148 research outputs found

    Reviewing Ligand-Based Rational Drug Design: The Search for an ATP Synthase Inhibitor

    Get PDF
    Following major advances in the field of medicinal chemistry, novel drugs can now be designed systematically, instead of relying on old trial and error approaches. Current drug design strategies can be classified as being either ligand- or structure-based depending on the design process. In this paper, by describing the search for an ATP synthase inhibitor, we review two frequently used approaches in ligand-based drug design: The pharmacophore model and the quantitative structure-activity relationship (QSAR) method. Moreover, since ATP synthase ligands are potentially useful drugs in cancer therapy, pharmacophore models were constructed to pave the way for novel inhibitor designs

    Computational Studies and Design of PPARγ and GLUT1 Inhibitors

    Get PDF
    The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor of the nuclear receptor superfamily that controls the expression of a variety of genes involved in fatty acid metabolism, adipogenesis, and insulin sensitivity. PPARγ is a target for insulin-sensitizing drugs, and it plays a significant function in prostate cancer. PPARγ antagonists have anti-proliferative effects in a broad range of hematopoietic and epithelial cell lines. The ligand binding domain (LBD) of PPARγ is large and has orthosteric and allosteric binding sites. Several PPARγ-ligand co-crystal structures show two bound molecules, one to the orthosteric pocket and a second to the allosteric site. We ran docking studies against the orthosteric and allosteric binding sites to determine the most favorable binding site for PPARγ antagonists. We found that Glide docking performed well in predicting PPARγ antagonist binding affinities, and that the allosteric site of PPARγ was the most favorable binding site for antagonists. We also investigated PPARγ ligand-protein interactions to better define a structural basis for the binding selectivity of PPARγ antagonists. We found that Phe282, Arg288, and Lys367 interact with antagonists more than with agonists and partial agonists. We then identified several potential PPARγ antagonists by virtual screening of the PPARγ allosteric pocket. The glucose transporter 1 (GLUT1) is a uniporter protein that facilitates the transport of glucose across the plasma membranes of mammalian cells. As GLUT1 is overexpressed in numerous tumors, this transporter is a potential target for cancer treatment. GLUT1 works through conformational switching from an outward-open (OOP) to an inward-open (IOP) conformation passing through an occluded conformation. We sought to determine which conformation is favored for ligand binding by molecular docking studies of known GLUT1 inhibitors with the different GLUT1 conformers. Our data revealed that the IOP is the preferred conformation and that residues Phe291, Phe379, Glu380, Trp388, and Trp412 may play important roles in ligand binding to GLUT1. To identify new chemotypes targeting GLUT1, we built a pharmacophore model and searched against an NCI compound database. Sixteen hit molecules with good docking scores were screened for GLUT1 inhibition and anti-proliferative activities. From these, we identified four compounds that inhibited cell viability in an HCT116 colon cancer cell line. We also determined that one of these, NSC295720, inhibited GLUT1 in a biochemical assay

    Exploring Allosteric Modulation of the Nuclear Receptor RORγt from a Drug Discovery Perspective

    Get PDF

    Development of a pseudoreceptor model for virtual screening

    Get PDF
    Im Rahmen dieser Arbeit wurde die Eignung von Pseudorezeptoren im virtuellen Screening untersucht. Hierzu wurde nach intensiver Auseinandersetzung mit bisher bekannten Konzepten ein neues Computerprogramm zur automatischen Konstruktion von Pseudorezeptormodellen entwickelt. Das Ziel von Pseudorezeptoren ist die Konstruktion eines alternativen, artifiziellen Wirtssystems aus bekannten Liganden eines Zielproteins, dessen dreidimensionale Struktur unbekannt ist. Der generierte Pseudorezeptor ist zu verstehen als die Menge aller Pseudoatome, die um die Ausgangssubstanz(en) projiziert werden. Bei multiplen Referenzliganden wird eine Gewichtung der Pseudoatome durchgeführt. Zudem wird ausschließlich von Distanz- und Winkelparametern Gebrauch gemacht, die aus Untersuchungen von Kokristall-strukturen gewonnenen wurden. Eine abschließende Kodierung generierter Pseudorezeptoren als 90-dimensionalen Korrelationsvektor wurde zum virtuellen Screening eingesetzt. In zwei retrospektiven Fallbeispielen wird gezeigt, dass die generierten Pseudorezeptoren für COX-2 und PPARα mit den realen Zuständen ihrer kokristallisierten Bindetaschen in den PDB Einträge 6cox und 2p54 kompatibel sind. Im retrospektiven virtuellen Screening in der Wirkstoffdatenbank COBRA (8.311 Moleküle) nach COX-2 Inhibitoren (136 Aktive) konnte eine Anreicherung der aktiven Strukturen in den ersten zwei Perzentilen gezeigt werden (54% der Aktiven). Zudem konnten 80% der aktiven Moleküle bereits nach Vorhersage von 10% Falsch-Positiven gefunden werden. Im Falle des retrospektiven Screenings nach 94 PPAR Liganden konnten 30% der aktiven Moleküle nach der Vorhersage von 10% Falsch-Positiven entdeckt. Nach 20% Falsch-Positiver wurden 46% der PPAR Liganden wieder gefunden. Weiterhin konnte mit den ligandenbasierten Informationen eines H4 Pseudorezeptors eine Justierung einer potentiellen Bindetasche des Histamin H4 Rezeptors aus einer molekularen Dynamiksimulation vorgenommen werden. Schließlich wurde in einem prospektiven virtuellen Screening nach Histamin H4 Liganden mit einem Pseudorezeptor zwei Strukturen mit unterschiedlichem Grundgerüst und einem Ki ~ 30 µM identifiziert.In this thesis, the suitability of pseudoreceptors for virtual screening applications was analyzed. An automated pseudoreceptor construction program was developed after known design principles had been thoroughly studied and compared. The aim of pseudoreceptor modelling is the construction of an alternative host system for known ligands of a given target protein in the absence of three-dimensional structure information. The constructed pseudoreceptor is represented as the sum of all pseudoatoms, which are projected around reference ligand(s). A weighting scheme is introduced, when pseudoreceptors are generated from multiple reference ligands. For pseudoatom placing distance and angle parameters from a survey of known co-crystal structures were used. For virtual screening pseudoreceptors were encoded as correlation vectors. It is demonstrated that the generated pseudoreceptors match with their respective co- crystallized binding pockets, taking COX-2 and PPAR-alpha as an example (PDB entries 6cox and 2p54). In a retrospective virtual screening in the drug collection COBRA (8,311 molecules) for COX-2 inhibitors (136 actives) high enrichment of ligands in the first two percentiles was yielded (54% of the actives). 80% of all active compounds were found after the prediction of only 10% false-positives. In a retrospective screening study for 94 PPAR ligands, 30% of the actives were found together with 10% false-positives. After the prediction of 20% false-positives, 46% of all PPAR ligands could be found. In addition, a putative binding pocket of the histamine H4 receptor from a molecular dynamics simulation could be adjusted using ligand-based information of a H4 pseudoreceptor. Finally, two micromolar ligands with different scaffolds were identified with a Ki ~ 30 µM by a pseudoreceptor-based prospective virtual screening for novel H4 ligands

    Part I. The Development of Non-secosteroidal Vitamin D Receptor Modulators Part II. The Development of a Universal GTP-ase Assay

    Get PDF
    The vitamin D receptor is a nuclear hormone receptor that regulates cell proliferation, cell differentiation, calcium homeostasis and immunomodulation. The receptor is activated by the vitamin D metabolite, 1,25-dihydroxyvitamin D3, which induces a cascade of events including the recruitment of coactivators that activate transcription of specific VDR target genes. Thousands of VDR agonists have been synthesized based on the secosteroid scaffold of 1,25-dihydroxyvitamin D3. However, most of these ligands are metabolically unstable, have sub-optimal drug-like properties, and induce hypercalcemia in vivo. The limited numbers of VDR antagonists reported bear the same secosteroid scaffold and thus exhibit similar problems encountered by VDR agonists. VDR has been implicated with many diseases including cancer, allergies, sarcoidosis and autoimmune diseases like Crohn’s disease. Therefore, the synthesis and biochemical evaluation of novel, non-secosteroidal modulators for VDR were developed and reported herein. First, VDR inhibitors were rationally designed to directly target the interactions between VDR and coactivator SRC2. A fluorescence polarization-based assay evaluated the binding of these molecules. Next, a high throughput screening campaign with the NIH chemical and genomics center (NCGC) identified GW0742 as a novel VDR anatagonist. Originally developed by GlaxoSmithKline as a selective PPARδ agonist, GW0742 was used as a scaffold for the synthesis of VDR inhibitors with decoupled activity towards PPARδ. Biochemical, cell-based, solubility and permeability assays determined drug-like properties of over 100 GW0742 analogs. Finally, secondary bile acids, which are known to bind VDR and modulate transcription without inducing hypercalcemia, lead to a study of phase 1 and phase 2 metabolites of lithocholic acid. In addition to biochemical and cell-based assays, a semi-quantitative real time polymerase chain reaction was used to confirm the ability of lithocholic acid derivatives to induce the transcription of VDR target genes. GTPases act as a molecular switches in which their “on” and “off” functions are triggered by the binding and hydrolysis of GTP. Due to their relationship to many diseases, numerous GTPase targeting drugs have been developed. One third of all drugs targeting proteins are either interacting with kinases (22% of drugs) or GTPases (15% of drugs). The growing interest in GTPase targeting drugs has promoted the development of assays that can efficiently test these compounds in a high throughput and inexpensive way. AviMed Pharmaceuticals, LLC, a local company founded by Dr. Daniel Sem, pursued the development of a universal kinase/GTPase assay kit that would be affordable and commercially available for industry and research labs to test potential drug candidates. The assay designed was based on previous research conducted by the founder and relies on a beta thiol substituted ATP (GTP for GTPases) that would be enzymatically hydrolyzed to produce ADP (GDP). The exposure of the thiol makes it nucleophilic and reactive towards thiol-sensitive fluorescent or calorimetric reagents such as Thiofluor 623. Herein, we report the synthesis of the assay reagents and the preliminary development of a universal, inexpensive, sensitive GTPase assay kit that directly detects the GTPβ-S hydrolysis product, GDPβ-Se

    Study of the mechanism of action of bioactive plants tarpenoids

    Get PDF
    2014-2015Natural products are small-molecule secondary metabolites displaying considerable structural complexity and “privileged scaffolds”. They are able to bind several endogenous targets eliciting biological effects as chemical weapons or to convey information from one organism to another. Nowadays, medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets. Therefore, the primary purpose of this PhD thesis has been a comprehensive characterization of the interactome profile and then the molecular mechanism of action of bioactive natural molecules. Achieving this in an effective, unbiased and efficient manner subsists as a significant challenge for the new era in drug discovery and optimization. Indeed, the full understanding of the mechanism of action of natural molecules could lead to a number of advantages: first of all, exploit their full therapeutic potential, the identification of side effects or toxicity, or the ability to set up target-based assays and to allow structure activity relationships studies to guide medicinal chemistry efforts towards lead optimization. In my research project, the attention was paid on ent-kaurane diterpenes, a class of natural terpenoids with a great structural variability and a wide spectrum of biological activities. Firstly, I focused on the determination of the interactome of a semi synthetic compound 15-ketoatractyligenin methyl ester. This compound has been previously reported to possess high antiproliferative activity against several solid tumor-derived cell lines. In this regard, I decided to investigate the mechanism of action of this actratylignin derivative researching first of all its molecular targets, responsible for the biological activity. In order to achieve this goal, I used a chemical proteomic approach first. This study led to the identification of PPARγ as the main cellular partner of this compound; achieved results were supported and validated through different biological assays. Subsequently, I studied another diterpene: oridonin. This molecule has been shown to have multiple biological activities. Among them, the anticancer activity has been repeatedly reported by many research groups. With the aim of expanding and validate our knowledge about this molecule, also seen the limitations of the fishing for partners method, I decided to use two orthogonal compound-centric proteomics approaches to define the possible protein target(s) of oridonin. Using this strategy HSP70 and nucleolin were identified. Therefore, several in vitro and in cell tests have been performed to validate the interaction of oridonin with these proteins, and to evaluate its effect on their activity. Some of these tests were developed and optimized during my period of research abroad at the Massachusset General Hospital- Center for System Biology -Harvard Medical School; in that twelve months period I expanded my knowledge into the techniques useful for the study of the mechanism of action of a small molecule, also applying experimental methods complementary to proteomics and focusing on the use of high-resolution intravital microscopy imaging for drug pharmacology. [edited by Author]XIV n.s

    Evaluation of in silico and in vitro screening methods for characterising endocrine disrupting chemical hazards

    Get PDF
    Anthropogenic activities have drastically altered chemical exposure, with traces of synthetic chemicals detected ubiquitously in the environment. Many of these chemicals are thought to perturb endocrine function, leading to declines in reproductive health and fertility, and increases in the incidence of cancer, metabolic disorders and diabetes. There are over 90 million unique chemicals registered under the Chemical Abstracts Service (CAS), of which only 308,000 were subject to inventory and/or regulation, in September 2013. However, as a specific aim of the EU REACH regulations, the UK is obliged to reduce the chemical safety initiatives reliance on in vivo apical endpoints, promoting the development and validation of alternative mechanistic methods. The human health cost of endocrine disrupting chemical (EDC) exposure in the EU, has been estimated at €31 billion per annum. In light of the EU incentives, this study aims to evaluate current in silico and in vitro tools for EDC screening and hazard characterisation; testing the hypothesis that in silico virtual screening accurately predicts in vitro mechanistic assays. Nuclear receptor binding interactions are the current focus of in silico and in vitro tools to predict EDC mechanisms. To the author’s knowledge, no single study has quantitatively assessed the relationship between in silico nuclear receptor binding and in vitro mechanistic assays, in a comprehensive manner. Tripos ® SYBYL software was used to develop 3D-molecular models of nuclear receptor binding domains. The ligand binding pockets of estrogen (ERα and ERβ), androgen (AR), progesterone (PR) and peroxisome proliferator activated (PPARγ) receptors were successfully modelled from X-ray crystal structures. A database of putative-EDC ligands (n= 378), were computationally ‘docked’ to the pseudo-molecular targets, as a virtual screen for nuclear receptor activity. Relative to in vitro assays, the in silico screen demonstrated a sensitivity of 94.5%. The SYBYL Surflex-Dock method surpassed the OECD Toolbox ER-Profiler, DfW and binary classification models, in correctly identifying endocrine active substances (EAS). Aiming to evaluate the current in vitro tools for endocrine MoA, standardised ERα transactivation (HeLa9903), stably transfected AR transactivation (HeLa4-11) assays in addition to novel transiently transfected reporter gene assays, predicted the mechanism and potency of test substances prioritised from the in silico results (n = 10 potential-EDCs and 10 hormone controls). In conclusion, in silico SYBYL molecular modelling and Surflex-Dock virtual screening sensitively predicted the binding of ERα/β, AR, PR and PPARγ potential EDCs, and was identified as a potentially useful regulatory tool, to support EAS hazard identification
    corecore