Evaluation of robenidine analog NCL195 as a novel broad-spectrum antibacterial agent

Abstract

The spread of multidrug resistance among bacterial pathogens poses a serious threat to public health worldwide. Recent approaches towards combating antimicrobial resistance include repurposing old compounds with known safety and development pathways as new antibacterial classes with novel mechanisms of action. Here we show that an analog of the anticoccidial drug robenidine (4,6-bis(2-((E)-4-methylbenzylidene)hydrazinyl)pyrimidin-2-amine; NCL195) displays potent bactericidal activity against Streptococcus pneumoniae and Staphylococcus aureus by disrupting the cell membrane potential. NCL195 was less cytotoxic to mammalian cell lines than the parent compound, showed low metabolic degradation rates by human and mouse liver microsomes, and exhibited high plasma concentration and low plasma clearance rates in mice. NCL195 was bactericidal against Acinetobacter spp and Neisseria meningitidis and also demonstrated potent activity against A. baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. in the presence of sub-inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA) and polymyxin B. These findings demonstrate that NCL195 represents a new chemical lead for further medicinal chemistry and pharmaceutical development to enhance potency, solubility and selectivity against serious bacterial pathogens.

Fig 1. Structure activity relationship between NCL812, NCL195 and NCL219.

Installation of a 4-tert-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.

Fig 2. Time kill and point of resistance assays.

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 S. pneumoniae D39 (A) or LB broth without supplementation for S. aureus Xen29 (B and C). Cultures were incubated statically at 37°C, 5% CO₂ (for S. pneumoniae D39) or at 37°C with agitation at 200 rpm (for S. aureus Xen29). Samples withdrawn at indicated times and plated on HBA overnight at 37°C, 5% CO₂ (for S. pneumoniae D39) or at 37°C with aeration (for S. aureus Xen29) for bacterial enumeration. For (D), S. aureus 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.

Fig 3. NCL812 compounds exert their antibacterial action on the cell membrane of S. pneumoniae and S. aureus.

(A and B), S. pneumoniae strain D39 exposed to 16 μg/ml NCL812 for 6 h exhibited significantly thicker cell membranes compared to untreated samples (A) (p < 0.0001; two-tailed unpaired t-test) and displayed significantly wider periplasmic space compared to untreated samples (B) (p < 0.001; two-tailed unpaired t-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. (C and D) NCL812 affects macromolecular synthesis (c) and ATP release (D) in S. aureus. (E and F), NCL Compounds dissipate the membrane potential of S. pneumoniae and S. aureus. Membrane potential measurements of S. pneumoniae D39 (E) and S. aureus ATCC49775 (F). 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 Materials and Methods.

Fig 4. NCL812 and NCL195 exhibit high plasma concentration and low plasma clearance rates.

Pharmacokinetic parameters for NCL812 (A) and NCL195 (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. (C), 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.

Fig 5. NCL195 demonstrates limited cytotoxicity to mammalian cell lines.

Real-time cell viability measurements for Hep G2 (A, B, C) and MDBK (D, E, F) 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).