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.

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.

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-β-carbomethoxy-3-β-(4-fluorophenyl)tropane2 (β-CFT), the non-competitive inhibitor MRS72923 and Zn2+ (ref. 4). We show how β-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.

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.

AcrB, a key component in bacterial efflux processes, exhibits distinct binding pockets that influence inhibitor interactions. In addition to the well-known distal binding pocket within the periplasmic domain, a noteworthy pocket amidst the transmembrane (TM) helices serves as an alternate binding site for inhibitors. The bacterial efflux mechanism involves a pivotal functional rotation of the TM protein, inducing conformational changes in each protomer and propelling drugs toward the outer membrane domain. Surprisingly, inhibitors binding to the TM domain display a preference for L protomers over T protomers. Metadynamics simulations elucidate that Lys940 in the TM domain of AcrB can adopt two conformations in L protomers, whereas the energy barrier for such transitions is higher in T protomers. This phenomenon results in stable inhibitor binding in l protomers. Upon a detailed analysis of unbinding pathways using random accelerated molecular dynamics and umbrella sampling, we have identified three distinct routes for ligand exit from the allosteric site, specifically involving regions within the TM domains─TM4, TM5, and TM10. To explore allosteric crosstalk, we focused on the following key residues: Val452 from the TM domain and Ala831 from the porter domain. Surprisingly, our findings reveal that inhibitor binding disrupts this communication. The shortest path connecting Val452 and Ala831 increases upon inhibitor binding, suggesting sabotage of the natural interdomain communication dynamics. This result highlights the intricate interplay between inhibitor binding and allosteric signaling within our studied system.

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.

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.

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.

β-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher\'s disease and are a major genetic risk factor for Parkinson\'s disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.

UNASSIGNED: Protein tyrosine phosphatases (PTPs), essential and evolutionarily highly conserved enzymes, govern cellular functions by modulating tyrosine phosphorylation, a pivotal post-translational modification for signal transduction. The recent strides in phosphatase drug discovery, leading to the identification of selective modulators for enzymes, restoring interest in the therapeutic targeting of protein phosphatases.
UNASSIGNED: The compilation of patents up to the year 2023 focuses on the efficacy of various classes of Tyrosine phosphatases and their inhibitors, detailing their chemical structure and biochemical characteristics. These findings have broad implications, as they can be applied to treating diverse conditions like cancer, diabetes, autoimmune disorders, and neurological diseases. The search for scientific articles and patent literature was conducted using well known different platforms to gather information up to 2023.
UNASSIGNED: The latest improvements in protein tyrosine phosphatase (PTP) research include the discovery of new inhibitors targeting specific PTP enzymes, with a focus on developing allosteric site covalent inhibitors for enhanced efficacy and specificity. These advancements have not only opened up new possibilities for therapeutic interventions in various disease conditions but also hold the potential for innovative treatments. PTPs offer promising avenues for drug discovery efforts and innovative treatments across a spectrum of health conditions.