Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound

Abstract

We have previously reported the inhibition of bacterial topoisomerase I activity by a fluoroquinophenoxazine compound (FP-11g) with a 6-bipiperidinyl lipophilic side chain that exhibited promising antituberculosis activity (MIC = 2.5 μM against Mycobacterium tuberculosis , SI = 9.8). Here, we found that the compound is bactericidal towards Mycobacterium smegmatis , resulting in greater than 5 Log 10 reduction in colony-forming units [cfu]/mL following a 10 h incubation at 1.25 μM (4X MIC) concentration. Growth inhibition (MIC = 50 μM) and reduction in cfu could also be observed against a clinical isolate of Mycobacterium abscessus . Stepwise isolation of resistant mutants of M . smegmatis was conducted to explore the mechanism of resistance. Mutations in the resistant isolates were identified by direct comparison of whole-genome sequencing data from mutant and wild-type isolates. These include mutations in genes likely to affect the entry and retention of the compound. FP-11g inhibits Mtb topoisomerase I and Mtb gyrase with IC 50 of 0.24 and 27 μM, respectively. Biophysical analysis showed that FP-11g binds DNA as an intercalator but the IC 50 for inhibition of Mtb topoisomerase I activity is >10 fold lower than the compound concentrations required for producing negatively supercoiled DNA during ligation of nicked circular DNA. Thus, the DNA-binding property of FP-11g may contribute to its antimycobacterial mechanism, but that alone cannot account for the observed inhibition of M tb topoisomerase I.

Novel and Structurally Diversified Bacterial DNA Gyrase Inhibitors Discovered through a Fluorescence-Based High-Throughput Screening Assay

“…For example, ethacridine and echinomycin are two DNA intercalators and bind to DNA with high affinity. 45,54 They also potently inhibited E. coli DNA gyrase activities (Figure S3c,d), although DNA intercalation/unwinding of pAB1_FL905 by these two intercalators interferes with the fluorescence signal detection (Figure S3a,b). In fact, many DNA intercalators are among the hits of this HTS campaign (Table S3).…”

“…Second, unlike previous HTS efforts mainly targeted the gyrB ATPase domain of bacterial gyrase, this HTS assay, employed relaxed DNA as the substrate, would identify inhibitors that affect all possible steps of the catalytic cycle of E. coli DNA gyrase, including DNA binding, DNA cleavage, strand passage, and DNA relegation. For example, ethacridine and echinomycin are two DNA intercalators and bind to DNA with high affinity. , They also potently inhibited E. coli DNA gyrase activities (Figure S3c,d), although DNA intercalation/unwinding of pAB1_FL905 by these two intercalators interferes with the fluorescence signal detection (Figure S3a,b). In fact, many DNA intercalators are among the hits of this HTS campaign (Table S3).…”

Type IA Topoisomerases as Targets for Infectious Disease Treatments

“…Derivative 11a with 6-methylpiperazinyl and 9-amino groups ( Figure 6 , IC 50 = 0.48 µM) showed broad spectrum antibacterial activity (MICs = 0.78–7.6 µM) against bacteria that include Gram-negative E. coli and M. tuberculosis . Derivative 11g with the 6-bipiperidinyl lipophilic substitution showed the most promising antitubercular activity (MIC = 2.5 µM, selectivity index (SI) = 9.8) and was also active (MIC = 50 µM) against a clinical isolate of Mycobacterium abscessus [ 114 , 115 ]. The mechanism of resistance in mycobacteria was investigated by stepwise isolation of resistant mutants in M. smegmatis followed by comparison of the whole-genome sequence of the original strain and mutant isolates.…”

“…The mechanism of resistance in mycobacteria was investigated by stepwise isolation of resistant mutants in M. smegmatis followed by comparison of the whole-genome sequence of the original strain and mutant isolates. Mutations in genes that affect compound entry and retention were identified instead of mutations in the topoisomerase I gene that might confirm the enzyme as the antibacterial target [ 115 ]. Biophysical analysis showed that DNA binding by fluoroquinophenoxazine 11g may contribute to the antibacterial mechanism, but could not account entirely for the direct binding and potent inhibition of the mycobacterial topoisomerase I [ 115 ].…”