DOCSLIB.ORG

Proteomic Analysis of Metacyclogenesis in Leishmania

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Proteomic Analysis of Metacyclogenesis in Leishmania e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183 Available online at www.sciencedirect.com ScienceDirect journal homepage: http://www.elsevier.com/locate/euprot Proteomic analysis of metacyclogenesis in Leishmania infantum wild-type and PTR1 null mutant 1 1 Wilfried Moreira , Danielle Légaré , Gina Racine, Gaétan Roy, ∗ Marc Ouellette Centre de Recherche en Infectiologie du CHU de Québec, Département de Microbiologie, Université Laval, Québec, QC, Canada a r a t i b s c l e i n f o t r a c t Article history: The parasite Leishmania exhibits a dimorphic life cycle with promastigotes found in the Received 23 May 2014 insect vector and amastigotes residing within mammalian macrophages. In the insect, pro- Received in revised form mastigotes undergo metacyclogenesis with two main stages, the procyclic and metacyclic 14 July 2014 stages. Reduced pterins, provided by the pteridine reductase PTR1, are important factors Accepted 17 July 2014 controlling metacyclogenesis. We applied here the liquid-based free-flow electrophore- Available online 8 August 2014 sis technique for the analysis of L. infantum wild-type and PTR1 null-mutant procyclic and metacyclic proteomes. Significant modulations in energy metabolism, antioxidant and Keywords: stress-related defences, genetic information processing, protein degradation and motility Leishmania were observed between procyclic and metacyclic forms, in addition to numerous hypothet- Reduced pterins ical proteins. Folates © 2014 The Authors. Published by Elsevier B.V. on behalf of European Proteomics Metacyclogenesis Association (EuPA). This is an open access article under the CC BY-NC-ND license Free-flow electrophoresis (http://creativecommons.org/licenses/by-nc-nd/3.0/). sequential development involves the rapid division of non- 1. Introduction infective procyclic promastigotes that ultimately leads to the appearance of non-dividing but highly infective metacyclic Leishmania species are affecting 12 million people world- promastigotes in the anterior parts of the digestive tract wide with 1.5–2 million new cases each year. These protozoa [1,2]. This cellular differentiation referred as metacyclogen- have a dimorphic life cycle and are found as flagellated esis is a key step for infectivity [2–4]. Indeed, it has been promastigotes within the sand fly midgut, and as intracel- shown that promastigote populations recovered from labo- lular non-motile amastigotes within macrophages of their ratory infected sand flies displayed a significant increase in vertebrate hosts. In the sand fly, promastigotes maturate infectivity in BALB/c mice as they develop temporally within and progress through a series of two distinct stages. This the flies [2]. Also, promastigotes recovered from the stationary ∗ Corresponding author at: Centre de Recherche en Infectiologie, CHU de Québec, 2705 Boul. Laurier, Québec, QC, Canada G1V 4G2. Tel.: +1 418 525 4444x48481; fax: +1 418 654 2715. E-mail address: [email protected] (M. Ouellette). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.euprot.2014.07.003 2212-9685/© 2014 The Authors. Published by Elsevier B.V. on behalf of European Proteomics Association (EuPA). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 172 e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183 phase of growth were found to be more infective in vitro 2. Material and methods than promastigotes isolated from the logarithmic phase [1–4]. The infectivity of stationary phase promastigotes has been 2.1. Cell culture, lectin-mediated purification and attributed among other things to changes in their surface characterization of metacyclics and procyclics enriched carbohydrates, including the major glycoconjugate lipophos- parasites phoglycan (LPG) coating molecule [4]. Changes in the structure of LPG allow the parasite to detach from the mucosa and Promastigotes of L. infantum wild-type (MHOM/MA/67/ITMAP- migrate to the proboscis for injection in the host tissue during −/− 263) and L. infantum PTR1 [18] were grown in SDM79 the blood meal. 9 medium as described previously [25] and parasites (10 cells) Metacyclogenesis is also accompanied by a number of in their exponential phase and stationary phase of growth morphological changes. In particular, procyclics possess a were pelleted by centrifugation (3000 rpm, 5 min). Pellets were flagella approximately one body length whereas metacyclics washed twice in Hepes–NaCl buffer (21 mM Hepes, 137 mM are shorter cells of reduced body width and flagella longer NaCl, 5 mM KCl, 0.7 mM Na2HPO4·H2O, and 6 mM dextrose, than one body length. Clear biochemical and molecular differ- pH 7.05). Metacyclic parasites were isolated by PNA agglu- ences have already been established during metacyclogenesis. tination (100 ␮g/ml, Sigma–Aldrich) as previously described These include a lower amount of total protein content asso- [2,26,27] from stationary phase populations whereas pro- ciated to a decrease in protein synthesis in metacyclics cyclics were collected from exponential phase cultures. compared to procyclics, as well as an increase in protease Enriched metacyclic and procyclic samples were visualized activities [1]. Secretory acid phosphatase and nuclease activ- by light microscopy (400×, result not shown) and were further ities have also been reported to be decreased in metacyclic characterized by real-time RTPCR to confirm procyclic and parasites [1]. metacyclic enrichment. Briefly, total RNAs from procyclic and Reduced pterins (tetrahydrobiopterin, THB) have been metacyclic parasites were isolated using the Qiagen RNeasy identified as an important factor controlling metacyclogenesis Mini Kit (Qiagen) according to the manufacturer’s instructions. as a decrease in the THB level seems to stimulate meta- At least 5 independent RNA preparations from the WT and cyclogenesis [5]. Leishmania is an auxotroph for pterins but DKO PTR1 procyclic and metacyclic parasites were prepared it possesses the biopterin transporter BT1 to supply for its and used in real-time RTPCR assays. Contaminating DNA was need in pterins [6–8]. After BT1-mediated uptake, pterins are digested with Turbo DNA-free Kit (Ambion). The quality and reduced into their active forms by the enzyme pteridine reduc- integrity of the starting RNA material was assessed with an tase 1 (PTR1) [9–11]. Lack of PTR1 leads to reduced levels of Agilent Technologies 2100 BioAnalyzer using the RNA 6000 intracellular THB, growth arrest and induction of metacycloge- Nano LabCHIP kit (Agilent). The cDNAs were generated from nesis. One of the earliest known cellular functions of THB was total RNAs using a Oligo (dT)12–18 following the protocol of as a growth factor for the parasite Crithidia fasciculata [12], and the manufacturer for Superscript II (Invitrogen). Real-time many studies have indicated that THB is indeed an essential quantitative RTPCR assays were carried out in a BioRad Cycler growth factor for Leishmania [13–16], although its exact role is using SYBR Green I (Molecular Probes). The reactions were still to be discovered (reviewed in [17]). THB was also shown to carried out in a final volume of 25 ␮l containing specific react readily with oxygen, superoxide, H2O2, and peroxynitrite primers, and iQ SYBR Green Supermix (Bio-Rad). Mixtures and it was demonstrated that it protects cells against oxidative ◦ were initially incubated at 95 C for 5 min and then cycled 35 damages [18,19] (and reviewed in [20,21]). PTR1 and reduced ◦ ◦ ◦ times at 95 C, 60 C for 15 s and 72 C for 20 s. All real-time pterins thus play important roles in Leishmania differentiation RTPCR data were normalized with the real-time amplification and life cycle in which the infective metacyclic form encoun- of GAPDH. The expression data are shown relative to the ters an impressive oxidative stress as it meets with the host data for the wild-type strain. Primer SHERP (LinJ23.1210) For- immune system. ward: 5 -GGACCAGATGAACAACGCCGCGG-3 ; Primer SHERP Few studies have investigated the proteome changes dur- Reverse: 5 -ACGAGCCGCCGCTTATCTTGTCC-3 ; Primer GAPDH ing the metacyclogenesis process in Leishmania [22,23] and (LinJ30.3000) Forward: 5 -GATCACGGTGGAGGCTGTG-3 ; none have assessed the role of reduced pterins in this cell Primer GAPDH Reverse: 5 -CCCTTGATGTGGCCCTCGG-3 . cycle transition. In this study, we enriched for metacyclic and procyclic parasites from wild-type and PTR1 null mutant Leish- 2.2. Protein sample preparation mania infantum strains [18]. Their respective proteins were subjected to free-flow electrophoresis (FFE), a liquid based Protein extractions were prepared from procyclic and meta- IEF separation technique. We identified more than 260 indi- cyclic parasites by incubating cell pellets in 300 ␮l of 2D vidual proteins differentially expressed. Important changes lysis buffer (7 M urea, 2 M thiourea, 3% (w/v) Chaps, 20 mM in energy metabolism, antioxidant, stress-related defenses DTT, 5 mM TCEP, 50 mM Tris-base, 0.5% IPG buffer pH 4–7, and proteins involved in motility were observed. A signifi- and 0.25% IPG buffer pH 3–10) supplemented with 10 ␮l of cant modulation in the expression of proteins involved in protease inhibitor cocktail (2 mM AEBSF, 14 ␮M E-64, 130 ␮M genetic information processing was also detected, support- Bestatin, 0.9 ␮M Leupeptin, 0.3 ␮M Aprotinin, and 1 mM EDTA) ing the notion that changes in the proteome during parasite’s (Sigma-Aldrich) for 2 h at room temperature with frequent differentiation and life cycle seem to be controlled by post- vortexing. The supernatants were collected after centrifuga- transcriptional mechanisms, including modulation of mRNA tion (10,000 rpm, 2 min) and protein contents were determined stability, regulation of translation and post-translational mod- using a 2-D Quant Kit (GE Healthcare). ifications [24]. e u p a o p e n p r o t e o m i c s 4 ( 2 0 1 4 ) 171–183 173 2.3.
Load more

Recommended publications
  • Kohen Curriculum Vitae
    Curriculum Vitae Amnon Kohen Department of Chemistry Tel: (319) 335-0234 University of Iowa FAX: (319) 335-1270 Iowa City, IA 52242 [email protected] EDUCATION AND PROFESSIONAL HISTORY Education D.Sc., Chemistry 1989-1994 Technion – Israel Institute of Technology, Haifa, Israel Advisor: Professor T. Baasov Topic: Mechanistic Studies of the Enzyme KDO8P Synthase B.Sc., Chemistry (with Honors) 1986-1989 Hebrew University, Jerusalem, Israel Positions Professor 2010-Present Department of Chemistry, University of Iowa, Iowa City, IA and Molecular and Cellular Biology Program, and MSTP faculty member Associate Professor 2005-2010 Department of Chemistry, University of Iowa, Iowa City, IA and Molecular and Cellular Biology Program Assistant Professor 1999-2005 Department of Chemistry, University of Iowa, Iowa City, IA Postgraduate Researcher 1997-1999 With Professor Judith Klinman Department of Chemistry, University of California, Berkeley Topic: Hydrogen Tunneling in Biology: Alcohol Dehydrogenases Postgraduate Fellow 1995-1997 With Professor Judith Klinman Department of Chemistry, University of California, Berkeley Topic: Hydrogen Tunneling in Biology: Glucose Oxidase Visiting Scholar Fall 1994 With Professor Karen S. Anderson Department of Pharmacology, Yale Medical School, New Haven, CT Kohen, A. Affiliations American Society for Biochemistry and Molecular Biology (ASBMB, 2013 - present) Center for Biocatalysis and Bioprocessing (CBB) University of Iowa (2000-Present) The Interdisciplinary Graduate Program in Molecular Biology, University of Iowa (2003- Present) American Association for the Advancement of Science (AAAS, 2011-present) American Chemical Society (1995-Present) Divisions: Organic Chemistry, Physical Chemistry, Biochemistry Sigma Xi (1997-Present) Protein Society (1996-1998) Honors and Awards • Career Development Award (University of Iowa- 2015-2016) • Graduate College Outstanding Faculty Mentor Award (2015).
    [Show full text]
  • Molecular Cloning, Expression and Enzymatic Assay of Pteridine Reductase 1 from Iranian Lizard Leishmania
    Iranian Biomedical Journal 14 (3): 97-102 (July 2010) Molecular Cloning, Expression and Enzymatic Assay of Pteridine Reductase 1 from Iranian Lizard Leishmania Bahram Kazemi*1,2, Farideh Tohidi3,4, Mojgan Bandehpour1 and Fatemeh Yarian1 1Cellular and Molecular Biology Research Center and 2Dept. of Parasitology and Mycology, Shahid Beheshti University, Tehran; 3Dept. of Parasitology and Mycology, Gorgan University of Medical Sciences, Gorgan; 4Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran Received 17 April 2010; revised 17 July 2010; accepted 24 July 2010 ABSTRACT Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively.
    [Show full text]
  • Design, Synthesis and Biological Evaluation of Novel Inhibitors of Trypanosoma Brucei Pteridine Reductase 1 Daniel Spinks, Han B
    MED DOI: 10.1002/cmdc.201000450 Design, Synthesis and Biological Evaluation of Novel Inhibitors of Trypanosoma brucei Pteridine Reductase 1 Daniel Spinks, Han B. Ong, Chidochangu P. Mpamhanga, Emma J. Shanks, David A. Robinson, Iain T. Collie, Kevin D. Read, Julie A. Frearson, Paul G. Wyatt, Ruth Brenk, Alan H. Fairlamb, and Ian H. Gilbert*[a] Genetic studies indicate that the enzyme pteridine reductase 1 chemistry and structure-based approaches, we were able to (PTR1) is essential for the survival of the protozoan parasite Try- derive compounds with potent activity against T. brucei PTR1 app m panosoma brucei. Herein, we describe the development and (K i = 7n ), which had high selectivity over both human and optimisation of a novel series of PTR1 inhibitors, based on ben- T. brucei dihydrofolate reductase. Unfortunately, these com- zo[d]imidazol-2-amine derivatives. Data are reported on 33 pounds displayed weak activity against the parasites. Kinetic compounds. This series was initially discovered by a virtual studies and analysis indicate that the main reason for the lack screening campaign (J. Med. Chem., 2009, 52, 4454). The inhibi- of cell potency is due to the compounds having insufficient tors adopted an alternative binding mode to those of the nat- potency against the enzyme, which can be seen from the low m m ural ligands, biopterin and dihydrobiopterin, and classical in- Km to Ki ratio (Km =25 n and Ki = 2.3 n , respectively). hibitors, such as methotrexate. Using both rational medicinal Introduction Human African trypanosomiasis (HAT) is a serious health prob- lem in sub-Saharan Africa, with an estimated 50 000 new infec- tions each year, and over 60 million people in 36 countries are at risk of infection.[1] HAT is a progressive and ultimately fatal disease.
    [Show full text]
  • Dihydropteridine Reductase
    John M. Whiteley et uf. : Dihydropteridine reductase Pteridines Vol. 4, 1993, pp. 159-173 Review Dihydropteridine Reductase John M. Whiteley§, Kottayil I. Varughesej:, Nguyen H. Xuong#, David A. Matthews, t and Charles E. Grimshawf §The Scripps Research Institute, La Jolla, CA 92037, USA., #University of California at San Diego, La Jolla, CA 92093-0317, U.SA., tAgouron Pharmaceuticals, Inc., San Diego, CA 92121, USA, and fThe Whittier Institute, La Jolla, CA 92037, USA. (Received August lO, 1993) Summary During the past decade numerous advances have been made in understanding the structure, mechanism and clinical properties of dihydropteridine reductase. An attempt is made here to delineate the current status of this essential enzyme by describing its structural features, its kinetic mechanism, the cloning and expression of both rat and human enzyme forms, the solution of their crystal structures, their classifica­ tion as members of a large family of short chain dehydrogenases, and finally a brief description is included indicating how current molecular biological applications have allowed the clinical definition of the aberrant form of phenylketonuria caused by a defective reductase. Key words: Dihydropteridine reductase, Quinonoid dihydrobiopterin, Crystal structure, Aberrant PKU, Gene expression, Mutagenesis Introduction and history in their heterocyclic nucleus of many centers for protonation, which often influence binding and reac­ Naturally occurring pteridines, which usually con­ tivity. Important metabolic functions of conjugated tain 2-amino and 4-hydroxyl substituents can be sep­ pteridine-mediated biological reactions include the arated into two distinct classes. One class contains one-carbon insertion reactions fundamental to pu­ the pterins of the folic acid series which possess rine biosynthesis (4.
    [Show full text]
  • One Scaffold, Three Binding Modes: Novel and Selective Pteridine Reductase 1 Inhibitors Derived from Fragment Hits Discovered by Virtual Screening†
    4454 J. Med. Chem. 2009, 52, 4454–4465 DOI: 10.1021/jm900414x One Scaffold, Three Binding Modes: Novel and Selective Pteridine Reductase 1 Inhibitors Derived from Fragment Hits Discovered by Virtual Screening† Chidochangu P. Mpamhanga, Daniel Spinks, Lindsay B. Tulloch, Emma J. Shanks, David A. Robinson, Iain T. Collie, Alan H. Fairlamb, Paul G. Wyatt, Julie A. Frearson, William N. Hunter, Ian H. Gilbert, and Ruth Brenk* Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K. Received March 31, 2009 The enzyme pteridine reductase 1 (PTR1) is a potential target for new compounds to treat human African trypanosomiasis. A virtual screening campaign for fragments inhibiting PTR1 was carried out. Two novel chemical series were identified containing aminobenzothiazole and aminobenzimidazole scaffolds, respectively. One of the hits (2-amino-6-chloro-benzimidazole) was subjected to crystal structure analysis and a high resolution crystal structure in complex with PTR1 was obtained, confirming the predicted binding mode. However, the crystal structures of two analogues (2-amino- benzimidazole and 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole) in complex with PTR1 revealed two alternative binding modes. In these complexes, previously unobserved protein movements and water-mediated protein-ligand contacts occurred, which prohibited a correct prediction of the binding modes. On the basis of the alternative binding mode of 1-(3,4-dichloro-benzyl)-2-amino-benzimidazole, derivatives
    [Show full text]
  • Evidence of Pyrimethamine and Cycloguanil Analogues As Dual Inhibitors of Trypanosoma Brucei Pteridine Reductase and Dihydrofolate Reductase
    pharmaceuticals Article Evidence of Pyrimethamine and Cycloguanil Analogues as Dual Inhibitors of Trypanosoma brucei Pteridine Reductase and Dihydrofolate Reductase Giusy Tassone 1,† , Giacomo Landi 1,†, Pasquale Linciano 2,† , Valeria Francesconi 3 , Michele Tonelli 3 , Lorenzo Tagliazucchi 2 , Maria Paola Costi 2 , Stefano Mangani 1 and Cecilia Pozzi 1,* 1 Department of Biotechnology, Chemistry and Pharmacy, Department of Excellence 2018–2022, University of Siena, via Aldo Moro 2, 53100 Siena, Italy; [email protected] (G.T.); [email protected] (G.L.); [email protected] (S.M.) 2 Department of Life Science, University of Modena and Reggio Emilia, via Campi 103, 41125 Modena, Italy; [email protected] (P.L.); [email protected] (L.T.); [email protected] (M.P.C.) 3 Department of Pharmacy, University of Genoa, Viale Benedetto XV n.3, 16132 Genoa, Italy; [email protected] (V.F.); [email protected] (M.T.) * Correspondence: [email protected]; Tel.: +39-0577-232132 † These authors contributed equally to this work. Abstract: Trypanosoma and Leishmania parasites are the etiological agents of various threatening Citation: Tassone, G.; Landi, G.; neglected tropical diseases (NTDs), including human African trypanosomiasis (HAT), Chagas disease, Linciano, P.; Francesconi, V.; Tonelli, and various types of leishmaniasis. Recently, meaningful progresses in the treatment of HAT, due to M.; Tagliazucchi, L.; Costi, M.P.; Trypanosoma brucei (Tb), have been achieved by the introduction of fexinidazole and the combination Mangani, S.; Pozzi, C. Evidence of therapy eflornithine–nifurtimox. Nevertheless, due to drug resistance issues and the exitance of Pyrimethamine and Cycloguanil animal reservoirs, the development of new NTD treatments is still required.
    [Show full text]
  • Methylated Spirits: Epigenetic Hypotheses of Psychiatric Disorders
    Trends in Psychopharmacology Methylated Spirits: Epigenetic Hypotheses of Psychiatric Disorders Stephen M. Stahl, MD, PhD NEW TREND IN various brain circuits and creating risk for develop- PSYCHOPHARMACOLOGY ing a symptom of a mental illness. Now comes the Our spirits may be regulated by the methylation role of epigenetic actions in mental illnesses. If nor- of our genes. Methylation, acetylation, and other mal genes make normal gene products but at the biochemical processes are the molecular switches wrong time, either being epigenetically expressed for turning genes on and off. There is evidence in neurons when they should be silenced or epi- now that certain behaviors, feelings, and psychiat- genetically silenced in neurons when they should ric symptoms may be modified by turning various be expressed, particularly under the influence of genes on or off. If classical genetics is the sequence environmental factors and stress, this, too, can of DNA that is inherited, then epigenetics is a par- contribute to inefficient information processing in allel process determining whether a given gene brain circuits, increasing the chance of develop- (ie, a sequence of DNA coding for transcription) is ing symptoms of a psychiatric disorder. Here we expressed into its RNA or is silenced. Epigenetics describe the role of epigenetics and methylomics is now entering psychiatry with the hypothesis (methylating or demethylating upstream genes that normal genes as well as risk genes can both and downstream molecules) in various psychiatric contribute to a mental disorder. That is, it has long disorders, emphasizing schizophrenia, and demon- been hypothesized that when “abnormal” genes strate whether your spirits can be truly methylated.
    [Show full text]
  • New Benzimidazole Derivatives As Inhibitors of Pteridine Reductase 1: Design, Molecular Docking Study and ADMET Prediction
    Journal of Applied Pharmaceutical Science Vol. 10(09), pp 030-039, September, 2020 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2020.10904 ISSN 2231-3354 New benzimidazole derivatives as inhibitors of Pteridine reductase 1: Design, molecular docking study and ADMET prediction Shraddha Phadke1*, Rakesh Somani2, Devender Pathak3 1Dr. L. H. Hiranandani College of Pharmacy, Thane, India. 2D. Y. Patil University School of Pharmacy, Navi Mumbai, India. 3Pharmacy College Saifai, Uttar Pradesh University of Medical Sciences, Etawah, India. ARTICLE INFO ABSTRACT Received on: 23/04/2020 Pteridine reductase 1 (PTR1) is a unique enzyme required for survival of Leishmania species, a causative organism for Accepted on: 14/06/2020 the disease leishmaniasis. We herein report the design, docking, and Absorption, Distribution, Metabolism, Excretion, Available online: 05/09/2020 Toxicity (ADMET) prediction studies of 2-substituted-5-[(6-substituted-1H-benzimidazol-2yl)methyl]azole derivatives (B1–B14) as PTR1 inhibitors. Molecular docking studies showed good binding interaction of the compounds with the active site of pteridine reductase from Leishmania Major, with compounds B5 and B12 showing docking scores Key words: of −61.5232 and −62.5897, respectively, which were comparable with the original ligand, dihydrobiopterin. Large Pteridine reductase 1, substituents on the azole ring, as well as substitutions on sixth position of the benzimidazole ring, were found to be leishmaniasis, benzimidazole favorable for interaction with PTR1 active site. Physicochemical properties, bioactivity prediction, and toxicity profiles derivatives, docking studies, of the compounds were studied using the Molinspiration and admetSAR web servers. All compounds followed Lipinski’s ADMET, druglikeness. rule of five and can be considered as good oral candidates.
    [Show full text]
  • Discovery of Potent Pteridine Reductase Inhibitors to Guide Antiparasite Drug Development
    Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development Antonio Cavazzuti*, Giuseppe Paglietti†, William N. Hunter‡, Francisco Gamarro§, Sandra Piras†, Mario Loriga†, Sergio Alleca†, Paola Corona†, Karen McLuskey‡, Lindsay Tulloch‡, Federica Gibellini*‡, Stefania Ferrari*, and Maria Paola Costi*¶ *Dipartimento di Scienze Farmaceutiche, Universita`di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy; †Dipartimento Farmaco Chimico Tossicologico, Universita`degli Studi di Sassari, via Muroni 23/a 07100 Sassari, Italy; ‡Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom; and §Instituto de Parasitologia y Biomedicina ‘‘Lopez-Neyra,’’ Consejo Superior de Investigaciones Cientificas, Parque Tecnolo´gico de Ciencias de la Salud, Avenida del Conocimiento s.n., 18100 Armilla, Granada, Spain Edited by Robert M. Stroud, University of California, San Francisco, CA, and approved November 26, 2007 (received for review May 10, 2007) Pteridine reductase (PTR1) is essential for salvage of pterins by Because trypanosomatids are auxotrophic for folates and pterins, parasitic trypanosomatids and is a target for the development of the inhibition of the enzymes depending on them should provide improved therapies. To identify inhibitors of Leishmania major and suitable treatments. However, antifolates are not used in the Trypanosoma cruzi PTR1, we combined a rapid-screening strategy therapy of trypanosomatid infections mainly because of
    [Show full text]
  • Drug Discovery Against Leishmaniasis
    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 265 Drug discovery against leishmaniasis Bio- and chemoinformatic guided strategies for target evaluation and hit identification ELISABET VIKEVED ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 ISBN 978-91-513-0521-9 UPPSALA urn:nbn:se:uu:diva-368499 2019 Dissertation presented at Uppsala University to be publicly examined in A1:107a, BMC, Husargatan 3, Uppsala, Friday, 1 February 2019 at 09:15 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Faculty examiner: Professor David Horn (The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee). Abstract Vikeved, E. 2019. Drug discovery against leishmaniasis. Bio- and chemoinformatic guided strategies for target evaluation and hit identification. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 265. 67 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0521-9. Leishmaniasis is a neglected tropical disease mainly affecting poor people in developing countries. It is caused by infections of flagellated protozoa belonging to genus Leishmania. The few available drugs are associated with problems such as low effectiveness, severe side effects and resistance development. The overall aim of this thesis is to aid in drug discovery against leishmaniasis – primarily using bio- and chemoinformtic approaches. In the first part of the thesis potential drug targets in Leishmania parasites were identified and hits against these targets were thereafter suggested. In paper I bioinformatics together with experimental work were used to evaluate lateral gene transfer (LGT) in genus Leishmania. LGTs of prokaryote origin often lack human homologs, and are therefore hypothesized to be valuable drug targets.
    [Show full text]
  • Homology Modelling, Molecular Docking, And
    Vadloori et al. BMC Res Notes (2018) 11:246 https://doi.org/10.1186/s13104-018-3354-1 BMC Research Notes RESEARCH NOTE Open Access Homology modelling, molecular docking, and molecular dynamics simulations reveal the inhibition of Leishmania donovani dihydrofolate reductase‑thymidylate synthase enzyme by Withaferin‑A Bharadwaja Vadloori1, A. K. Sharath2, N. Prakash Prabhu2 and Radheshyam Maurya1* Abstract Objective: Present in silico study was carried out to explore the mode of inhibition of Leishmania donovani dihydro- folate reductase-thymidylate synthase (Ld DHFR-TS) enzyme by Withaferin-A, a withanolide isolated from Withania somnifera. Withaferin-A (WA) is known for its profound multifaceted properties, but its antileishmanial activity is not well understood. The parasite’s DHFR-TS enzyme is diverse from its mammalian host and could be a potential drug target in parasites. Results: A 3D model of Ld DHFR-TS enzyme was built and verifed using Ramachandran plot and SAVES tools. The protein was docked with WA-the ligand, methotrexate (MTX)-competitive inhibitor of DHFR, and dihydrofolic acid (DHFA)-substrate for DHFR-TS. Molecular docking studies reveal that WA competes for active sites of both Hu DHFR and TS enzymes whereas it binds to a site other than active site in Ld DHFR-TS. Moreover, Lys 173 residue of DHFR-TS forms a H-bond with WA and has higher binding afnity to Ld DHFR-TS than Hu DHFR and Hu TS. The MD simulations confrmed the H-bonding interactions were stable. The binding energies of WA with Ld DHFR-TS were calculated using MM-PBSA. Homology modelling, molecular docking and MD simulations of Ld DHFR-TS revealed that WA could be a potential anti-leishmanial drug.
    [Show full text]
  • Exploiting the 2-Amino-1,3,4-Thiadiazole Scaffold
    This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Article http://pubs.acs.org/journal/acsodf Exploiting the 2‑Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery † ‡ ∇ § § § ∥ Pasquale Linciano, Alice Dawson, Ina Pöhner, David M. Costa, Monica S. Sa,́Anabela Cordeiro-da-Silva, , † ⊥ ⊥ ⊥ ⊥ ⊥ Rosaria Luciani, Sheraz Gul, Gesa Witt, Bernhard Ellinger, Maria Kuzikov, Philip Gribbon, ⊥ ⊥ ⊥ # # ∥∥ † §§ Jeanette Reinshagen, Markus Wolf, Birte Behrens, Veroniqué Hannaert, Paul A. M. Michels, , Erika Nerini, , ¶ ¶ ¶ § † † ⊥⊥ Cecilia Pozzi, Flavio di Pisa, Giacomo Landi, Nuno Santarem, Stefania Ferrari, Puneet Saxena, , † † †† ## †† †† Sandra Lazzari, Giuseppe Cannazza, Lucio H. Freitas-Junior, , Carolina B. Moraes, Bruno S. Pascoalino, †† †† †† ‡‡ ‡‡ Laura M. Alcantara,̂ Claudia P. Bertolacini, Vanessa Fontana, Ulrike Wittig, Wolfgang Müller, ∇ ○ ⧫ ‡ ¶ † † Rebecca C. Wade, , , William N. Hunter,*, Stefano Mangani,*, Luca Costantino,*, and Maria P. Costi*, † Dipartimento di Scienze della Vita, Universitàdegli Studi di Modena e Reggio Emilia, Via Campi 103, 41125 Modena, Italy ‡ Biological Chemistry & Drug Discovery, School of Life Sciences, The Wellcome Trust Building, University of Dundee, Dow Street, Dundee DD1 5EH, U.K. § ∥ Instituto de Investigaca̧õ e Inovaca̧õ em Saude,́ Instituto de Biologia Molecular e Celular,
    [Show full text]