Activity of Tricyclic Pyrrolopyrimidine Gyrase B Inhibitor against Mycobacterium abscessus

Abstract

Tricyclic pyrrolopyrimidines (TPPs) are a new class of antibacterials inhibiting the ATPase of DNA gyrase. TPP8, a representative of this class, is active against Mycobacterium abscessus in vitro. Spontaneous TPP8 resistance mutations mapped to the ATPase domain of M. abscessus DNA gyrase, and the compound inhibited DNA supercoiling activity of recombinant M. abscessus enzyme. Further profiling of TPP8 in macrophage and mouse infection studies demonstrated proof-of-concept activity against M. abscessus ex vivo and in vivo.

Keywords: DNA gyrase; NTM; SPR719; nontuberculous mycobacteria.

FIG 1

Structure and DNA gyrase inhibition activity of TPP8. (A) Structure of TPP8 and SPR719 (15, 20). (B) Effect of TPP8 and comparator compounds on the DNA supercoiling activity of recombinant M. abscessus ATCC 19977 DNA gyrase. Relaxed pBR322 plasmid was used as the substrate to measure the effect of compounds on the supercoiling activity of M. abscessus DNA gyrase as described previously (23). The conversion of relaxed (R) into supercoiled (SC) plasmid by DNA gyrase was visualized by agarose gel electrophoresis. OC, open circular plasmid. Lane 13, Gyrase -, reaction mix without added enzyme showing unaltered substrate. Lane 12, Gyrase +, reaction mix with added enzyme (without drug) showing conversion of relaxed plasmid into its supercoiled form. Lanes 1 to 11 show the effect of decreasing drug concentrations. The concentration ranges are as follows: TPP8, 1.5, 0.75, 0.37, 0.18, 0.09, 0.04, 0.02, 0.01, 0.005, 0.002, and 0.001 μM; SPR719, moxifloxacin (MXF), and clarithromycin (CLR), 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09, and 0.04 μM. The experiments were repeated three times independently, yielding similar results, and a representative example is shown. (C) Quantitative inhibition of DNA gyrase supercoiling activity by TPP8 and comparator drugs. The bands obtained from the three experiments represented in panel B were quantified by the Invitrogen iBright FL1000 imaging system to determine half-maximal inhibitory concentrations (IC₅₀) as described previously (23). Means and standard deviations are shown. TPP8 inhibited DNA gyrase with an IC₅₀ of 0.3 μM. SPR719 and MXF inhibited the enzyme with an IC₅₀ of 1 μM and 3 μM, respectively (23). IC₅₀ derived from panel C are indicated by asterisks in panel B. CLR, included as a negative control, did not affect the supercoiling activity of the enzyme.

FIG 2

Activity of TPP8 against M. abscessus growing in broth and in THP-1-derived macrophages. (A) To determine whether TPP8 displays bactericidal activity in vitro, 1-mL cultures of M. abscessus ATCC 19977 growing in Middlebrook 7H9 in tubes (11) were treated with MIC multiples of TPP8, SPR719, moxifloxacin (MXF), or clarithromycin (CLR). CFU were enumerated by plating samples on Middlebrook 7H10 agar. The growth kinetics of drug-free controls are shown on the left, and the effects of TPP8 and comparators on CFU reduction are shown after 3 days of treatment. As MICs measured in tubes can be different from those measured in 96-well plates, tube MICs were measured and used as the baseline in these experiments (11). They were as follows (with MIC values shown in Table 1 and determined by the broth microdilution method in parentheses): TPP8, 0.04 μM (0.02 μM); SPR719, 6 μM (1.5 μM); MXF, 6 μM (3 μM); CLR, 1.5 μM (3 μM). (B) To determine the activity against intracellular bacteria, THP-1 cells were prepared and differentiated into macrophages with phorbol-12-myristate-13-acetate for 24 h, and the resulting macrophages were infected with a multiplicity of infection of 10 for 3 h using M. abscessus ATCC 19977 as described previously (26) and treated with the same concentration range of TPP8, SPR719, MXF, or CLR as in panel A. Intracellular CFU were enumerated by plating samples on Middlebrook 7H10 agar after 3 days of treatment. Experiments in panels A and B were carried out three times independently, and the results are represented as mean values with standard deviations.

FIG 3

Pharmacokinetic profile and efficacy of TPP8 in mice. (A) Plasma concentration-time profile of TPP8 following a single intraperitoneal dose of 10 or 25 mg/kg in CD-1 mice. The MIC of TPP8 against M. abscessus K21 (Table 1), the strain used in our murine infection model, is indicated by a dotted line. (B) Efficacy of TPP8 and comparator compounds in a NOD SCID mouse model of M. abscessus K21 lung infection. Mouse lung and spleen CFU are shown 1 day after intranasal infection with M. abscessus K21 (D1), following daily intraperitoneal administration of 20% Solutol HS15 in phosphate-buffered saline, pH 7.4 (TPP8 vehicle), for 10 days (D11; DF, drug free), daily intraperitoneal administration of TPP8 (12.5 or 25 mg/kg), or daily oral administration of clarithromycin (CLR, 250 mg/kg formulated in 0.5% carboxymethyl cellulose), moxifloxacin (MXF, 200 mg/kg formulated in water), or SPR720 (SPR, 100 mg/kg formulated in 0.5% methylcellulose) for 10 days. Mean and standard deviation are shown for each treatment group (n = 6). Statistical significance of the results was analyzed by one-way analysis of variance multicomparison and Dunnett’s posttest: *, P < 0.01; **, P < 0.001. The experiment was carried out twice, and one representative data set is shown.