Structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases.

Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform specificity.

Development of a computational tool to rival experts in the prediction of sites of metabolism of xenobiotics by p450s.

The metabolism of xenobiotics--and more specifically drugs--in the liver is a critical process controlling their half-life. Although there exist experimental methods, which measure the metabolic stability of xenobiotics and identify their metabolites, developing higher throughput predictive methods is an avenue of research. It is expected that predicting the chemical nature of the metabolites would be an asset for designing safer drugs and/or drugs with modulated half-lives. We have developed IMPACTS (In-silico Metabolism Prediction by Activated Cytochromes and Transition States), a computational tool combining docking to metabolic enzymes, transition state modeling, and rule-based substrate reactivity prediction to predict the site of metabolism (SoM) of xenobiotics. Its application to sets of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 substrates and comparison to experts' predictions demonstrates its accuracy and significance. IMPACTS identified an experimentally observed SoM in the top 2 predicted sites for 77% of the substrates, while the accuracy of biotransformation experts' prediction was 65%. Application of IMPACTS to external sets and comparison of its accuracy to those of eleven other methods further validated the method implemented in IMPACTS.

Integrating medicinal chemistry, organic/combinatorial chemistry, and computational chemistry for the discovery of selective estrogen receptor modulators with Forecaster, a novel platform for drug discovery.

As part of a large medicinal chemistry program, we wish to develop novel selective estrogen receptor modulators (SERMs) as potential breast cancer treatments using a combination of experimental and computational approaches. However, one of the remaining difficulties nowadays is to fully integrate computational (i.e., virtual, theoretical) and medicinal (i.e., experimental, intuitive) chemistry to take advantage of the full potential of both. For this purpose, we have developed a Web-based platform, Forecaster, and a number of programs (e.g., Prepare, React, Select) with the aim of combining computational chemistry and medicinal chemistry expertise to facilitate drug discovery and development and more specifically to integrate synthesis into computer-aided drug design. In our quest for potent SERMs, this platform was used to build virtual combinatorial libraries, filter and extract a highly diverse library from the NCI database, and dock them to the estrogen receptor (ER), with all of these steps being fully automated by computational chemists for use by medicinal chemists. As a result, virtual screening of a diverse library seeded with active compounds followed by a search for analogs yielded an enrichment factor of 129, with 98% of the seeded active compounds recovered, while the screening of a designed virtual combinatorial library including known actives yielded an area under the receiver operating characteristic (AU-ROC) of 0.78. The lead optimization proved less successful, further demonstrating the challenge to simulate structure activity relationship studies.

Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

Introduction: Alzheimer's disease (AD) is characterized by neurotoxic immuno-inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aß) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses. Methods: We performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic-molecular mechanisms of cytokine-mediated and Aß-mediated neurotoxicities in AD.  Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small-molecule therapeutics for AD. Results: In response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aß is released as an early responder immunopeptide triggering an innate immunity cascade in which Aß exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon "self" neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane-penetrating attack by antimicrobial peptides (AMPs) such as Aß. After this self-attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aß, leading to a chronic self-perpetuating autoimmune cycle.  AD thus emerges as a brain-centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti-AD molecules capable of chemical modification into multi-site therapeutic modulators targeting AD's complex immunopathic-proteopathic pathogenesis. Discussion:  Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug-like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

Structural chemistry of the histone methyltransferases cofactor binding site.

Histone methyltransferases (HMTs) transfer a methyl group from the cofactor S-adenosyl methionine to lysine or arginine residues on histone tails, thereby regulating chromatin compaction, binding of effector proteins and gene transcription. HMTs constitute an emerging target class in diverse disease areas, and selective chemical probes are necessary for target validation. Potent and selective competitors of the substrate peptide have been reported, but the chemical tractability of the cofactor binding site is poorly understood. Here, a systematic analysis of this site across structures of 14 human HMTs or close homologues was conducted. The druggability, interaction hotspots, and diversity of the cofactor binding pocket were dissected. This analysis strongly suggests that this site is chemically tractable. General principles underlying tight binding and specific guidelines to achieve selective inhibition are presented.

Pharmacophore screening of the protein data bank for specific binding site chemistry.

A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

Evaluation of virtual screening as a tool for chemical genetic applications.

A collection of over 50,000 functionally annotated drugs, clinical candidates, and endogenous ligands was docked in silico against nine binding sites from seven protein targets, representing diverse function and structure, namely the sulfotransferases SULT1E1 and SULT1A3, the histone methyltransferase EHMT1, the histone acetyltransferase MYST3, and the nuclear hormone receptors ERalpha, PPARgamma, and TRbeta. For 5 of the 9 virtual screens, compounds that docked best to the receptors clearly recapitulated known biological functions of the genes or identified novel biology subsequently validated in a separate experimental study. In two cases, the hit list indicated some relevant but isolated biological functions which would probably have been ignored a priori, and selected compounds were completely unrelated to gene function for the last two virtual screens. This study demonstrates that virtual screening of pharmacologically annotated compound libraries can be used to derive target biology.

Discovery of indoline-based, natural-product-like compounds as probes of focal adhesion kinase signaling pathways.

With the goal of identifying small molecule modulators of protein-protein interactions, we developed a solid-phase synthesis method, which was then successfully utilized in a library generation of 164 aminoindoline-derived, natural-product-like compounds. This library and several other related intermediates synthesized during this project were then subjected to different biological assays in search of small molecule modulators of focal adhesion kinase (FAK)-mediated signaling pathways. This study included (i) an in vitro, full length FAK inhibition assay, (ii) a cell proliferation assay, and (iii) a wound healing assay. In FAK inhibition assay, eight library members (5-12) and three aminoindoline derivatives (13, 14, and 2) were identified as promising candidates. Compounds 13 and 2 inhibited the FAK activity by 25-45% at 10 microM. These two lead compounds also showed activity in a wound healing assay. To our knowledge, these aminoindoline-derived small molecules belong to a new family of FAK inhibitors.

Building skeletally diverse architectures on the Indoline Scaffold: the discovery of a chemical probe of focal adhesion kinase signaling networks.

Inspired by bioactive indoline alkaloid natural products, here, we report a divergent synthesis approach that led to skeletally diverse indoline alkaloid-inspired compounds. The natural product-inspired compounds obtained were then subjected to a series of in vitro and cellular assays to examine their properties as modulators of focal adhesion kinase (FAK) activity. This study resulted in the identification of a promising lead inhibitor of FAK (42), which also showed activity in a wound healing and cell invasion assay. The in silico study of the lead compound (42) was also undertaken.

Anaesthetic binding sites for etomidate and propofol on a GABAA receptor model.

Investigating the molecular basis of general anaesthetic activity at the GABA(A) ligand-gated ion channel is challenging due to the wide structural diversity among known general anaesthetics, and the lack of an experimental structure for the GABA(A) protein. In this molecular modelling study, two distinct binding cavities were identified within the beta(2) subunit of the transmembrane domain in a molecular model of the GABA(A) protein. The first, located near the centre of the alpha-helical bundle, contains Asn265 (TM2), which is essential for modulation by etomidate. The second, located near the TM1, TM3 and TM4 segments close to the membrane-extracellular interface, is capped by Met286 (TM3), a residue thought to be involved in the propofol binding site. Potential interactions of etomidate and propofol with other side-chains were also identified.

Molecular modelling of the GABAA ion channel protein.

The GABAA ion channel protein is central to the mechanism of action of general anaesthetics and thus to the phenomenon of human consciousness. A molecular model of the alpha1beta2gamma2 gamma-aminobutyric acid type-A (GABAA) ligand-gated ion channel protein has been constructed. The cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata and the X-ray crystal structure of the acetylcholine binding protein (AChBP) from Lymnaea stagnalis were used as starting templates for comparative modelling. Features of the modelling approach used in the development of this GABAA model include: (1) multiple sequence alignment of members of the Cys-loop superfamily; (2) the design and implementation of a quasi-ab initio loop modelling algorithm; (3) expansion of the transmembrane domain (TMD) ion pore to model the open-state of the GABAA channel; (4) hydrophobicity analysis of the TMD to refine the structure in regions involved in general anaesthetic binding. The final model of the alpha1beta2gamma2 GABAA protein agrees with available experimental data concerning general anaesthetics.

Methods for docking small molecules to macromolecules: a user's perspective. 2. Applications.

A large number of research articles describe novel methodologies of docking and/or scoring methods. An even larger number of publications report the successful use of these methods in the identification of novel hit molecules. What is less documented is the application of docking methods in other areas. We review herein the application of docking methods to not only hit identification but also to de novo design, fragment-based drug discovery, lead optimization, metabolism prediction, off-target binding, selectivity, protein structure prediction and drug-drug interaction.

Methods for docking small molecules to macromolecules: a user's perspective. 1. The theory.

Over the last two decades, computationally docking potential protein ligands (e.g., enzyme inhibitors) has become one of the most widely used strategies in computer aided drug design. While these docking methods were developed, some effort focused on their user-friendliness up to a point where they can be used by non-experts with nearly no training, somewhat hiding the underlying theory. However, basic knowledge is still required to avoid pitfalls and misinterpretations of docking experiments. Over the years, we have collected the common mistakes and necessary information for the proper use of docking programs. In this review, we compiled this data for non-experts in the field. In a first section, we present the theory of docking and scoring approaches as well as their limitations, followed by the most recent progress towards the consideration of protein flexibility, water molecules, metal ions, and covalent drugs. In a second section, we describe what we believe are the necessary steps to ensure optimal docking. More specifically, we present the selection of a docking program, available databases of small molecules, macromolecules and biological data, the necessary steps for the preparation of proteins and small molecules, and finally post docking analysis techniques. In the following sections, we compile the sources of biases and describe docking to nucleic acids.

Finding Inspiration in the Protein Data Bank to Chemically Antagonize Readers of the Histone Code.

Members of the Royal family of proteins are readers of the histone code that contain aromatic cages capable of recognizing specific sequences and lysine methylation states on histone tails. These binding modules play a key role in epigenetic signalling, and are part of a larger group of epigenetic targets that are becoming increasingly attractive for drug discovery. In the current study, pharmacophore representations of the aromatic cages forming the methyl-lysine (Me-Lys) recognition site were used to search the Protein Data Bank (PDB) for ligand binding pockets possessing similar chemical and geometrical features in unrelated proteins. The small molecules bound to these sites were then extracted from the PDB, and clustered based on fragments binding to the aromatic cages. The compounds collected are numerous and structurally diverse, but point to a limited set of preferred chemotypes; these include quaternary ammonium, sulfonium, and primary, secondary and tertiary amine moieties, as well as aromatic, aliphatic or orthogonal rings, and bicyclic systems. The chemical tool-kit identified can be used to design antagonists of the Royal family and related proteins.

Structural biology of human H3K9 methyltransferases.

UNLABELLED: SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.