Identification of a strong and specific antichlamydial N-acylhydrazone

Abstract

Sexually transmitted Chlamydia trachomatis is an extremely common infection and often leads to serious complications including infertility and pelvic inflammatory syndrome. Several broad-spectrum antibiotics are currently used to treat C. trachomatis. Although effective, they also kill beneficial vaginal lactobacilli. Two N-acylhydrazones, CF0001 and CF0002, have been shown previously to inhibit chlamydial growth without toxicity to human cells and Lactobacillus spp. Of particular significance, the rate of random mutation leading to resistance of these inhibitors appears to be extremely low. Here, we report three analogs of CF0001 and CF0002 with significantly stronger inhibitory effects on chlamydiae. Even though the new compounds (termed SF1, SF2 and SF3) displayed slightly decreased inhibition efficiencies for a rare Chlamydia variant selected for CF0001 resistance (Chlamydia muridarum MCR), they completely overcame the resistance when used at concentrations of 75–100 μM. Importantly, SF1, SF2 and SF3 did not shown any toxic effect on lactobacilli, whereas SF3 was also well tolerated by human host cells. An effort to isolate SF3-resistant variants was unsuccessful. By comparison, variants resistant to rifampin or spectinomycin were obtained from smaller numbers of chlamydiae. Our findings suggest that SF3 utilizes an antichlamydial mechanism similar to that of CF0001 and CF0002, and will be more difficult for chlamydiae to develop resistance to, potentially making it a more effective antichlamydial agent.

GrgA as a potential target of selective antichlamydials

“…Compared to the prototype antichlamydial BAH CF0001, SF3 [( E )- N ’-(3,5-dibromo-4-hydroxybenzylidene)-3,5-dinitrobenzohydrazide], a recently developed BAH, has a stronger antichlamydial activity, and can fully inhibit MCR [26]. At 80 μM, SF3 also achieved full inhibition of all three tested clonal populations (r8s6, r8s7 and r8s11) with S4(R51G GrgA) as the sole mutant allele although at lower concentrations it inhibited the wild-type MoPn more efficiently (Fig.…”

“…BAH belong to a novel group of selective antichlamydials [25, 26]. The genome of the rare BAH-resistant C. muridarum variant MCR carries four SNPs [25].…”

“…Compared to the prototype antichlamydial BAH CF0001, the recently-developed SF3 has a stronger antichlamydial activity while maintaining non-toxicity to mammalian cells and vaginal lactobacilli [26]. It has been shown previously that MCR can be inhibited completely by SF3 even though it is less susceptible than wild-type MoPn to lower concentrations of SF3 [26]. While it is expected that clonal recombinant populations carrying S4(R51G GrgA) as sole mutant allele demonstrate the same properties as MCR, these clonal populations will be more useful for identifying additional selective antichlamydials that interferes with a process involving GrgA.…”

“…Wild-type C. muridarum MoPn and the BAH-resistant variant MCR have been described previously [25]. MoPn_Rif R , MoPn_Spc R , MCR_LBM R and MCR_Rif R were derived by culturing MoPn and MCR in medium containing appropriate inhibitors (i.e., rifampin, spectinomycin or LBM415) at gradually increased concentrations starting at sub-MIC, as we previously outlined [25, 26, 28, 29].…”

“…Significantly, at concentrations above minimal inhibition concentrations, BAH have no adverse effects on animal cells or vaginal lactobacilli [25]. Another attractive feature of BAH is their extremely low spontaneous mutation rates leading to resistance [25, 26]. Although a C. muridarum variant termed MCR with a low-level of BAH resistance was initially isolated following a lengthy selection process, multiple repeated attempts to isolate additional resistant variants from mutagenized as well as non-mutagenized stocks of C. muridarum and C. trachomatis were unsuccessful [25, 26].…”