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.

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.

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.

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.

Operational models of allosteric modulation of G protein-coupled receptors / 药学学报

Since the Monod-Wyman-Changeux (MWC) model was initially proposed to explain the allosteric interactions between proteins and their ligands 50 years ago, there have been various models and hypotheses such as the induced-fit model on the interaction. These theoretical developments have been used broadly in the study of allosteric modulations of enzymes and receptors. In 1980, Lefkowitz and coworkers proposed a ternary complex model (TCM) for the regulatory mechanism of G protein-coupled receptors (GPCRs) that laid the theoretical foundation in the study of allosteric sites and ligands of GPCRs, the largest family of known receptors. The findings on how ligands interact with receptors to cause a functional response have significantly impacted the drug discovery field and accelerated the identification of allosteric modulators.