Abstract
Chlamydia trachomatis is an obligate intracellular human parasite responsible for the most common sexually transmitted bacterial infection world-wide. The largely asymptomatic infection frequently results in complications including ectopic pregnancy, infertility and reversible blindness. The mainstay therapy for chlamydia is the use of macrolide antibiotics such as azithromycin and erythromycin. While these drugs are currently effective, there are growing concerns about their efficacy due to the ever-increasing prevalence of the disease, coupled with the fact that the drugs are general antibacterials with no pre-designed specificity for Chlamydia which is an atypical bacterium. This necessitates the timely search for Chlamydia-targeted drugs with new mechanisms of action.
C. trachomatis high temperature requirement A protease (CtHtrA) is a serine protease identified to be crucial for the survival and virulence of Chlamydia in the human body, and a viable drug target. JO146 (Boc-Val-Pro-ValP(OPh)2), a mixture of two diastereomers with R-/S-Val at P1, was previously identified as a covalent inhibitor of CtHtrA (IC50 = 12.5 µM) that is selectively toxic to human and koala pathogenic Chlamydia species. However, JO146 is not potent enough and may also be susceptible to enzymatic degradation, being a peptide-based compound.
The present study explored different avenues to improve the potency and selectivity of JO146, as well as reduce its peptidic nature. The influence of the stereochemical difference between the two diastereomers of JO146 on their anti-chlamydial activity was examined. Inhibitors containing different covalent and noncovalent reversible transition state analogues of the phosphonate moiety were synthesized and tested in in vitro enzyme inhibition and Chlamydia cell culture assays. Analogues with significant anti-chlamydial activity were identified and further optimized by improving their binding affinity at the various subpockets of CtHtrA.
JO146-D2, the isomer with the physiologically relevant R-Val at P1 had significantly better anti-chlamydial activity over JO146-D1, the isomer with the S-Val at P1 (> 100-fold increase). Both compounds had excellent selectivity for CtHtrA over the potential off-target serine proteases trypsin and chymotrypsin. Noncovalent binders, which include the N-methylamide, valinol and N-methyl imidazole analogues of JO146 were generally devoid of activity, inferring the necessity of covalent interactions for activity. Of the tested reversible covalent inhibitors, only the prime side-binding α-ketobenzothiazole analogue displayed viable anti-chlamydial activity. Binding optimization to CtHtrA revealed isoleucine and tertiary leucine to be the most active residues at P1 and P3 respectively, yielding a new lead molecule Boc-Tle-Pro-IleP(OPh)2 which had approximately 1000-fold increase in cellular anti-chlamydial activity relative to JO146. Additionally, a good number of these inhibitors showed improved selectivity for CtHtrA over the structurally close human neutrophil elastase, compared to the lead molecule JO146.
In summary, more potent and potentially safe analogues of JO146 suitable for in vivo pre-clinical studies have been discovered. In addition, comprehensive structure activity relationships that would enable the design of clinically relevant inhibitors for the treatment of chlamydial infections were built in this study.