Integrative residue-intuitive machine learning and MD Approach to Unveil Allosteric Site and Mechanism for ß2AR.

Allosteric drugs offer a new avenue for modern drug design. However, the identification of cryptic allosteric sites presents a formidable challenge. Following the allostery nature of residue-driven conformation transition, we propose a state-of-the-art computational pipeline by developing a residue-intuitive hybrid machine learning (RHML) model coupled with molecular dynamics (MD) simulation, through which we can efficiently identify the allosteric site and allosteric modulator as well as reveal their regulation mechanism. For the clinical target ß2-adrenoceptor (ß2AR), we discover an additional allosteric site located around residues D792.50, F2826.44, N3187.45 and S3197.46 and one putative allosteric modulator ZINC5042. Using Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) and protein structure network (PSN), the allosteric potency and regulation mechanism are probed to further improve identification accuracy. Benefiting from sufficient computational evidence, the experimental assays then validate our predicted allosteric site, negative allosteric potency and regulation pathway, showcasing the effectiveness of the identification pipeline in practice. We expect that it will be applicable to other target proteins.

Finely ordered intracellular domain harbors an allosteric site to modulate physiopathological function of P2X3 receptors.

P2X receptors, a subfamily of ligand-gated ion channels activated by extracellular ATP, are implicated in various physiopathological processes, including inflammation, pain perception, and immune and respiratory regulations. Structural determinations using crystallography and cryo-EM have revealed that the extracellular three-dimensional architectures of different P2X subtypes across various species are remarkably identical, greatly advancing our understanding of P2X activation mechanisms. However, structural studies yield paradoxical architectures of the intracellular domain (ICD) of different subtypes (e.g., P2X3 and P2X7) at the apo state, and the role of the ICD in P2X functional regulation remains unclear. Here, we propose that the P2X3 receptor's ICD has an apo state conformation similar to the open state but with a less tense architecture, containing allosteric sites that influence P2X3's physiological and pathological roles. Using covalent occupancy, engineered disulfide bonds and voltage-clamp fluorometry, we suggested that the ICD can undergo coordinated motions with the transmembrane domain of P2X3, thereby facilitating channel activation. Additionally, we identified a novel P2X3 enhancer, PSFL77, and uncovered its potential allosteric site located in the 1α3ß domain of the ICD. PSFL77 modulated pain perception in P2rx3+/+, but not in P2rx3-/-, mice, indicating that the 1α3ß, a "tunable" region implicated in the regulation of P2X3 functions. Thus, when P2X3 is in its apo state, its ICD architecture is fairly ordered rather than an unstructured outward folding, enabling allosteric modulation of the signaling of P2X3 receptors.

Positive allosteric modulation of a GPCR ternary complex.

The activation of a G protein-coupled receptor (GPCR) leads to the formation of a ternary complex between agonist, receptor, and G protein that is characterized by high-affinity binding. Allosteric modulators bind to a distinct binding site from the orthosteric agonist and can modulate both the affinity and the efficacy of orthosteric agonists. The influence allosteric modulators have on the high-affinity active state of the GPCR-G protein ternary complex is unknown due to limitations on attempting to characterize this interaction in recombinant whole cell or membrane-based assays. Here, we use the purified M2 muscarinic acetylcholine receptor reconstituted into nanodiscs to show that, once the agonist-bound high-affinity state is promoted by the G protein, positive allosteric modulators stabilize the ternary complex that, in the presence of nucleotides, leads to an enhanced initial rate of signaling. Our results enhance our understanding of how allosteric modulators influence orthosteric ligand signaling and will aid the design of allosteric therapeutics.

Designed dualsteric modulators: A novel route for drug discovery.

Orthosteric and allosteric modulators, which constitute the majority of current drugs, bind to the orthosteric and allosteric sites of target proteins, respectively. However, the clinical efficacy of these agents is frequently compromised by poor selectivity or reduced potency. Dualsteric modulators feature two linked pharmacophores that bind to orthosteric and allosteric sites of the target proteins simultaneously, thereby offering a promising avenue to achieve both potency and specificity. In this review, we summarize recent structures available for dualsteric modulators in complex with their target proteins, elucidating detailed drug-target interactions and dualsteric action patterns. Moreover, we provide a design and optimization strategy for dualsteric modulators based on structure-based drug design approaches.

Structural Models of Human Norepinephrine Transporter Ensemble Reveal the Allosteric Sites and Ligand-Binding Mechanism.

The norepinephrine transporter (NET) plays a pivotal role in recycling norepinephrine (NE) from the synaptic cleft. However, the structures referring to the conformational heterogeneity of NET during the transport cycle remain poorly understood. Here, three structural models of NE bound to the orthosteric site of NET in outward-open (OOholo), outward-occluded (OCholo), and inward-open (IOholo) conformations were first obtained using the multistate structures of serotonin transporter as templates and further characterized through Gaussian-accelerated molecular dynamics and free energy reweighting. Analysis of the structures revealed eight potential allosteric sites on the functional-specific states of NET. One of the pharmacologically relevant pockets located at the extracellular vestibule was further verified by simulating the binding behaviors of a clinical trial drug χ-MrIA that is allosterically regulating NET. These structural and energetic insights into NET advanced our understanding of NE reuptake and paved the way for discovering novel molecules targeting the allosteric sites.

Molecular basis of inhibition of the amino acid transporter B0AT1 (SLC6A19).

The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B0AT1 inhibitors with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.

Labeling of CC Chemokine Receptor 2 with a Versatile Intracellular Allosteric Probe.

Interest in affinity-based probes (AfBPs) as novel tools to interrogate G protein-coupled receptors (GPCRs) has gained traction in recent years. AfBPs represent an interesting and more versatile alternative to antibodies. In the present study, we report the development and validation of AfBPs that target the intracellular allosteric pocket of CCR2, a GPCR of interest for the development of therapies targeting autoimmune and inflammatory diseases and also cancer. Owing to the two-step labeling process of these CCR2 AfBPs through the incorporation of a click handle, we were successful in applying our most efficient probe in a variety of in vitro experiments and making use of multiple different detection techniques, such as SDS-PAGE and LC/MS-based proteomics. Collectively, this novel probe shows high selectivity, versatility, and applicability. Hence, this is a valuable alternative for CCR2-targeting antibodies and other traditional tool compounds and could aid in target validation and engagement in drug discovery.

Insight of the molecular mechanism of inhibitors located at different allosteric sites regulating the activity of wild type and mutant KRAS (G12).

As the important hub of many cellular signaling networks, KRAS (Kirsten rat sarcoma viral oncogene homologue) has been identified as a tumor biomarker. It is the frequently mutated oncogene in human cancers, and KRAS protein activation caused by mutations, such as G12D, has been found in many human tumors tissues. Although, there are two specific allosteric sites (AS1 and AS2) on the KRAS protein that can be used as the targets for inhibitor development, the difference of regulatory mechanisms between two individual allosteric sites still not be reported. Here, using molecular dynamics simulations combined with molecular mechanics generalized born surface area (MM/GBSA) analysis, we found that both of the inhibitors, located at AS1 and AS2, were able to reduce the binding free energy between wild type, mutant KRAS (G12/D/V/S/C) and GTP remarkably, however the effect of inhibitors on the binding free energy between wild type, mutant KRAS and GDP was limited. In addition, the degree of decrease of binding free energy between KRAS and GTP caused by inhibitors at AS2 was significantly greater than that caused by inhibitors at AS1. Further analysis revealed that both inhibitors at AS1 and AS2 were able to regulate the fluctuation of Switch Ⅰ and Switch Ⅱ to expand the pocket of the orthosteric site (GTP binding site), thereby reducing the binding of KRAS to GTP. Noteworthy there was significant differences in the regulatory preferences on Switch Ⅰ and Switch Ⅱ between two type inhibitor. The inhibitor at AS2 mainly regulated Switch Ⅱ to affect the pocket of the orthosteric site, while the inhibitor at AS1 mainly expand the pocket of the orthosteric site by regulating the fluctuation of Switch Ⅰ. Our study compared the differences between two type inhibitors in regulating the KRAS protein activity and revealed the advantages of the AS2 as the small molecule drug target, aiming to provide theoretical guidance for the research of novel KRAS protein inhibitors.

Discovery of Triazolopyrimidine Derivatives as Selective P2X3 Receptor Antagonists Binding to an Unprecedented Allosteric Site as Evidenced by Cryo-Electron Microscopy.

The P2X3 receptor (P2X3R), an ATP-gated cation channel predominantly expressed in C- and Aδ-primary afferent neurons, has been proposed as a drug target for neurological inflammatory diseases, e.g., neuropathic pain, and chronic cough. Aiming to develop novel, selective P2X3R antagonists, tetrazolopyrimidine-based hit compound 9 was optimized through structure-activity relationship studies by modifying the tetrazole core as well as side chain substituents. The optimized antagonist 26a, featuring a cyclopropane-substituted triazolopyrimidine core, displayed potent P2X3R-antagonistic activity (IC50 = 54.9 nM), 20-fold selectivity versus the heteromeric P2X2/3R, and high selectivity versus other P2XR subtypes. Noncompetitive P2X3R blockade was experimentally confirmed by calcium influx assays. Cryo-electron microscopy revealed that 26a stabilizes the P2X3R in its desensitized state, acting as a molecular barrier to prevent ions from accessing the central pore. In vivo studies in a rat neuropathic pain model (spinal nerve ligation) showed dose-dependent antiallodynic effects of 26a, thus presenting a novel, promising lead structure.

Large-Scale Screening of Per- and Polyfluoroalkyl Substance Binding Interactions and Their Mixtures with Nuclear Receptors.

Despite their significant impact, comprehensive screenings and detailed analyses of per- and polyfluoroalkyl substance (PFAS) binding strengths at the orthosteric and allosteric sites of NRs are currently lacking. This study addresses this gap by focusing on the binding interaction analysis of both common and uncommon PFAS with the nuclear receptors (NRs) vitamin D receptor (VDR), peroxisome proliferator-activated receptor gamma (PPARγ), pregnane X receptor (PXR), and estrogen receptor alpha (ERα). Advanced docking simulations were used to screen 9507 PFAS chemicals at the orthosteric and allosteric sites of PPARγ, PXR, VDR, and ERα. All receptors exhibited strong binding interactions at the orthosteric and allosteric site with a significant number of PFAS. We verified the accuracy of the docking protocol through multiple docking controls and validations. A mixture modeling analysis indicates that PFAS can bind in various combinations with themselves and endogenous ligands simultaneously, to disrupt the endocrine system and cause carcinogenic responses. These findings reveal that PFAS can interfere with nuclear receptor activity by displacing endogenous or native ligands by binding to the orthosteric and allosteric sites. The purpose of this study is to explore the mechanisms through which PFAS exert their endocrine-disrupting effects, potentially leading to more targeted therapeutic strategies. Importantly, this study is the first to explore the binding of PFAS at allosteric sites and to model PFAS mixtures at nuclear receptors. Given the high concentration and persistence of PFAS in humans, this study further emphasizes the urgent need for further research into the carcinogenic mechanisms of PFAS and the development of therapeutic strategies that target nuclear receptors.

Structure of the human dopamine transporter and mechanisms of inhibition.

The neurotransmitter dopamine has central roles in mood, appetite, arousal and movement1. Despite its importance in brain physiology and function, and as a target for illicit and therapeutic drugs, the human dopamine transporter (hDAT) and mechanisms by which it is inhibited by small molecules and Zn2+ are without a high-resolution structural context. Here we determine the structure of hDAT in a tripartite complex with the competitive inhibitor and cocaine analogue, (-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane2 (ß-CFT), the non-competitive inhibitor MRS72923 and Zn2+ (ref. 4). We show how ß-CFT occupies the central site, approximately halfway across the membrane, stabilizing the transporter in an outward-open conformation. MRS7292 binds to a structurally uncharacterized allosteric site, adjacent to the extracellular vestibule, sequestered underneath the extracellular loop 4 (EL4) and adjacent to transmembrane helix 1b (TM1b), acting as a wedge, precluding movement of TM1b and closure of the extracellular gate. A Zn2+ ion further stabilizes the outward-facing conformation by coupling EL4 to EL2, TM7 and TM8, thus providing specific insights into how Zn2+ restrains the movement of EL4 relative to EL2 and inhibits transport activity.

Structural basis for CCR6 modulation by allosteric antagonists.

The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the molecular basis of CCR6 activation. Here, we present two inactive CCR6 structures in ternary complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/SQA1/OXM2. The oxomorpholine analogues, OXM1 and OXM2 are highly selective CCR6 antagonists which bind to an extracellular pocket and disrupt the receptor activation network. An energetically favoured U-shaped conformation in solution that resembles the bound form is observed for the active analogues. SQA1 is a squaramide derivative with close-in analogues reported as antagonists of chemokine receptors including CCR6. SQA1 binds to an intracellular pocket which overlaps with the G protein site, stabilizing a closed pocket that is a hallmark of inactive GPCRs. Minimal communication between the two allosteric pockets is observed, in contrast to the prevalent allosteric cooperativity model of GPCRs. This work highlights the versatility of GPCR antagonism by small molecules, complementing previous knowledge of CCR6 activation, and sheds light on drug discovery targeting CCR6.

FGF1 Suppresses Allosteric Activation of ß3 Integrins by FGF2: A Potential Mechanism of Anti-Inflammatory and Anti-Thrombotic Action of FGF1.

Several inflammatory cytokines bind to the allosteric site (site 2) and allosterically activate integrins. Site 2 is also a binding site for 25-hydroxycholesterol, an inflammatory lipid mediator, and is involved in inflammatory signaling (e.g., TNF and IL-6 secretion) in addition to integrin activation. FGF2 is pro-inflammatory and pro-thrombotic, and FGF1, homologous to FGF2, has anti-inflammatory and anti-thrombotic actions, but the mechanism of these actions is unknown. We hypothesized that FGF2 and FGF1 bind to site 2 of integrins and regulate inflammatory signaling. Here, we describe that FGF2 is bound to site 2 and allosterically activated ß3 integrins, suggesting that the pro-inflammatory action of FGF2 is mediated by binding to site 2. In contrast, FGF1 bound to site 2 but did not activate these integrins and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2 and that the anti-inflammatory action of FGF1 is mediated by blocking site 2. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of αvß3, suppressed ß3 integrin activation by FGF2 as effectively as WT FGF1.

Identification of aryl hydrocarbon receptor allosteric antagonists from clinically approved drugs.

The human aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, plays a pivotal role in a diverse array of pathways in biological and pathophysiological events. This position AhR as a promising target for both carcinogenesis and antitumor strategies. In this study we utilized computational modeling to screen and identify FDA-approved drugs binding to the allosteric site between α2 of bHLH and PAS-A domains of AhR, with the aim of inhibiting its canonical pathway activity. Our findings indicated that nilotinib effectively fits into the allosteric pocket and forms interactions with crucial residues F82, Y76, and Y137. Binding free energy value of nilotinib is the lowest among top hits and maintains stable within its pocket throughout entire (MD) simulations time. Nilotinib has also substantial interactions with F295 and Q383 when it binds to orthosteric site and activate AhR. Surprisingly, it does not influence AhR nuclear translocation in the presence of AhR agonists; instead, it hinders the formation of the functional AhR-ARNT-DNA heterodimer assembly, preventing the upregulation of regulated enzymes like CYP1A1. Importantly, nilotinib exhibits a dual impact on AhR, modulating AhR activity via the PAS-B domain and working as a noncompetitive allosteric antagonist capable of blocking the canonical AhR signaling pathway in the presence of potent AhR agonists. These findings open a new avenue for the repositioning of nilotinib beyond its current application in diverse diseases mediated via AhR.

Molecular basis of human noradrenaline transporter reuptake and inhibition.

Noradrenaline, also known as norepinephrine, has a wide range of activities and effects on most brain cell types1. Its reuptake from the synaptic cleft heavily relies on the noradrenaline transporter (NET) located in the presynaptic membrane2. Here we report the cryo-electron microscopy (cryo-EM) structures of the human NET in both its apo state and when bound to substrates or antidepressant drugs, with resolutions ranging from 2.5 Å to 3.5 Å. The two substrates, noradrenaline and dopamine, display a similar binding mode within the central substrate binding site (S1) and within a newly identified extracellular allosteric site (S2). Four distinct antidepressants, namely, atomoxetine, desipramine, bupropion and escitalopram, occupy the S1 site to obstruct substrate transport in distinct conformations. Moreover, a potassium ion was observed within sodium-binding site 1 in the structure of the NET bound to desipramine under the KCl condition. Complemented by structural-guided biochemical analyses, our studies reveal the mechanism of substrate recognition, the alternating access of NET, and elucidate the mode of action of the four antidepressants.

Prediction of Protein Allosteric Sites with Transfer Entropy and Spatial Neighbor-Based Evolutionary Information Learned by an Ensemble Model.

Allostery is one of the most direct and efficient ways to regulate protein functions. The diverse allosteric sites make it possible to design allosteric modulators of differential selectivity and improved safety compared with those of orthosteric drugs targeting conserved orthosteric sites. Here, we develop an ensemble machine learning method AllosES to predict protein allosteric sites in which the new and effective features are utilized, including the entropy transfer-based dynamic property, secondary structure features, and our previously proposed spatial neighbor-based evolutionary information besides the traditional physicochemical properties. To overcome the class imbalance problem, the multiple grouping strategy is proposed, which is applied to feature selection and model construction. The ensemble model is constructed where multiple submodels are trained on multiple training subsets, respectively, and their results are then integrated to be the final output. AllosES achieves a prediction performance of 0.556 MCC on the independent test set D24, and additionally, AllosES can rank the real allosteric sites in the top three for 83.3/89.3% of allosteric proteins from the test set D24/D28, outperforming the state-of-the-art peer methods. The comprehensive results demonstrate that AllosES is a promising method for protein allosteric site prediction. The source code is available at https://github.com/ChunhuaLab/AllosES.

Discovery and characterization of small-molecule TGR5 ligands with agonistic activity.

The Takeda G protein-coupled receptor 5 (TGR5) is activated endogenously by primary and secondary bile acids. This receptor is considered a candidate target for addressing inflammatory and metabolic disorders. We have targeted TGR5 with structure-based methods for ligand finding using the recently solved experimental structures, as well as structures obtained from molecular dynamics simulations. Through addressing the orthosteric as well as a putative allosteric site, we identified agonists and positive allosteric modulators. While the predicted binding locations were not in line with their efficacy, our work contributes activating small-molecule ligands that we have thoroughly characterized in vitro.

Lipid Trolling to Optimize A3 Adenosine Receptor-Positive Allosteric Modulators (PAMs).

A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs) (2,4-disubstituted-1H-imidazo[4,5-c]quinolin-4-amines) allosterically increase the Emax of A3AR agonists, but not potency, due to concurrent orthosteric antagonism. Following mutagenesis/homology modeling of the proposed lipid-exposed allosteric binding site on the cytosolic side, we functionalized the scaffold, including heteroatom substitutions and exocyclic phenylamine extensions, to increase allosteric binding. Strategically appended linear alkyl-alkynyl chains with terminal amino/guanidino groups improved allosteric effects at both human and mouse A3ARs. The chain length, functionality, and attachment position were varied to modulate A3AR PAM activity. For example, 26 (MRS8247, p-alkyne-linked 8 methylenes) and homologues increased agonist Cl-IB-MECA's Emax and potency ([35S]GTPγS binding). The putative mechanism involves a flexible, terminally cationic chain penetrating the lipid environment for stable electrostatic anchoring to cytosolic phospholipid head groups, suggesting "lipid trolling", supported by molecular dynamic simulation of the active-state model. Thus, we have improved A3AR PAM activity through rational design based on an extrahelical, lipidic binding site.

Crystal structure of the GluK1 ligand-binding domain with kainate and the full-spanning positive allosteric modulator BPAM538.

Kainate receptors play an important role in the central nervous system by mediating postsynaptic excitatory neurotransmission and modulating the release of the inhibitory neurotransmitter GABA through a presynaptic mechanism. To date, only three structures of the ligand-binding domain (LBD) of the kainate receptor subunit GluK1 in complex with positive allosteric modulators have been determined by X-ray crystallography, all belonging to class II modulators. Here, we report a high-resolution structure of GluK1-LBD in complex with kainate and BPAM538, which belongs to the full-spanning class III. One BPAM538 molecule binds at the GluK1 dimer interface, thereby occupying two allosteric binding sites simultaneously. BPAM538 stabilizes the active receptor conformation with only minor conformational changes being introduced to the receptor. Using a calcium-sensitive fluorescence-based assay, a 5-fold potentiation of the kainate response (100 µM) was observed in presence of 100 µM BPAM538 at GluK1(Q)b, whereas no potentiation was observed at GluK2(VCQ)a. Using electrophysiology recordings of outside-out patches excised from HEK293 cells, BPAM538 increased the peak response of GluK1(Q)b co-expressed with NETO2 to rapid application of 10 mM L-glutamate with 130 ± 20 %, and decreased desensitization determined as the steady-state/peak response ratio from 23 ± 2 % to 90 ± 4 %. Based on dose-response relationship experiments on GluK1(Q)b the EC50 of BPAM538 was estimated to be 58 ± 29 µM.

The value of protein allostery in rational anticancer drug design: an update.

INTRODUCTION: Allosteric drugs are advantageous. However, they still face hurdles, including identification of allosteric sites that will effectively alter the active site. Current strategies largely focus on identifying pockets away from the active sites into which the allosteric ligand will dock and do not account for exactly how the active site is altered. Favorable allosteric inhibitors dock into sites that are nearby the active sites and follow nature, mimicking diverse allosteric regulation strategies. AREAS COVERED: The following article underscores the immense significance of allostery in drug design, describes current allosteric strategies, and especially offers a direction going forward. The article concludes with the authors' expert perspectives on the subject. EXPERT OPINION: To select a productive venue in allosteric inhibitor development, we should learn from nature. Currently, useful strategies follow this route. Consider, for example, the mechanisms exploited in relieving autoinhibition and in harnessing allosteric degraders. Mimicking compensatory, or rescue mutations may also fall into such a thesis, as can molecular glues that capture features of scaffolding proteins. Capturing nature and creatively tailoring its mimicry can continue to innovate allosteric drug discovery.