Molecular modelling and simulation studies of the Mycobacterium tuberculosis multidrug efflux pump protein Rv1258c

Abstract

Mycobacterial efflux pumps play a major role in the emergence of antimycobacterial drug resistance. Of particular interest is the proteinaceous multi-drug efflux pump protein Rv1258c that encodes a tetracycline/aminoglycoside resistance (TAP-2)-like efflux pump which is active in susceptible and drug resistant Mycobacterium tuberculosis. Rv1258c is implicated in drug resistance to numerous antimycobacterials including first line drugs rifampicin and isoniazid as well as fluoroquinolone and aminoglycoside antibiotic classes. To date, compounds like verapamil and piperine have been shown to inhibit Rv1258c but no direct evidence for binding or mode of action exist. Therefore in the present study we generated an accurate 3D model of Rv1258c using MODELLER and validated its structure using molecular dynamic simulation studies with GROMACS software. The 3D-structures of Rv1258c and the homologous template 1pw4 were simulated within a POPE/POPG lipid bilayer and found to behave similar. Another important finding was the identification of one local energy minima state of the apo protein, which speaks to the flexibility of the protein and will be investigated further. Extraction of one of the open channel conformations of Rv1258c and blind docking of various structurally diverse putative inhibitors and substrates, allowed for the identification of a probable binding site. Spectinamide was found to bind to a different location on the outside surface of the protein suggesting its ability to avoid the efflux channel. We further identified 246 putative compounds that showed higher binding affinity values to Rv1258c compared to piperine and verapamil. Interaction analysis of the top 20 purchasable compounds identified crucial hydrogen bond interactions with Ser26, Ser45 and Glu243 as well as a pi-pi stacking interaction with Trp32 that accounted for the strong affinity of these compounds for Rv1258c. Future studies will entail purchasing a number of compounds for in vitro activity testing against Mycobacterium tuberculosis.

Molecular characterization of pre-extensive drug resistant Mycobacterium tuberculosis in Northeast Brazil

“…Regarding aminoglycosides, no mutations were found at codon 1401 of the rrs gene with 43.9% of isolates resistant to STR in the BACTEC system. However, mutations have also been described in other genes, such as rpsL and gidB 7 , and other genes related to efflux pumps 34 .…”

Protein–protein interaction of Rv0148 with Htdy and its predicted role towards drug resistance in Mycobacterium tuberculosis

“…The template and the target sequence were aligned using the malign program in Modeller software [31]. Homology model of Rv0148 was constructed using Modeller and the quality of the model was further analysed using PROCHECK [32], ProSA-web [33] and verify 3D [34]. The model was also validated using root mean square deviation (RMSD).…”

“…Docking was performed using NAD cofactor as the ligand with the predicted model of Rv0148 to understand its mode of interaction within the binding site. GOLD v5.2 (genetic optimization for ligand docking) is a widely used protein-ligand docking program [34]. GOLD uses a genetic algorithm methodology for protein-ligand docking that allows full ligand and partial protein flexibility.…”

Is the efflux pump inhibitor Verapamil a potential booster for isoniazid against Mycobacterium tuberculosis?

“…In the search for new treatments in cases of resistant TB, in which classical anti-TB drugs are not effective, the combinatory use of efflux pumps inhibitors (EPI), such as VP, has become the subject of some studies (Gupta et al, 2010a;Rodrigues et al, 2012;Cloete et al, 2018;Sharma et al, 2010). However, the experimental procedures for understanding EPI activity in M. tuberculosis are mostly limited to laboratory strains and few have been performed with clinical isolates (Machado et al, 2012), and divergence in results have been observed.…”