79 research outputs found

    In Silico

    Get PDF

    Integral membrane pyrophosphatases : A novel drug target for human pathogens?

    Get PDF
    Membrane-integral pyrophosphatases (mPPases) are found in several human pathogens, including Plasmodium species, the protozoan parasites that cause malaria. These enzymes hydrolyze pyrophosphate and couple this to the pumping of ions (H+ and/or Na+) across a membrane to generate an electrochemical gradient. mPPases play an important role in stress tolerance in plants, protozoan parasites, and bacteria. The solved structures of mPPases from Vigna radiata and Thermotoga maritima open the possibility of using structure-based drug design to generate novel molecules or repurpose known molecules against this enzyme. Here, we review the current state of knowledge regarding mPPases, focusing on their structure, the proposed mechanism of action, and their role in human pathogens. We also summarize different methodologies in structure-based drug design and propose an example region on the mPPase structure that can be exploited by these structure-based methods for drug targeting. Since mPPases are not found in animals and humans, this enzyme is a promising potential drug target against livestock and human pathogens. © 2016, Adrian Goldman, et al.Peer reviewe

    Integrating protein annotations for the in silico prioritization of putative drug target proteins in malaria

    Get PDF
    Current anti-malarial methods have been effective in reducing the number of malarial cases. However, these methods do not completely block the transmission of the parasite. Research has shown that repeated use of the current anti-malarial drugs, which include artemisinin-based drug combinations, might be toxic to humans. There have also been reports of an emergence of artemisinin-resistant parasites. Finding anti-malarial drugs through the drug discovery process takes a long time and failure results in a great financial loss. The failure of drug discovery projects can be partly attributed to the improper selection of drug targets. There is thus a need for an eff ective way of identifying and validating new potential malaria drug targets for entry into the drug discovery process. The availability of the genome sequences for the Plasmodium parasite, human host and the Anopheles mosquito vector has facilitated post-genomic studies on malaria. Proper utilizationof this data, in combination with computational biology and bioinformatics techniques, could aid in the in silico prioritization of drug targets. This study was aimed at extensively annotating the protein sequences from the Plasmodium parasites, H. sapiens and A. gambiae with data from di fferent online databases in order to create a resource for the prioritization of drug targets in malaria. Essentiality, assay feasibility, resistance, toxicity, structural information and druggability were the main target selection criteria which were used to collect data for protein annotations. The data was used to populate the Discovery resource (http://malport. bi.up.ac.za/) for the in silico prioritization of potential drug targets. A new version of the Discovery system, Discovery 2.0 (http://discovery.bi.up.ac.za/), has been developed using Java. The system contains new and automatically updated data as well as improved functionalities. The new data in Discovery 2.0 includes UniProt accessions, gene ontology annotations from the UniProt-GOA project, pathways from Reactome and Malaria Parasite Metabolic Pathways databases, protein-protein interactions data from. IntAct as well as druggability data from the DrugEBIlity resource hosted by ChEMBL. Users can access the data by searching with a protein identi er, UniProt accession, protein name or through the advanced search which lets users filter protein sequences based on different protein properties. The results are organized in a tabbed environment, with each tab displaying different protein annotation data. A sample investigation using a previously proposed malarial target, S-adenosyl-Lhomocysteine hydrolase, was carried out to demonstrate the diff erent categories of data available in Discovery 2.0 as well as to test if the available data is su fficient for assessment and prioritization of drug targets. The study showed that using the annotation data in Discovery 2.0, a protein can be assessed, in a species comparative manner, on the potential of being a drug target based on the selection criteria mentioned here. However, supporting data from literature is also needed to further validate the findings.Dissertation (MSc)--University of Pretoria, 2012.Biochemistryunrestricte

    Exploiting multiple hit-identification strategies to identify novel inhibitors of the anti-infective target DXPS

    Get PDF
    The coronavirus pandemic has raised awareness for infectious diseases, which also put a spotlight on the fight against anti-microbial resistance. A promising new target in this fight is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). Although it has been known for the past few decades, only a few promising inhibitors have been identified so far. For this thesis, multiple hit-identification strategies were pursued with a special focus on Mycobacterium tuberculosis and Plasmodium falciparum DXPS to find new inhibitors. For this thesis a focused fragment library was screened against DXPS in different biophysical assay, in collaboration with the company Atomwise, a virtual screening was performed and three previously identified hit classes were investigated in phenotypic assays against P. falciparum and synthetically optimized. In summary, this thesis has contributed to the identification of several new binders and inhibitors that have promising properties to continue their optimization into leads for drug development.Die Corona Pandemie hat dafĂŒr gesorgt, dass Infektionskrankheiten und damit auch der Kampf gegen antibiotikaresistente Keime ins Bewusstsein der Öffentlichkeit gerĂŒckt sind. Ein neues Target in diesem Kampf ist die 1-Deoxy-D-xylulose-5-phosphat Synthase (DXPS). Das Enzym ist bereits seit einigen Jahrzehnten bekannt, aber bisher wurden nur wenige Inhibitoren gefunden. In dieser Arbeit wurden verschiedene Hit-Identifikationsstrategien genutzt, um neue Inhibitoren gegen Mycobacterium tuberculosis und Plasmodium falciparum DXPS zu finden. DafĂŒr wurde eine fokussierte Fragmentbibliothek gegen DXPS in verschiedenen biophysikalischen Assays untersucht, ein HPLC-MS/MS-basierter DXPS Assay wurde etabliert, in Kooperation mit der Firma Atomwise wurde ein virtuelles Screening an DXPS durchgefĂŒhrt und drei bereits bekannte Hit-Klassen wurden im Rahmen dieser Arbeit in phĂ€notypischen Assays gegen P. falciparum getestet und synthetisch optimiert. Zusammengefasst hat diese Arbeit zur Identifikation mehrerer Binder und Inhibitoren beigetragen, die vielversprechende Eigenschaften aufweisen und weiter zu Lead-Verbindungen optimiert und somit fĂŒr die Medikamentenentwicklung genutzt werden können

    Lead optimization for new antimalarials and Successful lead identification for metalloproteinases: A Fragment-based approach Using Virtual Screening

    Get PDF
    Lead optimization for new antimalarials and Successful lead identification for metalloproteinases: A Fragment-based approach Using Virtual Screening Computer-aided drug design is an essential part of the modern medicinal chemistry, and has led to the acceleration of many projects. The herein described thesis presents examples for its application in the field of lead optimization and lead identification for three metalloproteins. DOXP-reductoisomerase (DXR) is a key enzyme of the mevalonate independent isoprenoid biosynthesis. Structure-activity relationships for 43 DXR inhibitors are established, derived from protein-based docking, ligand-based 3D QSAR and a combination of both approaches as realized by AFMoC. As part of an effort to optimize the properties of the established inhibitor Fosmidomycin, analogues have been synthesized and tested to gain further insights into the primary determinants of structural affinity. Unfortunately, these structures still leave the active Fosmidomycin conformation and detailed reaction mechanism undetermined. This fact, together with the small inhibitor data set provides a major challenge for presently available docking programs and 3D QSAR tools. Using the recently developed protein tailored scoring protocol AFMoC precise prediction of binding affinities for related ligands as well as the capability to estimate the affinities of structurally distinct inhibitors has been achieved. Farnesyltransferase is a zinc-metallo enzyme that catalyzes the posttranslational modification of numerous proteins involved in intracellular signal transduction. The development of farnesyltransferase inhibitors is directed towards the so-called non-thiol inhibitors because of adverse drug effects connected to free thiols. A first step on the way to non-thiol farnesyltransferase inhibitors was the development of an CAAX-benzophenone peptidomimetic based on a pharmacophore model. On its basis bisubstrate analogues were developed as one class of non-thiol farnesyltransferase inhibitors. In further studies two aryl binding and two distinct specificity sites were postulated. Flexible docking of model compounds was applied to investigate the sub-pockets and design highly active non-thiol farnesyltransferase inhibitor. In addition to affinity, special attention was paid towards in vivo activity and species specificity. The second part of this thesis describes a possible strategy for computer-aided lead discovery. Assembling a complex ligand from simple fragments has recently been introduced as an alternative to traditional HTS. While frequently applied experimentally, only a few examples are known for computational fragment-based approaches. Mostly, computational tools are applied to compile the libraries and to finally assess the assembled ligands. Using the metalloproteinase thermolysin (TLN) as a model target, a computational fragment-based screening protocol has been established. Starting with a data set of commercially available chemical compounds, a fragment library has been compiled considering (1) fragment likeness and (2) similarity to known drugs. The library is screened for target specificity, resulting in 112 fragments to target the zinc binding area and 75 fragments targeting the hydrophobic specificity pocket of the enzyme. After analyzing the performance of multiple docking programs and scoring functions forand the most 14 candidates are selected for further analysis. Soaking experiments were performed for reference fragment to derive a general applicable crystallization protocol for TLN and subsequently for new protein-fragment complex structures. 3-Methylsaspirin could be determined to bind to TLN. Additional studies addressed a retrospective performance analysis of the applied scoring functions and modification on the screening hit. Curios about the differences of aspirin and 3-methylaspirin, 3-chloroaspirin has been synthesized and affinities could be determined to be 2.42 mM; 1.73 mM und 522 ÎŒM respectively. The results of the thesis show, that computer aided drug design approaches could successfully support projects in lead optimization and lead identification. fragments in general, the fragments derived from the screening are docke

    Application of computer-aided drug design for identification of P. falciparum inhibitors

    Get PDF
    Malaria is a millennia-old disease with the first recorded cases dating back to 2700 BC found in Chinese medical records, and later in other civilizations. It has claimed human lives to such an extent that there are a notable associated socio-economic consequences. Currently, according to the World Health Organization (WHO), Africa holds the highest disease burden with 94% of deaths and 82% of cases with P. falciparum having ~100% prevalence. Chemotherapy, such as artemisinin combination therapy, has been and continues to be the work horse in the fight against the disease, together with seasonal malaria chemoprevention and the use of insecticides. Natural products such as quinine and artemisinin are particularly important in terms of their antimalarial activity. The emphasis in current chemotherapy research is the need for time and cost-effective workflows focussed on new mechanisms of action (MoAs) covering the target candidate profiles (TCPs). Despite a decline in cases over the past decades with, countries increasingly becoming certified malaria free, a stalling trend has been observed in the past five years resulting in missing the 2020 Global Technical Strategy (GTS) milestones. With no effective vaccine, a reduction in funding, slower drug approval than resistance emergence from resistant and invasive vectors, and threats in diagnosis with the pfhrp2/3 gene deletion, malaria remains a major health concern. Motivated by these reasons, the primary aim of this work was a contribution to the antimalarial pipeline through in silico approaches focusing on P. falciparum. We first intended an exploration of malarial targets through a proteome scale screening on 36 targets using multiple metrics to account for the multi-objective nature of drug discovery. The continuous growth of structural data offers the ideal scenario for mining new MoAs covering antimalarials TCPs. This was combined with a repurposing strategy using a set of orally available FDA approved drugs. Further, use was made of time- and cost-effective strategies combining QVina-W efficiency metrics that integrate molecular properties, GRIM rescoring for molecular interactions and a hydrogen mass repartitioning (HMR) molecular dynamics (MD) scheme for accelerated development of antimalarials in the context of resistance. This pipeline further integrates a complex ranking for better drug-target selectivity, and normalization strategies to overcome docking scoring function bias. The different metrics, ranking, normalization strategies and their combinations were first assessed using their mean ranking error (MRE). A version combining all metrics was used to select 36 unique protein-ligand complexes, assessed in MD, with the final retention of 25. From the 16 in vitro tested hits of the 25, fingolimod, abiraterone, prazosin, and terazosin showed antiplasmodial activity with IC50 2.21, 3.37, 16.67 and 34.72 ÎŒM respectively and of these, only fingolimod was found to be not safe with respect to human cell viability. These compounds were predicted active on different molecular targets, abiraterone was predicted to interact with a putative liver-stage essential target, hence promising as a transmission-blocking agent. The pipeline had a promising 25% hit rate considering the proteome-scale and use of cost-effective approaches. Secondly, we focused on Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) using a more extensive screening pipeline to overcome some of the current in silico screening limitations. Starting from the ZINC lead-like library of ~3M, hierarchical ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches with molecular docking and re-scoring using eleven scoring functions (SFs) were used. Later ranking with an exponential consensus strategy was included. Selected hits were further assessed through Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), advanced MD sampling in a ligand pulling simulations and (Weighted Histogram Analysis Method) WHAM analysis for umbrella sampling (US) to derive binding free energies. Four leads had better predicted affinities in US than LC5, a 280 nM potent PfDXR inhibitor with ZINC000050633276 showing a promising binding of -20.43 kcal/mol. As shown with fosmidomycin, DXR inhibition offers fast acting compounds fulfilling antimalarials TCP1. Yet, fosmidomycin has a high polarity causing its short half-life and hampering its clinical use. These leads scaffolds are different from fosmidomycin and hence may offer better pharmacokinetic and pharmacodynamic properties and may also be promising for lead optimization. A combined analysis of residues’ contributions to the free energy of binding in MM-PBSA and to steered molecular dynamics (SMD) Fmax indicated GLU233, CYS268, SER270, TRP296, and HIS341 as exploitable for compound optimization. Finally, we updated the SANCDB library with new NPs and their commercially available analogs as a solution to NP availability. The library is extended to 1005 compounds from its initial 600 compounds and the database is integrated to Mcule and Molport APIs for analogs automatic update. The new set may contribute to virtual screening and to antimalarials as the most effective ones have NP origin.Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 202

    Medicinal chemistry approaches to malaria drug discovery

    Get PDF
    Tese de doutoramento, FarmĂĄcia (QuĂ­mica FarmacĂȘutica e TerapĂȘutica), Universidade de Lisboa, Faculdade de FarmĂĄcia, 2016Malaria remains a major burden to global public health, causing nearly 600,000 deaths annually. Efforts to control malaria are hampered by parasite drug resistance, insecticide resistance in mosquitoes, and the lack of an effective vaccine. However antimalarial drugs are a mainstay in efforts to control and eventually eradicate this disease, thus the discovery of new antimalarials is critical. Antimalarial drug discovery is especially challenging due to the unique biology of malaria parasites, the scarcity of tools for identifying new drug targets, and the poorly understood mechanisms of action of existing antimalarials. Therefore, this work describes the use of different medicinal chemistry approaches to address unmet needs in antimalarial drug discovery. Part of this process includes ensuring that sufficient drug leads are available to prime the drug discovery pipeline, particularly those with novel modes of action in order to limit issues of cross-resistance with existing drugs. The first approach described in this thesis consists on the phenotypic-based hit to lead optimization designed to explore the antimalarial potential of the 3-piperidin-4-yl-1H-indole. The hit compound was identified in a phenotypic screen of ~2 million compounds against asexual blood stage P. falciparum. Three series of analogs were synthesized following a reagentbased diversity approach, in a total of 38 compounds, and screened for their blood stage antimalarial activity. The SAR shows that 3-piperidin-4-yl-1H-indole is intolerant to most Npiperidinyl modifications. Out of the analogs synthesized, three were active (2.19, 2.20 and 2.29). Furthermore, the (4-(1H-indol-3-yl)piperidin-1-yl)(pyridin-3-yl)methanone (2.29) showed in vitro antimalarial activity (EC50 values ∌3 ÎŒM), no cross-resistance with chloroquine, selectivity for the parasite, and lead-like properties (cLogP 20). These results allow the prioritization of the phenylalanyl tRNA synthetase (FRS), histidyl tRNA synthetase (HRS), alanyl tRNA synthetase (ARS), and prolyl tRNA synthetase (PRS) as the top four enzymes for further exploration as drug targets in blood stage malaria. aaSA compound treatment of P. falciparum increased eIF2α phosphorylation at 100X concentration, inducing the amino acid starvation pathway through direct inhibition of the corresponding PfaaRS, with few exceptions. Moreover, analogs were also active in vitro against liver stage P. berghei, with > 99% parasite growth inhibition at the higher concentration of 10 ÎŒM. Thus, underscoring the potential of the aaRS family as an attractive novel class of antimalarial drug targets. To further explore cytoplasmic prolyl tRNA synthetase (cPRS) as an antimalarial target, which we have previously identified as the long sought biochemical target of the antimalarial halofuginone (HFG), novel HFG based inhibitors were designed to exploit additional ligandprotein interactions in the active site of cPRS, and may serve as lead compounds in the preclinical development of a mechanistically unique class of malaria drugs with activity against both liver and blood stage life cycle stages. Furthermore, we aimed to characterize the biology of cPRS inhibition and resulting amino acid starvation response. Understanding the enzymeinhibitor complex formed by the different types of inhibitors (HFG and L-ProSA) would further elucidate on the deferential effect observed on the amino acid starvation response in mammalian cells, despite targeting the same enzyme. Thus, a two-step proteomic approach to isolate the protein complex using immunoprecipitation followed by identification of its components using mass spectrometry is proposed. Despite not being able to completely establish the protocol, the results in MCF-7 cells are consistent with the model proposed, thus further work needs to be done towards increasing the amount of the enzyme-inhibitor complex isolated to meet the detection requirements of the techniques used. Finally, to address the problem of limited target identification and validation tools for novel antimalarial compounds, the third aim of this thesis investigates the use of fluorescently labeled small molecules as a novel target discovery approach in malaria drug discovery efforts. A new methodology, which originated from our efforts to synthetize MayaFluors in a one-pot, two-step approach via BODIPY-OTf intermediate, was developed to label drugs with the BODIPY fluorophore. The method allows for substitution of either one or both of the canonical fluorides on the BODIPY dye with alkoxy ligands, under mild conditions. We successfully labeled a group of small molecules. Of these, two known drugs ((+) JQ1 and hydroxychloroquine) were evaluated for their activity and cellular localization. In both cases the labeled drug presented comparable activity to the parent drug. Furthermore, the fluorescently labeled antimalarial displayed a subcellular staining pattern in mammalian cells that is consistent with accumulation in acidic vesicles in the cytoplasm. Moreover, the probe was also tested in Plasmodium falciparum cultures, where results show subcellular staining pattern that seems consistent with accumulation in the food vacuole. Despite the identified limitation concerning the solvent compatibility of the method, this approach allows direct labeling of hydroxylfunctionalized drugs, which we believe may have broad applications for rapid and specific imaging of elusive biological targets in living cells. Taken together, in this thesis multiple medicinal chemistry approaches are explored in an effort to identify novel antimalarial chemotypes that act on underexploited targets. Furthermore, these results present new opportunities for malaria drug discovery to aid efforts in malaria control and eventual eradication

    IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

    Get PDF
    Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD
    • 

    corecore