A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA

Abstract

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

“…Intriguingly, and consistent with the known role of PknB in cell wall homeostasis, these inhibitors were able to significantly potentiate the effect of b-lactams in several types of mycobacteria and in the related Nocardia asteroides [236]. This synergistic concept has also been reported for kinase inhibitors of the nonessential L. monocytogenes PrkA and S. aureus Stk1 Hanks kinases [219,223,[237][238][239][240]. In L. monocytogenes, the broad-spectrum kinase inhibitor staurosporine, as well as some more selective repurposed human kinase inhibitors, was seen to inhibit PrkA activity and sensitize cells to b-lactam drugs [219,237].…”

“…This synergistic concept has also been reported for kinase inhibitors of the nonessential L. monocytogenes PrkA and S. aureus Stk1 Hanks kinases [219,223,[237][238][239][240]. In L. monocytogenes, the broad-spectrum kinase inhibitor staurosporine, as well as some more selective repurposed human kinase inhibitors, was seen to inhibit PrkA activity and sensitize cells to b-lactam drugs [219,237]. Similarly, several studies have identified Stk1-inhibiting molecules from small-molecule libraries that sensitize MRSA S. aureus strains to b-lactams [223,[238][239][240].…”

Comparative analysis of PknB inhibitors for reactivity and toxicity

“…Although modifications may reduce this compound's reactivity and toxicity, further evidence is needed to assess whether it will retain its effectiveness against Mycobacterium tuberculosis. Although YH-8 is reactive and has poor pharmacokinetic properties, several stable non-reactive inhibitors are known for PknB and other related bacterial kinases, including some which were in human trials for other diseases [15][16][17][29][30][31][32][33]. Improving YH-8 by reducing its reactivity and toxicity and incorporating structure-activity relationship data from the aforementioned inhibitors may lead to improvements in the properties of YH-8, leading to a viable clinical lead compound.…”

Defeating Antibiotic-Resistant Bacteria: Exploring Alternative Therapies for a Post-Antibiotic Era

“…Listeria monocytogenes mutants deficient in the PASTA kinase, PrkA, show impaired growth under nutrient-limiting conditions and reduced survival and replication in host cells when compared to the wild-type strain [63]. Moreover, deletion of homologous PASTA kinase in some species, for example L. monocytogenes and S. aureus, has been linked to increased susceptibility to β-lactam antibiotics [64][65][66]. This contrasts the homologous Mycobacterium tuberculosis PASTA kinase, PknB, which is essential for survival [67].…”