Species-Specific and Inhibitor-Dependent Conformations of LpxC: Implications for Antibiotic Design

LpxC is an essential enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different Gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against Gram-negative infections

High-Throughput Screening for Novel Inhibitors of <em>Neisseria gonorrhoeae</em> Penicillin-Binding Protein 2

<div><p>The increasing prevalence of <em>N. gonorrhoeae</em> strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for β-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of <em>N. gonorrhoeae</em> PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of <em>N. gonorrhoeae</em>. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of <em>N. gonorrhoeae</em>. It also provides a set of lead compounds for optimization of anti-gonococcal agents.</p> </div

Structures of seven compounds that exhibited antimicrobial activity against <i>N. gonorrhoeae</i>.

<p>Compound numbers correspond with those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044918#pone-0044918-t002" target="_blank">Table 2</a>.</p

Statistical parameters of the FP assay.

<p>The performance of the assay was determined during initial optimization and after the HTS assay. Pc is the mean FP signal of PBP 2-bound tracer control, Nc is the mean FP signal of free Bocillin-FL, and Dc is the FP signal in the presence of 100 µm penicillin G. S/N (signal-to-noise) and the Z factors are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044918#s2" target="_blank">Material and Methods</a>. (n = number of measurements). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044918#pone.0044918.s001" target="_blank">Fig. S1</a>).</p

Docking of two compounds into the structure of <i>N. gonorrhoeae</i> penicillin-binding protein 2.

<p>The main chain of PBP 2 is shown as a grey ribbon, residues of the active site motifs of PBPs are shown with green bonds and additional amino acids that are predicted to form interactions with the ligand are shown in blue. <b>A.</b> compound <b>2</b>. <b>B.</b> compound <b>7</b>. Figure prepared with MOE 2011.10 (Chemical Computing Group Inc.).</p

Initial HTS of 50,080 small lead compounds from DIVERSet ChemBridge Library was performed using cocktails of 10 different compounds (10X cocktails).

<p>Each plate contained duplicate samples of 10X cocktails with PBP 2 and Bocillin-FL. Data points denoted by the black dots represent the percent of inhibition for each 10X cocktail determined using the average of two sample FP measurements, and the means of free Bocillin-FL (Nc) and bound Bocillin–FL (Pc) controls recorded in quadruplicate for each corresponding plate. Data points of the displaced tracer controls (Dc - the FP of the Bocillin-FL - protein at 100 µM penicillin G), denoted by open circles, represent the percent of inhibition based on the average of four FP measurements. Fifty-two plates with 96×10X cocktails each and one plate with eight×10 X cocktails were screened. The box indicates the 58 cocktails that exhibited ≥80% inhibition of Bocillin-FL binding to PBP 2.</p

IC₅₀ values and antimicrobial activities of seven compounds identified by high-throughput screening against PBP 2 from <i>N. gonorrhoeae</i> strain FA19.

<p>IC₅₀ values and antimicrobial activities of seven compounds identified by high-throughput screening against PBP 2 from <i>N. gonorrhoeae</i> strain FA19.</p

IC₅₀ values of three representative hits from HTS for inhibition of acylation of PBP 2 by Bocillin-FL.

<p>IC₅₀s of the inhibitors were determined by SDS-PAGE-based concentration-response assays using a 0.05–1000 µM concentration range for the inhibitors with 1 µM PBP 2 and 10 µM of Bocillin-FL. Triton X-100 (0.01%) was included to eliminate promiscuous inhibitors. A. Compound 2 (in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044918#pone-0044918-t002" target="_blank">Table 2</a>). B. Compound 4. C. Compound 7. In all experiments, the data points represent the mean ± standard deviation for two replicate (compounds 4 and two) or four replicate (compound 7) experiments at each concentration of inhibitor.</p

Inhibition of FP of Bocillin-FL and PBP 2 by increasing amounts of penicillin G.

<p>Assays contained variable concentrations of penicillin G (0.05–1000 µM) with fixed concentrations of PBP 2 (1 µM) and Bocillin-FL (1 µM) with or without 10% DMSO. The solid line represents data for PBP 2 without DMSO and the dashed line is PBP 2 with DMSO. In all experiments, the data points represent the mean ± standard deviation of four replicate experiments at each concentration of penicillin G.</p

Optimization of the fluorescence polarization (FP) assay.

<p><b>A</b>. Fluorescence polarization (mP) of free Bocillin-FL at various concentrations from 0.002 to 4 µM. <b>B</b>. Binding experiments with Bocillin-FL (1 µM) and PBP 2 at various protein concentrations from 0.2 to 4 µM. Fluorescence of Bocillin-FL – PBP2 was measured in relative fluorescence units (RFU) (open circles). Maximum specific binding (triangles), <i>i.e.</i> assay window (ΔmP), was determined by FP. Assay window is defined as the difference between FP of protein-tracer sample and free-tracer, <i>i.e.</i> ΔmP  =  mP_s – mP_free. In all experiments, the data points represent the mean ± standard deviation of four replicate experiments at each concentration of Bocillin-FL or PBP 2.</p