Discovery of Selective Inhibitors Against EBNA1 via High Throughput In Silico Virtual Screening

Background: Epstein-Barr Virus (EBV) latent infection is associated with several human malignancies and is a causal agent of lymphoproliferative diseases during immunosuppression. While inhibitors of herpesvirus DNA polymerases, like gancyclovir, reduce EBV lytic cycle infection, these treatments have limited efficacy for treating latent infection. EBNA1 is an EBVencoded DNA-binding protein required for viral genome maintenance during latent infection. Methodology: Here, we report the identification of a new class of small molecules that inhibit EBNA1 DNA binding activity. These compounds were identified by virtual screening of 90,000 low molecular mass compounds using computational docking programs with the solved crystal structure of EBNA1. Four structurally related compounds were found to inhibit EBNA1-DNA binding in biochemical assays with purified EBNA1 protein. Compounds had a range of 20–100 mM inhibition of EBNA1 in fluorescence polarization assays and were further validated for inhibition using electrophoresis mobility shift assays. These compounds exhibited no significant inhibition of an unrelated DNA binding protein. Three of these compounds inhibited EBNA1 transcription activation function in cell-based assays and reduced EBV genome copy number when incubated with a Burkitt lymphoma cell line. Conclusions: These experiments provide a proof-of-principle that virtual screening can be used to identify specific inhibitor

Predicting drug metabolism: experiment and/or computation?

Drug metabolism can produce metabolites with physicochemical and pharmacological properties that differ substantially from those of the parent drug, and consequently has important implications for both drug safety and efficacy. To reduce the risk of costly clinical-stage attrition due to the metabolic characteristics of drug candidates, there is a need for efficient and reliable ways to predict drug metabolism in vitro, in silico and in vivo. In this Perspective, we provide an overview of the state of the art of experimental and computational approaches for investigating drug metabolism. We highlight the scope and limitations of these methods, and indicate strategies to harvest the synergies that result from combining measurement and prediction of drug metabolism.This is the accepted manuscript of a paper published in Nature Reviews Drug Discovery (Kirchmair J, Göller AH, Lang D, Kunze J, Testa B, Wilson ID, Glen RC, Schneider G, Nature Reviews Drug Discovery, 2015, 14, 387–404, doi:10.1038/nrd4581). The final version is available at http://dx.doi.org/10.1038/nrd458

Domain-based small molecule binding site annotation

BACKGROUND: Accurate small molecule binding site information for a protein can facilitate studies in drug docking, drug discovery and function prediction, but small molecule binding site protein sequence annotation is sparse. The Small Molecule Interaction Database (SMID), a database of protein domain-small molecule interactions, was created using structural data from the Protein Data Bank (PDB). More importantly it provides a means to predict small molecule binding sites on proteins with a known or unknown structure and unlike prior approaches, removes large numbers of false positive hits arising from transitive alignment errors, non-biologically significant small molecules and crystallographic conditions that overpredict ion binding sites. DESCRIPTION: Using a set of co-crystallized protein-small molecule structures as a starting point, SMID interactions were generated by identifying protein domains that bind to small molecules, using NCBI's Reverse Position Specific BLAST (RPS-BLAST) algorithm. SMID records are available for viewing at . The SMID-BLAST tool provides accurate transitive annotation of small-molecule binding sites for proteins not found in the PDB. Given a protein sequence, SMID-BLAST identifies domains using RPS-BLAST and then lists potential small molecule ligands based on SMID records, as well as their aligned binding sites. A heuristic ligand score is calculated based on E-value, ligand residue identity and domain entropy to assign a level of confidence to hits found. SMID-BLAST predictions were validated against a set of 793 experimental small molecule interactions from the PDB, of which 472 (60%) of predicted interactions identically matched the experimental small molecule and of these, 344 had greater than 80% of the binding site residues correctly identified. Further, we estimate that 45% of predictions which were not observed in the PDB validation set may be true positives. CONCLUSION: By focusing on protein domain-small molecule interactions, SMID is able to cluster similar interactions and detect subtle binding patterns that would not otherwise be obvious. Using SMID-BLAST, small molecule targets can be predicted for any protein sequence, with the only limitation being that the small molecule must exist in the PDB. Validation results and specific examples within illustrate that SMID-BLAST has a high degree of accuracy in terms of predicting both the small molecule ligand and binding site residue positions for a query protein

Generating Triangulated Macromolecular Surfaces by Euclidean Distance Transform

Macromolecular surfaces are fundamental representations of their three-dimensional geometric shape. Accurate calculation of protein surfaces is of critical importance in the protein structural and functional studies including ligand-protein docking and virtual screening. In contrast to analytical or parametric representation of macromolecular surfaces, triangulated mesh surfaces have been proved to be easy to describe, visualize and manipulate by computer programs. Here, we develop a new algorithm of EDTSurf for generating three major macromolecular surfaces of van der Waals surface, solvent-accessible surface and molecular surface, using the technique of fast Euclidean Distance Transform (EDT). The triangulated surfaces are constructed directly from volumetric solids by a Vertex-Connected Marching Cube algorithm that forms triangles from grid points. Compared to the analytical result, the relative error of the surface calculations by EDTSurf is <2–4% depending on the grid resolution, which is 1.5–4 times lower than the methods in the literature; and yet, the algorithm is faster and costs less computer memory than the comparative methods. The improvements in both accuracy and speed of the macromolecular surface determination should make EDTSurf a useful tool for the detailed study of protein docking and structure predictions. Both source code and the executable program of EDTSurf are freely available at http://zhang.bioinformatics.ku.edu/EDTSurf

Nicotinic acetylcholine receptors and their interactions with allosteric ligands

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels (pLGICs) expressed widely throughout the body, including in the peripheral nervous system, central nervous system and at the neuromuscular junction. nAChRs are of therapeutic interest due to their involvement in several pathophysiological conditions. The most widely expressed nAChR subtypes, α7 and α4β2 have attracted a lot of attention and many allosteric ligands have been pharmacologically and chemically characterised for these receptors. However, much remains to be understood about where and how these ligands bind to the receptors and modulate their function. This thesis has focussed on a set of transmembrane binding allosteric modulators for the α7 nAChR and sought to aid understanding of their interactions with their target receptor by building models of nAChRs in physiologically relevant states. A transmembrane error in the only example of a pLGIC structure determined in a native lipid membrane environment, the T. marmorata nAChR, has been corrected through modelling and refinement into previously determined electron cryo-microscopy density maps, in putative closed and open conformations. The refined models offer important reference structures for anyone working in the pLGIC field and here have been used as templates to model the α7 nAChR. A consensus docking protocol has been developed and was utilised in conjunction with the α7 models to predict binding modes for a set of allosteric modulators and provide insight into how they may elicit distinct pharmacology. Based on binding modes of allosteric modulators predicted by the consensus docking protocol, pharmacophores were generated for use in ligand-based virtual screening and allosteric modulators have been uncovered for α7 and α4β2 nAChRs from the existing pharmacopeia. Further to this, novel reactive chemical probes have been developed and synthesised to study the covalent incorporation of allosteric modulators into nAChRs

In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and the farnesoid x receptor (FXR)

In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and farnesoid X receptor (FXR)Montserrat Vaqué Marquès En aquesta tesis es pretén aplicar metodologies computacionals (generació de farmacòfors i docking proteïna lligand) en l'àmbit de la nutigenòmica (ciència que pretén entendre, a nivell molecular, com els nutrients afecten la salut). S'aplicaran metodologies in silico per entendre a nivell molecular com productes naturals com els compostos fenòlics presents en la nostra dieta, poden modular la funció d'una diana comportant un efect en la salut. Aquest efecte es creu que podria ser degut a la seva interacció directa amb proteïnes de vies de senyalització molecular o bé a la modificació indirecta de l'expressió gènica. Donat que el coneixement de l'estructura del complex lligand-receptor és bàsic per entendre el mecanisme d'acció d'aquests lligands s'aplica la metodologia docking per predir l'estructura tridimensional del complex. En aquest sentit, un dels programes de docking és AutoGrid/AutoDock (un dels més citats). No obstant, l'automatització d'AutoGrid/AutoDock no és trivial tan per (a) la cerca virtual en una llibreria de lligands contra un grup de possibles receptors, (b) l'ús de flexibilitat, i (c) realitzar un docking a cegues utilitzant tota la superfície del receptor. Per aquest motiu, es dissenya una interfície gràfica de fàcil ús per utilitzar AutoGrid/AutoDock. Blind Docking Tester (BDT) és una aplicació gràfica que s'executa sobre quatre programes escrits en Fortran i que controla les condicions de les execucions d'AutoGrid i AutoDock. BDT pot ser utilitzat per equips d'investigadors en el camp de la química i de ciències de la vida interessats en dur a terme aquest tipus d'experiments però que no tenen suficient habilitats en programació. En la modulació del metabolisme de la glucosa, treballs in vivio i in vitro en el nostre grup de recerca s'han atribuït els efectes beneficiosos de l'extracte de pinyol de raïm en induir captació de glucosa (punt crític pel manteniment de l'homeostasis de la glucosa). No obstant alguns compostos fenòlics no tenen efecte en la captació de la glucosa, d'altres l'inhibeixen reversiblement. En alguns casos aquesta inhibició és el resultat de la competició dels compostos fenòlics amb ATP pel lloc d'unió de l'ATP de la subunitat catalítica de la fosfatidil inositol 3-kinasa (PI3K). Estudis recents amb inhibidors específics d'isoforma han identificat la p110&#945; (la subunitat catalítica de PI3K&#945;) com la isoforma crucial per la captació de glucosa estimulada per insulina en algunes línies cel·lulars. Els programes computacionals han estat aplicats per tal de correlacionar l'activitat biològica dels compostos fenòlics amb informació estructural per obtenir una relació quantitativa estructura-activitat (3D-QSAR) i obtenir informació dels requeriments estructura-lligand per augmentar l'afinitat i/o selectivitat amb la diana (proteïna). Tot hi haver-se demostrat que l'adició d'extractes de compostos fenòlics en l'aliment pot tenir en general un benefici per la salut, s'ha de tenir en compte que l'estudi 3D-QSAR (construït a partir d'inhibidors sintètics de p110&#945;) prediu que algunes d'aquestes molècules poden agreujar la resistència a la insulina en individus susceptibles dificultant la capatació de glucosa en múscul i teixit adipós i, per tant, produir un efecte secundari indesitjat. Resultats en el nostre grup de recerca han demostrat que compostos fenòlics presents en extractes de llavor de raïm incrementen l'activitat del receptor "farnesoid x receptor" (FXR) de manera dosi depenent quan el lligand natural de FXR (CDCA) és present. Les metodologies in silico, docking i 3D-QSAR, han estat aplicades juntament amb dades biològiques d'agonistes no esteroidals de FXR que s'uneixen a un lloc d'unió proper però diferent al lligand esteroidal 6CDCA. Els resultats han mostrat que els compostos fenòlics no són capaços d'activar FXR per ells mateixos però poden afegir noves interaccions que estabilitzarien la conformació activa de FXR en presència del lligand natural CDCA. Els compostos fenòlics podrien induir canvis conformacionals específics que augmentarien l'activitat de FXR. In silico studies of the effect of phenolic compounds from grape seed extracts on the activity of phosphoinositide 3-kinase (PI3K) and farnesoid X receptor (FXR)Montserrat Vaqué Marquès This thesis was written with the aim of applying computational methods that have already been developed for molecular design and simulation (i.e. pharmacophore generation and protein-ligand docking) to nutrigenomics. So, in silico tools that are routinely used by the pharmaceutical industry to develop drugs have been used to understand, at the molecular level, how natural products such as phenolic compounds (i.e. molecules that are commonly found in fruits and vegetables) can improve health and prevent diseases. Therefore, we first focused on predicting the structure of protein-ligand complexes. The docking algorithms can use the individual structures from receptor and ligand to predict (1) whether they can form a complex and (2) if so, the structure of the resulting complex. This prediction can be made, for instance, with AutoGrid/AutoDock, the most cited docking software in the literature. The automation of AutoGrid/AutoDock is not trivial for tasks such as (1) the virtual screening of a library of ligands against a set of possible receptors; (2) the use of receptor flexibility and (3) making a blind-docking experiment with the whole receptor surface. Therefore, in order to circumvent these limitations, we have designed BDT (i.e. blind-docking tester; http://www.quimica.urv.cat/~pujadas/BDT), an easy-to-use graphic interface for using AutoGrid/AutoDock. BDT is a Tcl/Tk graphic front-end application that runs on top of four Fortran programs and which controls the conditions of the AutoGrid and AutoDock runs. As far as the modulation of the glucose metabolism is concerned, several in vivo and in vitro results obtained by our group have shown that grape seed procyanidin extracts (GSPE) stimulate glucose uptake in 3T3-L1 adipocytes and thus help to maintain their glucose homeostasis. In contrast, it is also well known that although some phenolic compounds do not affect glucose uptake, others reversibly inhibit it in several cell lines. Moreover, for at least some of these phenolic compounds, this inhibition is the result of their competition with ATP for the ATP-binding site in p110&#945; (i.e. the &#945; isoform of the catalytic subunit of phosphoinositide 3-kinase or PI3K&#945;). Furthermore, recent studies with isoform-specific inhibitors have identified p110&#945; as the crucial isoform for insulin-stimulated glucose-uptake in some cell lines. Therefore, although it has been proved that the addition of phenolic compound extracts to food can have an overall benefit on health, it should be taken into account that some of these molecules may exacerbate insulin resistance in susceptible individuals via impaired glucose uptake in muscle and adipose tissues and, therefore, produce an undesirable side effect. In this context, we have applied computational approaches (i.e. protein-ligand docking and 3D-QSAR) to predict the IC50 (i.e. the concentration that reduces the p110&#945; activity to 50%). Our results agree with previous experimental results and predict that some compounds are potential inhibitors of this enzyme. Recent results in our research group have demonstrated that the phenolic compounds in GSPE increase the activity of the farnesoid X receptor (i.e. FXR) in a dose-dependent way when the natural ligand of FXR (i.e. CDCA) is also present. The phenolic compounds might induce specific conformational changes that increase FXR activity and then contribute to cardioprotection through mechanisms that are independent of their intrinsic antioxidant capacities but that involve direct interaction with FXR to modulate gene expression. Taking into account this hypothesis a 3D-QSAR analysis was made in an attempt to understand how phenolic compounds activate FXR. So, our results explain why phenolic compounds cannot activate FXR by themselves and how they can add new interactions to stabilize the active conformation of FXR when its natural ligand (i.e. CDCA) is present. Therefore, we proposed a mechanism of FXR activation by dietary phenolic compounds in which they may enhance bile acid-bound FXR activity

Drug Design, Docking Studies, Synthesis and In-Vitro Evaluation of Certain Novel Isoxazole Incorporated Coumarin Derivatives as Potent α - Amylase Inhibitors

The present work was focused on the designing and synthesis of novel isoxazole derivatives incorporated with coumarin moiety having α-amylase enzyme inhibitory activity. For this, following approach has been adopted. PHASE I: LITERATURE REVIEW Literature survey showed that coumarin is a drug like scaffold and is a core skeleton for the active sites involved in enzyme inhibiton in Type 2 diabetes. It also revealed that isoxazole possess enzyme inhibition for Type 2 diabetes. PHASE II: DRUG DESIGN APPROACH It involves the following stages: Stage 1: Identification of target α-amylase was selected as the target enzyme as its inhibition will prevent carbohydrate hydrolysis. The target enzyme (1UA7) was downloaded from RCBs Protein Databank. Stage 2: Lead optimization Lead optimisation was done by computation of drug likness score. Isoxazole derivatives of coumarin were the desired compounds with good molecular properties and bioactivity score, ie., the compounds A1, A3, A4 and K1, K3, K5 showed good scores. PHASE III: SYNTHESIS AND PHYSICAL CHARACTERIZATION A) Synthesis of the designed compounds In this work, ten new compounds are designed in which Six different isoxazole derivatives were synthesized by using three aromatic aldehydes and three aromatic ketones with coumarin moiety. The first step involved the synthesis of 6- acetyl 7- hydroxy 4- methyl coumarin by acetylation. Chalcones were prepared from 6- acetyl 7- hydroxy 4-methyl coumarin by using different aromatic aldehydes and aromatic ketones. Finally, the chalcones were reacted with hydroxylamine hydrochloride to form isoxazoles. B) Physical characterization Melting point of all the newly synthesised compounds was determined by capillary tube method. Rf values were determined by fixing various suitable solvent system on precoated silicagel- G plates. PHASE IV: SPECTRAL STUDIES The structure of the synthesised compounds was established by using IR, 1H NMR, and Mass spectral data. PHASE V: EVALUATION OF BIOLOGICAL ACTIVITIES Evaluation of α-amylase inhibitory activity All the newly sysnthesised compounds were screened for in vitro α-amylase inhibitory activity. All compounds showed significant activity in inhibition of the α-amylase enzyme. Comparatively A1 and K1 showed good % of inhibition activity, while A3, A4, K3, K5 showed moderate activity. CONCLUSION • The present study establishes that computational tools help in minimizing the tedious process of drug discovery and delivers new drug candidate more quickly. • α-amylase enzyme was selected as target and virtual screening made selection of lead compounds easier and coumarin was selected as lead molecule. • From among the ten docked molecules, six molecules with good Binding affinity were chosen for further laboratory synthesis. Drug likeness was predicted insilico before proceeding for synthesis. • Compounds A1, A3, A4 and K1, K3, K5 were found to have significant binding score against target enzyme α-amylase. compared to standard drug Acarbose. The selected derivatives were planned for synthesis. • The proposed Six compounds of isoxazole derivatives with coumarin ring system were synthesised in good yield using the developed schemes. • All the reactions were monitored by TLC one spot technique and the structures of the synthesised compounds were confirmed by IR, 1H NMR, Mass spectra. • Compounds A1, K1 and K3 exhibited maximum α-amylase inhibitory activity. Among the synthesized compounds, A1 and K1 can be taken for further studies as the lead molecule and acute toxicity studies are to be done on these promising compounds

Identification and Characterization of Small Molecule Antagonists of the Human Papillomavirus Oncoproteins

Human papillomavirus (HPV) is the most common sexually transmitted pathogen, and is associated with almost all cervical cancers, about 20 percent of head and neck cancers and an array of other cancers. Current HPV vaccines offer preventative care, however, long-term benefits are unknown and the vaccines are not effective for therapy. High risk forms of HPV mediate cell transformation via two viral oncoproteins, E6 and E7, which lead to cell cycle disruption and cancer. E7 deregulates the cell cycle and abrogates other pathways mediated by the retinoblastoma protein, pRb. pRb is essential for regulating many cellular activities through its binding and inhibition of E2F transcription activators, and pRb inactivation leads to many cancers. pRb activity can be perturbed by viral oncoproteins, including HPV, that contain an LxCxE motif. E6 mediates cell transformation, in part, by forming a complex with the cellular E3 ligase E6-Association Protein (E6AP) to target p53 for degradation. To tackle these problems, we performed two high throughput solution screens of ~88,000 compounds to search for (1) compounds that inhibit the ability of HPV-E7 to disrupt pRb/E2F complexes and (2) small molecule inhibitors of the E6/E6AP interaction. The HPV-E7 screen led to the identification of thiadiazolidinedione compounds that bind to pRb with mid-high nanomolar affinity, are competitive with the binding of viral oncoproteins containing an LxCxE motif and are selectively cytotoxic in HPV positive cells alone and in mice. The HPV-E6 screen resulted in 30 inhibitors with in vitro IC50 values in the low-micromolar to mid-nanomolar range. Six of these compounds were shown to associate with HPV-E6, block p53 degradation and promote apoptosis in high risk HPV positive cells. These E6 and E7 inhibitors provide promising scaffolds for the development of therapies to treat HPV-mediated pathologies. An in silico screen was also done to identify small molecules that bind directly to E7 and prevent its interaction with E2F. This resulted in two compounds that prevented E7-mediated displacement of E2F from pRb, however, the IC50 values were very high, and not pursued further. The interaction between E7 and another cellular target, p300, was characterized by various biochemical and biophysical techniques. The interaction seemed very weak, different from the adenovirus E1A interaction with p300. This suggested that the E1A/p300 interaction would be easier to characterize

Prédictions de complexes protéine-ligand par arrimage moléculaire : développement et applications

Les protéines sont des entités intrinsèquement dynamiques et de nombreuses études ont démontré l’importance de cette propriété à leurs fonctions. Plus particulièrement, la flexibilité protéique est essentielle dans le processus de reconnaissance moléculaire. Lors de tels évènements, les protéines peuvent subir des changements conformationnels mineurs (déplacement de chaînes latérales des acides aminés), majeurs (déplacement de domaines entiers de la protéine) et/ou même se replier. Mes travaux de thèse ont permis de démontrer que de tels réarrangements mineurs sont fréquents et ont aussi permis d’élucider certaines causes potentielles physiques et chimiques. De plus, mes travaux ont démontré l’importance de considérer la flexibilité des chaînes latérales lors de simulations de tels évènements de reconnaissance moléculaire. Plusieurs méthodes computationnelles, dont la dynamique moléculaire et l’arrimage moléculaire, peuvent être utilisées pour prédire la liaison d’un ligand à sa cible. D’un côté, la dynamique moléculaire permet de considérer la flexibilité protéique à toute échelle, mais nécessite un pouvoir computationnel énorme. D’un autre côté, l’arrimage moléculaire restreint le nombre de degrés de liberté considérés, entre autres imposés par la flexibilité protéique. Mes travaux de thèse, en ce qui a attrait au développement de la méthode d’arrimage moléculaire appelée FlexAID, ont permis d’inclure une certaine flexibilité protéique intrinsèque limitant ainsi le nombre de degrés de liberté requis, tout en offrant la possibilité d’ajouter des degrés de liberté supplémentaire pour les mouvements de plus grande envergure ne pouvant être accommodés par cette plasticité protéique. De plus, mes travaux démontrent que FlexAID est compétitive aux autres méthodes dans le domaine et obtient de meilleures performances dans le scénario où les conformations des protéines sous la forme liée sont inconnues. Dans un autre ordre d’idées, les nombreuses simplifications introduites par un logiciel d’arrimage lui permettent d’être une méthode rapide et applicable à la découverte de nouvelles molécules ayant un effet thérapeutique potentiel. Lorsqu’une méthode de repointage est utilisée, les résultats de FlexAID en enrichissement de composés se rapprochent des performances d’autres logiciels couramment utilisés lors de criblage virtuel. Mes travaux portant sur le système biologique de la Matriptase-2 montrent que la méthode FlexAID peut être utilisée à la découverte de nouvelles petites molécules