Natures antibiotics: the potential of antimicrobial peptides as new drugs

Animals and plants make a variety of substances to prevent potentially lethal infections. These include small antibiotic proteins, or peptides, which target bacteria, fungi and viruses. Research into these peptides not only give us an insight into how we naturally prevent infections, but can also provide us with new drugs to treat the ever-increasing danger of infections caused by antibiotic-resistant bacteria.     

Combinations of anticancer drugs and immunotherapy

Immunotherapy (biological therapy) comprises such things as active specific immunotherapy ("cancer vaccines"), nonspecific immunostimulation with cytokines, and the inhibition of suppressor influences exerted or elicited by the tumor. Just as cancer chemotherapy began with the use of single agents and evolved into combination therapy, so immunotherapeutic agents have been combined with each other and with chemotherapy. The alkylating agent cyclophosphamide (Cytoxan; CYC) has been used for many years to inhibit tumor-derived suppressor influences in rodents, and has been exploited for the same use in humans. Combinations of CYC and cancer vaccines such as autologous tumor cells, Melacine, large multivalent immunogen (LMI), and Theratope have been tested with some success in humans for more than a decade. In this use, the CYC is a biological response modifier rather than an antitumor agent, intended to inhibit suppressor influences. CYC and low- to moderate-dose IL-2 has also been a useful regimen in treating human melanomas. IL-2 is itself a useful component of combination immunotherapy, such as with melanoma peptide vaccines, or with interferon -2b, (IFN-), as a dual combination or part of a biochemotherapy regimen. Several different combinations of drugs and biological agents have been used as biochemotherapy for melanoma, but although there are higher response (regression) rates the long-range survival benefits have been marginal, not justifying the severe toxicity. Combinations of 5-fluorouracil (5-FU) and IFN- or levamisole have had efficacy in colon and head and neck cancers, but here the biological agents have been biochemical modulators, not immunotherapy. Although experience with combinations of monoclonal antibodies and chemotherapy has been limited, it appears that trastuzumab (Herceptin) potentiates antitumor therapy in breast cancer but also increases the cardiotoxicity of those regimens.This article forms part of the Symposium in Writing on "Cellular immunity for cancer chemoimmunotherapy" in Volume 52 (2003)     

Application of non-steroidal anti-inflammatory drugs to enhance 5-fluorouracil efficacy in experimental systems

The elevated cyclooxygenase-2 (COX-2) expression has been shown to affect the carcinogenesis and tumor progression processes, including cell proliferation, motility and angiogenesis. COX-2 is overexpressed in approximately 80% of sporadic colorectal carcinomas and COX-2 enzyme is the best defined target of non-steroidal anti-inflammatory drugs (NSAIDs). In the chemotherapy of colorectal carcinomas 5-fluorouracil (5-FU) has been the most important of the basic drugs for more than 40 years. In order to improve the effectiveness of 5-FU therapy different biological modifiers i.e. inhibitors of its catabolism or activators of anabolism have been studied recently. The rate-limiting enzyme of 5-FU catabolism is dihydropyrimidine dehydrogenase (DPD) since more than 80% of the administered 5-FU is catabolized by DPD. Tumoral DPD has become of clinical interest because elevated intratumoral DPD can decrease the tumor response to 5-FU therapy. The main purpose of our experiments was to investigate the effect of COX inhibitors on the efficacy of 5-FU on high and low COX-2 expressing HCA-7 and HT-29 human colon adenocarcinoma cell lines, respectively, and also on xenografts derived from HT-29 cells. The cytotoxic and antitumor effects of 5-FU in the presence of low doses of indomethacin (non-selective COX-2 inhibitor) and that of NS-398 (highly selective COX-2 inhibitor) on HT-29 and HCA-7 cells and also on the HT-29 xenograft were investigated. In addition, our intention was to understand the mechanism(s) by which NSAIDs could enhance the cytotoxic effect of 5-FU. Our data indicated that the elevated COX-2 expression of HCA-7, the collagen-induced HT-29-C cells and of the HT-29 xenograft were associated with reduced 5-FU sensitivity. Based on the fact that at the same time DPD activity was also increased it might be conceivable that a possible explanation for the decrease of 5-FU sensitivity is the co-existence of high COX-2 and DPD activity. Indomethacin or NS-398 enhanced in a simultaneous and significant manner the sensitivity and cytotoxic effect of 5-FU on high COX-2 expressing cells and xenografts through the modulation of DPD - decrease of its mRNA expression and/or enzyme activity. Based on our results it could be presumable that 5-FU efficacy is limited by the COX-2 associated high DPD expression and activity in patients with colorectal cancer as well, therefore further clinical studies are warranted to decide if NSAIDs in the therapeutic protocol might improve the antitumor potency of 5-FU. Réti A. Application of non-steroidal anti-inflammatory drugs to enhance 5-fluorouracil efficacy in experimental systems.     

Neutrophil antimicrobial proteins enhance Shigella flexneri adhesion and invasion

Shigella flexneri is an enteric pathogen that causes massive inflammation and destruction of the human intestinal epithelium. Neutrophils are the first cells of the innate immune system recruited to the site of infection. These cells can attack microbes by phagocytosis, Neutrophil Extracellular Trap (NET) formation and degranulation. Here, we investigated how neutrophil degranulation affects virulence and show that exposure of Shigella to granular proteins enhances infection of epithelial cells. During this process, cationic granular proteins bind to the Shigella surface causing increased adhesion which ultimately leads to hyperinvasion. This effect is mediated by changes in the surface charge, since a lipopolysaccharide (LPS) mutant with a negative surface shows enhanced hyperinvasion compared with wild‐type Shigella. We propose that Shigella evolved to use host defence molecules to enhance its virulence and subvert the innate immune system.     

Detection of sulfa drugs and antibiotics in milk

A disc assay method for testing sulfa drugs and antibiotics in milk was developed wherein Bacillus megaterium ATCC 9855 was used as the test organism and Mueller-Hinton agar was used as the test substrate. Incubation was at 37 C for 4 to 5 hr. The test procedure is an improvement over the Bacillus subtilis-Antibiotic Medium No. 1 method, as described in Standard Methods for the Examination of Dairy Products, in that it is sensitive to eight sulfa drugs and to bacitracin without a significant change in sensitivity to eight other antibiotics commonly used for mastitis therapy.     

In vitro antimicrobial activity of propolis and synergism between propolis and antimicrobial drugs

The aim of this study was to investigate antimicrobial properties of ethanolic extract of 13 propolis (EEP) samples from different regions of Serbia against 39 microorganisms (14 resistant or multiresistant to antibiotics), and to determine synergistic activity between antimicrobials and propolis. Antimicrobial activity of propolis samples was evaluated by agar diffusion and agar dilution method. The synergistic action of propolis with antimicrobial drugs was assayed by the disc diffusion method on agar containing subinhibitory concentrations of propolis. Obtained results indicate that EEP, irrespectively of microbial resistance to antibiotics, showed significant antimicrobial activities against Gram-positive bacteria (MIC 0.078%–1.25% of EEP) and yeasts (0.16%–1.25%), while Gram-negative bacteria were less susceptible (1.25&%ndash;>5%). Enterococcus faecalis was the most resistant Gram-positive bacterium, Salmonella spp. the most resistant Gram-negative bacteria, and Candida albicans the most resistant yeast. EEP showed synergism with selected antibiotics, and displayed ability to enhance the activities of antifungals. The shown antimicrobial potential of propolis alone or in combination with certain antibiotics and antifungals is of potential medical interest.     

Synergism between fungal enzymes and bacterial antibiotics may enhance biocontrol

The interactions between biocontrol fungi and bacteria may play a key role in the natural process of biocontrol, although the molecular mechanisms involved are still largely unknown. Synergism can occur when different agents are applied together, and cell wall degrading enzymes (CWDEs) produced by fungi can increase the efficacy of bacteria. Pseudomonas spp. produce membrane-disrupting lipodepsipeptides (LDPs) syringotoxins (SP) and syringomycins (SR). SR are considered responsible for the antimicrobial activity, and SP for the phytotoxicity. CWDEs of Trichoderma spp. synergistically increased the toxicity of SP_25-A or SR_E purified from P. syringae against fungal pathogens. For instance, the fungal enzymes made Botrytis cinerea and other phytopathogenic fungi, normally resistant to SP_25-A alone, more susceptible to this antibiotic. Pseudomonas produced CWDEs in culture conditions that allow the synthesis of the LDPs. Purified bacterial enzymes and metabolites were also synergistic against fungal pathogens, although this mixture was less powerful than the combination with the Trichoderma CWDEs. The positive interaction between LDPs and CWDEs may be part of the biocontrol mechanism in some Pseudomonas strains, and co-induction of different antifungal compounds in both biocontrol bacteria and fungi may occur. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microbial transformations of antimicrobial quinolones and related drugs

The quinolones are an important group of synthetic antimicrobial drugs used for treating bacterial diseases of humans and animals. Microorganisms transform antimicrobial quinolones (including fluoroquinolones) and the pharmacologically related naphthyridones, pyranoacridones, and cinnolones to a variety of metabolites. The biotransformation processes involve hydroxylation of methyl groups; hydroxylation of aliphatic and aromatic rings; oxidation of alcohols and amines; reduction of carboxyl groups; removal of methyl, carboxyl, fluoro, and cyano groups; addition of formyl, acetyl, nitrosyl, and cyclopentenone groups; and cleavage of aliphatic and aromatic rings. Most of these reactions greatly reduce or eliminate the antimicrobial activity of the quinolones.     

Small molecules enhance the potency of natural antimicrobial peptides

The skin-associated microbiome plays an important role in general well-being and in a variety of treatable skin conditions. In this regard, endogenous antimicrobial peptides have both a direct and indirect role in determining the composition of the microbiota. We demonstrate here that certain small molecular species can amplify the antimicrobial potency of naturally occurring antimicrobial peptides. In this study, we have used niacinamide, a form of vitamin B3 naturally found in foods and widely used in cosmetic skincare products, and two of its structural analogs, to investigate their cooperativity with the human antimicrobial peptide LL37 on the bacterium Staphylococcus aureus. We observed a clear synergistic effect of niacinamide and, to some extent, N-methylnicotinamide, whereas isonicotinamide showed no significant cooperativity with LL37. Adaptively biased molecular dynamics simulations using simplified model membrane substrates and single peptides revealed that these molecules partition into the headgroup region of an anionic bilayer used to mimic the bacterial membrane. The simulated effects on the physical properties of the simulated model membrane are well correlated with experimental activity observed in real biological assays despite the simplicity of the model. In contrast, these molecules have little effect on zwitterionic bilayers that mimic a mammalian membrane. We conclude that niacinamide and N-methylnicotinamide can therefore potentiate the activity of host peptides by modulating the physical properties of the bacterial membrane, and to a lesser extent through direct interactions with the peptide. The level of cooperativity is strongly dependent on the detailed chemistry of the additive, suggesting an opportunity to fine-tune the behavior of host peptides.     

Combinations of protein-chemical complex structures reveal new targets for established drugs

Biological networks are powerful tools for predicting undocumented relationships between molecules. The underlying principle is that existing interactions between molecules can be used to predict new interactions. Here we use this principle to suggest new protein-chemical interactions via the network derived from three-dimensional structures. For pairs of proteins sharing a common ligand, we use protein and chemical superimpositions combined with fast structural compatibility screens to predict whether additional compounds bound by one protein would bind the other. The method reproduces 84% of complexes in a benchmark, and we make many predictions that would not be possible using conventional modeling techniques. Within 19,578 novel predicted interactions are 7,793 involving 718 drugs, including filaminast, coumarin, alitretonin and erlotinib. The growth rate of confident predictions is twice that of experimental complexes, meaning that a complete structural drug-protein repertoire will be available at least ten years earlier than by X-ray and NMR techniques alone.     

Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Pseudomonas aeruginosa is one of the major nosocomial pathogen that can causes a wide variety of acute and chronic infections P. aeruginosa is a dreaded bacteria not just because of the high intrinsic and acquired antibiotic resistance rates but also the biofilm formation and production of multiple virulence factors. We investigated the in vitro activities of antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin and colistin) and antimicrobial cationic peptides (AMPs; LL-37, CAMA: cecropin(1–7)-melittin A(2–9) amide, melittin, defensin and magainin-II) alone or in combination against biofilms of laboratory strain ATCC 27853 and 4 clinical strains of P. aeruginosa. The minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentrations (MBEC) were determined by microbroth dilution technique. The MBEC values of antibiotics and AMPs were 80–>5120 and 640–>640 mg/L, respectively. When combined with the LL-37 or CAMA at 1/10× MBEC, the MBEC values of antibiotics that active against biofilms, were decreased up to 8-fold. All of the antibiotics, and AMPs were able to inhibit the attachment of bacteria at the 1/10× MIC and biofilm formation at 1× or 1/10× MIC concentrations. Time killing curve studies showed 3-log₁₀ killing against biofilms in 24 h with almost all studied antibiotics and AMPs. Synergism were seen in most of the studied combinations especially CAMA/LL-37 + ciprofloxacin against at least one or two strains’ biofilms. Since biofilms are not affected the antibiotics at therapeutic concentrations, using a combination of antimicrobial agents including AMPs, or inhibition of biofilm formation by blocking the attachment of bacteria to surfaces might be alternative methods to fight with biofilm associated infections.     

Axitinib targeted cancer stemlike cells to enhance efficacy of chemotherapeutic drugs via inhibiting the drug transport function of ABCG2

Stemlike cells have been isolated by their ability to efflux Hoechst 33342 dye and are called the side population (SP). We evaluated the effect of axitinib on targeting cancer stemlike cells and enhancing the efficacy of chemotherapeutical agents. We found that axitinib enhanced the cytotoxicity of topotecan and mitoxantrone in SP cells sorted from human lung cancer A549 cells and increased cell apoptosis induced by chemotherapeutical agents. Moreover, axitinib particularly inhibited the function of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) and reversed ABCG2-mediated multidrug resistance (MDR) in vitro. However, no significant reversal effect was observed in ABCB1-, ABCC1- or lung resistance-related protein (LRP)-mediated MDR. Furthermore, in both sensitive and MDR cancer cells axitinib neither altered the expression of ABCG2 at the mRNA or protein levels nor blocked the phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2. In nude mice bearing ABCG2-overexpressing S1-M1-80 xenografts, axitinib significantly enhanced the antitumor activity of topotecan without causing additional toxicity. Taken together, these data suggest that axitinib particularly targets cancer stemlike cells and reverses ABCG2-mediated drug resistance by inhibiting the transporter activity of ABCG2.     

Antibiotic and nonantibiotic ionophores can alter bacterial adherence to mammalian cells

Epithelioid (HeLa) and fibroblastic (L) cells in culture incubated for 18 hr with the ionophores amphotericin B and amiloride were noted to bind significantly more and less bacteria, respectively, than control cells incubated without ionophores. These effects were related to dose and incubation length and were present at concentrations approximating those in vivo after administration of maximal doses of these drugs given to humans therapeutically. Electron microscopy of both receptor cell lines revealed increased length and number of cellular projections in the amphotericin-treated cells and flattening and loss of membrane individuality in the amiloride-treated cells. These findings could explain the differences in subsequent bacterial binding. The ionophores nifedipine and verapamil which block calcium transport in cells which have calcium channels did not alter bacterial binding to these receptor cells or bacterial binding to calcium channel-containing myoblasts (in culture). These data suggest that certain ionophores could alter bacterial colonization and infection in the host indirectly by altering bacterial binding; however, the clinical significance of these findings remains to be determined.