Simple Synthesis of a Heterocyclophane Exhibiting Anti-c-Met Activity by Acting as a Hatch Blocking Access to the Active Site*.

A simple approach to the synthesis of heterocyclophane consisting of two 4,4'-bithiazoles has been developed in mild conditions. The heterocyclophane with two short chains was conveniently prepared by Hantzsch thiazoles synthesis using the reaction of 3-tert-butoxycarbonyl-3-azapentanethiocarboxamide with 1,4-dibromobutane-2,3-dione in methanol under reflux for only 15 min. Amino groups at the linkers of this heterocyclophane can be functionalized to give acylated and carbamate derivatives. Their properties as protein kinase inhibitors were investigated, and one of the heterocyclophanes exhibited specific anti-activity for c-mesenchymal epithelial transition factor (IC50 =603 nm), among seven types of protein kinases investigated. The computational site identification by ligand competitive saturation method was used to determine why the one heterocyclophane exhibited strong anti-activity for c-mesenchymal epithelial transition factor.

FFParam: Standalone package for CHARMM additive and Drude polarizable force field parametrization of small molecules.

Accurate force-field (FF) parameters are key to reliable prediction of properties obtained from molecular modeling (MM) and molecular dynamics (MD) simulations. With ever-widening applicability of MD simulations, robust parameters need to be generated for a wider range of chemical species. The CHARMM General Force Field program (CGenFF, https://cgenff.umaryland.edu/) is a tool for obtaining initial parameters for a given small molecule based on analogy with the available CGenFF parameters. However, improvement of these parameters is often required and performing their optimization remains tedious and time consuming. In addition, tools for optimization of small molecule parameters in the context of the Drude polarizable FF are not yet available. To overcome these issues, the FFParam package has been designed to facilitate the parametrization process. The package includes a graphical user interface (GUI) created using Qt libraries. FFParam supports Gaussian and Psi4 for performing quantum mechanical calculations and CHARMM and OpenMM for MM calculations. A Monte Carlo simulated annealing (MCSA) algorithm has been implemented for automated fitting of partial atomic charge, atomic polarizabilities and Thole scale parameters. The LSFITPAR program is called for automated fitting of bonded parameters. Accordingly, FFParam provides all the features required for generation and analysis of CHARMM and Drude FF parameters for small molecules. FFParam-GUI includes a text editor, graph plotter, molecular visualization, and text to table converter to meet various requirements of the parametrization process. It is anticipated that FFParam will facilitate wider use of CGenFF as well as promote future use of the Drude polarizable FF.

eBDIMS server: protein transition pathways with ensemble analysis in 2D-motion spaces.

SUMMARY: Understanding how proteins transition between different conformers, and how conformers relate to each other in terms of structure and function, is not trivial. Here, we present an online tool for transition pathway generation between two protein conformations using Elastic Network Driven Brownian Dynamics Importance Sampling, a coarse-grained simulation algorithm, which spontaneously predicts transition intermediates trapped experimentally. In addition to path-generation, the server provides an interactive 2D-motion landscape graphical representation of the transitions or any additional conformers to explore their structural relationships. AVAILABILITY AND IMPLEMENTATION: eBDIMS is available online: http://ebdims.biophysics.se/ or as standalone software: https://github.com/laura-orellana/eBDIMS, https://github.com/cabergh/eBDIMS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine.

Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the ß8-ß9 loop in the extracellular ligand-binding domain. The ß8-ß9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the ß8-ß9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.

Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.

The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate-gated chloride channel (GluClcryst ) as a structurally accessible model for GABAA receptors. In contrast to native-like controls, GluClcryst exhibits classical activation by its neurotransmitter ligand L-glutamate. The modified channel is also sensitive to allosteric modulators associated with human GABAA receptors, and to site-directed mutations predicted to alter channel opening.

Stabilization of the GluCl ligand-gated ion channel in the presence and absence of ivermectin.

Improving our understanding of the mechanisms and effects of anesthetics is a critically important part of neuroscience. The currently dominant theory is that anesthetics and similar molecules act by binding to Cys-loop receptors in the postsynaptic terminal of nerve cells and potentiate or inhibit their function. Although structures for some of the most important mammalian channels have still not been determined, a number of important results have been derived from work on homologous cationic channels in bacteria. However, partly due to the lack of a nervous system in bacteria, there are a number of questions about how these results relate to higher organisms. The recent determination of a structure of the eukaryotic chloride channel, GluCl, is an important step toward accurate modeling of mammalian channels, because it is more similar in function to human Cys-loop receptors such as GABAAR or GlyR. One potential issue with using GluCl to model other receptors is the presence of the large ligand ivermectin (IVM) positioned between all five subunits. Here, we have performed a series of microsecond molecular simulations to study how the dynamics and structure of GluCl change in the presence versus absence of IVM. When the ligand is removed, subunits move at least 2 Å closer to each other compared to simulations with IVM bound. In addition, the pore radius shrinks to 1.2 Å, all of which appears to support a model where IVM binding between subunits stabilizes an open state, and that the relaxed nonIVM conformations might be suitable for modeling other channels. Interestingly, the presence of IVM also has an effect on the structure of the important loop C located at the neurotransmitter-binding pocket, which might help shed light on its partial agonist behavior.

Assessment of homology templates and an anesthetic binding site within the γ-aminobutyric acid receptor.

BACKGROUND: Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. METHODS: The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and ß2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. RESULTS: Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between α and ß subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. CONCLUSION: Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.

Exploring the Conformational Impact of Glycine Receptor TM1-2 Mutations Through Coarse-Grained Analysis and Atomistic Simulations.

Pentameric ligand-gated ion channels (PLGICs) are a family of proteins that convert chemical signals into ion fluxes through cellular membranes. Their structures are highly conserved across all kingdoms from bacteria to eukaryotes. Beyond their classical roles in neurotransmission and neurological disorders, PLGICs have been recently related to cell proliferation and cancer. Here, we focus on the best characterized eukaryotic channel, the glycine receptor (GlyR), to investigate its mutational patterns in genomic-wide tumor screens and compare them with mutations linked to hyperekplexia (HPX), a Mendelian neuromotor disease that disrupts glycinergic currents. Our analysis highlights that cancer mutations significantly accumulate across TM1 and TM2, partially overlapping with HPX changes. Based on 3D-clustering, conservation, and phenotypic data, we select three mutations near the pore, expected to impact GlyR conformation, for further study by molecular dynamics (MD). Using principal components from experimental GlyR ensembles as framework, we explore the motions involved in transitions from the human closed and desensitized structures and how they are perturbed by mutations. Our MD simulations show that WT GlyR spontaneously explores opening and re-sensitization transitions that are significantly impaired by mutations, resulting in receptors with altered permeability and desensitization properties in agreement with HPX functional data.

Prediction and validation of protein intermediate states from structurally rich ensembles and coarse-grained simulations.

Protein conformational changes are at the heart of cell functions, from signalling to ion transport. However, the transient nature of the intermediates along transition pathways hampers their experimental detection, making the underlying mechanisms elusive. Here we retrieve dynamic information on the actual transition routes from principal component analysis (PCA) of structurally-rich ensembles and, in combination with coarse-grained simulations, explore the conformational landscapes of five well-studied proteins. Modelling them as elastic networks in a hybrid elastic-network Brownian dynamics simulation (eBDIMS), we generate trajectories connecting stable end-states that spontaneously sample the crystallographic motions, predicting the structures of known intermediates along the paths. We also show that the explored non-linear routes can delimit the lowest energy passages between end-states sampled by atomistic molecular dynamics. The integrative methodology presented here provides a powerful framework to extract and expand dynamic pathway information from the Protein Data Bank, as well as to validate sampling methods in general.

Conformational gating dynamics in the GluCl anion-selective chloride channel.

Cys-loop receptors are central to propagation of signals in the nervous system. The gating of the membrane-spanning pore is triggered by structural rearrangements in the agonist-binding site, located some 50 Å away from the pore. A sequential conformational change, propagating from the ligand-binding site to the pore, has been proposed to govern gating in all Cys-loop receptors. Here, we identify structural and dynamic components of the conformational gating in the eukaryotic glutamate-gated chloride channel (GluCl) by means of molecular dynamics (MD) simulations with and without the l-glutamate agonist bound. A significant increase in pore opening and accompanying hydration is observed in the presence of glutamate. Potential of mean force calculations reveal that the barrier for ion passage drops from 15 kcal/mol to 5-10 kcal/mol with the agonist bound. This appears to be explained by agonist binding that leads to significant changes in the intersubunit hydrogen-bonding pattern, which induce a slight tilt of the extracellular domain relative to the transmembrane domain in the simulations. This rearrangement is subtle, but correspond to the direction of the quaternary twist observed as a key difference between open and closed X-ray structures. While the full reversible gating is still a much slower process, the observed structural dynamics sheds new light on the early stages of how the agonist influences the extracellular domain, how the extracellular domain interacts with the transmembrane domain, and how changes in the transmembrane domain alter the free energy of ion passage.