10 research outputs found
A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA
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
The effect of the N-terminal HEAT repeats of the A-subunit on methylation.
<p>(<b>A</b>) Schematic representation of internal truncations of the N-terminal HEAT repeats to reduce the length of the A-subunit N-terminal structure. (<b>B</b>) Normalized concentrations of PP2Ac assembled with full-length or truncated A-subunits used for kinetic analysis of PP2A methylation in (<b>C</b>). (<b>C</b>) Kinetics of methylation of PP2A core enzymes with varied length of the A-subunit. Results from 6–7 independent experiments were summarized and the average K<sub>m</sub> ± std. dev. was calculated and shown in the table on the right. (<b>D</b>) Correlation between the number of deleted N-terminal HEAT repeats and the K<sub>m</sub> of methylation of PP2A core enzyme by LCMT-1. Statistical test of this correlation was performed and the P value was calculated and shown. (<b>E</b>) Structure of the core enzyme portion of PP2A holoenzyme (PDB ID: 2NPP). The A-subunit, PP2Ac, and PP2Ac-tail are colored green, blue, and magenta, respectively. The positions of the HEAT-repeats where truncations were made are indicated by numbers.</p
Electrostatic interactions between the A-subunit and LCMT-1.
<p>(<b>A</b>) Structural model of the PP2A core enzyme-LCMT-1 complex built from the structure of the PP2Ac-LCMT-1 complex and the A-subunit with morphed conformation. The electrostatic potential for the A-subunit (left panel) and LCMT-1 (right panel) is shown. The corresponding regions on the A-subunit and LCMT-1 for potential electrostatic interactions are circled (i and ii). The A-subunit residues that directly contact LCMT-1 in the model, Arg183 and Arg258, are shown. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086955#pone.0086955.s003" target="_blank">Movie S1</a>. (<b>B</b>) The effect of the A-subunit mutations to Arg183 and Arg258 on methylation of the PP2A core enzyme. Results from 5–7 independent experiments were summarized and the average K<sub>m</sub> ± std. dev. and the P value were calculated and shown. <b>(C)</b> FRET assay measured changes in the distance between the N- and C-termini of the A-subunit in the core enzyme prior to and after addition of an excess molar amount of LCMT-1 or a stoichiometric amount of PR70 (108–575). Representative results were shown with mean ± SEM calculated from triplicate.</p
Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation
<div><p>The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.</p></div
Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation
The A-subunit enhances PP2A methylation and interaction with LCMT-1.
<p>(<b>A</b>) Influence of the A-subunit, mini-A, and PTPA on PP2A methylation, tested by addition of these proteins in a stoichiometric amount to PP2Ac, prior to determination of the kinetics of PP2A methylation. Representative results were shown with mean ± SEM calculated from triplicate (upper figure panel). Statistical analysis of 4–12 independent experiments was performed and the average K<sub>m</sub> ± std. dev. and the P value were calculated and summarized in the table below. (<b>B</b>) Binding affinity between LCMT-1, preloaded with SAH, and unmethylated, methylated PP2A core enzyme or free PP2Ac, measured by ITC.</p
Shortening of PP2Ac-tail enhanced methylation.
<p>(<b>A</b>) Schematic representation of Δ294–298 truncation of PP2Ac and the range of PP2Ac-tail mobility. PP2Ac is shown as blue sphere with two small red spheres representing catalytic metal ions. The PP2Ac-tail is shown in cyan. The cyan spheres surrounding PP2Ac indicate the range of movement of PP2Ac-tail. Note that the Δ294–298 truncation reduces the space for the PP2Ac-tail movement. (<b>B</b>) Kinetics of methylation of PP2Ac Δ294–298 and full-length PP2Ac either as the free catalytic subunit or as the core enzymes assembled with the A-subunit with internal deletion of increasing number of N-terminal HEAT repeats. Results from 3–9 independent experiments were summarized and the average K<sub>m</sub> ± std. dev. and the P value were calculated and shown. (<b>C</b>) Kinetics of methylation of PP2Ac Δ294–298 associated with the A-subunit with internal deletion of increasing number of N-terminal HEAT repeats (left) and comparison of the full-length PP2Ac and PP2Ac Δ294–298 in correlation of the K<sub>m</sub> of methylation and the number of N-terminal HEAT repeats of the A-subunit deleted in the core enzymes (right). Statistical test of this correlation was performed and P values were calculated and shown.</p