Abstract
Leishmania drug design follows the typical path of the flow of genetic information: By analyzing genome information and considering infection-specific RNA and protein expression, potential targets for drug design and vaccine development are identified. Therefore, to implement successful intervention strategies against Leishmania infection, specific features of the process are critical; herein they are described, including specific genome information, good vaccine targets, and classical as well as innovative drug targeting strategies. In addition, a combination of software and web sites has been structured here with references and tools for rapid analysis to rank and examine new target structures in Leishmania.
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References
World Health Organization, September 2016.
Peacock CS, Seeger K, Harris D, Murphy L, et al. Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet. 2007;39(7):839–47.
Otranto D, Dantas-Torres F. The prevention of canine leishmaniasis and its impact on public health. Trends Parasitol. 2013;29(7):339–45.
Kaye P, Scott P. Leishmaniasis: complexity at the host-pathogen interface. Nat Rev Microbiol. 2011;9(8):604–15.
Marsden PD. Mucosal leishmaniasis (“espundia” Escomel, 1911). Trans R Soc Trop Med Hyg. 1986;80(6):859–76.
Dougall AM, Alexander B, Holt DC, Harris T, et al. Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia. Int J Parasitol. 2011;41(5):571–9.
Alvar J, Velez ID, Bern C, Herrero M, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 2012;7(5):e35671.
Bates PA. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int J Parasitol. 2007;37(10):1097–106.
Ivens AC, Peacock CS, Worthey EA, Murphy L, et al. The genome of the kinetoplastid parasite, Leishmania major. Science. 2005;309(5733):436–42.
Raymond F, Boisvert S, Roy G, Ritt JF, et al. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res. 2012;40(3):1131–47.
Coughlan S, Mulhair P, Sanders M, Schonian G, et al. The genome of Leishmania adleri from a mammalian host highlights chromosome fission in Sauroleishmania. Sci Rep. 2017;7:43747.
Remadi L, Haouas N, Chaara D, Slama D, et al. Clinical presentation of cutaneous leishmaniasis caused by Leishmania major. Dermatology. 2016;232(6):752–9.
do Rego Lima LV, Santos Ramos PK, Campos MB, dos Santos TV, et al. Preclinical diagnosis of American visceral leishmaniasis during early onset of human Leishmania (L.) infantum chagasi-infection. Pathog Glob Health. 2014;108(8):381–4.
Castro LS, Franca Ade O, Ferreira Ede C, Hans Filho G, et al. Leishmania infantum as a causative agent of cutaneous leishmaniasis in the state of Mato Grosso Do Sul, Brazil. Rev Inst Med Trop Sao Paulo. 2016;58:23.
Alves-Ferreira EV, Toledo JS, De Oliveira AH, Ferreira TR, et al. Differential gene expression and infection profiles of cutaneous and mucosal Leishmania braziliensis isolates from the same patient. PLoS Negl Trop Dis. 2015;9(9):e0004018.
Farias LH, Rodrigues AP, Silveira FT, Seabra SH, et al. Phosphatidylserine exposure and surface sugars in two Leishmania (Viannia) braziliensis strains involved in cutaneous and mucocutaneous leishmaniasis. J Infect Dis. 2013;207(3):537–43.
Gomes CM, de Paula NA, Cesetti MV, Roselino AM, Sampaio RN. Mucocutaneous leishmaniasis: accuracy and molecular validation of noninvasive procedures in a L. (V.) braziliensis-endemic area. Diagn Microbiol Infect Dis. 2014;79(4):413–8.
Avila-Garcia M, Mancilla-Ramirez J, Segura-Cervantes E, Farfan-Labonne B, et al. Transplacental transmission of cutaneous Leishmania mexicana strain in BALB/c mice. Am J Trop Med Hyg. 2013;89(2):354–8.
Galindo-Sevilla N, Soto N, Mancilla J, Cerbulo A, et al. Low serum levels of dehydroepiandrosterone and cortisol in human diffuse cutaneous leishmaniasis by Leishmania mexicana. Am J Trop Med Hyg. 2007;76(3):566–72.
Picado A, Ostyn B, Singh SP, Uranw S, et al. Risk factors for visceral leishmaniasis and asymptomatic Leishmania donovani infection in India and Nepal. PLoS One. 2014;9(1):e87641.
Morales CA, Palacio J, Rodriguez G, Camargo YC. Zosteriform cutaneous leishmaniasis due to Leishmania (Viannia ) panamensis and Leishmania (Viannia ) braziliensis: report of three cases. Biomedica. 2014;34(3):340–4.
Ives A, Ronet C, Prevel F, Ruzzante G, et al. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science. 2011;331(6018):775–8.
Gupta AK, Srivastava S, Singh A, Singh S. De novo whole-genome sequence and annotation of a Leishmania strain isolated from a case of post-kala-azar dermal Leishmaniasis. Genome Announc. 2015;3(4):e00809.
Mirzaei A, Schweynoch C, Rouhani S, Parvizi P. Diversity of Leishmania species and of strains of Leishmania major isolated from desert rodents in different foci of cutaneous leishmaniasis in Iran. Trans R Soc Trop Med Hyg. 2014;108(8):502–12.
Peters W, Bryceson A, Evans DA, Neal RA, et al. Leishmania infecting man and wild animals in Saudi Arabia. 8. The influence of prior infection with Leishmania arabica on challenge with L. major in man. Trans R Soc Trop Med Hyg. 1990;84(5):681–9.
Eslami G, Hajimohammadi B, Jafari AA, Mirzaei F, et al. Molecular identification of Leishmania tropica infections in patients with cutaneous leishmaniasis from an endemic central of Iran. Trop Biomed. 2014;31(4):592–9.
Kwakye-Nuako G, Mosore MT, Duplessis C, Bates MD, et al. First isolation of a new species of Leishmania responsible for human cutaneous leishmaniasis in Ghana and classification in the Leishmania enriettii complex. Int J Parasitol. 2015;45(11):679–84.
Yamamoto ES, Campos BL, Jesus JA, Laurenti MD, et al. The effect of ursolic acid on Leishmania (Leishmania) amazonensis is related to programed cell death and presents therapeutic potential in experimental cutaneous leishmaniasis. PLoS One. 2015;10(12):e0144946.
Coelho AC, Trinconi CT, Costa CH, Uliana SR. In vitro and in vivo miltefosine susceptibility of a Leishmania amazonensis isolate from a patient with diffuse cutaneous leishmaniasis. PLoS Negl Trop Dis. 2014;8(7):e2999.
Eliseev LN, Strelkova MV, Zherikhina II. The characteristics of the epidemic activation of a natural focus of zoonotic cutaneous leishmaniasis in places with a sympatric dissemination of Leishmania major, L. turanica and L. gerbilli. Med Parazitol (Mosk). 1991;3:24–9.
Negera E, Gadisa E, Hussein J, Engers H, et al. Treatment response of cutaneous leishmaniasis due to Leishmania aethiopica to cryotherapy and generic sodium stibogluconate from patients in Silti, Ethiopia. Trans R Soc Trop Med Hyg. 2012;106(8):496–503.
Akuffo HO, Fehniger TE, Britton S. Differential recognition of Leishmania aethiopica antigens by lymphocytes from patients with local and diffuse cutaneous leishmaniasis. Evidence for antigen-induced immune suppression. J Immunol. 1988;141(7):2461–6.
Longoni SS, Marin C, Sanchez-Moreno M. Excreted Leishmania peruviana and Leishmania amazonensis iron-superoxide dismutase purification: specific antibody detection in Colombian patients with cutaneous leishmaniasis. Free Radic Biol Med. 2014;69:26–34.
Isnard A, Shio MT, Olivier M. Impact of Leishmania metalloprotease GP63 on macrophage signaling. Front Cell Infect Microbiol. 2012;2:72.
Hassani K, Shio MT, Martel C, Faubert D. Absence of metalloprotease GP63 alters the protein content of Leishmania exosomes. PLoS One. 2014;9(4):e95007.
Depledge DP, Evans KJ, Ivens AC, Aziz N, et al. Comparative expression profiling of Leishmania: modulation in gene expression between species and in different host genetic backgrounds. PLoS Negl Trop Dis. 2009;3(7):e476.
Gupta SK, Bencurova E, Srivastava M, Pahlavan P. Improving re-annotation of annotated eukaryotic genomes. In: Big data analytics in genomics. Cham: Springer; 2016. p. 171–95.
Gupta SK, Kupper M, Ratzka C, Feldhaar H, et al. Scrutinizing the immune defence inventory of Camponotus floridanus applying total transcriptome sequencing. BMC Genomics. 2015;16:540.
Torres F, Arias-Carrasco R, Caris-Maldonado JC, Barral A, et al. LeishDB: a database of coding gene annotation and non-coding RNAs in Leishmania braziliensis. Database. 2017;2017:bax047. https://doi.org/10.1093/database/bax047. [1758-0463 (Electronic)]
Pigott DM, Bhatt S, Golding N, Duda KA, et al. Global distribution maps of the leishmaniases. elife. 2014;3
Aurrecoechea C, Barreto A, Basenko EY, Brestelli J, et al. EuPathDB: the eukaryotic pathogen genomics database resource. Nucleic Acids Res. 2017;45(D1):D581–d591.
Aslett M, Aurrecoechea C, Berriman M, Brestelli J, et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38(Database issue):D457–62.
Logan-Klumpler FJ, De Silva N, Boehme U, Rogers MB, et al. GeneDB—an annotation database for pathogens. Nucleic Acids Res. 2012;40(Database issue):D98–108.
Gazestani VH, Yip CW, Nikpour N, Berghuis N, et al. TrypsNetDB: an integrated framework for the functional characterization of trypanosomatid proteins. PLoS Negl Trop Dis. 2017;11(2):e0005368.
Saunders EC, MacRae JI, Naderer T, Ng M, et al. LeishCyc: a guide to building a metabolic pathway database and visualization of metabolomic data. Methods Mol Biol. 2012;881:505–29.
Dikhit MR, Moharana KC, Sahoo BR, Sahoo GC, et al. LeishMicrosatDB: open source database of repeat sequences detected in six fully sequenced Leishmania genomes. Database. 2014;2014:bau078. https://doi.org/10.1093/database/bau078.
Patel P, Mandlik V, Singh S. LmSmdB: an integrated database for metabolic and gene regulatory network in Leishmania major and Schistosoma mansoni. Genom Data. 2016;7:115–8.
Real F, Vidal RO, Carazzolle MF, Mondego JM, Costa GG, Herai RH, et al. The genome sequence of Leishmania (Leishmania) amazonensis: functional annotation and extended analysis of gene models. DNA Res. 2013;20(6):567–81.
Rana S, Dikhit MR, Rani M, Moharana KC, Sahoo GC, Das P. CPDB: cysteine protease annotation database in Leishmania species. Integr Biol (Camb). 2012;4(11):1351–7.
Dikhit MR, Nathasharma YP, Patel L, Rana SP, et al. A comparative protein function analysis database of different Leishmania strains. Bioinformation. 2011;6(1):20–2.
Waugh B, Ghosh A, Bhattacharyya D, Ghoshal N, et al. In silico work flow for scaffold hopping in Leishmania. BMC Res Notes. 2014;7:802.
http://www.ebi.ac.uk/compneur-srv/biomodels-main/MODEL1507180059
Chavali AK, Whittemore JD, Eddy JA, Williams KT, et al. Systems analysis of metabolism in the pathogenic trypanosomatid Leishmania major. Mol Syst Biol. 2008;4:177.
Hernandez-Santana YE, Ontoria E, Gonzalez-Garcia AC, Quispe-Ricalde MA, et al. The challenge of stability in high-throughput gene expression analysis: comprehensive selection and evaluation of reference genes for BALB/c mice spleen samples in the Leishmania infantum infection model. PLoS One. 2016;11(9):e0163219.
Patino LH, Ramirez JD. RNA-seq in kinetoplastids: a powerful tool for the understanding of the biology and host-pathogen interactions. Infect Genet Evol. 2017;49:273–82.
Kima PE. Leishmania molecules that mediate intracellular pathogenesis. Microbes Infect. 2014;16(9):721–6.
Clough E, Barrett T. The gene expression omnibus database. Methods Mol Biol. 2016;1418:93–110.
Beattie L, d’El-Rei Hermida M, Moore JW, Maroof A, et al. A transcriptomic network identified in uninfected macrophages responding to inflammation controls intracellular pathogen survival. Cell Host Microbe. 2013;14(3):357–68.
Fernandes MC, Dillon LA, Belew AT, Bravo HC. Dual transcriptome profiling of Leishmania-infected human macrophages reveals distinct reprogramming signatures. MBio. 2016;7(3):e00027.
Christensen SM, Dillon LA, Carvalho LP, Passos S, et al. Meta-transcriptome profiling of the human-Leishmania braziliensis cutaneous lesion. PLoS Negl Trop Dis. 2016;10(9):e0004992.
Kumar D, Singh R, Bhandari V, Kulshrestha A, et al. Biomarkers of antimony resistance: need for expression analysis of multiple genes to distinguish resistance phenotype in clinical isolates of Leishmania donovani. Parasitol Res. 2012;111(1):223–30.
Schriefer A, Wilson ME, Carvalho EM. Recent developments leading toward a paradigm switch in the diagnostic and therapeutic approach to human leishmaniasis. Curr Opin Infect Dis. 2008;21(5):483–8.
Braun P, Tasan M, Dreze M, Barrios-Rodiles M, et al. An experimentally derived confidence score for binary protein-protein interactions. Nat Methods. 2009;6(1):91–7.
Gupta SK, Gross R, Dandekar T. An antibiotic target ranking and prioritization pipeline combining sequence, structure and network-based approaches exemplified for Serratia marcescens. Gene. 2016;591(1):268–78.
Kaltdorf M, Srivastava M, Gupta SK, Liang C, et al. Systematic identification of anti-fungal drug targets by a metabolic network approach. Front Mol Biosci. 2016;3:22.
Walker DM, Oghumu S, Gupta G, McGwire BS, et al. Mechanisms of cellular invasion by intracellular parasites. Cell Mol Life Sci. 2014;71(7):1245–63.
Remmele CW, Luther CH, Balkenhol J, Dandekar T, et al. Integrated inference and evaluation of host-fungi interaction networks. Front Microbiol. 2015;6:764.
Kotlyar M, Pastrello C, Pivetta F, Lo Sardo A, et al. In silico prediction of physical protein interactions and characterization of interactome orphans. Nat Methods. 2015;12(1):79–84.
Bader JS, Chaudhuri A, Rothberg JM, Chant J. Gaining confidence in high-throughput protein interaction networks. Nat Biotechnol. 2004;22(1):78–85.
Lieke T, Nylen S, Eidsmo L, McMaster WR, et al. Leishmania surface protein gp63 binds directly to human natural killer cells and inhibits proliferation. Clin Exp Immunol. 2008;153(2):221–30.
Ammari MG, Gresham CR, McCarthy FM, Nanduri B. HPIDB 2.0: a curated database for host-pathogen interactions. Database. 2016;2016:baw103. https://doi.org/10.1093/database/baw103.
Durmus Tekir S, Cakir T, Ardic E, Sayilirbas AS, et al. PHISTO: pathogen-host interaction search tool. Bioinformatics. 2013;29(10):1357–8.
Rezende AM, Folador EL, Resende D de M, Ruiz JC. Computational prediction of protein-protein interactions in Leishmania predicted proteomes. PLoS One. 2012;7(12):e51304.
Gazestani VH, Nikpour N, Mehta V, Najafabadi HS, et al. A protein complex map of Trypanosoma brucei. PLoS Negl Trop Dis. 2016;10(3):e0004533.
Akhoon BA, Slathia PS, Sharma P, Gupta SK, et al. In silico identification of novel protective VSG antigens expressed by Trypanosoma brucei and an effort for designing a highly immunogenic DNA vaccine using IL-12 as adjuvant. Microb Pathog. 2011;51(1–2):77–87.
Murray HW, Berman JD, Davies CR, Saravia NG. Advances in leishmaniasis. Lancet. 2005;366(9496):1561–77.
Rezvan H, Moafi M. An overview on Leishmania vaccines: a narrative review article. Vet Res Forum. 2015;6(1):1–7.
Kedzierski L. Leishmaniasis vaccine: where are we today? J Glob Infect Dis. 2010;2(2):177–85.
Ahuja SS, Reddick RL, Sato N, Montalbo E, et al. Dendritic cell (DC)-based anti-infective strategies: DCs engineered to secrete IL-12 are a potent vaccine in a murine model of an intracellular infection. J Immunol. 1999;163(7):3890–7.
Gupta SK, Smita S, Sarangi AN, Srivastava M, et al. In silico CD4+ T-cell epitope prediction and HLA distribution analysis for the potential proteins of Neisseria meningitidis serogroup B--a clue for vaccine development. Vaccine. 2010;28(43):7092–7.
Costa CH, Peters NC, Maruyama SR, de Brito EC Jr, et al. Vaccines for the leishmaniases: proposals for a research agenda. PLoS Negl Trop Dis. 2011;5(3):e943.
Zaph C, Uzonna J, Beverley SM, Scott P. Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites. Nat Med. 2004;10(10):1104–10.
Brito RC, Guimaraes FG, Velloso JP, Correa-Oliveira R, et al. Immunoinformatics features linked to Leishmania vaccine development: data integration of experimental and in silico studies. Int J Mol Sci. 2017;18(2)
Del Tordello E, Serruto D. Functional genomics studies of the human pathogen Neisseria meningitidis. Brief Funct Genomics. 2013;12(4):328–40.
Gorringe AR, Pajon R. Bexsero: a multicomponent vaccine for prevention of meningococcal disease. Hum Vaccin Immunother. 2012;8(2):174–83.
Martin NG, Snape MD. A multicomponent serogroup B meningococcal vaccine is licensed for use in Europe: what do we know, and what are we yet to learn? Expert Rev Vaccines. 2013;12(8):837–58.
Gupta SK, Srivastava M, Akhoon BA, Smita S, et al. Identification of immunogenic consensus T-cell epitopes in globally distributed influenza-A H1N1 neuraminidase. Infect Genet Evol. 2011;11(2):308–19.
Gupta SK, Singh A, Srivastava M, Gupta SK, et al. In silico DNA vaccine designing against human papillomavirus (HPV) causing cervical cancer. Vaccine. 2009;28(1):120–31.
Ranjbar MM, Gupta SK, Ghorban K, Nabian S, et al. Designing and modeling of complex DNA vaccine based on tropomyosin protein of Boophilus genus tick. Appl Biochem Biotechnol. 2015;175(1):323–39.
Gupta SK, Srivastava M, Akhoon BA, Gupta SK, et al. In silico accelerated identification of structurally conserved CD8+ and CD4+ T-cell epitopes in high-risk HPV types. Infect Genet Evol. 2012;12(7):1513–8.
Baloria U, Akhoon BA, Gupta SK, Sharma S, et al. In silico proteomic characterization of human epidermal growth factor receptor 2 (HER-2) for the mapping of high affinity antigenic determinants against breast cancer. Amino Acids. 2012;42(4):1349–60.
Singh KP, Verma N, Akhoon BA, Bhatt V, et al. Sequence-based approach for rapid identification of cross-clade CD8+ T-cell vaccine candidates from all high-risk HPV strains. 3 Biotech. 2016;6(1):39.
Luo H, Lin Y, Gao F, Zhang CT, et al. DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements. Nucleic Acids Res. 2014;42(Database issue):D574–80.
Ravooru N, Ganji S, Sathyanarayanan N, Nagendra HG. In silico analysis of hypothetical proteins unveils putative metabolic pathways and essential genes in Leishmania donovani. Front Genet. 2014;5:291.
Jeong H, Mason SP, Barabasi AL, Oltvai ZN. Lethality and centrality in protein networks. Nature. 2001;411(6833):41–2.
Zirkel J, Cecil A, Schäfer F, Rahlfs S, et al. Analyzing thiol-dependent redox networks in the presence of methylene blue and other antimalarial agents with RT-PCR-supported in silico modeling. Bioinform Biol Insights. 2012;6:287–302.
Akhoon BA, Gupta SK, Dhaliwal G, Srivastava M, et al. Virtual screening of specific chemical compounds by exploring E.Coli NAD+−dependent DNA ligase as a target for antibacterial drug discovery. J Mol Model. 2011;17(2):265–73.
Srivastava M, Gupta SK, Abhilash PC, Singh N. Structure prediction and binding sites analysis of curcin protein of Jatropha curcas using computational approaches. J Mol Model. 2012;18(7):2971–9.
Srivastava M, Akhoon BA, Gupta SK, Gupta SK. Development of resistance against blackleg disease in Brassica oleracea var. botrytis through in silico methods. Fungal Genet Biol. 2010;47(10):800–8.
Akhoon BA, Gupta SK, Verma V, Dhaliwal G, et al. In silico designing and optimization of anti-breast cancer antibody mimetic oligopeptide targeting HER-2 in women. J Mol Graph Model. 2010;28(7):664–9.
Akhoon BA, Singh KP, Varshney M, Gupta SK, et al. Understanding the mechanism of atovaquone drug resistance in Plasmodium falciparum cytochrome b mutation Y268S using computational methods. PLoS One. 2014;9(10):e110041.
Gupta SK, Gupta SK, Smita S, Srivastava M, et al (2011) Computational analysis and modeling the effectiveness of ‘Zanamivir’ targeting neuraminidase protein in pandemic H1N1 strains. Infect Genet Evol 11 (5):1072–1082.
Song CM, Lim SJ, Tong JC. Recent advances in computer-aided drug design. Brief Bioinform. 2009;10(5):579–91.
Leelananda SP, Lindert S. Computational methods in drug discovery. Beilstein J Org Chem. 2016;12:2694–718.
Sliwoski G, Kothiwale S, Meiler J, Lowe EW Jr. Computational methods in drug discovery. Pharmacol Rev. 2014;66(1):334–95.
Field MC, Horn D, Fairlamb AH, Ferguson MA, et al. Anti-trypanosomatid drug discovery: an ongoing challenge and a continuing need. Nat Rev Microbiol. 2017;15(4):217–31.
Sundar S, Sinha PK, Rai M, Verma DK, et al. Comparison of short-course multidrug treatment with standard therapy for visceral leishmaniasis in India: an open-label, non-inferiority, randomised controlled trial. Lancet. 2011;377(9764):477–86.
Coulibaly B, Pritsch M, Bountogo M, Meissner PE, et al. Efficacy and safety of triple combination therapy with artesunate-amodiaquine-methylene blue for falciparum malaria in children: a randomized controlled trial in Burkina Faso. J Infect Dis. 2015;211(5):689–97.
Kunz M, Liang C, Nilla S, Cecil A, et al. The drug-minded protein interaction database (DrumPID) for efficient target analysis and drug development. Database. 2016;2016:baw041. https://doi.org/10.1093/database/baw041.
Fliri AF, Loging WT, Volkmann RA. Cause-effect relationships in medicine: a protein network perspective. Trends Pharmacol Sci. 2010;31(11):547–55.
Iorio F, Saez-Rodriguez J, di Bernardo D. Network based elucidation of drug response: from modulators to targets. BMC Syst Biol. 2013;7:139.
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Gupta, S.K., Dandekar, T. (2018). Bioinformatics in Leishmania Drug Design. In: Ponte-Sucre, A., Padrón-Nieves, M. (eds) Drug Resistance in Leishmania Parasites. Springer, Cham. https://doi.org/10.1007/978-3-319-74186-4_13
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