Multiple Binding Sites Contribute to the Mechanism of Mixed Agonistic and Positive Allosteric Modulators of the Cannabinoid CB1 Receptor

The cannabinoid CB1 receptor (CB1R) is an abundant metabotropic G‐protein‐coupled receptor that has been difficult to address therapeutically because of CNS side effects exerted by orthosteric drug candidates. Recent efforts have focused on developing allosteric modulators that target CB1R. Compounds from the recently discovered class of mixed agonistic and positive allosteric modulators (Ago‐PAMs) based on 2‐phenylindoles have shown promising functional and binding properties as CB1R ligands. Here, we identify binding modes of both the CP 55,940 agonist and GAT228, a 2‐phenylindole allosteric modulator, by using our metadynamics simulation protocol, and quantify their affinity and cooperativity by atomistic simulations. We demonstrate the involvement of multiple adjunct binding sites in the Ago‐PAM characteristics of the 2‐phenylindole modulators and explain their ability to compete with orthosteric agonists at higher concentrations. We validate these results experimentally by showing the contribution of multiple sites on the allosteric binding of ZCZ011, another homologous member of the class, together with the orthosteric agonist.     

Self-Assembly of Coordinative Supramolecular Polygons with Open Binding Sites

The design and synthesis of coordinative supramolecular polygons with open binding sites is described. Coordination-driven self-assembly of 2,6-bis(pyridin-4-ylethynyl)pyridine with 60° and 120° organoplatinum acceptors results in quantitative formation of a supramolecular rhomboid and hexagon, respectively, both bearing open pyridyl binding sites. The structures were determined by multinuclear (³¹P and ¹H) NMR spectroscopy and electrospray ionization (ESI) mass spectrometry, along with a computational study.     

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.     

5-羟色胺转运蛋白显像剂的研究进展

中枢神经系统5-羟色胺神经元功能异常,特别是突触前膜的5-羟色胺转运蛋白（SERT）密度的变化常导致复杂的精神紊乱疾病。SERT的正电子发射断层（PET）和单光子发射断层（SPECT）活体显像剂有助于研究该系统的变化与精神紊乱疾病的关系,以及精神紊乱病人疗效的监测。本文综述了近年来SERT显像剂的最新研究进展,并指出了今后该类显像剂的发展趋势。     

Study on the Binding of Chloride Ion to Hemoglobin Using Electromotive Force Method

The electromotive force of the concentration cell, in which both half‐cells contain the same concentration of sodium chloride, and one of which also contains hemoglobin (Hb), was measured on the isoelectric point of Hb (pH = 6.70). On the basis of Scatchard equation, a stepwise binding model and an improved calculation method were presented. Using the new calculation method, the number of the chloride‐binding sites on Hb molecule and the corresponding binding constants were calculated. The results show that there are three classes of binding groups on a Hb molecule, the number of the binding sites and the corresponding binding constants are n₁ = 1, K⁰₁ = 245; n₂ = 8, K⁰₂ = 3.50; n₃ = 8, K⁰₃ = 1.91, respectively. The factors of influencing the interaction between Cl^? and Hb molecule were clarified, and that the differences between our results and the results of computer modeling, as well as the results of molecular dynamics simulation were also discussed.     

Allosteric Binding of MDMA to the Human Serotonin Transporter (hSERT) via Ensemble Binding Space Analysis with ΔG Calculations,Induced Fit Docking and Monte Carlo Simulations

Despite the recent promising results of MDMA (3,4-methylenedioxy-methamphetamine) as a psychotherapeutic agent and its history of misuse, little is known about its molecular mode of action. MDMA enhances monoaminergic neurotransmission in the brain and its valuable psychoactive effects are associated to a dual action on the 5-HT transporter (SERT). This drug inhibits the reuptake of 5-HT (serotonin) and reverses its flow, acting as a substrate for the SERT, which possesses a central binding site (S1) for antidepressants as well as an allosteric (S2) one. Previously, we characterized the spatial binding requirements for MDMA at S1. Here, we propose a structure-based mechanistic model of MDMA occupation and translocation across both binding sites, applying ensemble binding space analyses, electrostatic complementarity, and Monte Carlo energy perturbation theory. Computed results were correlated with experimental data (r = 0.93 and 0.86 for S1 and S2, respectively). Simulations on all hSERT available structures with Gibbs free energy estimations (ΔG) revealed a favourable and pervasive dual binding mode for MDMA at S2, i.e., adopting either a 5-HT or an escitalopram-like orientation. Intermediate ligand conformations were identified within the allosteric site and between the two sites, outlining an internalization pathway for MDMA. Among the strongest and more frequent interactions were salt bridges with Glu494 and Asp328, a H-bond with Thr497, a π-π with Phe556, and a cation-π with Arg104. Similitudes and differences with the allosteric binding of 5-HT and antidepressants suggest that MDMA may have a distinctive chemotype. Thus, our models may provide a framework for future virtual screening studies and pharmaceutical design and to develop hSERT allosteric compounds with a unique psychoactive MDMA-like profile.     

Druggable Allosteric Sites in β-Propeller Lectins

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a β-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing ¹⁹F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure–activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol^?1 HA^?1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens.     

Interfacial Sites in Ag Supported Layered Double Oxide for Dehydrogenation Coupling of Ethanol to n-Butanol

Upgrading of ethanol to n-butanol through dehydrogenation coupling has received increasing attention due to the wide application of n-butanol. But the enhancement of ethanol dehydrogenation and followed coupling to produce high selectivity to n-butanol is still highly desired. Our previous work has reported an acid-base-Ag synergistic catalysis, with Ag particles supported on Mg and Al-containing layered double oxides (Ag/MgAl-LDO). Here, Ag-LDO interfaces have been manipulated for dehydrogenation coupling of ethanol to n-butanol by tailoring the size of Ag particles and the interactions between Ag and LDO. It has been revealed that increasing the population of surface Ag sites at Ag-LDO interfaces promotes not only the dehydrogenation of ethanol to acetaldehyde but also the subsequent aldol condensation of generated acetaldehyde. A selectivity of up to 76 % to n-butanol with an ethanol conversion of 44 % has been achieved on Ag/LDO with abundant interfacial Ag sites, much superior to the state-of-the-art catalysts.     

Competitive Binding of Drugs to the Multiple Binding Sites on Human Serum Albumin: A calorimetric study

A multiple-site competitive model has been developed to evaluate quantitatively the equilibrium competition of drugs that bind to multiple classes of binding sites on human serum albumin (HSA). The equations, which are based on the multiple-class binding site model, assume that competition exists at individual sites, that the binding parameters for drug or drug competitor pertain to individual sites, and also that the binding parameters for drug or competitor at any given site are independent of drug or competitor bound at other sites. For the drug-competitor pairs, ethacrynic acid (EA) -caproic acid (C6), -lauric acid (C12), and -palmitic acid (C16), the reaction heat of EA binding to HSA was measured in the absence and the presence of fatty acids at the molar ratio of 3:1 with HSA at pH 7.4 and 37°C by isothermal titration microcalorimetry. The calorimetric titration data induced by the presence of fatty acids were directly compaired to the computer simulation curves by the corresponding multiple-site competititve models, which were precedently calculated from binding parameters of EA and fatty acids. In the case of EA-C12 or -C16 competitive binding, EA binding at the first and the second classes of binding sites on HSA were instantaneously inhibited by C12 or C16, resulting that the binding constant of the first class of binding sites of EA were decreased and that the second class of binding sites on HSA entirely disappeared. In the competition between EA and C6, the first class of binding sites of EA was diminished by C6, resulting in the decrease of the binding constants and the number of binding sites in the first class of EA, whereas, the second class of binding sites was unaffected. The multiple-site competitive model assuming site-site competition could be directly comparable to the calorimetric data and be suitable to account for the competitive processes for drugs bound to the multiple-class of binding sites on HSA.This revised version was published online in November 2005 with corrections to the Cover Date.     

Zincbindpredict—Prediction of Zinc Binding Sites in Proteins

Background: Zinc binding proteins make up a significant proportion of the proteomes of most organisms and, within those proteins, zinc performs rôles in catalysis and structure stabilisation. Identifying the ability to bind zinc in a novel protein can offer insights into its functions and the mechanism by which it carries out those functions. Computational means of doing so are faster than spectroscopic means, allowing for searching at much greater speeds and scales, and thereby guiding complimentary experimental approaches. Typically, computational models of zinc binding predict zinc binding for individual residues rather than as a single binding site, and typically do not distinguish between different classes of binding site—missing crucial properties indicative of zinc binding. Methods: Previously, we created ZincBindDB, a continuously updated database of known zinc binding sites, categorised by family (the set of liganding residues). Here, we use this dataset to create ZincBindPredict, a set of machine learning methods to predict the most common zinc binding site families for both structure and sequence. Results: The models all achieve an MCC ≥ 0.88, recall ≥ 0.93 and precision ≥ 0.91 for the structural models (mean MCC = 0.97), while the sequence models have MCC ≥ 0.64, recall ≥ 0.80 and precision ≥ 0.83 (mean MCC = 0.87), with the models for binding sites containing four liganding residues performing much better than this. Conclusions: The predictors outperform competing zinc binding site predictors and are available online via a web interface and a GraphQL API.     

Synergistic Nitrogen Binding Sites in a Metal-Organic Framework for Efficient N2/O2 Separation

Porous materials with d₃ electronic configuration open metal sites have been proved to be effective adsorbents for N₂ capture and N₂/O₂ separation. However, the reported materials remain challenging to address the trade-off between adsorption capacity and selectivity. Herein, we report a robust MOF, MIL-102Cr, that features two binding sites, can synergistically afford strong interactions for N₂ capture. The synergistic adsorption site exhibits a benchmark Q_st of 45.0 kJ mol⁻¹ for N₂ among the Cr-based MOFs, a record-high volumetric N₂ uptake (31.38 cm³ cm⁻³), and highest N₂/O₂ selectivity (13.11) at 298 K and 1.0 bar. Breakthrough experiments reveal that MIL-102Cr can efficiently capture N₂ from a 79/21 N₂/O₂ mixture, providing a record 99.99 % pure O₂ productivity of 0.75 mmol g⁻¹. In situ infrared spectroscopy and computational modelling studies revealed that a synergistic adsorption effect by open Cr(III) and fluorine sites was accountable for the strong interactions between the MOF and N₂.     

Phosphate-Rich Biomimetic Peptides Shed Light on High-Affinity Hyperphosphorylated Uranyl Binding Sites in Phosphoproteins

Some phosphoproteins such as osteopontin (OPN) have been identified as high-affinity uranyl targets. However, the binding sites required for interaction with uranyl and therefore involved in its toxicity have not been identified in the whole protein. The biomimetic approach proposed here aimed to decipher the nature of these sites and should help to understand the role of the multiple phosphorylations in UO₂²⁺ binding. Two hyperphosphorylated cyclic peptides, pS₁₆₈ and pS₁₃₆₈ containing up to four phosphoserine (pSer) residues over the ten amino acids present in the sequences, were synthesized with all reactions performed in the solid phase, including post-phosphorylation. These β-sheet-structured peptides present four coordinating residues from four amino acid side chains pointing to the metal ion, either three pSer and one glutamate in pS₁₆₈ or four pSer in pS₁₃₆₈ . Significantly, increasing the number of pSer residues up to four in the cyclodecapeptide scaffolds produced molecules with an affinity constant for UO₂²⁺ that is as large as that reported for osteopontin at physiological pH. The phosphate-rich pS₁₃₆₈ can thus be considered a relevant model of UO₂²⁺ coordination in this intrinsically disordered protein, which wraps around the metal ion to gather four phosphate groups in the UO₂²⁺ coordination sphere. These model hyperphosphorylated peptides are highly selective for UO₂²⁺ with respect to endogenous Ca²⁺, which makes them good starting structures for selective UO₂²⁺ complexation.     

Identification and Characterization of a Single High‐Affinity Fatty Acid Binding Site in Human Serum Albumin

A single high‐affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐C₁₂ fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site‐specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA‐binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow‐sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X‐ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug‐protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.     

Binding neutral guests to concave surfaces of molecular hosts. Directional association of water and methylene chloride with hosts containing only cyclic urea binding sites

This paper is concerned with studies of weak intermolecular interactions in molecular inclusion type systems involving uncharged host and guest entities. Three new complexes of synthetic organic ligands with water and methylene chloride have been characterized by single-crystal X-ray diffraction. The hosts are composed of three cyclic urea units whose carbonyl groups are held in convergent positions by bonding their attached nitrogens to one another through two (noncyclic ligand) or three (macrocyclic ligand) rigid spacer units. Conformational organization is further enforced by an aliphatic bridge between two of the phenylene spacers in the macrocyclic hosts and an additional dimerization of the open-chain ligand. The host species were found to be particularly suitable to interact with proton donating H₂O and CH₂Cl₂ guest moieties, as their molecular surface contains appropriately sized polar cavities lined with the carbonyl functions. Association between the interacting components in these complexes is stabilized by O–HO and C–HO hydrogen bonds. In the corresponding crystal structures additional molecules of the solvent are located between units of the complex. The significance of preorganization of the host structure to an efficient guest binding is emphasized by an observation that no stable complexes of a similar but unbridged macrocyclic ligand could be crystallized from the same solvent. The structural features of the inclusion compounds are described in detail, and the host-guest interaction scheme is compared to that observed in complexes of 18-crown-6 with neutral guests. Supplementary Data relating to this article are deposited with the British Library as Supplementary Publication No. SUP 82039 (98 pages)     

Thermophoresis in Nanoliter Droplets to Quantify Aptamer Binding

Biomolecule interactions are central to pharmacology and diagnostics. These interactions can be quantified by thermophoresis, the directed molecule movement along a temperature gradient. It is sensitive to binding induced changes in size, charge, or conformation. Established capillary measurements require at least 0.5 μL per sample. We cut down sample consumption by a factor of 50, using 10 nL droplets produced with acoustic droplet robotics (Labcyte). Droplets were stabilized in an oil–surfactant mix and locally heated with an IR laser. Temperature increase, Marangoni flow, and concentration distribution were analyzed by fluorescence microscopy and numerical simulation. In 10 nL droplets, we quantified AMP‐aptamer affinity, cooperativity, and buffer dependence. Miniaturization and the 1536‐well plate format make the method high‐throughput and automation friendly. This promotes innovative applications for diagnostic assays in human serum or label‐free drug discovery screening.     

Replacement of Water Molecules in a Phosphate Binding Site by Furanoside‐Appended lin‐Benzoguanine Ligands of tRNA‐Guanine Transglycosylase (TGT)

The enzyme tRNA‐guanine transglycosylase has been identified as a drug target for the foodborne illness shigellosis. A key challenge in structure‐based design for this enzyme is the filling of the polar ribose‐34 pocket. Herein, we describe a novel series of ligands consisting of furanoside‐appended lin‐benzoguanines. They were designed to replace a conserved water cluster and differ by the functional groups at C(2) and C(3) of the furanosyl moiety being either OH or OMe. The unfavorable desolvation of Asp102 and Asp280, which are located close to the ribose‐34 pocket, had a significant impact on binding affinity. While the enzyme has tRNA as its natural substrate, X‐ray co‐crystal structures revealed that the furanosyl moieties of the ligands are not accommodated in the tRNA ribose‐34 site, but at the location of the adjacent phosphate group. A remarkable similarity of the position of the oxygen atoms in these two structures suggests furanosides as a potential phosphate isoster.     

Binding of topotecan to a nicked DNA oligomer in solution

Topotecan (TPT) is in clinical use as an antitumor agent. It acts by binding to the covalent complex formed between nicked DNA and topoisomerase I, and inserts itself into the single-strand nick, thereby inhibiting the religation of the nick and acting as a poison. A crystal structure analysis of the ternary complex has shown how the drug binds (B. L. Staker, K. Hjerrild, M. D. Feese, C. A. Behnke, A. B. Burgin, L. Stewart, Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 15 387-15 392), but has left a number of unanswered questions. Herein, we use NMR spectroscopy and molecular modeling to show that the solution structure of a complex of TPT with nicked natural DNA is similar, but not identical to the crystal conformation, and that other geometries are of very low population. We also show that the lactone form of TPT binds approximately 40 times more strongly than the ring-opened carboxylate.     

埃铬青R与牛血清白蛋白的结合反应

在ＰＨ２．４含０．５％ＮａＣｌ的溶液中，牛血清白蛋白与埃铬青Ｒ（ＥＣＲ）发生相互作用而导致溶颜色发生变化。测得这一反应的表观结合常数为５．２×１０＾５；最大结合数为３８；桑德尔灵敏度指数为０．７５μｇ／ｃｍ＾２．