Integration of Bioinformatics and in vitro Analysis Reveal Anti-leishmanial Effects of Azithromycin and Nystatin

Author(s): Irum Jehangir , Syed Farhan Ahmad* , Maryam Jehangir , Anwar Jamal , Momin Khan .

Journal Name: Current Bioinformatics

Volume 14 , Issue 5 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Background: Leishmaniasis is the major cause of mortality in under-developed countries. One of the main problems in leishmaniasis is the limited number of drug options, resistance and side effects. Such a situation requires to study the new chemical series with anti-leishmanial activity.

Objective: To assess the anti-leishmanial activity of antibacterial and antifungal drugs.

Methods: We have applied an integrative approach based on computational and in vitro methods to elucidate the efficacy of different antibacterial and antifungal drugs against Leishmania tropica (KWH23). Firstly these compounds were analyzed using in silico molecular docking. This analysis showed that the nystatin and azithromycin interacted with the active site amino acids of the target protein leishmanolysin. The nystatin, followed by azithromycin, produced the lowest binding energies indicating their inhibitive activity against the target. The efficacy of the docked drugs was further validated in vitro which showed that our bioinformatics based predictions completely agreed with experimental results. Stock solutions of drugs, media preparation and parasites cultures were performed according to the standard in-vitro protocol.

Results: We found that the half maximal inhibitory concentration (IC50) value of dosage form of nystatin (10,000,00 U) and pure nystatin was 0.05701 µM and 0.00324 µM respectively. The IC50 value of combined azithromycin and nystatin (dosage and pure form) was 0.156 µg/ml and 0.0023 µg /ml (0.00248 µM) respectively. It was observed that IC50 value of nystatin is better than azithromycin and pure form of drugs had significant activity than the dosage form of drugs.

Conclusion: From these results, it was also proven that pure drugs combination result is much better than all tested drugs results. The results of both in vitro and in silico studies clearly indicated that comparatively, nystatin is the potential candidate drug in combat against Leishmania tropica.

Keywords: Drugs, Leishmania tropica, Docking, leishmanolysin, IC50, in vitro.

Allison M. Leishmaniasis The Cambridge world history of human disease. Cambridge University Press 1993; pp. 832-4.
Murray HW, Berman JD, Davies CR, Saravia NG. Advances in leishmaniasis. Lancet 2005; 366(9496): 1561-77.
Alvar J, Aparicio P, Aseffa A, et al. The relationship between leishmaniasis and AIDS: the second 10 years. Clin Microbiol Rev 2008; 21(2): 334-59.
Mahmoudzadeh-Niknam H, Kiaei SS, Iravani D. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice. Korean J Parasitol 2007; 45(2): 103-9.
Alvar J, Vélez ID, Bern C, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 2012; 7(5): e35671.
Pérez-Victoria JM, Pérez-Victoria FJ, Parodi-Talice A, et al. Alkyl-lysophospholipid resistance in multidrug-resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like transporter modulator. Antimicrob Agents Chemother 2001; 45(9): 2468-74.
Armijos RX, Weigel MM, Calvopina M, Hidalgo A, Cevallos W, Correa J. Safety, immunogenecity, and efficacy of an autoclaved Leishmania amazonensis vaccine plus BCG adjuvant against New World cutaneous leishmaniasis. Vaccine 2004; 22(9-10): 1320-6.
Coler RN, Reed SG. Second-generation vaccines against leishmaniasis. Trends Parasitol 2005; 21(5): 244-9.
Croft SL, Sundar S, Fairlamb AH. Drug resistance in leishmaniasis. Clin Microbiol Rev 2006; 19(1): 111-26.
Bianchini G, Bocedi A, Ascenzi P, Gavuzzo E, Mazza F, Aschi M. Molecular dynamics simulation of Leishmania major surface metalloprotease GP63 (leishmanolysin). Proteins 2006; 64(2): 385-90.
Yao C, Donelson JE, Wilson ME. The major surface protease (MSP or GP63) of Leishmania sp. Biosynthesis, regulation of expression, and function. Mol Biochem Parasitol 2003; 132(1): 1-16.
Schlagenhauf E, Etges R, Metcalf P. The crystal structure of the Leishmania major surface proteinase leishmanolysin (GP63). Structure 1998; 6(8): 1035-46.
Yiallouros I, Kappelhoff R, Schilling O, et al. Activation mechanism of pro-astacin: role of the pro-peptide, tryptic and autoproteolytic cleavage and importance of precise amino-terminal processing. J Mol Biol 2002; 324(2): 237-46.
Leite ACL, Espíndola JWP, de Oliveira Cardoso MV, de Oliveira Filho GB. Privileged Structures in the design of Potential Drug Candidates for Neglected Diseases. Curr Med Chem 2017.
Salas JM, Caballero AB, Esteban-Parra GM, Méndez-Arriaga JM. Leishmanicidal and trypanocidal activity of metal complexes with 1, 2, 4-Triazolo[1, 5-A]pyrimidines: Insights on their therapeutic potential against leishmaniasis and chagas disease. Curr Med Chem 2017; 24(25): 2796-806.
Loureiro I, Faria J, Santarem N, et al. Potential drug targets in the pentose phosphate pathway of trypanosomatids. Curr Med Chem 2018; 25(39): 5239-65.
Rahim F. An in silico development of selective inhibitor for histamine receptors. Biotechnology 2010; 9(2): 157-63.
Levin NMB, Pintro VO, Bitencourt-Ferreira G, de Mattos BB, de Castro Silvério A, de Azevedo WF Jr. Development of CDK-targeted scoring functions for prediction of binding affinity. Biophys Chem 2018; 235: 1-8.
Xavier MM, Heck GS, Avila MB, et al. SAnDReS a computational tool for statistical analysis of docking results and development of scoring functions. Comb Chem High Throughput Screen 2016; 19(10): 801-12.
Amaral MEA, Nery LR, Leite CE, de Azevedo WF Jr, Campos MM. Pre-clinical effects of metformin and aspirin on the cell lines of different breast cancer subtypes. Invest New Drugs 2018; 36(5): 782-96.
Kaur J, Kumar P, Tyagi S, et al. In silico screening, structure-activity relationship, and biologic evaluation of selective pteridine reductase inhibitors targeting visceral leishmaniasis. Antimicrob Agents Chemother 2011; 55(2): 659-66.
Westbrook J, Feng Z, Chen L, et al. The protein data bank and structural genomics. Nucleic Acids Res 2003; 31(1): 489-91.
Schlagenhauf E, Etges R, Metcalf P. The crystal structure of the Leishmania major surface proteinase leishmanolysin (GP63). Structure 1998; 6(8): 1035-46.
Morris GM, Goodsell DS, Halliday RS, et al. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 1998; 19(14): 1639-62.
Ngan CH, Hall DR, Zerbe B, Grove LE, Kozakov D, Vajda S. FTSite: high accuracy detection of ligand binding sites on unbound protein structures. Bioinformatics 2012; 28(2): 286-7.
Huey R, Morris GM, Olson AJ, Goodsell DS. A semiempirical free energy force field with charge-based desolvation. J Comput Chem 2007; 28(6): 1145-52.
Wallace AC, Laskowski RA, Thornton JM. LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions. Protein Eng 1995; 8(2): 127-34.
The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.
Huang CC, Couch GS, Pettersen EF, Ferrin TE. Chimera: An extensible molecular modeling application constructed using standard components. Pac Symp Biocomput 1996; 1: 724.
de Oliveira-Silva F, de Morais-Teixeira E, Rabello A. Antileishmanial activity of azithromycin against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi. Am J Trop Med Hyg 2008; 78(5): 745-9.
Protocols for handling and working with leishmania species (Working with Leishmania for Dummies). 2008, Mottram laboratories.
de Azevedo WF Jr, Soares MB. Selection of targets for drug development against protozoan parasites. Curr Drug Targets 2009; 10(3): 193-201.
Das BB, Ganguly A, Majumder HK. DNA topoisomerases of Leishmania: the potential targets for anti-leishmanial therapy. Adv Exp Med Biol 2008; 625: 103-15.
Carrero-Lérida J, Pérez-Moreno G, Castillo-Acosta VM, Ruiz-Pérez LM, González-Pacanowska D. Intracellular location of the early steps of the isoprenoid biosynthetic pathway in the trypanosomatids Leishmania major and Trypanosoma brucei. Int J Parasitol 2009; 39(3): 307-14.
Andrés FF, Stanley W, Carlos M. Current Advances in Computational Strategies for Drug Discovery in Leishmaniasis, Current Topics in Tropical Medicine, Dr. Alfonso Rodriguez-Morales (Ed.), 2012; InTech.
Talele TT, Khedkar SA, Rigby AC. Successful applications of computer aided drug discovery: moving drugs from concept to the clinic. Curr Top Med Chem 2010; 10(1): 127-41.
Jorgensen WL. The Many Roles of Computation in Drug Discovery. Science 2004; 303(5665): 1813-8.
Ogungbe IV, Ng JD, Setzer WN. Interactions of antiparasitic alkaloids with Leishmania protein targets: a molecular docking analysis. Future Med Chem 2013; 5(15): 1777-99.
Singh S, Vijaya Prabhu S, Suryanarayanan V, Bhardwaj R, Singh SK, Dubey VK. Molecular docking and structure-based virtual screening studies of potential drug target, CAAX prenyl proteases, of Leishmania donovani. J Biomol Struct Dyn 2016; 34(11): 2367-86.
Mutlu O. In silico molecular modeling and docking studies on the leishmanial tryparedoxin peroxidase. Braz Arch Biol Technol 2014; 57(2): 244-52.
Morris GM, Goodsell DS, Halliday RS, et al. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 1998; 19(14): 1639-62.
Huey R, Morris GM, Olson AJ, Goodsell DS. A semiempirical free energy force field with charge-based desolvation. J Comput Chem 2007; 28(6): 1145-52.
Goodsell DS, Morris GM, Olson AJ. Automated docking of flexible ligands: applications of AutoDock. J Mol Recognit 1996; 9(1): 1-5.
Thomsen R, Christensen MH. MolDock: A new technique for high-accuracy molecular docking. J Med Chem 2006; 49(11): 3315-21.
Cantin L, Chamberland S. In vitro evaluation of the activities of azithromycin alone and combined with pyrimethamine against Toxoplasma gondii. Antimicrob Agents Chemother 1993; 37(9): 1993-6.
Ohrt C, Willingmyre GD, Lee P, Knirsch C, Milhous W. Assessment of azithromycin in combination with other antimalarial drugs against Plasmodium falciparum in vitro. Antimicrob Agents Chemother 2002; 46(8): 2518-24.
Blais J, Garneau V, Chamberland S. Inhibition of Toxoplasma gondii protein synthesis by azithromycin. Antimicrob Agents Chemother 1993; 37(8): 1701-3.
Tateda K, Ishii Y, Matsumoto T, et al. Direct evidence for antipseudomonal activity of macrolides: exposure-dependent bactericidal activity and inhibition of protein synthesis by erythromycin, clarithromycin, and azithromycin. Antimicrob Agents Chemother 1996; 40(10): 2271-5.
Krolewiecki A, Leon S, Scott P, Abraham D. Activity of azithromycin against Leishmania major in vitro and in vivo. Am J Trop Med Hyg 2002; 67(3): 273-7.
Ali SA, Iqbal J. Nabeel, et al. Leishmanicidal activity of Nystatin (mycostatin): a potent polyene compound. J Pak Med Assoc 1997; 47(10): 246-8.
Michel GW. Nystatin. Anal Profiles Drug Subst 1977; 6: 341-421.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [450 - 459]
Pages: 10
DOI: 10.2174/1574893614666181217142344
Price: $58

Article Metrics

PDF: 20