Combination of Repurposed Drug Diosmin with Amoxicillin-Clavulanic acid Causes Synergistic Inhibition of Mycobacterial Growth

Anju Choorakottayil Pushkaran,Anil Kumar Vasudevan,Chethampadi Gopi Mohan,Sudarslal Sadasivan Nair,Muralidharan Vanuopadath,Shantikumar V Nair,Raja Biswas,Vivek Vinod

doi:10.1038/s41598-019-43201-x

Abstract

Effective therapeutic regimens for the treatment of tuberculosis (TB) are limited. They are comprised of multiple drugs that inhibit the essential cellular pathways in Mycobacterium tuberculosis (Mtb). The present study investigates an approach which enables a combination of Amoxicillin-Clavulanic acid (AMC) and a repurposed drug for its synergistic effect towards TB treatment. We identified Diosmin (DIO), by targeting the active site residues of L,D-transpeptidase (Ldt) enzymes involved in Mtb cell wall biosynthesis by using a structure-based drug design method. DIO is rapidly converted into aglycone form Diosmetin (DMT) after oral administration. Binding of DIO or DMT towards Ldt enzymes was studied using molecular docking and bioassay techniques. Combination of DIO (or DMT) and AMC exhibited higher mycobactericidal activity against Mycobacterium marinum as compared to individual drugs. Scanning electron microscopy study of M. marinum treated with AMC-DIO and AMC-DMT showed marked cellular leakage. M. marinum infected Drosophila melanogaster fly model showed an increased fly survival of ~60% upon treatment with a combination of AMC and DIO (or DMT). Finally, the enhanced in vitro antimicrobial activity of AMC-DIO was validated against Mtb H37Ra and a MDR clinical isolate. Our results demonstrate the potential for AMC and DIO (or DMT) as a synergistic combination for the treatment of TB.

Highlights

Enzymes involved in the Mycobacterium tuberculosis (Mtb) cell wall biosynthetic pathway are absent in eukaryotic hosts, and are attractive targets for TB drug development
We proved that both DIO and DMT interact with LdtMt1 and LdtMt2 enzymes using molecular docking technique and in vitro bioassay
The in vivo efficacy of AMC-DIO or AMC-DMT combination of drugs was validated in M. marinum infected D. melanogaster fly model

Summary

Introduction

Enzymes involved in the Mtb cell wall biosynthetic pathway are absent in eukaryotic hosts, and are attractive targets for TB drug development. Www.nature.com/scientificreports cross-linkages (classical and non-classical) are observed in Mtb. In the classical type of PG cross-linkage, the carboxyl group of D-Ala[4] is linked to meso-DAP3 of the neighbouring pentapeptide (D-Ala4→meso-DAP3)[6]. Enterococcus faecium, Mycobacterium species and related species contain non-classical type PG cross-linkage formed between the neighbouring meso-DAP3 residues, which are catalysed by the L,D-transpeptidase (Ldt) enzymes leading to β-lactam resistance[6,8,9,10,11]. Drug repurposing/repositioning involves identifying novel druggable targets for the existing drugs and this includes Linezolid, Simvastatin, Metformin, Colesevelam, Zidovudine[25,26]. The main objective of the present study involves repurposing of FDA approved drugs for TB combination treatment, by integrating in silico, in vitro and in vivo techniques. The efficacy of AMC-DIO combination against Mtb and an MDR-Mtb clinical isolate was assessed

Methods

Results

Conclusion