Rationally designed transmembrane peptide mimics of the multidrug transporter protein Cdr1 act as antagonists to selectively block drug efflux and chemosensitize azole-resistant clinical isolates of Candida albicans.

Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.

Mechanism of action of novel synthetic dodecapeptides against Candida albicans.

BACKGROUND: Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α,ß-didehydrophenylalanine (∆Phe) exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans and other yeast and filamentous pathogenic fungi. In the present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated. METHODS: The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, Reactive Oxygen Species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using Scanning and Transmission Electron Microscopy. RESULTS AND CONCLUSIONS: The MIC of the peptides against C. albicans and other yeast and filamentous fungal pathogens ranged between 3.91 and 250µM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans. GENERAL SIGNIFICANCE: The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.

Enhancing Antimicrobial Peptide Potency through Multivalent Presentation on Coiled-Coil Nanofibrils.

Antibiotic-resistant microbes have become a global health threat. New delivery systems that enhance the efficacy of antibiotics and/or overcome the resistances can help combat them. In this context, we present FF03, a fibril-forming α-helical coiled-coil peptide that functions as an efficient scaffold for the multivalent presentation of the weakly cationic antimicrobial peptide (AMP) IN4. The resulting IN4-decorated FF03 coiled-coil fibrils (FF03 + IN4) are nonhemolytic and noncytotoxic and show enhanced antimicrobial activity relative to unconjugated IN4 and standard antibiotics against several bacterial strains. Scanning electron microscopy analysis shows that FF03 + IN4 nanofibers disrupt methicillin-resistant Staphylococcus aureus membranes, indicating a surface-level mode of action. Furthermore, transmission electron microscopy and circular dichroism studies indicate that decoration of the FF03 scaffold with IN4 does not alter the secondary-structure propensity or fibril-forming properties of FF03. Thus, the approach reported herein provides a new peptidic scaffold for the multivalent presentation of AMPs to obtain nonhemolytic and noncytotoxic antimicrobial systems with improved efficacy relative to the unconjugated AMP analogues.

Coassembly Generates Peptide Hydrogel with Wound Dressing Material Properties.

Multicomponent self-assembly of peptides is a powerful strategy to fabricate novel functional materials with synergetic properties that can be used for several nanobiotechnological applications. In the present study, we used a coassembly strategy to generate an injectable ultrashort bioactive peptide hydrogel formed by mixing a dipeptide hydrogelator with a macrophage attracting short chemotactic peptide ligand. Coassembly does not impede hydrogelation as shown by cryo-transmission electron microscopy (cryo-TEM), scanning electron microscopy, and rheology. Biocompatibility was shown by cytotoxicity assays and confocal microscopy. The hydrogels release the entrapped skin antibiotic ciprofloxacin, among others, in a slow and continuous manner. Such bioinspired advanced functional materials can find applications as wound dressing materials to treat chronic wound conditions like diabetic foot ulcer.

C-Terminal Fragment, Aß39-42-Based Tetrapeptides Mitigates Amyloid-ß Aggregation-Induced Toxicity.

Since the introduction of acetyl cholinesterase inhibitors as the first approved drugs by the US Food and Drug Administration for Alzheimer's disease (AD) in clinics, less than satisfactory success in the design of anti-AD agents has impelled the scientists to also focus toward inhibition of Aß aggregation. Considering the specific binding of fragments for their parent peptide, herein, we synthesized more than 40 new peptides based on a C-terminus tetrapeptide fragment of Aß1-42. Initial screening by MTT cell viability assay and supportive results by ThT fluorescence assay led us to identify a tetrapeptide showing complete inhibition for Aß1-42 aggregation. Peptide 20 displayed 100% cell viability at 20 µM concentration, while at lower concentrations of 10 and 2 µM 76.6 and 70% of cells were viable. Peptide 20 was found to restrict the conformational transition of Aß1-42 peptide toward ß-sheet structure. Inhibitory activity of tetrapeptide 20 was further evidenced by the absence of Aß1-42 aggregates in electron microscopy. Peptide 20 and other significantly active tetrapeptide analogues could prove imperative in the future design of anti-AD agents.

"A novel highly stable and injectable hydrogel based on a conformationally restricted ultrashort peptide".

Nanostructures including hydrogels based on peptides containing non protein amino acids are being considered as platform for drug delivery because of their inherent biocompatibility and additional proteolytic stability. Here we describe instantaneous self-assembly of a conformationally restricted dipeptide, LeuΔPhe, containing an α,ß-dehydrophenylalanine residue into a highly stable and mechanically strong hydrogel, under mild physiological aqueous conditions. The gel successfully entrapped several hydrophobic and hydrophilic drug molecules and released them in a controlled manner. LeuΔPhe was highly biocompatible and easily injectable. Administration of an antineoplastic drug entrapped in the gel in tumor bearing mice significantly controlled growth of tumors. These characteristics make LeuΔPhe an attractive candidate for further development as a delivery platform for various biomedical applications.

C-Terminal Fragment, Aß32-37, Analogues Protect Against Aß Aggregation-Induced Toxicity.

Amyloid-ß aggregation is a major etiological phenomenon in Alzheimer's disease. Herein, we report peptide-based inhibitors that diminish the amyloid load by obviating Aß aggregation. Taking the hexapeptide fragment, Aß32-37, as lead, more than 40 new peptides were synthesized. Upon evaluation of the newly synthesized hexapeptides as inhibitors of Aß toxicity by the MTT-based cell viability assay, a number of peptides exhibited significant Aß aggregation inhibitory activity at sub-micromolar concentration range. A hexapeptide (1) showed complete mitigation of Aß toxicity in the cell culture assay at 2 µM. In the ThT fluorescence assay, upon incubation of Aß with this peptide, we observed no increase in the ThT fluorescence relative to control. The secondary structure estimation by circular dichroism spectroscopy and morphological examination by transmission electron microscopy further confirmed the results.