Namfon Pantarat

Combating multidrug-resistant Gram-negative bacteria with structurally nanoengineered antimicrobial peptide polymers

With the recent emergence of reports on resistant Gram-negative ‘superbugs’, infections caused by multidrug-resistant (MDR) Gram-negative bacteria have been named as one of the most urgent global health threats due to the lack of effective and biocompatible drugs. Here, we show that a class of antimicrobial agents, termed ‘structurally nanoengineered antimicrobial peptide polymers’ (SNAPPs) exhibit sub-μM activity against all Gram-negative bacteria tested, including ESKAPE and colistin-resistant and MDR (CMDR) pathogens, while demonstrating low toxicity. SNAPPs are highly effective in combating CMDR Acinetobacter baumannii infections in vivo, the first example of a synthetic antimicrobial polymer with CMDR Gram-negative pathogen efficacy. Furthermore, we did not observe any resistance acquisition by A. baumannii (including the CMDR strain) to SNAPPs. Comprehensive analyses using a range of microscopy and (bio)assay techniques revealed that the antimicrobial activity of SNAPPs proceeds via a multimodal mechanism of bacterial cell death by outer membrane destabilization, unregulated ion movement across the cytoplasmic membrane and induction of the apoptotic-like death pathway, possibly accounting for why we did not observe resistance to SNAPPs in CMDR bacteria. Overall, SNAPPs show great promise as low-cost and effective antimicrobial agents and may represent a weapon in combating the growing threat of MDR Gram-negative bacteria.

Role and mechanism of phosphatidylinositol‐specific phospholipase C in survival and virulence of Cryptococcus neoformans

Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans. To investigate the role of phosphatidylinositol‐specific phospholipase C (PI‐PLC) in Plb1 secretion, we identified two putative PI‐PLC‐encoding genes in C. neoformans var. grubii (PLC1 and PLC2), and created Δplc1 and Δplc2 deletion mutants. In Δplc1, which expressed less PI‐PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37°C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Δplc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen‐activated protein kinase (MAPK) in the presence of cell wall‐perturbing agents. In contrast to Δplc2, which was as virulent as WT, Δplc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25°C, demonstrating that mechanism(s) independent of the 37°C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.

Maculatin 1.1 disrupts Staphylococcus aureus lipid membranes via a pore mechanism.

ABSTRACT Maculatin 1.1 (Mac1) showed potent activity against Staphylococcus aureus with an MIC of 7 μM. The mode of action of Mac1 was investigated by combining assays with S. aureus cells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs of S. aureus incubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans by S. aureus was observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores in S. aureus membranes.

Heterocyclic dithiocarbazate iron chelators: Fe coordination chemistry and biological activity

The iron coordination and biological chemistry of a series of heterocyclic dithiocarbazate Schiff base ligands is reported with regard to their activity as Fe chelators for the treatment of Fe overload and also cancer. The ligands are analogous to tridentate heterocyclic hydrazone and thiosemicarbazone chelators we have studied previously which bear NNO and NNS donor sets. The dithiocarbazate Schiff base ligands in this work also are NNS chelators and form stable low spin ferric and ferrous complexes and both have been isolated. In addition an unusual hydroxylated ligand derivative has been identified via an Fe-induced oxidation reaction. X-ray crystallographic and spectroscopic characterisation of these complexes has been carried out and also the electrochemical properties have been investigated. All Fe complexes exhibit totally reversible Fe(III/II) couples in mixed aqueous solvents at potentials higher than found in analogous thiosemicarbazone Fe complexes. The ability of the dithiocarbazate Schiff base ligands to mobilise Fe from cells and also to prevent Fe uptake from transferrin was examined and all ligands were effective in chelating intracellular Fe relative to known controls such as the clinically important Fe chelator desferrioxamine. The Schiff base ligands derived from 2-pyridinecarbaldehyde were non-toxic to SK-N-MC neuroepithelioma (cancer) cells but those derived from the ketones 2-acetylpyridine and di-2-pyridyl ketone exhibited significant antiproliferative activity.

Multimerization of a Proline-Rich Antimicrobial Peptide, Chex-Arg20, Alters Its Mechanism of Interaction with the Escherichia coli Membrane.

A3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization.

Cellular Uptake of the Antitumor Agent Dp44mT Occurs via a Carrier/Receptor-Mediated Mechanism

The chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) shows potent and selective anticancer and antimetastatic activity. However, the mechanism by which it is initially transported into cells to induce cytotoxicity is unknown. Hence, the current investigation examined the cellular uptake of 14C-Dp44mT relative to two structurally related ligands, namely the aroylhydrazone 14C-pyridoxal isonicotinoyl hydrazone (14C-PIH) and the thiosemicarbazone 14C-2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (14C-Bp4eT). In marked contrast to the cellular uptake of 14C-PIH and 14C-Bp4eT, which were linear as a function of concentration, 14C-Dp44mT uptake was saturable using SK-N-MC neuroepithelioma cells (Bmax, 4.28 × 107 molecules of chelator/cell; and Kd, 2.45 μM). Together with the fact that 14C-Dp44mT uptake was temperature-dependent and significantly (P < 0.01) decreased by competing unlabeled Dp44mT, these observations indicated a saturable transport mechanism consistent with carrier/receptor–mediated transport. Other unlabeled ligands that shared the saturated N4 structural moiety with Dp44mT significantly (P < 0.01) inhibited 14C-Dp44mT uptake, illustrating its importance for carrier/receptor recognition. Nevertheless, unlabeled Dp44mT most markedly decreased 14C-Dp44mT uptake, demonstrating that the putative carrier/receptor shows high selectivity for Dp44mT. Interestingly, in contrast to 14C-Dp44mT, uptake of its Fe complex [Fe(14C-Dp44mT)2] was not saturable as a function of concentration and was much greater than the ligand alone, indicating an alternate mode of transport. Studies examining the tissue distribution of 14C-Dp44mT injected intravenously into a mouse tumor model demonstrated the 14C label was primarily identified in the excretory system. Collectively, these findings examining the mechanism of Dp44mT uptake and its distribution and excretion have clinical implications for its bioavailability and uptake in vivo.

Membrane transport and intracellular sequestration of novel thiosemicarbazone chelators for the treatment of cancer.

Iron is a critical nutrient for DNA synthesis and cellular proliferation. Targeting iron in cancer cells using specific chelators is a potential new strategy for the development of novel anticancer agents. One such chelator, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), possesses potent and selective anticancer activity (J Med Chem 50:3716–3729, 2007). To elucidate the mechanisms of its potent antitumor activity, Bp4eT was labeled with 14C. Its efficacy was then compared with the 14C-labeled iron chelator pyridoxal isonicotinoyl hydrazone (PIH), which exhibits low anticancer activity. The ability of these ligands to permeate the cell membrane and their cellular retention was examined under various conditions using SK-N-MC neuroepithelioma cells. The rate of [14C]PIH uptake into cells was significantly (p < 0.001) lower than that of [14C]Bp4eT at 37°C, indicating that the increased hydrophilicity of [14C]PIH reduced membrane permeability. In contrast, the efflux of [14C]PIH was significantly (p < 0.05) higher than that of [14C]Bp4eT, leading to increased cellular retention of [14C]Bp4eT. In addition, the uptake and release of the 14C-labeled chelators was not reduced by metabolic inhibitors, indicating that these processes were energy-independent. No significant differences were evident in the uptake of [14C]Bp4eT at 37 or 4°C, demonstrating a temperature-independent mechanism. Furthermore, adjusting the pH of the culture medium to model the tumor microenvironment did not affect [14C]Bp4eT membrane transport. It can be concluded that [14C]Bp4eT more effectively permeated the cell membrane and evaded rapid efflux in contrast to [14C]PIH. This property, in part, accounts for the more potent anticancer activity of Bp4eT relative to PIH.

A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.

Effect of Caspofungin on Metabolite Profiles of Aspergillus Species Determined by Nuclear Magnetic Resonance Spectroscopy

ABSTRACT Invasive aspergillosis remains a potentially life-threatening infection, the incidence of which is increasing. Current methods used to determine the susceptibilities of Aspergillus strains to antifungal drugs are often unreliable. Nuclear magnetic resonance (NMR) spectroscopy can identify the metabolic complement of microorganisms while monitoring nutrient utilization from the incubation medium. We used 600-MHz 1H NMR spectroscopy to monitor the metabolic responses of five Aspergillus species cultured in RPMI 1640-2% glucose-morpholinepropanesulfonate buffer to various concentrations of the antifungal drugs amphotericin B (AMB) and caspofungin. The metabolic endpoint (MEP) was determined from nutrient and metabolite resonances, measured as a function of the drug concentration, and was defined as a ≥50% reduction in nutrient consumption or metabolite production. MICs were evaluated by a modification of Clinical and Laboratory Standards Institute broth microdilution method M27-A, and minimal effective concentrations (MECs) were determined by microscopic examination of fungal hyphae. For AMB, the MEPs coincided with the MICs. For caspofungin, the MEPs agreed with the MECs for several Aspergillus strains, but the effect of drug pressure was more complex for others. Expansion of the MEP definition to include any significant changes in metabolite production resulted in agreement with the MEC in most cases. Paradoxical metabolic responses were observed for several Aspergillus strains at either high or low caspofungin concentrations and for one Aspergillus terreus strain with AMB. NMR spectroscopy proved to be a powerful tool for detecting the subtle effects of drug pressure on fungal metabolism and has the potential to provide an alternative method for determining the susceptibilities of Aspergillus species to antifungal drugs.

In Vitro Antifungal Activities of Bis(Alkylpyridinium)Alkane Compounds against Pathogenic Yeasts and Molds

ABSTRACT Ten bis(alkylpyridinium)alkane compounds were tested for antifungal activity against 19 species (26 isolates) of yeasts and molds. We then determined the MICs and minimum fungicidal concentrations (MFCs) of four of the most active compounds (compounds 1, 4, 5, and 8) against 80 Candida and 20 cryptococcal isolates, in comparison with the MICs of amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin, using Clinical Laboratory and Standards Institutes broth microdulition M27-A3 (yeasts) or M38-A2 (filamentous fungi) susceptibility protocols. The compounds were more potent against Candida and Cryptococcus spp. (MIC range, 0.74 to 27.9 μg/ml) than molds (0.74 to 59.7 μg/ml). MICs against Exophiala were 0.37 to 5.9 μg/ml and as low as 1.48 μg/ml for Scedosporium but ≥25 μg/ml for zygomycetes, Aspergillus, and Fusarium spp. Compounds 1, 4, 5, and 8 exhibited good fungicidal activity against Candida and Cryptococcus, except for Candida parapsilosis (MICs of >44 μg/ml). Geometric mean (GM) MICs were similar to those of amphotericin B and lower than or comparable to fluconazole GM MICs but 10- to 100-fold greater than those for the other azoles. GM MICs against Candida glabrata were <1 μg/ml, significantly lower than fluconazole GM MICs (P < 0.001) and similar to those of itraconazole, posaconazole, and voriconazole (GM MIC range of 0.4 to 1.23 μg/ml). The GM MIC of compound 4 against Candida guilliermondii was lower than that of fluconazole (1.69 μg/ml versus 7.48 μg/ml; P = 0.012). MICs against Cryptococcus neoformans and Cryptococcus gattii were similar to those of fluconazole. The GM MIC of compound 4 was significantly higher for C. neoformans (3.83 μg/ml versus 1.81 μg/ml for C. gattii; P = 0.015). This study has identified clinically relevant in vitro antifungal activities of novel bisalkypyridinium alkane compounds.