噬菌体展示肽在血管导向治疗方面的应用

介绍了一种利用噬菌体肽库的新技术－体内噬菌体展示（n vivo phage display)。这项技术是在活的动物体内进行的肽库筛选。将肽库通过静脉注射到动物体内,因为血管分子内皮的异质性,噬菌体可以选择性地导向不同组织,这样就可以筛到与特定组织特异结合的噬菌体展示肽。动物实验表明,前凋亡小肽和细胞毒素与导各肽偶联后 治疗效果。这项技术应用于临床,一定有助于肿瘤等疾病的导向治疗和造影技术的发展。     

The probability of HIV infection in a new host and its reduction with microbicides

We use a simple mathematical model to estimate the probability and its time dependence that one or more HIV virions successfully infect target cells. For the transfer of a given number of virions to target cells we derive expressions for the probability P(inf), of infection. Thus, in the case of needlestick transfer we determine P(inf) and an approximate time window for post-exposure prophylaxis (PEP). For heterosexual transmission, where the transfer process is more complicated, a parameter gamma is employed which measures the strength of the infection process. For the smaller value of gamma, P(inf) is from 6x10(-5) to 0.93 or from 7.82x10(-6) to 0.29, where the lower figures are for the transfer of 100 virions and the upper figures are for the transfer of 4.4 million virions. We estimate the reductions in P(inf) which occur with a microbicide of a given efficacy. It is found that reductions may be approximately as stated when the number of virions transferred is less than about 10(5), but declines to zero for viral loads above that number. It is concluded that PEP should always be applied immediately after a needlestick incident. Further, manufacturers of microbicides should be encouraged to investigate and report their effectiveness at various transferred viral burdens.     

Antimicrobial peptide mimics for improved therapeutic properties

The relatively recent recognition of the major role played by antimicrobial peptides (AMPs) in sustaining an effective host response to immune challenges was greatly influenced by studies of amphibian peptides. AMPs are also widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from molecular simplicity to low-resistance swift-kill of a broad range of microbial cells. However, the peptide formula per se, represents less than ideal drug candidates, namely because of poor bioavailability issues, potential immunogenicity, optional toxicity and high production costs. To address these issues, synthetic peptides have been designed, reproducing the critical peptide biophysical characteristic in unnatural sequence-specific oligomers. Thus, the use of peptidomimetics to overcome the limitations inherent to peptides physical characteristics is becoming an important and promising approach for improving the therapeutic potential of AMPs. Here, we review most recent advances in the design strategies and the biophysical properties of the main classes of mimics to natural AMPs, emphasizing the importance of structure-activity relationship studies in fine-tuning of their physicochemical attributes for improved antimicrobial properties.     

Antimicrobial peptide buforin I inhibits tissue factor-initiated coagulation

The enhanced extrinsic blood coagulation following septic shock often manifests cardiovascular complications. The upregulated monocytic tissue factor (mTF) was shown to be a primary contributor to the extrinsic hypercoagulation following acute bacterial endotoxin (LPS) infection. A single-stage clotting assay monitors TF-initiated coagulation. We herein demonstrate a novel anticoagulant activity of antimicrobial peptide Buforin I (BF I) in offsetting LPS-induced mTF hypercoagulation in THP-1 cells, which was confirmed in a cell-free in vitro model, showing that BF I effectively blocked rabbit brain thromboplastin (rbTF) procoagulant activity. Upon inclusion of 25 microM BF I into human plasma, the prolonged prothrombin time (PT) was consistent with the depressed TF-initiated coagulation. In a two-stage chromogenic assay monitoring S-2288 hydrolysis, BF I significantly inhibited not only mTF- but also rbTF-catalyzed FVII activation accompanied by the diminished FVIIa formation. The inhibition by BF I of FVII activation accounted for its novel anticoagulant activity in offsetting mTF-initiated hypercoagulation.     

Antimicrobial peptide resistance mechanisms of human bacterial pathogens

The critical role played by antimicrobial peptides (AMPs) in mammalian innate immunity is increasingly recognized. Bacteria differ in their intrinsic susceptibility to AMPs, and the relative resistance of some important human pathogens to these defense molecules is now appreciated as an important virulence phenotype. Experimental analysis has identified diverse mechanisms of bacterial AMP resistance including altered cell surface charge, active efflux, production of proteases or trapping proteins, and modification of host cellular processes. The contribution of these resistance mechanisms to pathogenesis is confirmed through direct comparison of wild-type bacteria and AMP-sensitive mutants using in vivo infection models. Knowledge of the molecular basis of bacterial AMP resistance may provide new targets for antimicrobial therapy of human infectious diseases.     

Designing allosteric peptide ligands targeting a globular protein

Patented signal analytic algorithms applied to hydrophobically transformed, numerical amino acid sequences have previously been used to design short, protein-targeted, L or D retro-inverso peptides. These peptides have demonstrated allosteric and/or indirect agonist effects on a variety of G-protein and tyrosine kinase coupled membrane receptors with 30% to over 80% hit rates. Here we extend these approaches to a globular protein target. We designed eight peptide ligands targeting an ELISA antibody responsive protein, beta-galactosidase, betaGAL. Three of the eight 14mer peptides allosterically activated betaGAL with ELISA methodology. Using Bayesian statistics, this 38% hit rate would have occurred 2 x 10(-9) by chance. These peptides demonstrated binding site competitive or noncompetitive interactions, suggesting allosteric site multiplicity with respect to their betaGAL binding-mediated ELISA signal. Kinetic studies demonstrated the temperature dependence of the betaGAL peptide binding functions. Using the van't Hoff relation, we found evidence for enthalpy-entropy compensation. This relation is often found for hydrophobic interactions in aqueous media, and is consistent with the postulated hydrophobic series encoding underlying our protein-targeted, peptide design methods. It appears that our algorithmic, hydrophobic autocovariance eigenvector template approach to the design of allosteric peptides targeting membrane receptors may also be applicable to the design of peptide ligands targeting nonmembrane involved globular proteins.     

Structure-based design of an agonistic peptide targeting Fas

A small agonistic peptide FRAP-4 (WEWT, Fas reactive peptide-4) that binds to the human Fas molecule was discovered using our computer screening strategy named the Amino acid Complement Wave (ACW) method, which is based on the complementarities of interacting amino acids between comprehensive testing peptides and a target protein surface pocket. In silico docking studies demonstrated the specific interaction of FRAP-4 with the main Fas ligand (FasL) binding domain in the Fas molecule. An octamer of this peptide produced by carboxyl terminal linkages of polylysine branches (MAP), (FRAP-4)₈-MAP, effectively induced apoptosis in human ovarian cancer cell line NOS4 cells that was associated with the activation of caspases-8, -9 and -3, and the cleavage of PARP. Alanine substitution of the N-terminal W in FRAP-4 resulted in complete loss of FasL-mimetic action of (FRAP-4)₈-MAP, suggesting that the aromatic functionality at the N-terminal position W appears to play an essentially important role in Fas binding ability. These observations indicate that the FasL-mimetic peptide should serve as an excellent starting point for the design of effective compounds with FasL-mimetic activity. Furthermore, the ACW method for the structure-based design of optimized small peptides against receptor molecules such as Fas could open new avenues for the development of peptide mimetic and nonpeptidic organic forms to generate novel effective pharmaceuticals.     

Selection and characterization of tenascin C targeting peptide

Since tenascin C is a factor expressed highly in the tumor-associated matrix, it would be a desirable first step for targeting the tumor-specific microenvironment. In fact, a high level of tenascin C expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. Therefore, the targeted binding of tenascin C in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. We isolated a peptide that bound to tenascin C by phage display peptide library selection, and the selected peptide specifically recognized tenascin C protein in xenograft mouse tissue. We also observed exclusive staining of tenascin C by the selected peptide in tumor patient tissues. Moreover, the peptide reduced tenascin C-induced cell rounding and migration. We propose that the tenascin C targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now includingHomo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAsin vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1,ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a dauntingprospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately,no reliable method has been available to measure levels of specificmRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless,we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cellsare taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAsin human cancer xenografts in a mouse model. The oncogenes cyclinD1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experimentsprovide a proof-of-principle for noninvasive detection of oncogeneexpression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Novel antiviral agents targeting HIV entry and transmission

Studies of the mechanism of HIV entry and transmission have identified multiple new targets for drug development. A range of inhibitors have demonstrated potent antiretroviral activity by interfering with CD4-gp120 interaction, coreceptor binding or viral-cell fusion in preclinical and clinical studies. One of these agents, fusion inhibitor enfuvirtide, is already in clinical use. Here we review the progress in the development of specific entry inhibitors as novel therapeutics. The potential of entry inhibitors as topical microbicides to block HIV transmission is also discussed. Foundation items: NIH (AI065413 and AI041346) and the 973 Program (2006CB504200) for financial support.     

Antimicrobial activity of an abiotic host defense peptide mimic

Bacterial drug resistance is emerging as one of the most significant challenges to human health. Antimicrobial peptides (AMPs), which are produced by many tissues and cell types of invertebrates, insects, and humans, as part of their innate immune system, have attracted considerable interest as alternative antibiotics. Interest in novel mimics of AMPs has increased greatly over the last few years. This report details a new AMP mimic, based on phenylene ethynylene, with improved antimicrobial activity and selectivity. Screening against a large set of bacterial and other organisms demonstrates broad spectrum antimicrobial activity including activity against antibiotic resistant bacterial like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) as well as activity against yeast (Candida albicans) and fungus (Stachybotrys chartarum). Bacterial resistance development studies using Staphylococcus aureus show a rapid increase in MIC for conventional antibiotics, ciprofloxacin and norfloxacin. In sharp contrast, no change in MIC was observed for the AMP mimic. Cytotoxicity experiments show that the AMP mimic acts preferentially on microbes as opposed to mammalian red blood cells, 3T3 fibroblasts, and HEPG2 cells. In vivo experiments determined the maximum tolerated dose (MTD) to be 10 mg/kg suggesting a therapeutic window is available. These studies indicate that nonpeptidic amphiphilic AMP mimics could be developed as potential new treatments for antibiotic-resistant bacterial infections.     

Antimicrobial peptide modulation in a differentiated reconstructed gingival epithelium

Gingival innate immunity has been studied by using biopsies and normal or transformed epithelial cell monolayers. To overcome individual biological variabilities and as a physiological alternative, we have proposed using a reconstructed tissue equivalent. In this study, we investigated the functionality and the stage of differentiation of a reconstructed human gingival epithelium. We also characterized this epithelium at the molecular level to investigate its differentiation stage compared with native human gingival epithelium. The expression levels and localization of markers related to proteins and lipids of well-differentiated stratified epithelium, such as cytokeratins, cornified envelope proteins and enzymes, or to factors in lipid synthesis and trafficking were examined. Immunohistochemistry revealed similar localization patterns in both types of epithelia and mRNA quantification showed a close resemblance of their expression profiles. We further revealed that, like native gingiva, reconstructed gingival epithelium was able to respond to pro-inflammatory or lipopolysaccharide stimuli by producing antimicrobial peptides hβD-2, hβD-3 or LL-37. Finally, we demonstrated that reconstructed human gingival epithelium, as a model, was good enough to be proposed as a functional equivalent for native human gingival epithelium in order to study the regulation of gingival innate immunity against periodontal infections. This investigation was supported by a grant from Pierre Fabre Oral Care.     

Antimicrobial peptide genes in Bacillus strains from plant environments

The presence of the antimicrobial peptide (AMP) biosynthetic genes srfAA (surfactin), bacA (bacylisin), fenD (fengycin), bmyB (bacyllomicin), spaS (subtilin), and ituC (iturin) was examined in 184 isolates of Bacillus spp. obtained from plant environments (aerial, rhizosphere, soil) in the Mediterranean land area of Spain. Most strains had between two and four AMP genes whereas strains with five genes were seldom detected and none of the strains had six genes. The most frequent AMP gene markers were srfAA, bacA, bmyB, and fenD, and the most frequent genotypes srfAA-bacA-bmyB and srfAAbacA- bmyB-fenD. The dominance of these particular genes in Bacillus strains associated with plants reinforces the competitive role of surfactin, bacyllomicin, fengycin, and bacilysin in the fitness of strains in natural environments. The use of these AMP gene markers may assist in the selection of putative biological control agents of plant pathogens.     

Antimicrobial peptides with cell-penetrating peptide properties and vice versa

Antimicrobial peptides (AMPs) are a group of peptides that are active against a diverse spectrum of microorganisms. Due to their mode of action, AMPs are a promising class of molecules that could overcome the problems of increasing resistance of bacteria to conventional antibiotics. Furthermore, AMPs are strongly membrane-active and some are able to translocate into cells without the necessity for permanent membrane permeabilization. This feature has brought them into focus for use as transport vectors in the context of drug delivery. Since the plasma membrane restricts transport of bioactive substances into cells, great research interest lies in the development of innovative ways to overcome this barrier and to increase bioavailability. In this context, peptide-based transport systems, such as cell-penetrating peptides (CPPs), have come into focus, and their efficiency has been demonstrated in many different applications. However, more recently, also some AMPs have been used as efficient vectors for intracellular translocation of various active molecules. This review summarizes recent efforts in this interesting field of drug delivery. Moreover, some examples of the application of CPPs as efficient antimicrobial substances will be discussed.