Small-Molecule Inhibitors of Haemophilus influenzae IgA1 Protease

Abstract

Newly identified, non-typable H. influenza strains represent a serious threat to global health. Due to the increasing prevalence of antibiotic resistance, virulence factors have emerged as potential therapeutic targets that would be less likely to promote resistance. IgA1 proteases are secreted virulence factors of many Gram-negative human pathogens. These enzymes play important roles in tissue invasion as well as evasion of the immune response, yet there has been limited work on pharmacological inhibitors. Here we report the discovery of the first small molecule, non-peptidic inhibitors of H. influenzae IgA1 proteases. We screened over 47,000 compounds in a biochemical assay using recombinant protease, and identified a hit compound with micromolar potency. Preliminary SAR produced additional inhibitors, two of which showed improved inhibition and selectivity for IgA protease over other serine proteases. We further showed dose-dependent inhibition against four different IgA1 protease variants collected from clinical isolates. These data support further development of IgA protease inhibitors as potential therapeutics for antibacterialresistant H. influenza strains. The newly-discovered inhibitors also represent valuable probes for exploring the roles of these proteases in bacterial colonization, invasion, and infection of mucosal tissues.

High-Throughput Screening for the Discovery of Enzyme Inhibitors

“…Similarly, fluorescence ,− ,,− ,− ,,,,,,,,​,,,,,,− and/or absorbance-based ,,,,,,,,,,,,− assays were extensively used, with other detection methods (luminescence, ,, radioactivity, ,, and mass spectrometry ,, ) playing a much less significant role, consistent with the wider literature (Figure ). The overwhelming majority of studies used X-ray crystallography ,,,− ,− ,− ,,,,,,,​,− ,− ,− ,− ,,− ,,…”

“…The identified campaigns − (Table ) had 75 examples of hit to lead development. In many cases few details of the screening library are available, so for the purposes of this study lead development from a fragment was considered to have occurred where the initial hit had a molecular weight of 300 Da or less (a typical upper molecular weight limit used in fragment screening campaigns).…”

“…Analysis of these 75 campaigns (Table ) showed that kinases, ,,,,,,,,, phosphatases, ,,, phosphodiesterases, ,,,,, and pyrophosphatases, proteases ,,,,, and peptidases ,,,,, were particular common drug targets, with cancer, ,,,− ,,,,,− ,,,,​− ,,− ,− ,,− ,, bacterial infection, ,,,,,,,− ,, viral infection, ,,, and inflammation ,,,,,, being the most common disease types targeted, consistent with the analysis of the wider literature (Figure ). Similarly, fluorescence ,…”

IgA: Structure, Function, and Developability

“…A more detailed understanding of the molecular basis of IgA1 hinge cleavage by IgA1 proteases may have therapeutic application. For example, following earlier work to identify possible inhibitors for IgA1 protease [118,119], small molecule non-peptidic inhibitors for H. influenzae IgA1 protease have recently been described in the first steps towards development of potential therapeutics for antibiotic-resistant H. influenzae strains [120]. Further, it has been proposed that IgA1 proteases may have utility as therapeutic options to degrade pathogenic immune complexes of aberrantly glycosylated IgA1 in IgA nephropathy, a common cause of kidney disease [121,122].…”

Alternative Treatment Strategies for Secondary Bacterial and Fungal Infections Associated with COVID-19

“…IgA protease in H. influenzae is a virulence factor secreted by various gram-negative bacteria and is a promising target for new drugs for managing antibiotic-resistant H. influenzae strains [ 131 ]. Apart from targeting essential and unique enzymes in pathogens, structural variations in a host enzyme and pathogen enzyme are another aspect to limit microbial infection.…”