Virtual Screening of Interaction of rpoB gene to Secondary-Line Anti- Leprosy Drugs for Rifampicin Resistant M. leprae using In-Silico Approach

Author Name(s): Mallika Lavania, Itu Singh, Ravindra P Turankar, Utpal Sengupta
Author Email: mallika.lavania@leprosymission.in

Abstract

Leprosy, caused by the pathogen Mycobacterium leprae, is still a public health threat in some parts of the world. It has been recently noted that several M. leprae isolates have developed secondary resistance to WHO recommended MDT especially to its bactericidal drug, Rifampicin. In this work, in silico molecular docking was performed using Molegro virtual Docker docking server, in order to find the binding interaction and other properties of fluoroquinolones and minocycline which are used as secondary line drugs for M. leprae rpoB gene, resistant to Rifampicin and could be of great help in selection of secondary line of anti-leprosy drugs using rpoB coded protein as a potential target.

Introduction

Leprosy, a chronic infectious disease, is caused by an obligate intracellular pathogen- Mycobacterium leprae (M. leprae). Although the prevalence of this disease has significantly gone down after the introduction of WHO regimen of Multidrug Therapy (MDT), the incidence remains a constant peril with approximately 211, 973 cases reported globally in 2015 out of which 127,334 cases were reported from India alone 1,2. Multi-drug therapy (MDT) includes Rifampicin (RIF), Dapsone and Clofazimine in 1982 by WHO 3. RIF is being used for the treatment of mycobacterial diseases such as tuberculosis and leprosy due to its efficient antimicrobial action. However, after its use for more than 3 decades’ drug resistance to RIF in leprosy is being reported4-8. Considering that the resistance to RIF might pose a problem in the treatment of leprosy, a study on  RIF and its interaction with resistant M. leprae mutants at the level of structure and function of bacterial DNA-dependent RNA polymerase (RNAP) and its involvement in the modulation of complex physiological pathways will be of utmost importance9 Mutations in the rpoB gene encoding the β subunit of RNA polymerase have been reported to result in resistance to RIF in several mycobacterial species including M. leprae10,11. Resistance to RIF is almost entirely coupled to mutations within a 279-bp region of the rpoB gene, called the RIF resistance-determining region (RRDR).

Since the late 1990s, multi-drug resistant (MDR) isolates of M. leprae, resistant to RIF and DDS, have emerged, the importance of Ofloxacin has been a focus for the treatment of MDR leprosy12. Fluoroquinolones (FQs) are bactericidal antibiotics currently in use as second-line drugs in the treatment of TB and leprosy13. FQs inhibit type II DNA topoisomerases, including DNA gyrase and topoisomerase IV14. However, as M. leprae possess only DNA gyrase15, it acts as the sole target for FQs. Recently, use of Ofloxacin has been very common for treatment of other infections and therefore there is an urgent reason to the search for newer potent anti-leprosy drugs and is a challenge for scientific community.

During the past 20 years, a number of newer antimicrobial agents, among quinolones (e.g., Pefloxacin, Ofloxacin, Sparfloxacin, and Moxifloxacin), the macrolides (e.g., clarithromycin), and the tetracyclines (e.g., Minocycline), exhibited fairly good in vivo activity against experimental mouse footpad infections with M. leprae and in human trials16,17. Among these newer antimicrobial agents, Moxifloxacin has been demonstrated to display very powerful bactericidal activity against M. leprae that is virtually identical to that of rifampicin18.

Minocycline, an alkylated amino-tetracycline, widely used for bacterial infections, has also shown to have bactericidal activity against M. leprae foot pad growth in mice19. Furthermore, in vitro studies20 have also shown that minocycline inhibited the metabolic activity of M. leprae.

Lacking direct evidence for the mechanisms of M.leprae’s resistance to most of the anti-leprosy drugs, our current understanding is based on studies carried out in M. tuberculosis21 other bacteria, and a few studies with M. leprae genes in surrogate hosts22.

This study focusses here the bioinformatics approach for the in silico virtual screening of some anti-mycobacterial agents (Ofloxacin, Sparfloxacin, Moxifloxacin, and Minocycline) against rpoB protein in M.leprae using Molegro Virtual Docker 2011.5.0 (MVD). Generally, all RRDR mutations are coupled with different level of RIF resistance among clinical isolates. However, the Ser456Leu substitution, a most commonly found mutation, is correlated with a high level of resistance with molecular as well as mouse foot pad studies. Therefore, in the present study we used this mutant sequence to dock with secondary line of drugs to determine   the best possible treatment for the RIF resistant M. leprae. In the present investigation, we screened fluoroquinolone such as Ofloxacin, Sparfloxacin, and Moxifloxacin and Minocycline based on better Interaction energy and MolDock score, to find out their efficiency on resistant strain through in silico study.

Conclusion

In conclusion, we are suggesting that, a fast, efficient and promising alternative bioinformatics based procedure for the virtual screening and discovery of best drug for proteins is very essential for determining its anti-M. leprae activity. We hence conclude that determining the in silico predictions by the binding efficacies of inhibitory compounds with DRDR region of new M. leprae isolates can be a fast method to select the drug of choice for an effective line of in treatment of leprosy for its further eradication. 

Acknowledgements

We gratefully thank Dr. Masanori Matsuoka, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan for providing reference strains for current study. We extend our special thanks to BioAxis DNA Research Centre (P) Ltd Hyderabad, India for protein structure modeling and docking experiments. We thank Dr. Annamma S John, Head Research and Training, TLMTI for continued support and encouragement.

Financial Support: This work was supported by funding from The Leprosy Mission Trust India and TLM England and Wales.

Conflict of Interest: None to declare.

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