4-Chloroisocoumarins as Chlamydial Protease Inhibitors and Anti-Chlamydial Agents.

4-Chloroisocoumarin compounds have broad inhibitory properties against serine proteases. Here, we show that selected 3-alkoxy-4-chloroisocoumarins preferentially inhibit the activity of the conserved serine protease High-temperature requirement A of Chlamydia trachomatis. The synthesis of a new series of isocoumarin-based scaffolds has been developed, and their anti-chlamydial properties were investigated. The structure of the alkoxy substituent was found to influence the potency of the compounds against High-temperature requirement A, and modifications to the C-7 position of the 3-alkoxy-4-chloroisocoumarin structure attenuate anti-chlamydial properties.

HtrA, fatty acids, and membrane protein interplay in Chlamydia trachomatis to impact stress response and trigger early cellular exit.

Chlamydia trachomatis is an intracellular bacterial pathogen that undergoes a biphasic developmental cycle, consisting of intracellular reticulate bodies and extracellular infectious elementary bodies. A conserved bacterial protease, HtrA, was shown previously to be essential for Chlamydia during the reticulate body phase, using a novel inhibitor (JO146). In this study, isolates selected for the survival of JO146 treatment were found to have polymorphisms in the acyl-acyl carrier protein synthetase gene (aasC). AasC encodes the enzyme responsible for activating fatty acids from the host cell or synthesis to be incorporated into lipid bilayers. The isolates had distinct lipidomes with varied fatty acid compositions. A reduction in the lipid compositions that HtrA prefers to bind to was detected, yet HtrA and MOMP (a key outer membrane protein) were present at higher levels in the variants. Reduced progeny production and an earlier cellular exit were observed. Transcriptome analysis identified that multiple genes were downregulated in the variants especially stress and DNA processing factors. Here, we have shown that the fatty acid composition of chlamydial lipids, HtrA, and membrane proteins interplay and, when disrupted, impact chlamydial stress response that could trigger early cellular exit. IMPORTANCE: Chlamydia trachomatis is an important obligate intracellular pathogen that has a unique biphasic developmental cycle. HtrA is an essential stress or virulence protease in many bacteria, with many different functions. Previously, we demonstrated that HtrA is critical for Chlamydia using a novel inhibitor. In the present study, we characterized genetic variants of Chlamydia trachomatis with reduced susceptibility to the HtrA inhibitor. The variants were changed in membrane fatty acid composition, outer membrane proteins, and transcription of stress genes. Earlier and more synchronous cellular exit was observed. Combined, this links stress response to fatty acids, membrane proteins, and HtrA interplay with the outcome of disrupted timing of chlamydial cellular exit.

Optimization of peptide-based inhibitors targeting the HtrA serine protease in Chlamydia: Design, synthesis and biological evaluation of pyridone-based and N-Capping group-modified analogues.

The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) is responsible for the most common bacterial sexually transmitted infection and is the leading cause of preventable blindness, representing a major global health burden. While C. trachomatis infection is currently treatable with broad-spectrum antibiotics, there would be many benefits of a chlamydia-specific therapy. Previously, we have identified a small-molecule lead compound JO146 [Boc-Val-Pro-ValP(OPh)2] targeting the bacterial serine protease HtrA, which is essential in bacterial replication, virulence and survival, particularly under stress conditions. JO146 is highly efficacious in attenuating infectivity of both human (C. trachomatis) as well as koala (C. pecorum) species in vitro and in vivo, without host cell toxicity. Herein, we present our continuing efforts on optimizing JO146 by modifying the N-capping group as well as replacing the parent peptide structure with the 2-pyridone scaffold at P3/P2. The drug optimization process was guided by molecular modelling, enzyme and cell-based assays. Compound 18b from the pyridone series showed improved inhibitory activity against CtHtrA by 5-fold and selectivity over human neutrophil elastase (HNE) by 109-fold compared to JO146, indicating that 2-pyridone is a suitable bioisostere of the P3/P2 amide/proline for developing CtHtrA inhibitors. Most pyridone-based inhibitors showed superior anti-chlamydial potency to JO146 especially at lower doses (25 and 50 µM) in C. trachomatis and C. pecorum cell culture assays. Modifications of the N-capping group of the peptidyl inhibitors did not have much influence on the anti-chlamydial activities, providing opportunities for more versatile alterations and future optimization. In summary, we present 2-pyridone based analogues as a new generation of non-peptidic CtHtrA inhibitors, which hold better promise as anti-chlamydial drug candidates.