Developing Synergistic Drug Combinations To Restore Antibiotic Sensitivity in Drug-Resistant Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis. We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S ribosomal RNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis. Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant.These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

Rv0684/fusA1, an Essential Gene, Is the Target of Fusidic Acid and Its Derivatives in Mycobacterium tuberculosis

“…Earlier, we observed that resistance to FA in M. tuberculosis occurs due to the mutation in fusA1-encoded elongation factor G (EF-G). 14 To investigate whether FusA1 is the primary target of FA, we evaluated whether conditional depletion of FusA1 in M. tuberculosis would sensitize M. tuberculosis to the growth inhibitory effects of FA and its analogs: it would also confirm whether FA retains on-target activity against FusA1 in whole M. tuberculosis cells. To achieve this, we constructed an anhydrotetracycline (ATc)-regulated f usA1 conditional knockdown mutant in the Tet-OFF configuration, as described previously.…”

Antimycobacterial Activity, Synergism, and Mechanism of Action Evaluation of Novel Polycyclic Amines against Mycobacterium tuberculosis

“…Unfortunately, mainly due to extensive efflux by bacterial efflux pumps (specifically Rv1258c in M. tuberculosis ), spectinomycin lacks significant antibacterial activity [ 15 , 40 , 41 ]. The activity of spectinomycin is increased substantially under conditions where efflux pump activity is inhibited [ 42 ] making it a useful agent to screen for possible efflux pump inhibitory activity using synergism assays. All compounds apart from compounds 14 and 15 , which were excluded due to inherent antimycobacterial activity, were screened for synergism with spectinomycin using a checkerboard assay.…”

“…Possible drug interactions of the various test compounds and spectinomycin were investigated utilizing a slightly modified standard two-dimensional (2D) checkerboard assay [ 42 ]. Serially diluted drugs were dispensed along the x -axis and y -axis of 96-well microtiter plates, respectively, at a starting concentration 100 times the final concentration.…”

“…Serially diluted drugs were dispensed along the x -axis and y -axis of 96-well microtiter plates, respectively, at a starting concentration 100 times the final concentration. The assays were performed as previously described [ 42 ], and results were reported as a percentage growth inhibition at various drug concentration combinations.…”

Intracellular Accumulation of Novel and Clinically Used TB Drugs Potentiates Intracellular Synergy

“…6A and C ). Drug interactions were assessed by checkerboard dilution in a 96-well format with slight modification of the method described by Omollo et al ( 51 ). PhX1 was serially diluted in RPMI medium along the x axis (columns 3 to 11), with synergistic concentrations ranging from 25 to 0.19 μM, while WHN296 was serially diluted across the y axis (rows B to H), with synergistic concentrations ranging from 100 to 0.15 μM.…”