Pre-Clinical In Vitro and In Vivo testing of Novel Antimicrobials for the Treatment of Bacterial Infections

Abstract

New molecules are urgently required for the treatment of multidrug-resistant (MDR)- Gram-positive (GPB) and Gram-negative bacteria (GNB) infections. In the latter, these efforts are further impeded due to the presence of an outer membrane (OM) in GNB, which prevents many antibiotics from gaining entry to reach their target sites. This challenge has led to the current shortfall of new antimicrobials with novel mechanisms of action to treat MDR-GNB infections in particular. The present study aimed to identify promising lead compounds with new modes of action for pharmaceutical development to treat MDR bacterial infections, particularly GNB infections. Here, we focused on four novel antibiotic entities, the robenidine analogues NCL195 and NCL179 and benzguinol A and B, which each exhibited potent antimicrobial activity against MDR-GPB as stand-alone entities and were active against MDRGNB when combined with adjuvants that perturb the OM. We previously reported in vitro activity for the robenidine analogue NCL195 against MDR-GPB with minimum inhibitory concentrations (MIC) from 1-4 μg/mL. Here, we found partially synergistic effects of NCL195 in combination with ethylenediaminetetraacetic acid (EDTA) or polymyxin B nonapeptide (PMBN) and fully synergistic activity of NCL195 when combined with sub-inhibitory concentrations of polymyxin (PMB) or colistin against all tested GNB isolates, whereas NCL195 alone had no activity. We further used fluorescence-based membrane potential measurements to clearly show the dual mechanism of action of the NCL195 + colistin/PMB combination against Escherichia coli. Moreover, we used transmission electron microscopy (TEM) to demonstrate the effect of NCL195 alone and in combination with colistin on GPB (Staphylococcus aureus) and GNB (E. coli and Pseudomonas aeruginosa) membrane morphology, respectively. We observed specific membrane damage and the presence of mesosome-like membrane structures in TEM images of S. aureus cells treated with NCL195 that were not present in untreated bacteria. No membrane morphology changes were observed in E. coli and P. aeruginosa exposure to NCL195 alone, whereas the NCL195+colistin combination treatment caused significantly more cell membrane damage compared to colistin alone. TEM images mirrored the in vitro antimicrobial activity of NCL195 against S. aureus and synergistic interaction of NCL195 + colistin against E. coli and P. aeruginosa. We also showed that NCL195 exhibited limited in vitro cytotoxicity (16 μg/mL) to mammalian cell lines, and low haemolytic activity (128 μg/mL) to human erythrocytes. Moreover, intraperitoneal (IP) treatment (50 mg/kg, 2 x injections, 4 h apart) of NCL195 alone, oral treatment (50 mg/kg, 4 x injections, 4 h apart) of NCL195 alone and the combination of oral NCL195 (50 mg/kg, 4 x infections, 4 h apart) with IP colistin (0.125-4 mg/kg, two-fold increasing, 4 x injections, 4 h apart) were safe to mice. Furthermore, we showed promising in vivo efficacy against MDR-GPB human pathogens when NCL195 was administered systemically (50 mg/kg x 2 injections, 4 h apart) and orally (50 mg/kg x 4 injections, 4 h apart) in bioluminescent mouse sepsis models, with greater potency observed via the latter route of administration. Subsequently, we found that the combination of oral NCL195 (50 mg/kg, 4 x injections, 4 h apart) with different IP doses of colistin (0.125, 0.25, 0.5, 1 or 2 mg/kg, 4 x injections, 4 h apart) resulted in a dose-dependent significant reduction in infectious load when mice were challenged with both colistin-susceptible and -resistant bioluminescent E. coli and prolonged survival times compared to treatment with colistin alone at similar concentrations in a GNB sepsis model. We further investigated the potential of another analogue of robenidine (NCL179) to expand chemical diversity of this potentially new class of antimicrobial to treat MDR bacterial infections. We showed that NCL179 exhibited potent bactericidal activity against a wide range of GPB pathogens (MICs from 1-4 μg/mL) and synergistic activity combined with subinhibitory concentrations of colistin against GNB (MICs return 0.5-4 μg/mL), whereas NCL179 alone had no activity. NCL179 exhibited limited in vitro toxicity (16 μg/mL) to mammalian cell lines, and low haemolytic activity (32 μg/mL) to human erythrocytes. Oral treatment of mice with NCL179 (50 mg/kg, 4 x injections, 4 h apart) was safe and significantly reduced S. aureus populations in vivo and prolonged survival times in a mouse sepsis model. We also extended our research into further biological characterisation of the “lost antibiotic” unguinol and further chemical diversification of related nidulin-family fungal natural products which identified two semisynthetic derivatives, benzguinols A and B. Excellent in vitro activity against many clinical GPB pathogens (MICs, 0.25-1 μg/mL) and synergistic activity in combination with sub-inhibitory concentrations of colistin against GNB reference strains (MICs, 1-2 μg/mL) were observed, whereas the benzguinols alone had no activity against GNB. Moreover, benzguinol A and B showed low in vitro toxicity to mammalian cell lines (32 μg/mL), and low haemolytic activity (128 μg/mL) to human erythrocytes. Furthermore, IP treatment of benzguinol A or B (20 mg/kg, 4 x injections, 4 h apart) exhibited systemic safety in mice and significantly reduced bacterial loads and prolonged survival times compared to vehicle-only treated mice in a bioluminescent S. aureus murine sepsis challenge model. In conclusion, NCL195, NCL179 and benzguinols A and B are viable candidates for further pre-clinical development for specific treatment of MDR-GPB and GNB infections either as stand-alone antibiotics or in combination with sub-inhibitory concentrations of colistin, respectively. They represent excellent scaffolds for further medicinal chemistry development to improve potency.