonist ligand, indicating that glutamate acts as a partial inverse agonist to this mutant. Fluorescence energy transfer analysis of T789Y^6.43 suggested that the glutamate-induced reduction of the activity originated not from the dimeric rearrangement but from conformational changes within each protomer. Double mutational analysis showed that the specific interaction between Tyr-789^6.43 and Gly-831^7.45 in T789Y^6.43 mutant was important for this phenotype. Therefore, the present study is consistent with the notion that the metabotropic glutamate receptor shares a common activation mechanism with family 1 GPCRs, where rearrangement between helices VI and VII causes the active state formation.     

Discovery,synthesis, and structure–activity relationships of 2-aminoquinazoline derivatives as a novel class of metabotropic glutamate receptor 5 negative allosteric modulators

A virtual screening approach using various in silico methodologies led to the discovery of 2-(m-tolylamino)-7,8-dihydroquinazolin-5(6H)-one (1) as a moderately active negative allosteric modulator (NAM) of the metabotropic glutamate receptor subtype 5 (mGluR5) showing high selectivity against the subtype mGluR1. Modifications of the parent compound by rational design yielded a series of highly potent derivatives which will serve as valuable starting points for further hit-to-lead optimization efforts toward a suitable drug candidate for the treatment of l-DOPA induced dyskinesia.     

The anatomy of mammalian sweet taste receptors

All sweet‐tasting compounds are detected by a single G‐protein coupled receptor (GPCR), the heterodimer T1R2‐T1R3, for which no experimental structure is available. The sweet taste receptor is a class C GPCR, and the recently published crystallographic structures of metabotropic glutamate receptor (mGluR) 1 and 5 provide a significant step forward for understanding structure‐function relationships within this family. In this article, we recapitulate more than 600 single point site‐directed mutations and available structural data to obtain a critical alignment of the sweet taste receptor sequences with respect to other class C GPCRs. Using this alignment, a homology 3D‐model of the human sweet taste receptor is built and analyzed to dissect out the role of key residues involved in ligand binding and those responsible for receptor activation. Proteins 2017; 85:332–341. © 2016 Wiley Periodicals, Inc.     

Allosteric Effects of Sodium Ion Binding on Activation of the M3 Muscarinic G-Protein-Coupled Receptor

G-protein-coupled receptors (GPCRs) are important membrane proteins that mediate cellular signaling and represent primary targets for about one-third of currently marketed drugs. Recent x-ray crystallographic studies identified distinct conformations of GPCRs in the active and inactive states. An allosteric sodium ion was found bound to a highly conserved D2.50 residue in inactive GPCRs, whereas the D2.50 allosteric pocket became collapsed in active GPCR structures. However, the dynamic mechanisms underlying these observations remain elusive. In this study, we aimed to understand the mechanistic effects of sodium ion binding on dynamic activation of the M3 muscarinic GPCR through long-timescale accelerated molecular dynamics (aMD) simulations. Results showed that with the D2.50 residue deprotonated, the M3 receptor is bound by an allosteric sodium ion and confined mostly in the inactive state with remarkably reduced flexibility. In contrast, the D2.50-protonated receptor does not exhibit sodium ion binding to the D2.50 allosteric site and samples a significantly larger conformational space. The receptor activation is captured and characterized by large-scale structural rearrangements of the transmembrane helices via dynamic hydrogen bond and salt bridge interactions. The residue motions are highly correlated during receptor activation. Further network analysis revealed that the allosteric signaling between residue D2.50 and key residues in the intracellular, extracellular, and orthosteric pockets is significantly weakened upon sodium ion binding.     

Structural Determinants of Allosteric Agonism and Modulation at the M4 Muscarinic Acetylcholine Receptor: IDENTIFICATION OF LIGAND-SPECIFIC AND GLOBAL ACTIVATION MECHANISMS*

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M₄ mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M₁/M₄ mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M₄ mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile⁹³ and Lys⁹⁵ as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe¹⁸⁶ in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp⁴³² in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr⁴³⁹ and Tyr⁴⁴³, were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.     

Endogenous Benzodiazepine Site Peptide Ligands Operating Bidirectionally In Vivo in Neurogenesis and Thalamic Oscillations

By binding to the benzodiazepine site, diazepam binding inhibitor (DBI) is associated with negative allosteric modulation (NAM) of GABA_A receptors (Costa and Guidotti in Life Sci 49:325–344, 1991). However, the demonstration of a true physiological role of DBI and its fragments has only recently been reported. Based on DBI gain- and loss-of-function experiments in vivo, DBI and its fragment ODN were found to promote neurogenesis in the subventricular zone in vivo. Acting as NAM on GABA_A receptors of precursor cells, DBI counteracted the inhibitory effect of GABA and thereby enhanced the proliferation of these cells (Alfonso et al. in Cell Stem Cell 10:76–87, 2012). Conversely and most remarkably, in similar gain- and loss-of-function experiments in the thalamus, the DBI gene products acted as positive allosteric modulators (PAM) of GABA_A receptors in prolonging the duration of IPSCs, an effect which was specific for GABA transmission within the reticular nucleus (nRT) (Christian et al. in Neuron 78:1063–1074, 2013). Since intra-nRT potentiation of GABA transmission by benzodiazepine drugs exerts powerful anti-oscillatory effects, DBI might be endogenously effective by modulating seizure susceptibility. It remains to be seen by which mechanism both NAM and PAM activity can arise from the Dbi gene. Nevertheless, the results open new perspectives on the regionally distinct endogenous modulation of GABA transmission via the benzodiazepine site.     

Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca²⁺ ([Ca²⁺]_o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca²⁺-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca²⁺-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca²⁺ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca²⁺ ([Ca²⁺]_i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca²⁺-sensing capability largely intact. Mutating the predicted Ca²⁺-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca²⁺]_o and [Gd³⁺]_o but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca²⁺]_o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.     

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).     

Role of the extracellular amino terminus and first membrane-spanning helix of dopamine D1 and D5 receptors in shaping ligand selectivity and efficacy

Transmembrane (TM) helices of human D1-like dopaminergic receptors (hD1R and hD5R) harbor the same residues implicated in ligand binding and activation of catecholamine G protein-coupled receptors (GPCRs). Yet, hD1R and hD5R naturally display the distinct functional properties shared by wild type and constitutively active mutant GPCRs, respectively. Interestingly, we show in the present study that a class of synthetic phenylbenzazepine agonists containing a methyl on the azepine ring exhibited lower affinity for the more constitutively activated hD5R. These results cannot be explained by the “allosteric ternary complex model” postulating a higher agonist affinity for constitutively active GPCRs. We have also explored the functional role of distinct extracellular amino terminus (NT) and TM1 regions of hD1R and hD5R using a chimerical approach. Of these two regions, our studies suggest that TM1 predominantly shapes D1-like ligand affinity and selectivity. Additionally, NT and TM1 of hD1R and hD5R play no role in receptor constitutive activity but differentially modulate dopamine-mediated responsiveness. The TM1 exchange mediated drastic changes in intrinsic efficacy and activity of phenylbenzazepine drugs displaying partial agonism at hD1R and hD5R. Phenylbenzazepines were converted into strong partial agonists or full agonists in cells expressing hD1R-TM1^D5 chimera while being switched from full agonists to partial agonists and partial agonists to antagonists in cells harboring hD5R-TM1^D1 chimera. TM1 exchange had no effect on antipsychotic-mediated inverse agonism. In summary, our study shows that NT and TM1 of D1-like receptors control ligand binding and agonist-induced activation, poising these regions as important structural determinants for catecholamine GPCR function.     

Conformational change of the transmembrane helices II and IV of metabotropic glutamate receptor involved in G protein activation

G protein-coupled receptors are classified into several families on the basis of their amino acid sequences and the members of the same family exhibit sequence similarity but those of different families do not. In family 1 GPCRs such as rhodopsin and adrenergic receptor, extensive studies have revealed the stimulus-dependent conformational change of the receptor: the rearrangement of transmembrane helices III and VI is essential for G protein activation. In contrast, in family 3 GPCRs such as metabotropic glutamate receptor (mGluR), the inter-protomer relocation upon ligand binding has been observed but there is much less information about the structural changes of the transmsmbrane helices and the cytoplasmic domains. Here we identified constitutively active mutation sites at the cytoplasmic borders of helices II and IV of mGluR8 and successfully inhibited the G protein activation ability by engineering disulfide cross-linking between these cytoplasmic regions. The analysis of all possible single substitution mutants of these residues revealed that some steric interactions around these sites would be important to keep the receptor protein inactive. These results provided the model that the conformational changes at the cytoplasmic ends of helices II and IV of mGluR are involved in the efficient G protein coupling.     

Determinants of Endogenous Ligand Specificity Divergence among Metabotropic Glutamate Receptors

To determine the structural origins of diverse ligand response specificities among metabotropic glutamate receptors (mGluRs), we combined computational approaches with mutagenesis and ligand response assays to identify specificity-determining residues in the group I receptor, mGluR1, and the group III receptors, mGluR4 and mGluR7. Among these, mGluR1 responds to l-glutamate effectively, whereas it binds weakly to another endogenous ligand, l-serine-O-phosphate (l-SOP), which antagonizes the effects of l-glutamate. In contrast, mGluR4 has in common with other group III mGluR that it is activated with higher potency and efficacy by l-SOP. mGluR7 differs from mGluR4 and other group III mGluR in that l-glutamate and l-SOP activate it with low potency and efficacy. Enhanced versions of the evolutionary trace (ET) algorithm were used to identify residues that when swapped between mGluR1 and mGluR4 increased the potency of l-SOP inhibition relative to the potency of l-glutamate activation in mGluR1 mutants and others that diminished the potency/efficacy of l-SOP for mGluR4 mutants. In addition, combining ET identified swaps from mGluR4 with one identified by computational docking produced mGluR7 mutants that respond with dramatically enhanced potency/efficacy to l-SOP. These results reveal that an early functional divergence between group I/II and group III involved variation at positions primarily at allosteric sites located outside of binding pockets, whereas a later divergence within group III occurred through sequence variation both at the ligand-binding pocket and at loops near the dimerization interface and interlobe hinge region. They also demonstrate the power of ET for identifying allosteric determinants of evolutionary importance.     

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

TBPB and 77-LH-28-1 are selective agonists of the M₁ muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M₁ mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.     

4-(1-Phenyl-1H-pyrazol-4-yl)quinolines as novel, selective and brain penetrant metabotropic glutamate receptor 4 positive allosteric modulators

4-(1-Phenyl-1H-pyrazol-4-yl)quinoline (1) was identified by screening the Lundbeck compound collection, and characterized as having mGlu4 receptor positive allosteric modulator properties. Compound 1 is selective over other mGlu receptors and a panel of GPCRs, ion channels and enzymes, but has suboptimal lipophilicity and high plasma and brain non-specific binding. In view of the challenges at the hit-to-lead stage previously reported in the development of mGlu4 receptor positive allosteric modulators (PAMs), a thorough structure-mGlu4 PAM activity relationship study was conducted to interrogate the chemical tractability of this chemotype. The central pyrazole ring tolerates the addition of one or two methyl groups. The C-7 position of the quinoline ring provides a site tolerant to hydrophilic substituents, enabling the design of diverse analogs with good in vitro mGlu4 PAM potency and efficacy, as well as improved microsomal turnover in vitro, compared to 1. In spite of the excellent ligand efficiency of 1 (LE=0.43), optimization of in vitro potency for this series reached a plateau around EC(50)=200 nM.     

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.     

Differences in Allosteric Communication Pipelines in the Inactive and Active States of a GPCR

G-protein-coupled receptors (GPCRs) are membrane proteins that allosterically transduce the signal of ligand binding in the extracellular (EC) domain to couple to proteins in the intracellular (IC) domain. However, the complete pathway of allosteric communication from the EC to the IC domain, including the role of individual amino acids in the pathway is not known. Using the correlation in torsion angle movements calculated from microseconds-long molecular-dynamics simulations, we elucidated the allosteric pathways in three different conformational states of β₂-adrenergic receptor (β₂AR): 1), the inverse-agonist-bound inactive state; 2), the agonist-bound intermediate state; and (3), the agonist- and G-protein-bound fully active state. The inactive state is less dynamic compared with the intermediate and active states, showing dense clusters of allosteric pathways (allosteric pipelines) connecting the EC with the IC domain. The allosteric pipelines from the EC domain to the IC domain are weakened in the intermediate state, thus decoupling the EC domain from the IC domain and making the receptor more dynamic compared with the other states. Also, the orthosteric ligand-binding site becomes the initiator region for allosteric communication in the intermediate state. This finding agrees with the paradigm that the nature of the agonist governs the specific signaling state of the receptor. These results provide an understanding of the mechanism of allosteric communication in class A GPCRs. In addition, our analysis shows that mutations that affect the ligand efficacy, but not the binding affinity, are located in the allosteric pipelines. This clarifies the role of such mutations, which has hitherto been unexplained.