The Use of Chlorobenzene as a Probe Molecule in Molecular Dynamics Simulations

We map ligand binding sites on protein surfaces in molecular dynamics simulations using chlorobenzene as a probe molecule. The method was validated on four proteins. Two types of affinity maps that identified halogen and hydrophobic binding sites on proteins were obtained. Our method could prove useful for the discovery and development of halogenated inhibitors

The Application of Ligand-Mapping Molecular Dynamics Simulations to the Rational Design of Peptidic Modulators of Protein–Protein Interactions

A computational ligand-mapping approach to detect protein surface pockets that interact with hydrophobic moieties is presented. In this method, we incorporated benzene molecules into explicit solvent molecular dynamics simulations of various protein targets. The benzene molecules successfully identified the binding locations of hydrophobic hot-spot residues and all-hydrocarbon cross-links from known peptidic ligands. They also unveiled cryptic binding sites that are occluded by side chains and the protein backbone. Our results demonstrate that ligand-mapping molecular dynamics simulations hold immense promise to guide the rational design of peptidic modulators of protein–protein interactions, including that of stapled peptides, which show promise as an exciting new class of cell-penetrating therapeutic molecules

Enantioselective Synthesis of Chromanones via a Peptidic Phosphane Catalyzed Rauhut–Currier Reaction

The enantioselective intramolecular Rauhut–Currier reaction has been developed using a bifunctional dipeptidic phosphane catalyst, providing a direct access to biologically active α-methylene-δ-valerolactones in high yields and enantiomeric excesses. The novel catalyst is accessible in only four steps from commercial sources and exhibits unusual binding selectivities for a small molecule, suggesting the possibility for long-range interactions between the catalyst and the substrate

High Content Screening of Diverse Compound Libraries Identifies Potent Modulators of Tubulin Dynamics

Tubulin modulating agents such as the taxanes are among the most effective antimitotic cancer drugs, although resistance and toxicity present significant problems in their clinical use. However, most tubulin modulators are derived from complex natural products, which can make modification of their structure to address these problems difficult. Here, we report the discovery of new antimitotic compounds with simple structures that can be rapidly synthesized, through the phenotypic screening of a diverse compound library for the induction of mitotic arrest. We first identified a compound, which induced mitotic arrest in human cells at submicromolar concentrations. Its simple structure enabled rapid exploration of activity, defining a biphenylacetamide moiety required for activity, A family of analogues was synthesized, yielding optimized compounds that caused mitotic arrest and cell death in the low nanomolar range, comparable to clinically used antimitotic agents. These compounds can be synthesized in 1–3 steps and good yields. We show that one such compound targets tubulin, partially inhibiting colchicine but not vinblastine binding, suggesting that it acts allosterically to the known colchicine-binding site. Thus, our results exemplify the use of phenotypic screening to identify novel antimitotic compounds from diverse chemical libraries and characterize a family of biphenylacetamides (biphenabulins) that show promise for further development

Identification of Key Residues That Confer <i>Rhodobacter sphaeroides</i> LPS Activity at Horse TLR4/MD-2

<div><p>The molecular determinants underpinning how hexaacylated lipid A and tetraacylated precursor lipid IVa activate Toll-like receptor 4 (TLR4) are well understood, but how activation is induced by other lipid A species is less clear. Species specificity studies have clarified how TLR4/MD-2 recognises different lipid A structures, for example tetraacylated lipid IVa requires direct electrostatic interactions for agonism. In this study, we examine how pentaacylated lipopolysaccharide from <i>Rhodobacter sphaeroides</i> (RSLPS) antagonises human TLR4/MD-2 and activates the horse receptor complex using a computational approach and cross-species mutagenesis. At a functional level, we show that RSLPS is a partial agonist at horse TLR4/MD-2 with greater efficacy than lipid IVa. These data suggest the importance of the additional acyl chain in RSLPS signalling. Based on docking analysis, we propose a model for positioning of the RSLPS lipid A moiety (RSLA) within the MD-2 cavity at the TLR4 dimer interface, which allows activity at the horse receptor complex. As for lipid IVa, RSLPS agonism requires species-specific contacts with MD-2 and TLR4, but the R2 chain of RSLA protrudes from the MD-2 pocket to contact the TLR4 dimer in the vicinity of proline 442. Our model explains why RSLPS is only partially dependent on horse TLR4 residue R385, unlike lipid IVa. Mutagenesis of proline 442 into a serine residue, as found in human TLR4, uncovers the importance of this site in RSLPS signalling; horse TLR4 R385G/P442S double mutation completely abolishes RSLPS activity without its counterpart, human TLR4 G384R/S441P, being able to restore it. Our data highlight the importance of subtle changes in ligand positioning, and suggest that TLR4 and MD-2 residues that may not participate directly in ligand binding can determine the signalling outcome of a given ligand. This indicates a cooperative binding mechanism within the receptor complex, which is becoming increasingly important in TLR signalling.</p></div

Concise Copper-Catalyzed Synthesis of Tricyclic Biaryl Ether-Linked Aza-Heterocyclic Ring Systems

A new method for the synthesis of tricyclic biaryl ether-linked ring systems incorporating seven-, eight-, and nine-membered ring amines is presented. In the presence of catalytic quantities of copper(I), readily accessible acyclic precursors undergo an intramolecular carbon–oxygen bond-forming reaction facilitated by a “templating” chelating nitrogen atom. The methodology displays a broad substrate scope, is practical, and generates rare and biologically interesting tricyclic heteroaromatic products that are difficult to access by other means

RSLPS requires specific residues within horse MD-2 and TLR4, yet is independent of CD14.

<p>HEK293 cells were transiently transfected with combinations of human and horse TLR4 and MD-2, with or without horse CD14, and reporter constructs NF-κB-luc and phRG-TK. Cells were stimulated 48 hours later for 6 hours with 100 ng/ml RSLPS, 10 ng/ml ECLPS, 100 ng/ml RSLPS+10 ng/ml ECLPS, or medium alone. Data are from a representative experiment (n = 3 experiments) and expressed as triplicate mean ±SEM for that experiment, relative to the maximum ECLPS response. A) Cells were transfected with different combinations of human and horse TLR4 and MD-2. B) Horse TLR4/MD-2 was transfected with and without CD14. C) MD-2 mutants were transfected with horse TLR4/CD14. D) TLR4 mutants were transfected with horse MD-2/CD14.</p

Chemical structures of lipid A derivatives.

<p>A) Lipid A from <i>E. coli</i>. B) Lipid A synthesis intermediate lipid IVa. C) Lipid A from <i>Rhodobacter sphaeroides</i>.</p

RSLA and lipid IVa sit differently within the MD-2 pocket.

<p>A) Docking models of RSLA and lipid IVa bound to horse TLR4/MD-2 were overlaid to assess ligand and receptor positioning. The acyl chains of RSLA (blue) sit more deeply in the MD-2 (pink) pocket than lipid IVa (green), and the R2 chain of RSLA protrudes from the MD-2 pocket to contact TLR4* (grey). The 1-PO₄ is also moved away from TLR4 due to lowering of the diglucosamine backbone. B) Overlay of RSLA (blue; horse model), lipid IVa (green; horse model) and lipid A (red; human crystal) in situ in the MD-2 pocket. TLR4 and MD-2 have been removed for clarity. The PO₄ groups and acyl chains of all three ligands sit somewhat differently to one another within the pocket. Lipid A and RSLA appear to occupy a similar volume within the pocket.</p

RSLPS activity requires the presence of both R385 and P442 in horse TLR4.

<p>HEK293 cells were transiently transfected with combinations of human and horse TLR4 and MD-2, together with horse CD14 and reporter constructs NF-κB-luc and phRG-TK. Cells were stimulated 48 hours later for 6 hours. Data are from a representative experiment (n = 3 experiments) and expressed as triplicate mean ±SEM for that experiment, relative to the maximum ECLPS response. A) TLR4 point mutants were transfected with horse MD-2/CD14 and stimulated with 100 ng/ml RSLPS, 10 ng/ml ECLPS, 100 ng/ml RSLPS+10 ng/ml ECLPS or medium alone. B) TLR4 point mutants were transfected with horse MD-2/CD14 and stimulated with 1 µg/ml lipid IVa, 1 µg/ml lipid IVa+10 ng/ml ECLPS, or medium alone.</p