Design, synthesis and biological evaluation of P2-modified proline analogues targeting the HtrA serine protease in Chlamydia

Abstract

High temperature requirement A (HtrA) serine proteases have emerged as a novel class of antibacterial target, which are crucial in protein quality control and are involved in the pathogenesis of a wide array of bacterial infections. Previously, we demonstrated that HtrA in Chlamydia is essential for bacterial survival, replication and virulence. Here, we report a new series of proline (P2)-modified inhibitors of Chlamydia trachomatis HtrA (CtHtrA) developed by proline ring expansion and Cγ-substitutions. The structure-based drug optimization process was guided by molecular modelling and in vitro pharmacological evaluation of inhibitory potency, selectivity and cytotoxicity. Compound 25 from the first-generation 4-substituted proline analogues increased antiCtHtrA potency and selectivity over human neutrophil elastase (HNE) by approximately 6-and 12-fold, respectively, relative to the peptidic lead compound 1. Based on this compound, second-generation substituted proline residues containing 1,2,3triazole moieties were synthesized by regioselective azide-alkyne click chemistry. Compound 49 demonstrated significantly improved antichlamydial activity in whole cell assays, diminishing the bacterial infectious progeny below the detection limit at the lowest dose tested. Compound 49 resulted in approximately 9-and 22-fold improvement in the inhibitory potency and selectivity relative to 1, respectively. To date, compound 49 is the most potent HtrA inhibitor developed against Chlamydia spp. a Relative inhibition against CtHtrA is the ratio of IC50 CtHtrA(1) to IC50 CtHtrA (compound).b Relative inhibition against HNE is the ratio of IC50 HNE (1) to IC50 HNE (compound).

Inhibition of Listeria Monocytogenes HtrA Protease with Camostat, Gabexate and Nafamostat Mesylates and the Binding Mode of the Inhibitors

“…JO146 exhibited more potent antichlamydial activity than JCP83 [ 35 ]. Subsequently, by considering JO146 as a template, modifications were made at the P1, P2 and P3 positions and new analogues were generated; some of the compounds exhibited improved antichlamydial activity than the parent compound [ 36 – 38 ].…”

“…In a recent study, P2 proline-modified analogues of JO146 were designed and one of the developed compounds exhibited potent anti-chlamydial activity in bacterial cell assays. The antiCtHtrA potency and selectivity of this compound over human neutrophil elastase improved by approximately 9- and 22-fold, respectively, as compared to JO146 [ 38 ].…”

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

“…This IC 50 value is then compared to a positive control (JO146 was used for 2a-g, and 2g was used for 6a-h), giving the 'relative potency'. This is the established method for assessing CtHtrA inhibitory potency [9,11,12]. Table 1 shows the inhibitory properties of these compounds relative to the known inhibitor JO146 (Figure 1).…”

“…Successive optimization studies have investigated the effects of replacing various amino acids within the JO146 structure, and substitution on the pyrrolidine ring, with some improvement to the inhibitory activity without loss of anti-chlamydial properties [11,12]. However, off-target effects have been observed through the inhibition of Human Leukocyte Elastase (HLE) [9,[11][12][13], a serine protease that has been implicated in the pathogenesis of Chronic Obstructive Pulmonary Disease [14]. Thus, we hypothesized that HLE inhibitors may exhibit inhibitory activity against CtHtrA and that these chemical scaffolds may serve as a source of new lead compounds as CtHtrA inhibitors.…”

“…We also sought to investigate the anti-chlamydial properties of the newly synthesized 4-chloroisocoumarins. Successive optimization studies have investigated the effects of replacing various amino acids within the JO146 structure, and substitution on the pyrrolidine ring, with some improvement to the inhibitory activity without loss of anti-chlamydial properties [11,12]. However, off-target effects have been observed through the inhibition of Human Leukocyte Elastase (HLE) [9,[11][12][13], a serine protease that has been implicated in the pathogenesis of Chronic Obstructive Pulmonary Disease [14].…”