NCL812 compounds exert their antibacterial action on the cell membrane of <i>S</i>. <i>pneumoniae</i> and <i>S</i>. <i>aureus</i>.

<p>(<b>A and B</b>), <i>S</i>. <i>pneumoniae</i> strain D39 exposed to 16 μg/ml NCL812 for 6 h exhibited significantly thicker cell membranes compared to untreated samples (<b>A</b>) (<i>p</i> < 0.0001; two-tailed unpaired <i>t</i>-test) and displayed significantly wider periplasmic space compared to untreated samples (<b>B</b>) (<i>p</i> < 0.001; two-tailed unpaired <i>t</i>-test. Data presented are an example from 12 different bacterial cells, each with at least 10 measurements per bacteria for both treated and untreated samples. (<b>C and D</b>) NCL812 affects macromolecular synthesis (<b>c</b>) and ATP release (<b>D</b>) in <i>S</i>. <i>aureus</i>. (<b>E and F</b>), NCL Compounds dissipate the membrane potential of <i>S</i>. <i>pneumoniae</i> and <i>S</i>. <i>aureus</i>. Membrane potential measurements of <i>S</i>. <i>pneumoniae</i> D39 (<b>E</b>) and <i>S</i>. <i>aureus</i> ATCC49775 (<b>F</b>). Bacterial suspensions were exposed to 16 μg/ml NCL812, NCL195, NCL219, or ampicillin (control) for 5 min after which DiOC₂(3) was added and the fluorescence monitored until it plateaued. Cells were then re-energized with 0.5% glucose and the establishment of a membrane potential was measured as an increase in fluorescence until it plateaued. The membrane potential was then disrupted by the addition of the proton ionophore (CCCP). Data presented is representative of two experiments. For full description, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183457#sec002" target="_blank">Materials and Methods</a>.</p

Antimicrobial activity of NCL195 against Gram-negative bacteria alone and in combination with EDTA or polymyxin B.

<p>Antimicrobial activity of NCL195 against Gram-negative bacteria alone and in combination with EDTA or polymyxin B.</p

NCL812 and NCL195 exhibit high plasma concentration and low plasma clearance rates.

<p>Pharmacokinetic parameters for NCL812 (<b>A</b>) and NCL195 (<b>B</b>) after IV administration at a dose of 5 mg/kg to male CD1 mice (n = 8 per compound), indicating that both compounds exhibited high plasma concentrations and long apparent terminal elimination half-lives. (<b>C</b>), Pharmacokinetic parameters for NCL195 after IP administration at a dose of 43 mg/kg to male CD1 mice (n = 2 per time point), indicating that NCL195 was rapidly absorbed after dosing, and plasma concentrations remained above 3–4 μg/ml for the initial 7.5 h post-dose period.</p

Structure activity relationship between NCL812, NCL195 and NCL219.

<p>Installation of a 4-<i>tert</i>-butyl and a C-methyl imine moiety provided NCL219 with considerably enhanced hydrolytic stability while retaining the excellent antimicrobial activity of NCL812, while guanidine to 2,4,6-triaminopyrimindine bioisosteric modification yielded NCL195, which allowed potency and drug-like character enhancement.</p

Physicochemical and metabolism parameters for NCL812, NCL195 and NCL219 in human and mouse liver microsomes.

<p>Physicochemical and metabolism parameters for NCL812, NCL195 and NCL219 in human and mouse liver microsomes.</p

NCL195 demonstrates limited cytotoxicity to mammalian cell lines.

<p>Real-time cell viability measurements for Hep G2 (<b>A, B, C</b>) and MDBK (<b>D, E, F</b>) cells after treatment with 2 or 8 μg/ml NCL812, NCL195 or NCL219. Cell viability was measured every 60 min for 20 h at 37°C and 5% CO₂ on a Cytation 5 Cell Imaging Multi-Mode Reader (BioTek) using the RealTime-Glo MT Cell Viability Assay reagent (Promega). Data are means (± s.e.m.) relative light units (RLU) for each treatment per time point (in duplicate).</p

MIC range values for NCL812, NCL195 and NCL219 compared to MIC₉₀ values for daptomycin and ampicillin against <i>S</i>. <i>pneumoniae</i>, <i>S</i>. <i>aureus</i> and vancomycin resistant enterococci (VRE).

<p>MIC range values for NCL812, NCL195 and NCL219 compared to MIC₉₀ values for daptomycin and ampicillin against <i>S</i>. <i>pneumoniae</i>, <i>S</i>. <i>aureus</i> and vancomycin resistant enterococci (VRE).</p

Time kill and point of resistance assays.

<p>NCL195 was prepared at 2× and 4× MIC (using 4× MIC of ampicillin (or 4× MIC of daptomycin) and normal saline as controls) in either LB broth supplemented with 3% lysed horse blood and 5% horse serum for <i>S</i>. <i>pneumoniae</i> D39 (<b>A</b>) or LB broth without supplementation for <i>S</i>. <i>aureus</i> Xen29 (<b>B and C</b>). Cultures were incubated statically at 37°C, 5% CO₂ (for <i>S</i>. <i>pneumoniae</i> D39) or at 37°C with agitation at 200 rpm (for <i>S</i>. <i>aureus</i> Xen29). Samples withdrawn at indicated times and plated on HBA overnight at 37°C, 5% CO₂ (for <i>S</i>. <i>pneumoniae</i> D39) or at 37°C with aeration (for <i>S</i>. <i>aureus</i> Xen29) for bacterial enumeration. For (<b>D</b>), <i>S</i>. <i>aureus</i> Xen29 was grown in 2 ml LB broth the presence of 0.5×MIC, 0.75×MIC, 1×MIC, 1.5×MIC, 2×MIC, and 4×MIC of NCL195, using 1×MIC, 4×MIC and 8×MIC of daptomycin as control.</p