Developing Allosteric Inhibitors of SARS-CoV-2 RNA-dependent RNA polymerase.

The use of Fpocket and virtual screening techniques enabled us to identify potential allosteric druggable pockets within the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Of the compounds screened, compound 1 was identified as a promising inhibitor, lowering a SARS-CoV-2 RdRp activity to 57% in an enzymatic assay at 10 µM concentration. The structure of compound 1 was subsequently optimized in order to preserve or enhance inhibitory activity. This involved the substitution of problematic ester and aromatic nitro groups with more inert functionalities. The N,N'-diphenylurea scaffold with two NH groups was identified as essential for the compound's activity but also exhibited high toxicity in Calu-3 cells. To address this issue, a scaffold hopping approach was employed to replace the urea core with potentially less toxic urea isosteres. This approach yielded several structural analogues with notable activity, specifically 2,2'-bisimidazol (in compound 55 with residual activity RA = 42%) and (1H-imidazol-2-yl)urea (in compounds 59 and 60, with RA = 50 and 28%, respectively). Despite these advances, toxicity remained a major concern. These compounds represent a promising starting point for further structure-activity relationship studies of allosteric inhibitors of SARS-CoV-2 RdRp, with the goal of reducing their cytotoxicity and improving aqueous solubility.

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.

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.

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.

Advances in SHP2 tunnel allosteric inhibitors and bifunctional molecules.

SHP2 is a non-receptor tyrosine phosphatase encoded by PTPN11, which performs the functions of regulating cell proliferation, differentiation, apoptosis, and survival through removing tyrosine phosphorylation and modulating various signaling pathways. The overexpression of SHP2 or its mutations is related to developmental diseases and several cancers. Numerous allosteric inhibitors with striking inhibitory potency against SHP2 allosteric pockets have recently been identified, and several SHP2 tunnel allosteric inhibitors have been applied in clinical trials to treat cancers. However, based on clinical results, the efficacy of single-agent treatments has been proven to be suboptimal. Most clinical trials involving SHP2 inhibitors have adopted drug combination strategies. This review briefly discusses the research progress on SHP2 allosteric inhibitors and pathway-dependent drug combination strategies for SHP2 in cancer therapy. In addition, we summarize the current bifunctional molecules of SHP2 and elaborate on the design and structural optimization strategies of these bifunctional molecules in detail, offering further direction for the research on novel SHP2 inhibitors.

Synthesis, In Silico Prediction, and In Vitro Evaluation of Anti-tumor Activities of Novel 4'-Hydroxybiphenyl-4-carboxylic Acid Derivatives as EGFR Allosteric Site Inhibitors.

INTRODUCTION: Allosteric inhibition of EGFR Tyrosine Kinase (TK) is currently among the most attractive approaches for designing and developing anti-cancer drugs to avoid chemoresistance exhibited by clinically approved ATP-competitive inhibitors. The current work aimed to synthesize new biphenyl-containing derivatives that were predicted to act as EGFR TK allosteric site inhibitors based on molecular docking studies. METHOD: A new series of 4'-hydroxybiphenyl-4-carboxylic acid derivatives, including hydrazine-1-carbothioamide (S3-S6) and 1,2,4-triazole (S7-S10) derivatives, were synthesized and characterized using IR, 1HNMR, 13CNMR, and HR-mass spectroscopy. Compound S4 had a relatively high pharmacophore-fit score, indicating that it may have biological activity similar to the EGFR allosteric inhibitor reference, and it scored a relatively low ΔG against EGFR TK allosteric site, indicating a high likelihood of drug-receptor complex formation. Compound S4 was cytotoxic to the three cancer cell lines tested, particularly HCT-116 colorectal cancer cells, with an IC50 value comparable to Erlotinib. Compound S4 induced the intrinsic apoptotic pathway in HCT-116 cells by arresting them in the G2/M phase. RESULT: All of the new derivatives, including S4, met the in silico requirements for EGFR allosteric inhibitory activity. CONCLUSION: Compound S4 is a promising EGFR tyrosine kinase allosteric inhibitor that warrants further research.

Discovery and development of novel 10,12-disubstituted aloperine derivatives against HCoV-OC43 by targeting allosteric site of host TMPRSS2.

By inducing steric activation of the 10CH bond with a 12-acyl group to form a key imine oxime intermediate, 20 novel (10S)-10,12-disubstituted aloperine derivatives were successfully synthesized and assessed for their antiviral efficacy against HCoV-OC43. Of them, compound 3i exhibited the moderate activities against HCoV-OC43, as well as against the SARS-CoV-2 variant EG.5.1 with the comparable EC50 values of 4.7 and 4.1 µM. A mechanism study revealed that it inhibited the protease activity of host TMPRSS2 by binding to an allosteric site, rather than the known catalytic center, different from that of camostat. Also, the combination of compound 3i and molnupiravir, as an RdRp inhibitor, showed an additive antiviral effect against HCoV-OC43. The results provide a new binding mode and lead compound for targeting TMPRSS2, with an advantage in combating broad-spectrum coronavirus.

Mechanistic insights into sodium ion-mediated ligand binding affinity and modulation of 5-HT2B GPCR activity: implications for drug discovery and development.

PURPOSE: The G-protein coupled receptor (GPCR) family, implicated in neurological disorders and drug targets, includes the sensitive serotonin receptor subtype, 5-HT2B. The influence of sodium ions on ligand binding at the receptor's allosteric region is being increasingly studied for its impact on receptor structure. METHODS: High-throughput virtual screening of three libraries, specifically the Asinex-GPCR library, which contains 8,532 compounds and FDA-approved (2466 compounds) and investigational compounds (2731)) against the modeled receptor [4IB4-5HT2BRM] using the standard agonist/antagonist (Ergotamine/Methysergide), as previously selected from our studies based on ADMET profiling, and further on basis of binding free energy a single compound - dihydroergotamine is chosen. RESULTS: This compound displayed strong interactions with the conserved active site. Ions influence ligand binding, with stronger interactions (3-H-bonds and 1-π-bond around 3.35 Å) observed when an agonist and ions are present. Ions entry is guided by conserved motifs in helices III, IV, and VII, which regulate the receptor. Dihydroergotamine, the selected drug, showed binding variance based on ions presence/absence, affecting amino acid residues in these motifs. DCCM and PCA confirmed the stabilization of ligands, with a greater correlation (∼46.6%-PC1) observed with ions. Dihydroergotamine-modified interaction sites within the receptor necessary for activation, serving as a potential 5HT2BRM agonist. RDF analysis showed the sodium ions density around the active site during dihydroergotamine binding. CONCLUSION: Our study provides insights into sodium ion mobility's role in controlling ligand binding affinity in 5HT2BR, offering therapeutic development insights.

Structure, cooperativity and inhibition of the inosine 5'-monophosphate-specific phosphatase from Saccharomyces cerevisiae.

The nucleoside inosine is a main intermediate of purine nucleotide catabolism in Saccharomyces cerevisiae and is produced via the dephosphorylation of inosine monophosphate (IMP) by IMP-specific 5'-nucleotidase 1 (ISN1), which is present in many eukaryotic organisms. Upon transition of yeast from oxidative to fermentative growth, ISN1 is important for intermediate inosine accumulation as purine storage, but details of ISN1 regulation are unknown. We characterized structural and kinetic behavior of ISN1 from S. cerevisiae (ScISN1) and showed that tetrameric ScISN1 is negatively regulated by inosine and adenosine triphosphate (ATP). Regulation involves an inosine-binding allosteric site along with IMP-induced local and global conformational changes in the monomer and a tetrameric re-arrangement, respectively. A proposed interaction network propagates local conformational changes in the active site to the intersubunit interface, modulating the allosteric features of ScISN1. Via ATP and inosine, ScISN1 activity is likely fine-tuned to regulate IMP and inosine homeostasis. These regulatory and catalytic features of ScISN1 contrast with those of the structurally homologous ISN1 from Plasmodium falciparum, indicating that ISN1 enzymes may serve different biological purposes in different organisms.

Identifying Potential Ligand Binding Sites on Glycogen Synthase Kinase 3 Using Atomistic Cosolvent Simulations.

Glycogen synthase kinase 3 ß (GSK3ß) is a serine/threonine kinase that phosphorylates several protein substrates in crucial cell signaling pathways. Owing to its therapeutic importance, there is a need to develop GSK3ß inhibitors with high specificity and potency. One approach is to find small molecules that can allosterically bind to the GSK3ß protein surface. We have employed fully atomistic mixed-solvent molecular dynamics (MixMD) simulations to identify three plausible allosteric sites on GSK3ß that can facilitate the search for allosteric inhibitors. Our MixMD simulations narrow down the allosteric sites to precise regions on the GSK3ß surface, thereby improving upon the previous predictions of the locations of these sites.

Identification of novel aminopyrimidine derivatives for the treatment of mutant NSCLC.

Starting from the binding mode of allosteric EGFR inhibitor JBJ-04-125-02 and the key pharmacophore of the third-generation EGFR inhibitors, we designed and synthesized a novel series of EGFR inhibitors, represented by (R)-N-(4-((2-aminopyrimidin-4-yl)amino)phenyl)-2-(5-(4-(4-methylpiperazin-1-yl)phenyl)-1-oxoisoindolin-2-yl)-2-phenylacetamide (6q). Docking study demonstrated that top compound 6q spanned orthosteric and allosteric sites of EGFR, and formed three key H-bonds with the residues Asp855, Lys745, and Met793 located in two sites. Biological evaluation indicated that compound 6q showed potential inhibitory activity against Ba/F3-EGFRL858R/T790M/C797S and Ba/F3-EGFRDel19/T790M/C797S cells, with IC50 values of 0.42 µM and 0.41 µM, respectively. Furthermore, compound 6q showed excellent activity against mutant NSCLC cell line NCI-H1975-EGFRL858R/T790M/C797S cells, with IC50 value of 0.82 µM which was superior to that of osimertinib (IC50 = 2.94 µM), JBJ-04-125-02 (IC50 = 3.66 µM), and coadministration of JBJ-04-125-02 and osimertinib (IC50 = 1.25 µM). Cell cycle arrest and cell apoptosis assay indicated that compound 6q could promote apoptosis of NCI-H1975-EGFRL858R/T790M/C797S cells at the concentration of 0.8 µM and no obvious cell cycle arrest was found.

Pharmacologically targeting intracellular allosteric sites of GPCRs for drug discovery.

G-protein-coupled receptors (GPCRs) are a family of cell surface proteins that can sense a variety of extracellular stimuli and mediate multiple signaling transduction pathways involved in human physiology. Recent advances in GPCR structural biology have revealed a relatively conserved intracellular allosteric site in multiple GPCRs, which can be utilized to modulate receptors from the inside. This novel intracellular site partially overlaps with the G-protein and ß-arrestin coupling sites, providing a novel avenue for biological intervention. Here, we review evidence available for GPCR structures complexed with intracellular small-molecule allosteric modulators, elucidating drug-target interactions and allosteric mechanisms. Moreover, we highlight the potential of intracellular allosteric modulators in achieving biased signaling, which provides insights into biased allosteric mechanisms.

Harnessing the druggability at orthosteric and allosteric sites of PD-1 for small molecule discovery by an integrated in silico pipeline.

The PD-1/PD-L1 interaction is a promising target for small molecule inhibitors in cancer immunotherapy, but targeting this interface has been challenging. While efforts have been made to identify compounds that target the orthosteric sites, no reports have explored the potential of small molecules to target the allosteric region of PD-1. Therefore, our study aims to establish a pipeline to identify small molecules that can effectively bind to either the orthosteric or allosteric pockets of PD-1. We categorized the PD-1 interface into two hot-spot zones (P-and N-zones) based on extensive analysis of its structural, dynamical, and energetic properties. These zones correspond to the orthosteric and allosteric PPI sites, respectively, targeted by monoclonal antibodies. We used a guided virtual screening workflow to identify hits from ∼7 million compounds library, which were then clustered based on structural similarity and assessed by interaction fingerprinting. The selective and diverse chemical representatives were subjected to MD simulations and binding energetics calculations to filter out false positives and identify actual binders. Binding poses metadynamics calculations confirmed the stability of the final hits in the pocket. This study emphasizes the need for an integrated pipeline that uses molecular dynamics simulations and binding energetics to identify potential binders for the dynamic PD-1/PD-L1 interface, due to the lack of small molecule co-crystals. Only a few potential binders were discovered from a large pool of molecules targeting both the allosteric and orthosteric zones. Our results suggest that the allosteric site has more potential than the orthosteric site for inhibitor design. The identified "computational hits" hold potential as starting points for in vitro evaluations followed by hit-to-lead optimization. Overall, this study represents an effort to establish a computational pipeline for exploring and enriching both the allosteric and orthosteric sites of PPI interfaces, "a tough but indispensable nut to crack".

Allosteric site variants affect GTP hydrolysis on Ras.

RAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4, and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.

Discovery of a Potential Allosteric Site in the SARS-CoV-2 Spike Protein and Targeting Allosteric Inhibitor to Stabilize the RBD Down State Using a Computational Approach.

BACKGROUND: The novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a worldwide public health crisis. At present, the development of effective drugs and/or related therapeutics is still the most urgent and important task for combating the virus. The viral entry and associated infectivity mainly rely on its envelope spike protein to recognize and bind to the host cell receptor angiotensin-converting enzyme 2 (ACE2) through a conformational switch of the spike receptor binding domain (RBD) from inactive to active state. Thus, it is of great significance to design an allosteric inhibitor targeting spike to lock it in the inactive and ACE2-inaccessible state. OBJECTIVE: This study aims to discover the potential broad-spectrum allosteric inhibitors capable of binding and stabilizing the diverse spike variants, including the wild type, Delta, and Omicron, in the inactive RBD down state. METHODS: In this work, we first detected a potential allosteric pocket within the SARS-CoV-2 spike protein. Then, we performed large-scale structure-based virtual screening by targeting the putative allosteric pocket to identify allosteric inhibitors that could stabilize the spike inactive state. Molecular dynamics simulations were further carried out to evaluate the effects of compound binding on the stability of spike RBD. RESULTS: Finally, we identified three potential allosteric inhibitors, CPD3, CPD5, and CPD6, against diverse SARS-CoV-2 variants, including Wild-type, Delta, and Omicron variants. Our simulation results showed that the three compounds could stably bind the predicted allosteric site and effectively stabilize the spike in the inactive state. CONCLUSION: The three compounds provide novel chemical structures for rational drug design targeting spike protein, which is expected to greatly assist in the development of new drugs against SARS-CoV-2.

CavityPlus 2022 Update: An Integrated Platform for Comprehensive Protein Cavity Detection and Property Analyses with User-friendly Tools and Cavity Databases.

Ligand binding sites provide essential information for uncovering protein functions and structure-based drug discovery. To facilitate cavity detection and property analysis process, we developed a comprehensive web server, CavityPlus in 2018. CavityPlus applies the CAVITY program to detect potential binding sites in a given protein structure. The CavPharmer, CorrSite, and CovCys tools can then be applied to generate receptor-based pharmacophore models, identify potential allosteric sites, or detect druggable cysteine residues for covalent drug design. While CavityPlus has been widely used, the constantly evolving knowledge and methods make it necessary to improve and extend its functions. This study presents a new version of CavityPlus, CavityPlus 2022 through a series of upgrades. We upgraded the CAVITY tool to greatly speed up cavity detection calculation. We optimized the CavPharmer tool for fast speed and more accurate results. We integrated the newly developed CorrSite2.0 into the CavityPlus 2022 web server for its improved performance of allosteric site prediction. We also added a new CavityMatch module for drug repurposing and protein function studies by searching similar cavities to a given cavity from pre-constructed cavity databases. The new version of CavityPlus is freely available at http://pkumdl.cn:8000/cavityplus/.

An Atomic Level Investigation of Sodium Ions Regulating Agonist and Antagonist Binding in the Active Site of a Novel Target 5HT2BR Against Drug-Resistant Epilepsy.

The study investigates the movement of sodium ions inside the ligand-binding pocket of the class-A GPCR serotonin receptor (5HT2BR), a primary target for modern drugs. The available PDBs are mutant chimeras, so a 3D structure is modeled and validated by structural similarity (84.05%), Ramachandran favorable residues (93.01%), and clash score. Using MD simulations (500 ns), the ion active site is tracked in the presence and absence of ions and ligands. The ions enter the active site along helices III, VI, and VII, and the primary residue (ASP3.32) interacts with ions via H-bond (stronger- ~2.4 Å). The radial distribution function around ASP3.32 rises promptly at intermediate distances (2 Å < r < 4 Å), suggesting a higher probability of finding sodium ions at these distances. The ions stabilize the receptor at a better RMSD and promote stronger interactions (3-H-bonds, 1-π-bond~3.35 Å) with the agonist, and not the antagonist (no H-bond). Simulating unrestrained ligands further confirms this pattern, suggesting that ions might promote agonist binding but not be a prerequisite for antagonist action. The study highlights the mechanistic evaluation of sodium ions mobility in 5HT2BR modulation and ligand binding, showing potential in drug development.

In Silico Design of New Dual Inhibitors of SARS-CoV-2 MPRO through Ligand- and Structure-Based Methods.

The viral main protease is one of the most attractive targets among all key enzymes involved in the life cycle of SARS-CoV-2. Considering its mechanism of action, both the catalytic and dimerization regions could represent crucial sites for modulating its activity. Dual-binding the SARS-CoV-2 main protease inhibitors could arrest the replication process of the virus by simultaneously preventing dimerization and proteolytic activity. To this aim, in the present work, we identified two series' of small molecules with a significant affinity for SARS-CoV-2 MPRO, by a hybrid virtual screening protocol, combining ligand- and structure-based approaches with multivariate statistical analysis. The Biotarget Predictor Tool was used to filter a large in-house structural database and select a set of benzo[b]thiophene and benzo[b]furan derivatives. ADME properties were investigated, and induced fit docking studies were performed to confirm the DRUDIT prediction. Principal component analysis and docking protocol at the SARS-CoV-2 MPRO dimerization site enable the identification of compounds 1b,c,i,l and 2i,l as promising drug molecules, showing favorable dual binding site affinity on SARS-CoV-2 MPRO.

Binding Modes of Xanthine-Derived Selective Allosteric Site Inhibitors of MTHFD2.

Methylenetetrahydrofolate dehydrogenase (MTHFD2) is a mitochondrial enzyme involved in 1 C metabolism that is upregulated in various cancer cells, but absent in normal proliferating cells. Xanthine derivatives are the first selective inhibitors of MTHFD2 which bind to its allosteric site. Xanthine derivatives (including the co-crystallized inhibitors) were herein interrogated by molecular/induced-fit docking, MM-GBSA binding free energy calculations and molecular dynamics simulations in both MTHFD2 and MTHFD1 (a close homolog expressed in healthy cells). The gained insights from our in silico protocol allowed us to study binding mode, key protein-ligand interactions and dynamic movement of the allosteric inhibitors, correlating with their experimental binding affinities, biological activities and selectivity for MTHFD2. The reported conformational changes with MTHFD2 upon binding of xanthine derivatives were furthermore evaluated and confirmed by RMSF analyses of the MD simulation trajectories. The results reported herein are expected to benefit in the rational design of selective MTHFD2 allosteric inhibitors.

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands.

Allosteric regulation is critical for the functioning of G protein-coupled receptors (GPCRs) and their signaling pathways. Endogenous allosteric regulators of GPCRs are simple ions, various biomolecules, and protein components of GPCR signaling (G proteins and ß-arrestins). The stability and functional activity of GPCR complexes is also due to multicenter allosteric interactions between protomers. The complexity of allosteric effects caused by numerous regulators differing in structure, availability, and mechanisms of action predetermines the multiplicity and different topology of allosteric sites in GPCRs. These sites can be localized in extracellular loops; inside the transmembrane tunnel and in its upper and lower vestibules; in cytoplasmic loops; and on the outer, membrane-contacting surface of the transmembrane domain. They are involved in the regulation of basal and orthosteric agonist-stimulated receptor activity, biased agonism, GPCR-complex formation, and endocytosis. They are targets for a large number of synthetic allosteric regulators and modulators, including those constructed using molecular docking. The review is devoted to the principles and mechanisms of GPCRs allosteric regulation, the multiplicity of allosteric sites and their topology, and the endogenous and synthetic allosteric regulators, including autoantibodies and pepducins. The allosteric regulation of chemokine receptors, proteinase-activated receptors, thyroid-stimulating and luteinizing hormone receptors, and beta-adrenergic receptors are described in more detail.