Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1β in Cellular and Animal Infection Models

Abstract

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1β. Furthermore, inhibition of LasBmediated IL-1β activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen.

Inhibitors of the Elastase LasB for the treatment of Pseudomonas aeruginosa lung infections

“…Therefore, the vast majority of the studied inhibitors contain zinc-binding groups (ZBGs), such as thiols 23,24 , hydroxamates 25 , carboxylic acids 26 , tropolones 27 or 3-hydroxypyridine-4(1H)-thiones 28 .…”

Inhibitors of the Elastase LasB for the Treatment of Pseudomonas aeruginosa Lung Infections

“…LasB or pseudolysin is a zinc-dependent metalloenzyme with an additional metal cation (Ca 2+ ) as a cofactor in the active site . Therefore, the vast majority of the studied inhibitors contain zinc-binding groups (ZBGs), such as thiols, , hydroxamates, carboxylic acids, tropolones, or 3-hydroxypyridine-4­(1 H )-thiones . We have extensively explored these ZBGs in the past few years and used those findings as the basis for the current work. − …”

Facile Production of the Pseudomonas aeruginosa Virulence Factor LasB in Escherichia coli for Structure‐Based Drug Design

“…Everett et al have recently reported that the analysis of LasB activity of clinical isolates is so far only possible in the supernatant and accordingly only in a more complex mixture. [18] For an in vitro activity assay, the clinical PA strains would have to be available in the first place and secrete sufficient LasB under laboratory conditions. To address these issues we have developed a high-yielding production protocol for recombinant LasB (LasB*) in E. coli, which allows the rapid purification of LasB*.…”

“…This poses a serious obstacle to attempts to exploit LasB for the development of antivirulence, or pathoblocker, compounds. LasB is a promising, validated target for drug development against serious PA infections, and high‐affinity inhibitors with outstanding selectivity over human off‐targets have very recently been reported [16–18] . From a medicinal chemistry perspective, extracellular targets are particularly attractive given that the drug candidates do not have to cross the Gram‐negative cell membrane to reach their site of action.…”

“…Everett et al . have recently reported that the analysis of LasB activity of clinical isolates is so far only possible in the supernatant and accordingly only in a more complex mixture [18] . For an in vitro activity assay, the clinical PA strains would have to be available in the first place and secrete sufficient LasB under laboratory conditions.…”