Angela M. Kavanagh

Activity and Predicted Nephrotoxicity of Synthetic Antibiotics Based on Polymyxin B

The polymyxin lipodecapeptides colistin and polymyxin B have become last resort therapies for infections caused by highly drug-resistant Gram-negative bacteria. Unfortunately, their utility is compromised by significant nephrotoxicity and polymyxin-resistant bacterial strains. We have conducted a systematic activity–toxicity investigation by varying eight of the nine polymyxin amino acid free side chains, preparing over 30 analogues using a novel solid-phase synthetic route. Compounds were tested against a panel of Gram-negative bacteria and counter-screened for in vitro cell toxicity. Promising compounds underwent additional testing against primary kidney cells isolated from human kidneys to better predict their nephrotoxic potential. Many of the new compounds possessed equal or better antimicrobial potency compared to polymyxin B, and some were less toxic than polymyxin B and colistin against mammalian HepG2 cells and human primary kidney cells. These initial structure–activity and structure–toxicity studies set the stage for further improvements to the polymyxin class of antibiotics.

Antibacterial serrulatane diterpenes from the Australian native plant Eremophila microtheca

Chemical investigations of the aerial parts of the Australian plant Eremophila microtheca resulted in the isolation of three serrulatane diterpenoids, 3-acetoxy-7,8-dihydroxyserrulat-14-en-19-oic acid (1), 3,7,8-trihydroxyserrulat-14-en-19-oic acid (2) and 3,19-diacetoxy-8-hydroxyserrulat-14-ene (3) as well as the previously reported compounds verbascoside (4) and jaceosidin (5). Acetylation and methylation of the major serrulatane diterpenoid 2 afforded 3,8-diacetoxy-7-hydroxyserrulat-14-en-19-oic acid (6) and 3,7,8-trihydroxyserrulat-14-en-19-oic acid methyl ester (7), respectively. The antibacterial activity of 1-7 was assessed against a panel of Gram-positive and Gram-negative bacterial isolates. All of the serrulatane compounds exhibited moderate activity against Streptococcus pyogenes (ATCC 12344) with minimum inhibitory concentrations (MICs) ranging from 64-128 μg/mL. Serrulatane 1 demonstrated activity against all Gram-positive bacterial strains (MICs 64-128 μg/mL) except for Enterococcus faecalis and Enterococcus faecium. This is the first report of natural products from E. microtheca.

Contribution of Amphipathicity and Hydrophobicity to the Antimicrobial Activity and Cytotoxicity of β-Hairpin Peptides

Bacteria have acquired extensive resistance mechanisms to protect themselves against antibiotic action. Today the bacterial membrane has become one of the “final frontiers” in the search for new compounds acting on novel targets to address the threat of multi-drug resistant (MDR) and XDR bacterial pathogens. β-Hairpin antimicrobial peptides are amphipathic, membrane-binding antibiotics that exhibit a broad range of activities against Gram-positive, Gram-negative, and fungal pathogens. However, most members of the class also possess adverse cytotoxicity and hemolytic activity that preclude their development as candidate antimicrobials. We examined peptide hydrophobicity, amphipathicity, and structure to better dissect and understand the correlation between antimicrobial activity and toxicity, membrane binding, and membrane permeability. The hydrophobicity, pI, net charge at physiological pH, and amphipathic moment for the β-hairpin antimicrobial peptides tachyplesin-1, polyphemusin-1, protegrin-1, gomesin, arenicin-3, and thanatin were determined and correlated with key antimicrobial activity and toxicity data. These included antimicrobial activity against five key bacterial pathogens and two fungi, cytotoxicity against human cell lines, and hemolytic activity in human erythrocytes. Observed antimicrobial activity trends correlated with compound amphipathicity and, to a lesser extent, with overall hydrophobicity. Antimicrobial activity increased with amphipathicity, but unfortunately so did toxicity. Of note, tachyplesin-1 was found to be 8-fold more amphipathic than gomesin. These analyses identify tachyplesin-1 as a promising scaffold for rational design and synthetic optimization toward an antibiotic candidate.

Mucin Binding Reduces Colistin Antimicrobial Activity

ABSTRACT Colistin has found increasing use in treating drug-resistant bacterial lung infections, but potential interactions with pulmonary biomolecules have not been investigated. We postulated that colistin, like aminoglycoside antibiotics, may bind to secretory mucin in sputum or epithelial mucin that lines airways, reducing free drug levels. To test this hypothesis, we measured binding of colistin and other antibiotics to porcine mucin, a family of densely glycosylated proteins used as a surrogate for human sputum and airway mucin. Antibiotics were incubated in dialysis tubing with or without mucin, and concentrations of unbound antibiotics able to penetrate the dialysis tubing were measured over time using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The percentage of antibiotic measured in the dialysate after 4 h in the presence of mucin, relative to the amount without mucin, was 15% for colistin, 16% for polymyxin B, 19% for tobramycin, 52% for ciprofloxacin, and 78% for daptomycin. Antibiotics with the strongest mucin binding had an overall polybasic positive charge, whereas those with comparatively little binding were less basic. When comparing MICs measured with or without added mucin, colistin and polymyxin B showed >100-fold increases in MICs for multiple Gram-negative bacteria. Preclinical evaluation of mucin binding should become a standard procedure when considering the potential pulmonary use of new or existing antibiotics, particularly those with a polybasic overall charge. In the airways, mucin binding may reduce the antibacterial efficacy of inhaled or intravenously administered colistin, and the presence of sub-MIC effective antibiotic concentrations could result in the development of antibiotic resistance.

A new antibiotic with potent activity targets MscL

The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections.

An azido-oxazolidinone antibiotic for live bacterial cell imaging and generation of antibiotic variants.

Graphical abstract

Metronidazole-triazole conjugates: Activity against Clostridium difficile and parasites

Metronidazole has been used clinically for over 50 years as an antiparasitic and broad-spectrum antibacterial agent effective against anaerobic bacteria. However resistance to metronidazole in parasites and bacteria has been reported, and improved second-generation metronidazole analogues are needed. The copper catalysed Huigsen azide-alkyne 1,3-dipolar cycloaddition offers a way to efficiently assemble new libraries of metronidazole analogues. Several new metronidazole-triazole conjugates (Mtz-triazoles) have been identified with excellent broad spectrum antimicrobial and antiparasitic activity targeting Clostridium difficile, Entamoeba histolytica and Giardia lamblia. Cross resistance to metronidazole was observed against stable metronidazole resistant C. difficile and G. lamblia strains. However for the most potent Mtz-triazoles, the activity remained in a therapeutically relevant window.

Short cationic lipopeptides as effective antibacterial agents: Design, physicochemical properties and biological evaluation.

The spread of drug-resistant bacteria has imparted a sense of urgency in the search for new antibiotics. In an effort to develop a new generation of antibacterial agents, we have designed de novo charged lipopeptides inspired by natural antimicrobial peptides. These short lipopeptides are composed of cationic lysine and hydrophobic lipoamino acids that replicate the amphiphilic properties of natural antimicrobial peptides. The resultant lipopeptides were found to self-assemble into nanoparticles. Some were effective against a variety of Gram-positive bacteria, including strains resistant to methicillin, daptomycin and/or vancomycin. The lipopeptides were not toxic to human kidney and liver cell lines and were highly resistant to tryptic degradation. Transmission electron microscopy analysis of bacteria cells treated with lipopeptide showed membrane-damage and lysis with extrusion of cytosolic contents. With such properties in mind, these lipopeptides have the potential to be developed as new antibacterial agents against drug-resistant Gram-positive bacteria.

Redesigned spider peptide with improved antimicrobial and anticancer properties

Gomesin, a disulfide-rich antimicrobial peptide produced by the Brazilian spider Acanthoscurria gomesiana, has been shown to be potent against Gram-negative bacteria and to possess selective anticancer properties against melanoma cells. In a recent study, a backbone cyclized analogue of gomesin was shown to be as active but more stable than its native form. In the current study, we were interested in improving the antimicrobial properties of the cyclic gomesin, understanding its selectivity toward melanoma cells and elucidating its antimicrobial and anticancer mode of action. Rationally designed analogues of cyclic gomesin were examined for their antimicrobial potency, selectivity toward cancer cells, membrane-binding affinity, and ability to disrupt cell and model membranes. We improved the activity of cyclic gomesin by ∼10-fold against tested Gram-negative and Gram-positive bacteria without increasing toxicity to human red blood cells. In addition, we showed that gomesin and its analogues are more toxic toward melanoma and leukemia cells than toward red blood cells and act by selectively targeting and disrupting cancer cell membranes. Preference toward some cancer types is likely dependent on their different cell membrane properties. Our findings highlight the potential of peptides as antimicrobial and anticancer leads and the importance of selectively targeting cancer cell membranes for drug development.

Structure–Activity and −Toxicity Relationships of the Antimicrobial Peptide Tachyplesin-1

Tachyplesin-1 (TP1; 1) is a cationic β-hairpin antimicrobial peptide with a membranolytic mechanism of action. While it possesses broad-spectrum, potent antimicrobial activity, 1 is highly hemolytic against mammalian erythrocytes, which precludes it from further development. In this study, we report a template-based approach to investigate the structure-function and structure-toxicity relationships of each amino acid of 1. We modulated charge and hydrophobicity by residue modification and truncation of the peptide. Antimicrobial activity was then assessed against six key bacterial pathogens and two fungi, with toxicity profiled against mammalian cells. The internal disulfide bridge Cys7-Cys12 of 1 was shown to play an important role in broad-spectrum antimicrobial activity against all pathogenic strains tested. Novel peptides based on the progenitor were then designed, including 5 (TP1[F4A]), 12 (TP1[I11A]), and 19 (TP1[C3A,C16A]). These had 26- to 64-fold improved activity/toxicity indices and show promise for further development. Structural studies of 5 (TP1[F4A]) and 12 (TP1[I11A]) identified a conserved β-hairpin secondary structure motif correlating with their very high stablility in mouse and human plasma. Membrane binding affinity determined by surface plasmon resonance confirmed their selectivity toward bacterial membranes, but the degree of membrane binding did not correlate with the degree of hemolysis, suggesting that other factors may drive toxicity.