Identification of Mutations Associated With Macozinone-Resistant in Mycobacterium Tuberculosis

Abstract

Macozinone is identified as a drug candidate and is currently under clinical development for the treatment of tuberculosis, but the mutations conferring resistance to Macozinone remain inadequately characterized. Herein, we investigated the Macozinone -resistance -associated mutations through selecting resistant isolates in vitro. Macozinone-resistant isolates were obtained through induction in vitro. The level of Macozinone -resistant strains was confirmed by MABA test. PCR sequencing analysis was carried out on dprE1 gene. Whole Genome Sequencing was performed to identify mutations associated with Macozinone -resistance. The totals of isolates obtained at Macozinone concentrations of 6.4ng/ml, 25.6ng/ml, 50ng/ml and 100ng/ml were 49, 20, 20 and 4 respectively. Among the 49 strains obtained by 6.4ng/ml Macozinone only one strain had C387S mutation in dprE1. C387S is only occurred in high-level resistant isolates (MIC > 500ng/ml). Meanwhile high-level resistance to Macozinone can occur in strains induced at 6.4ng/ml and the frequency of occurrence is low (1/49, 2.04%). The MIC90 of other strains except the strains carrying C387S mutation is at the same level (11.5ng/ml > MIC90 > 2ng/ml). The G61A or G248A mutations in dprE1 was discovered for the first time. Other gene mutations (rv0678, rrs, mbtF, rv2956, et al) were found in low-level resistant strains.Conclusions: High-level resistant isolates can be produced at low concentration of Macozinone. C387S mutation in dprE1 is directly related to high-level resistance.There may be new mechanisms involved in Macozinone-resistance independent of dprE1 mutations.

The evolution of antibiotic resistance is associated with collateral drug phenotypes inMycobacterium tuberculosis

“…In line with earlier findings, mutations in rv0678 produced cross resistance between CFZ and BDQ 17 . Our study also supports these prior findings that mutations in rv0678 provide resistance to PBTZ-169 (i.e., Macozinone) 32,56 . We also identify a broader range of antibiotics to which mutations in rv0678 provide low-level resistance including Q203.…”

Mutations in rv0678 confer low-level resistance to benzothiazinone DprE1 inhibitors in M. tuberculosis

“…Resistance is also observed in Mtb strains directly overexpressing the mmpS5/L5 operon, suggesting that efflux of BTZs is the relevant mechanism of resistance. Consistent with our findings, another recent study isolated PBTZ169-resistant mutants on progressively higher drug concentrations and identified a number of clones harboring mutations in rv0678 (55). Although the authors do not test the individual impact of each rv0678 mutation, the results presented here suggest that these are likely to be bona fide , low-level PBTZ169 resistance mutations.…”

How Mycobacterium tuberculosis drug resistance has shaped anti-tubercular drug discovery

“…Mutations in Rv0678 lead to upregulation of the MmpL5/ MmpS5 efflux system and increased efflux of the drug. Since this system also effluxes other drug classes, including azoles, clofazimine and macozinone (Hartkoorn et al, 2014;Chen et al, 2022;Guo et al, 2022), the appearance of these mutations in clinical isolates will lead to cross-resistance to multiple antimycobacterial classes. Similarly mutations in pepQ result in resistance to other agents such as macozinone (Chen et al, 2022;Guo et al, 2022), This underscores the need to determine mechanisms of resistance for new agents for both low-level and high-level resistance.…”

“…Since this system also effluxes other drug classes, including azoles, clofazimine and macozinone (Hartkoorn et al, 2014;Chen et al, 2022;Guo et al, 2022), the appearance of these mutations in clinical isolates will lead to cross-resistance to multiple antimycobacterial classes. Similarly mutations in pepQ result in resistance to other agents such as macozinone (Chen et al, 2022;Guo et al, 2022), This underscores the need to determine mechanisms of resistance for new agents for both low-level and high-level resistance. In addition, since Rv0678 mutations occur in clinical isolates (Andries et al, 2014), mutant strains with these SNPs should form part of any clinical isolate panel used for routine testing during drug discovery.…”