A Structure-Based Allosteric Modulator Design Paradigm.

Allosteric modulators, by targeting the less-conserved allosteric sites, represent an innovative strategy in drug discovery. These modulators have a distinctive advantage over orthosteric ligands that attach to the conserved, functional orthosteric sites. However, in structure-based drug design, it remains unclear whether allosteric protein structures determined without orthosteric ligand binding are suitable for allosteric drug screening. In this study, we performed large-scale conformational samplings of six representative allosteric proteins uncomplexed ( apo) and complexed ( holo) with orthosteric ligands to explore the effect of orthosteric site binding on the conformational dynamics of allosteric sites. The results, coupled with the redocking evaluation of allosteric modulators to their apo and holo proteins using their MD trajectories, indicated that orthosteric site binding had an effect on the dynamics of the allosteric sites and allosteric modulators preferentially bound to their holo proteins. According to the analysis data, we constructed a new correlation model for quantifying the allosteric site change driven by substrate binding to the orthosteric site. These results highlight the strong demand to select holo allosteric proteins as initial inputs in structure-based allosteric drug screening when the distance between orthosteric and allosteric sites in the protein is below 5 Å, which is expected to contribute to allosteric drug discovery.

Functional and computational studies reveal that the allosteric inhibition mechanism is conserved between glutamate and neutral amino acid transporters of the SLC1 family, and identify a novel allosteric inhibitor.

Development of allosteric modulators of GPCRs for treatment of CNS disorders

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.

Unraveling the mystery of protein allostery has been one of the greatest challenges in both structural and computational biology. However, recent advances in computational methods, particularly molecular dynamics (MD) simulations, have led to its utility as a powerful and popular tool for the study of protein allostery. By capturing the motions of a protein's constituent atoms, simulations can enable the discovery of allosteric hot spots and the determination of the mechanistic basis for allostery. These structural and dynamic studies can provide a foundation for a wide range of applications, including rational drug design and protein engineering. In our laboratory, the use of MD simulations and network analysis assisted in the elucidation of the allosteric hotspots and intracellular signal transduction of G protein-coupled receptors (GPCRs), primarily on one of the adenosine receptor subtypes, A2A adenosine receptor (A2AAR). In this chapter, we describe a method for calculating the map of allosteric signal flow in different GPCR conformational states and illustrate how these concepts have been utilized in understanding the mechanism of GPCR allostery. These structural studies will provide valuable insights into the allosteric and orthosteric modulations that would be of great help to design novel drugs targeting GPCRs in pathological states.

Recently determinedstructures of class C G protein-coupledreceptors(GPCRs) revealed the location of allosteric binding sites and openednew opportunities for the discovery of novel modulators. In this work,molecular docking screens for allosteric modulators targeting themetabotropic glutamate receptor 5 (mGlu5) were performed.The mGlu5 receptor is activated by the main excitatoryneurotransmitter of the nervous central system, L-glutamate, and mGlu5 receptor activity can be allosterically modulated by negativeor positive allosteric modulators. The mGlu5 receptor isa promising target for the treatment of psychiatric and neurodegenerativediseases, and several allosteric modulators of this GPCR have beenevaluated in clinical trials. Chemical libraries containing fragment-(1.6 million molecules) and lead-like (4.6 million molecules) compoundswere docked to an allosteric binding site of mGlu5 identifiedin X-ray crystal structures. Among the top-ranked compounds, 59 fragmentsand 59 lead-like compounds were selected for experimental evaluation.Of these, four fragment- and seven lead-like compounds were confirmedto bind to the allosteric site with affinities ranging from 0.43 to8.6 μM, corresponding to a hit rate of 9%. The four compoundswith the highest affinities were demonstrated to be negative allostericmodulators of mGlu5 signaling in functional assays. Theresults demonstrate that virtual screens of fragment- and lead-likechemical libraries have complementary advantages and illustrate howaccess to high-resolution structures of GPCRs in complex with allostericmodulators can accelerate lead discovery.

The rational search for allosteric modulators and the allosteric mechanisms of these modulators in the presence of evolutionary mutations, including resistant ones, is a relatively unexplored field. Here, we established novel in silico approaches and applied to SARS-CoV-2 main protease (Mpro). First, we identified six potential allosteric modulators (SANC00302, SANC00303, SANC00467, SANC00468, SANC00469, SANC00630) from the South African Natural Compounds Database (SANCDB) bound to the allosteric pocket of Mpro that we determined in our previous work. We also checked the stability of these compounds against Mpro of laboratory strain HCoV-OC43 and identified differences due to residue changes between the two proteins. Next, we focused on understanding the allosteric effects of these modulators on each protomer of the reference Mpro protein, while incorporating the symmetry problem in the functional homodimer. In general, asymmetric behavior of multimeric proteins is not commonly considered in computational analysis. We introduced a novel combinatorial approach and dynamic residue network (DRN) analysis algorithms to examine patterns of change and conservation of critical nodes, according to five independent criteria of network centrality (betweenness centrality (BC), closeness centrality (CC), degree centrality (DC), eigencentrality (EC) and katz centrality (KC)). The relationships and effectiveness of each metric in characterizing allosteric behavior were also investigated. We observed highly conserved network hubs for each averaged DRN metric on the basis of their existence in both protomers in the absence and presence of all ligands, and we called them persistent hubs (residues 17, 111, 112 and 128 for averaged BC; 6, 7, 113, 114, 115, 124, 125, 126, 127 and 128 for averaged CC; 36, 91, 146, 150 and 206 for averaged DC; 7, 115 and 125 for EC; 36, 125 and 146 for KC). We also detected ligand specific signal changes some of which were in or around functional residues (i.e. chameleon switch PHE140). Using EC persistent hubs and ligand introduced hubs we identified a residue communication path between allosteric binding site and catalytic site. Finally, we examined the effects of the mutations on the behavior of the protein in the presence of selected potential allosteric modulators and investigated the ligand stability. The hit compounds showed various levels of stability in the presence of SARS-CoV-2 Mpro mutations, being most stable in A173V, N274D and R279C, and least stable in R60C, N151D V157I, C160S and A255V. SANC00468 was the most stable compound in the 43 mutant protein systems. We further used DRN metric analysis to define cold spots as being those regions that are least impacted, or not impacted, by mutations. One crucial outcome of this study was to show that EC centrality hubs form an allosteric communication path between the allosteric ligand binding site to the active site going through the interface residues of Domain I and II; and this path was either weakened or lost in the presence of some of the mutations. Overall, the results of this study revealed crucial aspects that need to be considered in drug discovery in COVID-19 specifically and in general for rational computational drug design purposes.

Understanding mechanisms of allosteric regulation remains elusive for the SARS-CoV-2 spike protein, despite the increasing interest and effort in discovering allosteric inhibitors of the viral activity and interactions with the host receptor ACE2. The challenges of discovering allosteric modulators of the SARS-CoV-2 spike proteins are associated with the diversity of cryptic allosteric sites and complex molecular mechanisms that can be employed by allosteric ligands, including the alteration of the conformational equilibrium of spike protein and preferential stabilization of specific functional states. In the current study, we combine conformational dynamics analysis of distinct forms of the full-length spike protein trimers and machine-learning-based binding pocket detection with the ensemble-based ligand docking and binding free energy analysis to characterize the potential allosteric binding sites and determine structural and energetic determinants of allosteric inhibition for a series of experimentally validated allosteric molecules. The results demonstrate a good agreement between computational and experimental binding affinities, providing support to the predicted binding modes and suggesting key interactions formed by the allosteric ligands to elicit the experimentally observed inhibition. We establish structural and energetic determinants of allosteric binding for the experimentally known allosteric molecules, indicating a potential mechanism of allosteric modulation by targeting the hinges of the inter-protomer movements and blocking conformational changes between the closed and open spike trimer forms. The results of this study demonstrate that combining ensemble-based ligand docking with conformational states of spike protein and rigorous binding energy analysis enables robust characterization of the ligand binding modes, the identification of allosteric binding hotspots, and the prediction of binding affinities for validated allosteric modulators, which is consistent with the experimental data. This study suggested that the conformational adaptability of the protein allosteric sites and the diversity of ligand bound conformations are both in play to enable efficient targeting of allosteric binding sites and interfere with the conformational changes.

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.

This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

The N-methyl-D-aspartate receptor (NMDAR) is a Na(+) and Ca(2+) permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimer's disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure-activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.

Characterisation of bis(4-aminoquinoline)s as α1A adrenoceptor allosteric modulators

Differing Activity Profiles of the Stereoisomers of 2,3,5,6TMP-TQS, a Putative Silent Allosteric Modulator of α7 nAChR

mGluR5, which belongs to the G-protein-coupled receptor superfamily, is believed to be associated with many human diseases, such as a wide range of neurological disorders, gastroesophageal reflux disease, and cancer. Comparing with compounds that target on the orthosteric binding site, significant roles have been established for mGluR5 negative allosteric modulators (NAMs) due to their higher subtype selectivity and more suitable pharmacokinetic profiles. Nevertheless, to date, none of them have come to market for various reasons. In this study, a 3D quantitative pharmacophore model was generated by using the HypoGen module in Discovery Studio 4.0. With several validation methods ultilized, the optimal pharmacophore model Hypo2 was selected to discover potential mGluR5 NAMs from natural products. Two hundred and seventeen potential NAMs were obtained after being filtered by Lipinski’s rule (≥4). Then, molecular docking was used to refine the pharmacophore-based screening results and analyze the binding mode of NAMs and mGluR5. Three compounds, aglaiduline, 5-O-ethyl-hirsutanonol, and yakuchinone A, with good ADMET properties, acceptable Fit value and estimated value, and high docking score, were reserved for a molecular dynamics simulation study. All of them have stability of ligand binding. From our computational results, there might exhibit drug-like negative allosteric moderating effects on mGluR5 in these natural products. This work provides a reliable method for discovering mGluR5 NAMs from natural products.

The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon-counting imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca2+-ATPase, a pharmacologically important pump that transports Ca2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca2+-ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca2+-ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca2+-ATPase-phospholamban complex. Time-correlated single photon-counting imaging experiments using the nonhydrolyzable ATP analog β,γ-Methyleneadenosine 5'-triphosphate (AMP-PCP) showed ATP is an allosteric modulator of sarcoplasmic reticulum Ca2+-ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca2+-dependent shifts in the structural populations of sarcoplasmic reticulum Ca2+-ATPase, and it does not increase the pump's affinity for Ca2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca2+-ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca2+ concentrations at which sarcoplasmic reticulum Ca2+-ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca2+-ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca2+-ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca2+-ATPase activation by its substrate and a synthetic allosteric modulator.