Resistance or tolerance to traditional antibiotics is a challenging issue in antimicrobial chemotherapy. Moreover, traditional bactericidal antibiotics kill only actively growing bacterial cells, whereas nongrowing metabolically inactive cells are tolerant to and therefore “persist” in the presence of legacy antibiotics. Here, we report that the diarylurea derivative PQ401, previously characterized as an inhibitor of the insulin-like growth factor I receptor, kills both antibiotic-resistant and nongrowing antibiotic-tolerant methicillin-resistant Staphylococcus aureus (MRSA) by lipid bilayer disruption. PQ401 showed several beneficial properties as an antimicrobial lead compound, including rapid killing kinetics, low probability for resistance development, high selectivity to bacterial membranes compared to mammalian membranes, and synergism with gentamicin. In contrast to well-studied membrane-disrupting cationic antimicrobial low-molecular-weight compounds and peptides, molecular dynamic simulations supported by efficacy data demonstrate that the neutral form of PQ401 penetrates and subsequently embeds into bacterial lipid bilayers more effectively than the cationic form. Lastly, PQ401 showed efficacy in both the Caenorhabditis elegans and Galleria mellonella models of MRSA infection. These data suggest that PQ401 may be a lead candidate for repurposing as a membrane-active antimicrobial and has potential for further development as a human antibacterial therapeutic for difficult-to-treat infections caused by both drug-resistant and -tolerant S. aureus. IMPORTANCE Membrane-damaging antimicrobial agents have great potential to treat multidrug-resistant or multidrug-tolerant bacteria against which conventional antibiotics are not effective. However, their therapeutic applications are often hampered due to their low selectivity to bacterial over mammalian membranes or their potential for cross-resistance to a broad spectrum of cationic membrane-active antimicrobial agents. We discovered that the diarylurea derivative compound PQ401 has antimicrobial potency against multidrug-resistant and multidrug-tolerant Staphylococcus aureus. PQ401 selectively disrupts bacterial membrane lipid bilayers in comparison to mammalian membranes. Unlike cationic membrane-active antimicrobials, the neutral form of PQ401 rather than its cationic form exhibits maximum membrane activity. Overall, our results demonstrate that PQ401 could be a promising lead compound that overcomes the current limitations of membrane selectivity and cross-resistance. Also, this work provides deeper insight into the design and development of new noncharged membrane-targeting therapeutics to combat hard-to-cure bacterial infections.

Increasing microbial resistance, coupled with a lack of new antimicrobial discovery, has led researchers to refocus on antimicrobial peptides (AMPs) as novel therapeutic candidates. Significantly, the less toxic cecropins have gained widespread attention for potential antibacterial agent development. However, the narrow activity spectrum and long sequence remain the primary limitations of this approach. In this study, we truncated and modified cecropin 4 (41 amino acids) by varying the charge and hydrophobicity balance to obtain smaller AMPs. The derivative peptide C18 (16 amino acids) demonstrated high antibacterial activity against Gram-negative and Gram-positive bacteria, as well as yeasts. Moreover, C18 demonstrated a minimal inhibitory concentration (MIC) of 4 µg/mL against the methicillin-resistant Staphylococcus aureus (MRSA) and showed synergy with daptomycin with a fractional inhibition concentration index (FICI) value of 0.313. Similar to traditional cecropins, C18 altered the membrane potential, increased fluidity, and caused membrane breakage at 32 µg/mL. Importantly, C18 eliminated 99% persisters at 10 × MIC within 20 min and reduced the biofilm adherence by ~40% and 35% at 32 and 16 µg/mL. Besides, C18 possessed a strong binding ability with DNA at 7.8 μM and down-regulated the expression of virulence factor genes like agrA, fnb-A, and clf-1 by more than 5-fold (p < 0.05). Interestingly, in the Galleria mellonella model, C18 rescued more than 80% of larva infected with the MRSA throughout 120-h post-infection at a single dose of 8 mg/kg (p < 0.05). In conclusion, this study provides a reference for the transformation of cecropin to derive small peptides and presents C18 as an attractive therapeutic candidate to be developed to treat severe MRSA infections.

The formation of persister cells is associated with recalcitrance and infections. In this study, we examined the antimicrobial property of alpha mangostin, a natural xanthone molecule, against methicillin-resistant Staphylococcus aureus (MRSA) persisters and biofilm. The MIC of alpha mangostin against MRSA persisters was 2 µg/ml, and activity was mediated by causing membrane permeabilization within 30 min of exposure. The membrane activity of alpha mangostin was further studied by fast-killing kinetics of MRSA persiste r cells and found that the compound exhibited 99.99% bactericidal activity within 30 min. Furthermore, alpha mangostin disrupted established MRSA biofilms and inhibited bacterial attachment as biofilm formation. Alpha mangostin down-regulated genes associated with the formation of persister cells and biofilms, such as norA, norB, dnaK, groE, and mepR, ranging from 2 to 4-folds. Alpha mangostin at 16 μg/ml was non-toxic (&gt; 95% cell survival) to liver-derived HepG2 and lung-derived A549 cells, similarly. Still, alpha mangostin exhibited 50% cell lysis of human RBC at 16 μg/ml. Interestingly, alpha mangostin was effective in vivo at increasing the survival up to 75% (p&lt;0.0001) of Galleria mellonella larvae infected with MRSA persister for 120 h. In conclusion, we report that alpha mangostin is active against MRSA persisters and biofilms, and these data further our understanding of the antistaphylococcal activity and toxicity of this natural compound.