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.

Keywords: Listeria monocytogenes; PASTA Kinase; aminofurazans; antibiotics; bacterial protein kinase; bacterial signal transduction; drug discovery; drug screening; β-lactams.

Figure 1.

Library screen identifies GSK690693 as a compound that sensitizes L. monocytogenes to ceftriaxone. A, scatter plot representing percent growth inhibition of WT L. monocytogenes in the presence of a combination of a sublethal dose (1 μg/ml) of the β-lactam ceftriaxone and each compound in the screen. The solid black line represents the library mean (μ), and the dashed black line and gray line represent two (2σ) and three (3σ) S.D. above the library mean, respectively. The cyan, green, and red data points represent staurosporine, GSK690693, and other compounds from the IPA family, respectively. B, skeletal structure of GSK690693.

Figure 2.

GSK690693 potentiates the inhibitory action of ceftriaxone in a dose-dependent manner. Growth curves of WT L. monocytogenes grown in the presence of 2.5 μg/ml of ceftriaxone and increasing concentrations (μm) of GSK690693. Curves are representative of 3 independent trials.

Figure 3.

GSK690693 inhibits the PrkA kinase domain in vitro. A, autoradiography blot of purified PrkA kinase domain from L. monocytogenes and the nonspecific phosphoacceptor substrate MBP in the presence or absence of GSK690693. Blot is representative of 3 independent trials. B (top), GSK690693 docked in silico into the threaded model of the kinase domain of PrkA (bottom) crystal structure of GSK690693 bound to human AKT (PDB ID 3d0e). Gatekeeper methionine and xDF residues are represented as sticks.

Figure 4.

IPAs potentiate ceftriaxone activity to varying degrees. A, dose-response curves of L. monocytogenes growth versus ceftriaxone in the presence and absence of 10 μm IPAs. Curves are representative of 3 independent trials. B, autoradiography blot of purified PrkA kinase domain and MBP in the presence or absence of GSK690693 or SB-747651A. The blot is representative of 3 independent trials.

Figure 5.

Residues of the back pocket play a role in GSK690693 selectivity. A, autoradiography blot of purified Stk1 kinase domain from S. aureus and MBP in the presence or absence of GSK690693. B, stick figure representation of the amino acids that constitute the back pocket of the PrkA (cyan) and Stk1 (violet) kinase domains. GSK690693 (green sticks) is docked into the back pocket, which is represented by the translucent cavity surface. C, autoradiography blot of purified WT Stk1, F150T mutant, S62A/M73V/L85I triple mutant, and MBP in the presence or absence of GSK690693. Blots are representative of 3 independent trials.