Piperidine-4-Carboxamides Target DNA Gyrase in Mycobacterium abscessus

Abstract

New, more-effective drugs for the treatment of lung disease caused by nontuberculous mycobacteria (NTM) are needed. Among NTM opportunistic pathogens, Mycobacterium abscessus is the most difficult to cure and intrinsically multidrug resistant. In a whole-cell screen of a compound collection active against Mycobacterium tuberculosis, we previously identified the piperidine-4-carboxamide (P4C) MMV688844 (844) as a hit against M. abscessus. Here, we identified a more potent analog of 844 and showed that both the parent and improved analog retain activity against strains representing all three subspecies of the M. abscessus complex. Furthermore, P4Cs showed bactericidal and antibiofilm activity. Spontaneous resistance against the P4Cs emerged at a frequency of 10^-8/CFU and mapped to gyrA and gyrB encoding the subunits of DNA gyrase. Biochemical studies with recombinant M. abscessus DNA gyrase showed that P4Cs inhibit the wild-type enzyme but not the P4C-resistant mutant. P4C-resistant strains showed limited cross-resistance to the fluoroquinolone moxifloxacin, which is in clinical use for the treatment of macrolide-resistant M. abscessus disease, and no cross-resistance to the benzimidazole SPR719, a novel DNA gyrase inhibitor in clinical development for the treatment of mycobacterial diseases. Analyses of P4Cs in recA promoter-based DNA damage reporter strains showed induction of recA promoter activity in the wild type but not in the P4C-resistant mutant background. This indicates that P4Cs, similar to fluoroquinolones, cause DNA gyrase-mediated DNA damage. Together, our results show that P4Cs present a novel class of mycobacterial DNA gyrase inhibitors with attractive antimicrobial activities against the M. abscessus complex.

Keywords: DNA gyrase; MMV688844; Mycobacterium abscessus; NTM; nontuberculous mycobacteria.

FIG 1

Structures of piperidine-4-carboxamides 844 and 844-TFM, and the DNA gyrase inhibitors moxifloxacin (MXF) and SPR719.

FIG 2

Bactericidal and antibiofilm activity of 844 and 844-TFM against M. abscessus. (A) Exponentially growing suspension cultures of M. abscessus subsp. abscessus ATCC 19977 were treated with 1×, 4×, and 8× MIC of 844 or 844-TFM, moxifloxacin (MXF), SPR719, or clarithromycin (CLR), and CFU were enumerated by plating samples on agar after 2 and 3 days. (B) Exponentially growing biofilm cultures were treated with 1×, 4×, and 8× MIC of 844, 844-TFM, MXF, SPR719, or CLR, and surface-attached CFU were enumerated by suspending bacteria and plating on agar after 2 and 3 days. MXF, SPR719, and CLR are included for comparison (MXF, SPR719) or as control (CLR). Experiments in panels A and B were carried out three times independently, and the results are represented as mean values with the standard deviations displayed as error bars. A two-way ANOVA multiple comparison test was performed using GraphPad Prism 8 software to compare treated groups with untreated day 0 CFU.

FIG 3

Complementation of P4C-resistant M. abscessus with wild-type gyrAB. P4C-resistant M. abscessus subsp. abscessus ATCC 19977 884-TFM^r-1 harboring a D91N mutation in GyrA (Table 2) was transformed with plasmid pMV262 not carrying an insert (pMV262-empty; control) or with pMV262 carrying wild-type gyrAB constitutively expressed from the hsp60 promoter. Cultures were either grown without drug (DF, drug free) or treated with MIC of 844 (8 μM) or 844-TFM (1.5 μM) for 2 days (D2), and growth was measured by OD₆₀₀ determination. Clarithromycin (CLR) treatment at MIC (2 μM) was used as control. The experiments were carried out three times independently, and the results are represented as mean values with the standard deviations displayed as error bars. A two-way ANOVA with Sidak’s multiple comparison test was performed to compare the two groups using GraphPad Prism 8 software.

FIG 4

Effect of 844 and 844-TFM on supercoiling activity of M. abscessus wild type and GyrA D91N mutant recombinant DNA gyrase. Relaxed pBR322 plasmid was used as substrate to measure the effect of the P4Cs on the supercoiling activity of wild type or GyrA D91N mutant recombinant DNA gyrase derived from M. abscessus subsp. abscessus ATCC 19977 and M. abscessus subsp. abscessus ATCC 19977 844-TFM^r-1, respectively (Table 2). (A) The conversion of relaxed (R) into supercoiled (SC) plasmid was visualized by agarose gel electrophoresis. OC, open circular plasmid; Gyrase −, reaction without added enzyme showing unaltered substrate; Gyrase +, drug-free reaction with added enzyme showing conversion of relaxed plasmid into its supercoiled form. Triangles indicate drug containing reactions with drug concentrations ranging from 50, 25, 12.5, 6.2, 3.1, 1.5, 0.7, 0.39, 0.19, 0.09, to 0.05 μM. Moxifloxacin (MXF) and SPR719 were used as positive and clarithromycin (CLR) as negative controls. The bands presenting supercoiled plasmid were quantified by Invitrogen iBright FL1000 imaging system to determine IC₅₀ for the inhibitors. (B) Percent inhibition was calculated in reference to drug-free reactions. IC₅₀ values were determined using nonregression model fit of GraphPad Prism 8.0.1 software. 884 and 844-TFM inhibited wild-type DNA gyrase with IC₅₀s of 4.6 μM and 1.9 μM. No inhibition of activity was observed when GyrA D91N mutant gyrase was used. MXF and SPR719 inhibited wild-type gyrase with IC₅₀s of 17 μM and 2.3 μM, respectively. The D91N mutant version of DNA gyrase was inhibited by SPR719 with the same IC₅₀ as the wild-type enzyme. The IC₅₀ of MXF was higher than 50 μM for the mutant enzyme. The experiments were repeated three times, independently yielding the same results and a representative example is shown in panel A. Means and standard deviations are shown in panel B.

FIG 5

Effect of 844 and 844-TFM on recA promoter-driven LuxCDABE operon expression in wild-type and P4C-resistant gyrA D91N mutant M. abscessus. (A) Exponentially growing cultures of wild-type M. abscessus subsp. abscessus ATCC 19977 (black, Mab WT) or M. abscessus subsp. abscessus ATCC 19977 884-TFM^r-1 (Table 2), carrying the D91N allele of gyrA (red, Mab D91N), both harboring a LuxCDABE expressed under the control of the DNA damage inducible recA promoter, were treated with increasing concentration of 844 or 844-TFM for 4 h, and relative luminescence (RLU) was measured as a readout for recA promoter activity. The experiments were also carried out for moxifloxacin (MXF) and SPR719 for comparison and for clarithromycin (CLR) as negative control. (B) Growth inhibitory effects of the drugs used in panel A determined via OD₆₀₀ measurement after treatment of cultures for 3 days. (A, B) experiments were carried out three times independently, and the results are represented as mean values with the standard deviations displayed as error bars.

FIG 6

Concentration-time profile and plasma stability of 884-TFM. (A) Concentration-time profile of 884-TFM following a single intravenous (i.v.), subcutaneous (s.c.), or oral (p.o.) dose of 5, 25, and 25 mg/kg, respectively, to CD-1 mice. MIC indicates 884-TFM MIC (=3.5 μM) (Table 1) for M. abscessus subsp. abscessus K21, the strain we employ in our murine infection model to determine efficacy of drugs (43). (B) Compound stability in mouse, rabbit, monkey, and human plasma over 24 h. For panels A and B, mean and standard deviations (n = 3) are shown.