We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-lM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.

ESKAPE pathogens are notorious in causing nosocomial infections and escaping current antibiotic treatments. There has been a dramatic increase in nosocomial infections accompanied with a decrease in the number of antibiotics developed, leading to significant increase in morbidity and mortality among patients. In an attempt to combat this problem, derivatives of ciprofloxacin, rifabutin and beta-lactam antibiotics were synthesized and tested against the ESKAPE pathogens. From minimum inhibitory concentration assays, 4 ciprofloxacin analogs and 8 beta-lactam analogs were found to be effective against multiple bacterial species. Additionally, 12 rifabutin analogs and 23 beta-lactam analogs were potent against single bacterial species, primarily toward methicillin-resistant Staphylococcus aureus (MRSA) at a concentration of ≤ 25 µg mL-1. Based on the effectiveness against methicillin-resistant Staphylococcus aureus (MRSA), three rifabutin analogs were selected for further testing. Two rifabutin analogs (DU644 and DU645) were found to possess between a one to twofold mean increase of inhibitory activities, while the other rifabutin analogs (DU650) demonstrated up to a twofold decrease of inhibitory activity when compared to the parent drug. These compounds were then examined for their bactericidal and antibiofilm activities against MRSA. From these assays, we found that DU644 and DU645 were 4 times more bactericidal and antibiofilm against MRSA when compared to the parent drug. In addition, rpoB mutation validation results confirmed that modification of these rifabutin derivatives at the C3 and C4 positions, and bearing an imidazolyl ring carrying substituted spiropiperidyl ring, did not change their mechanism of action towards the beta-subunit of RNA polymerase. Cytotoxicity testing performed using human hepatocellular carcinoma epithelial cells (hepG2) showed that at concentrations ranged from 1.25 µg mL-1 to 25 µg mL-1, DU644 and DU645 showed very low toxicity. Collectively, structural drugs modifications of these obsolete drugs are able to restore their antibacterial activities against MRSA, which is notable as the most infectious nosocomial pathogen. Therefore, further development and application of rifabutin analogs might be beneficial for medical use to combat MRSA infections.

The rapid progression of Klebsiella pneumoniae towards antibiotic resistance is a significant concern, primarily due to its protective extracellular polysaccharide (EPS) capsule that shields the bacteria from host immunity. Our previous research demonstrated that antimicrobial peptides could disrupt the EPS capsule of K. pneumoniae. Further analysis identified Bac7 (1-35), a proline-rich antimicrobial peptide (PrAMP), as having the greatest ability to aggregate with the K. pneumoniae EPS capsule, exhibiting potent antimicrobial activity. However, the relationship between key features facilitating EPS and membrane interactions, as well as antimicrobial efficacy, remains poorly understood. Here, we used natural PrAMPs from diverse organisms to investigate their interactions with the cell envelope of K. pneumoniae. Apidaecin Cd3+, Tur1A, and PR-39 peptides demonstrated activity against all tested strains, with a minimum inhibitory concentration ≤ 1 µg/mL. These peptides shared a proline content exceeding 36% and a charge greater than +5. Active PrAMPs induced membrane depolarization in K. pneumoniae, with the extent of depolarization directly correlating with peptide charge, suggesting membrane depolarization as a potential mechanism for PrAMP entry into the cell. Checkerboard assays of active PrAMPs with PepC, an inactive peptide, suggested the membrane actions of PrAMPs have potential to rescue a therapeutic unable to access the bacterial membrane. Consistent with our findings with bac7(1-35) truncated analogs, both active and inactive PrAMPs aggregated with K. pneumoniae EPS, suggesting that the antimicrobial activities and polysaccharide aggregation potential of this class of peptides can be studied independently. Furthermore, the treatment of biofilms with active peptides revealed unique structure-based biofilm changes, with Tur1A causing more structural collapse than PR-39. Our findings highlight a potential membrane mediated peptide uptake into the cell which is dependent on the charge of the peptide. Differential biofilm interactions between similar peptides and EPS aggregation of inactive peptides warrant these attributes of PrAMPs to be further studied independently.