Novel Cyclic Lipopeptides Fusaricidin Analogs for Treating Wound Infections.

Both acute and chronic cutaneous wounds are often difficult to treat due to the high-risk for bacterial contamination. Once hospitalized, open wounds are at a high-risk for developing hospital-associated infections caused by multi drug-resistant bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. Treating these infections is challenging, not only because of antibiotic resistance, but also due to the production of biofilms. New treatment strategies are needed that will help in both stimulating the wound healing process, as well as preventing and eliminating bacterial wound infections. Fusaricidins are naturally occurring cyclic lipopeptides with antimicrobial properties that have shown to be effective against a variety of fungi and Gram-positive bacteria, with low toxicity. Continuing with our efforts toward the identification of novel cyclic lipopeptides Fusaricidin analogs, herein we report the synthesis and evaluation of the antimicrobial activity for two novel cyclic lipopeptides (CLP), CLP 2605-4 and CLP 2612-8.1 against methicillin resistant S. aureus and P. aeruginosa, respectively, in in vivo porcine full thickness wound model. Both CLPs were able to reduce bacterial counts by approximately 3 log CFU/g by the last assessment day. Peptide 2612-8.1 slightly enhanced the wound healing, however, wounds treated with peptide 2605-4, have shown higher levels of inflammation and impaired wound healing process. This study highlights the importance of identifying new antimicrobials that can combat bacterial infection while not impeding tissue repair.

Actinomycin D Down-regulates SOX2 Expression and Induces Death in Breast Cancer Stem Cells.

BACKGROUND/AIM: One of the major hurdles in the treatment of breast cancers is the inability of anti-cancer drugs to eliminate the breast cancer stem cells (BCSCs) population, which leads to disease relapse. The dearth in anti-cancer drugs that target BCSCs can be attributed to the absence of in vitro screening models that can not only recapitulate the tumor microenvironment consisting of BCSCs but also preserve the 3-dimensional (3D) architecture of in vivo tumors. MATERIALS AND METHODS: In our present study, we have developed a 3D cell culture system that shows: (i) enrichment of BCSCs, (ii) increased drug resistance, and (iii) generation of hypoxic conditions similar to tumors. RESULTS: Using this model, we were able to screen a FDA-approved diversity set and identify as well as validate actinomycin D as a potential anti-breast cancer agent. Interestingly, we show that actinomycin D specifically targets and down-regulates the expression of the stem cell transcription factor, Sox-2. Additionally, down-regulation of Sox-2 leads to depletion of the stem-cell population resulting in the inability of breast cancer cells to initiate tumor progression. CONCLUSION: This study demonstrates the utility of an in vivo-like 3D cell culture system for the identification and validation of anti-cancer agents that will have a better probability of success in the clinic.

Activity of lipo-cyclic γ-AApeptides against biofilms of Staphylococcus epidermidis and Pseudomonas aeruginosa.

Antibiotic resistant bacterial infection is currently a serious public concern. Their ability to form biofilms further complicates the treatment. Herein we investigated the activity of lipo-cyclic γ-AApeptides against both planktonic cells and biofilms of Staphylococcus epidermidis and Pseudomonas aeruginosa, in comparison to those of the conventional antibiotic ciprofloxacin. Our results suggest that these lipo-cyclic γ-AApeptides exhibit comparable or enhanced performance compared to ciprofloxacin in the prevention of biofilm formation for both Gram-positive and Gram-negative bacteria, providing a potential alternative treatment and prevention for indwelling device-related infections.

Short antimicrobial lipo-α/γ-AA hybrid peptides.

The last two decades have seen the rise of antimicrobial peptides (AMPs) to combat emerging antibiotic resistance. Herein we report the solid-phase synthesis of short lipidated α/γ-AA hybrid peptides. This family of lipo-chimeric peptidomimetics displays potent and broad-spectrum antimicrobial activity against a range of multi-drug resistant Gram-positive and Gram-negative bacteria. These lipo-α/γ-AA hybrid peptides also demonstrate high biological specificity, with no hemolytic activity towards red blood cells. Fluorescence microscopy suggests that these lipo-α/γ-AA chimeric peptides can mimic the mode of action of AMPs and kill bacterial pathogens via membrane disintegration. As the composition of these chimeric peptides is simple, therapeutic development could be economically feasible and amenable for a variety of antimicrobial applications.

The development of antimicrobial a-AApeptides that suppress proinflammatory immune responses.

Herein we describe the development of a new class of antimicrobial and anti-inflammatory peptidomimetics: cyclic lipo-α-AApeptides. They have potent and broad-spectrum antibacterial activity against a range of clinically relevant pathogens, including both multidrug-resistant Gram-positive and Gram-negative bacteria. Fluorescence microscopy suggests that cyclic lipo-α-AApeptides kill bacteria by disrupting bacterial membranes, possibly through a mechanism similar to that of cationic host-defense peptides (HDPs). Furthermore, the cyclic lipo-α-AApeptide can mimic cationic host-defense peptides by antagonizing Toll-like receptor 4 (TLR4) signaling responses and suppressing proinflammatory cytokines such as tumor necrosis factor-α (TNF-α). Our results suggest that by mimicking HDPs, cyclic lipo-α-AApeptides could emerge as a new class of antibiotic agents that directly kill bacteria, as well as novel antiinflammatory agents that act through immunomodulation.

Lipidated cyclic γ-AApeptides display both antimicrobial and anti-inflammatory activity.

Antimicrobial peptides (AMPs) are host-defense agents capable of both bacterial membrane disruption and immunomodulation. However, the development of natural AMPs as potential therapeutics is hampered by their moderate activity and susceptibility to protease degradation. Herein we report lipidated cyclic γ-AApeptides that have potent antibacterial activity against clinically relevant Gram-positive and Gram-negative bacteria, many of which are resistant to conventional antibiotics. We show that lipidated cyclic γ-AApeptides mimic the bactericidal mechanism of AMPs by disrupting bacterial membranes. Interestingly, they also harness the immune response and inhibit lipopolysaccharide (LPS) activated Toll-like receptor 4 (TLR4) signaling, suggesting that lipidated cyclic γ-AApeptides have dual roles as novel antimicrobial and anti-inflammatory agents.

AApeptides as a new class of antimicrobial agents.

Antibiotic resistance is an increasing public health concern around the world, and is recognized as one of the greatest threats facing humankind in the 21(st) century. Natural antimicrobial peptides (AMPs) are small cationic amphiphilic peptides found in virtually all living organisms, and play a key role in the defense against bacterial infections. Compared with conventional antibiotics, which target specific metabolic processes, AMPs are able to adopt globally amphipathic conformations, and kill bacteria through disruption of their membranes. As such, AMPs do not readily induce drug-resistance. However, AMPs are associated with intrinsic drawbacks such as low-to-moderate activity, susceptibility to enzymatic degradation, and inconvenience for optimization. Recently, we have developed a new class of peptidomimetics termed "AApeptides". Such peptide mimics are highly resistant to protease degradation and are straightforward for chemical diversification and development. Our current studies show that AApeptides with globally amphipathic structures can mimic the bactericidal mechanism of AMPs, and display potent and broad-spectrum activity against both Gram-positive and -negative multi-drug-resistant bacteria. In this review, we summarize our current findings of antimicrobial AApeptides, and discuss potential future directions on the development of more potent and specific analogues.

Lipo-γ-AApeptides as a new class of potent and broad-spectrum antimicrobial agents.

There is increasing demand to develop antimicrobial peptides (AMPs) as next generation antibiotic agents, as they have the potential to circumvent emerging drug resistance against conventional antibiotic treatments. Non-natural antimicrobial peptidomimetics are an ideal example of this, as they have significant potency and in vivo stability. Here we report for the first time the design of lipidated γ-AApeptides as antimicrobial agents. These lipo-γ-AApeptides show potent broad-spectrum activities against fungi and a series of Gram-positive and Gram-negative bacteria, including clinically relevant pathogens that are resistant to most antibiotics. We have analyzed their structure-function relationship and antimicrobial mechanisms using membrane depolarization and fluorescent microscopy assays. Introduction of unsaturated lipid chain significantly decreases hemolytic activity and thereby increases the selectivity. Furthermore, a representative lipo-γ-AApeptide did not induce drug resistance in S. aureus, even after 17 rounds of passaging. These results suggest that the lipo-γ-AApeptides have bactericidal mechanisms analogous to those of AMPs and have strong potential as a new class of novel antibiotic therapeutics.

Cellular translocation of a γ-AApeptide mimetic of Tat peptide.

Cell-penetrating peptides including the trans-activating transcriptional activator (Tat) from HIV-1 have been used as carriers for intracellular delivery of a myriad of cargoes including drugs, molecular probes, DNAs and nanoparticles. Utilizing fluorescence flow cytometry and confocal fluorescence microscopy, we demonstrate that a γ-AApeptide mimetic of Tat (48-57) can cross the cell membranes and enter the cytoplasm and nucleus of cells, with efficiency comparable to or better than that of Tat peptide (48-57). Deletion of the four side chains of the γ-AApeptide attenuates translocation capability. We also establish that the γ-AApeptide is even less toxic than the Tat peptide against mammalian cells. In addition to their low toxicity, γ-AApeptides are resistant to protease degradation, which may prove to be advantageous over α-peptides for further development of molecular transporters for intracellular delivery.

Cellular uptake of an α-AApeptide.

Some short and cationic peptides such as the Tat peptide can cross the cell membrane and function as vectors for intracellular delivery. Here we show that an α-AApeptide is able to penetrate the membranes of living cells from an extracellular environment and enter the endosome and cytoplasm of cells. The efficiency of the cellular uptake is comparable to a Tat peptide (48-57) of the same length and is unexpectedly superior to an α-peptide with identical functional groups. The mechanism of uptake is similar to that of the Tat peptide and is through endocytosis by an energy-dependent pathway. Due to the easy synthesis of the α-AApeptides, their resistance to proteolytic hydrolysis, and their low cytotoxicity, α-AApeptides represent a new class of transporters for the delivery of drugs.

Lipidated peptidomimetics with improved antimicrobial activity.

We report a series of lipidated α-AApeptides that mimic the structure and function of natural antimicrobial lipopeptides. Several short lipidated α-AApeptides show broad-spectrum activity against a range of clinically related Gram-positive and Gram-negative bacteria as well as fungus. Their antimicrobial activity and selectivity are comparable or even superior to the clinical candidate pexiganan as well as previously reported linear α-AApeptides. The further development of lipidated α-AApeptides will lead to a new class of antibiotics to combat drug resistance.

Identification of γ-AApeptides with potent and broad-spectrum antimicrobial activity.

We report the identification of a new class of antimicrobial peptidomimetics-γ-AApeptides with potent and broad-spectrum activity, including clinically-relevant strains that are unresponsive to most antibiotics. They are also not prone to select for drug-resistance.

Non-hemolytic α-AApeptides as antimicrobial peptidomimetics.

We report a new class of peptide mimetics, α-AApeptides, that display broad-spectrum activity against both Gram-negative and Gram-positive bacteria and fungi. With non-hemolytic activity, resistance to protease hydrolysis, and easy sequence programmability, α-AApeptides may emerge as a novel class of antibiotics.

Association of acenaphthoporphyrins with liposomes for the photodynamic treatment of leishmaniasis.

Acenaphthoporphyrins are potential photosensitizers for photodynamic therapy, but their hydrophobicity limits their potential. Liposomes have been widely investigated as delivery vehicles that can transport hydrophobic drugs in biological systems. Here we study the association of acenaphthoporphyrins with liposomes made up of dimyristoyl phosphatidylcholine (DMPC), and to liposomes made up of a mixture of DMPC, cholesterol (Chol) and distearoyl phosphatidylglycerol (DSPG) in a 2:1:0.8 molar ratio to evaluate how liposome composition affects association constants. In liposomes consisting only of DMPC, the smaller monoacenaphthoporphyrin had the largest association constant of 5.5 x 10(4) m(-1) while the larger adj-diacenaphthoporphyrin and opp-diacenaphthoporphyrin (ODP) had smaller association constants at 1.8 x 10(4) and 1.5 x 10(4) m(-1), respectively. The addition of liposomal Chol and DSPG has little effect on the magnitudes of the association constants. Polarization studies show that the acenaphthoporphyrins are driven far into the lipid bilayer to minimize polar-nonpolar interactions. Confocal microscopy confirms that the DMPC liposomes transport the porphyrins into promastigotes of Leishmania tarentolae. The compounds associated with DMPC:Chol:DSPG liposomes are effective in vitro against axenic and intracellular amastigotes of the pathogenic Leishmania panamensis. The effectiveness of the compounds is enhanced upon exposure of cultures to visible light.