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author:("Shi, wenchuan")
1.  Selective Membrane Disruption: Mode of Action of C16G2, a Specifically Targeted Antimicrobial Peptide ▿ 
The specifically targeted antimicrobial peptide (STAMP) C16G2 was developed to target the cariogenic oral pathogen Streptococcus mutans. Because the design of this peptide was novel, we sought to better understand the mechanism through which it functioned. Compared to antimicrobial peptides (AMPs) with wide spectra of activity, the STAMP C16G2 has demonstrated specificity for S. mutans in a mixed-culture environment, resulting in the complete killing of S. mutans while having minimal effect on the other streptococci. In the current study, we sought to further confirm the selectivity of C16G2 and also compare its membrane activity to that of melittin B, a classical toxic AMP, in order to determine the STAMP's mechanism of cell killing. Disruption of S. mutans cell membranes by C16G2 was demonstrated by increased SYTOX green uptake and ATP efflux from the cells similar to those of melittin B. Treatment with C16G2 also resulted in a loss of membrane potential as measured by DiSC(3)5 fluorescence. In comparison, the individual moieties of C16G2 demonstrated no specificity and limited antimicrobial activity compared to those of the STAMP C16G2. The data suggest that C16G2 has a mechanism of action similar to that of traditional AMPs and kills S. mutans through disruption of the cell membrane, allowing small molecules to leak out of the cell, which is followed by a loss of membrane potential and cell death. Interestingly, this membrane activity is rapid and potent against S. mutans, but not other noncariogenic oral streptococci.
PMCID: PMC3122425  PMID: 21518845
2.  Rapid Probing of Biological Surfaces with a Sparse-Matrix Peptide Library 
PLoS ONE  2011;6(8):e23551.
Finding unique peptides to target specific biological surfaces is crucial to basic research and technology development, though methods based on biological arrays or large libraries limit the speed and ease with which these necessary compounds can be found. We reasoned that because biological surfaces, such as cell surfaces, mineralized tissues, and various extracellular matrices have unique molecular compositions, they present unique physicochemical signatures to the surrounding medium which could be probed by peptides with appropriately corresponding physicochemical properties. To test this hypothesis, a naïve pilot library of 36 peptides, varying in their hydrophobicity and charge, was arranged in a two-dimensional matrix and screened against various biological surfaces. While the number of peptides in the matrix library was very small, we obtained “hits” against all biological surfaces probed. Sequence refinement of the “hits” led to peptides with markedly higher specificity and binding activity against screened biological surfaces. Genetic studies revealed that peptide binding to bacteria was mediated, at least in some cases, by specific cell-surface molecules, while examination of human tooth sections showed that this method can be used to derive peptides with highly specific binding to human tissue.
PMCID: PMC3156232  PMID: 21858167
3.  Design and Characterization of an Acid-Activated Antimicrobial Peptide 
Chemical biology & drug design  2009;75(1):127-132.
Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/ remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals creation of an acidic environment favors growth of acid enduring and acid generating species, which causes further reduction in the plaque pH. In this study we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype “acid-activated peptides” (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries.
PMCID: PMC2790279  PMID: 19878192
Targeted antimicrobial therapy; pH dependent antimicrobial activity; biofilm; Streptococcus mutans
4.  Systematic Approach to Optimizing Specifically Targeted Antimicrobial Peptides against Streptococcus mutans▿  
Previously we reported a novel strategy of “targeted killing” through the design of narrow-spectrum molecules known as specifically targeted antimicrobial peptides (STAMPs) (R. Eckert et al., Antimicrob. Agents Chemother. 50:3651-3657, 2006; R. Eckert et al., Antimicrob. Agents Chemother. 50:1480-1488, 2006). Construction of these molecules requires the identification and the subsequent utilization of two conjoined yet functionally independent peptide components: the targeting and killing regions. In this study, we sought to design and synthesize a large number of STAMPs targeting Streptococcus mutans, the primary etiologic agent of human dental caries, in order to identify candidate peptides with increased killing speed and selectivity compared with their unmodified precursor antimicrobial peptides (AMPs). We hypothesized that a combinatorial approach, utilizing a set number of AMP, targeting, and linker regions, would be an effective method for the identification of STAMPs with the desired level of activity. STAMPs composed of the Sm6 S. mutans binding peptide and the PL-135 AMP displayed selectivity at MICs after incubation for 18 to 24 h. A STAMP where PL-135 was replaced by the B-33 killing domain exhibited both selectivity and rapid killing within 1 min of exposure and displayed activity against multispecies biofilms grown in the presence of saliva. These results suggest that potent and selective STAMP molecules can be designed and improved via a tunable “building-block” approach.
PMCID: PMC2863653  PMID: 20211885
5.  Design and activity of a ‘dual-targeted’ antimicrobial peptide 
Numerous reports have indicated the important role of human normal flora in the prevention of microbial pathogenesis and disease. Evidence suggests that infections at mucosal surfaces result from the outgrowth of subpopulations or clusters within a microbial community and are not linked to one pathogenic organism alone. To preserve the protective normal flora while treating the majority of infective bacteria in the community, a tuneable therapeutic is necessary that can discriminate between benign bystanders and multiple pathogenic organisms. Here we describe the proof-of-principle for such a multitargeted antimicrobial: a multiple-headed specifically-targeted antimicrobial peptide (MH-STAMP). The completed MH-STAMP, M8(KH)-20, displays specific activity against targeted organisms in vitro (Pseudomonas aeruginosa and Streptococcus mutans) and can remove both species from a mixed planktonic culture with little impact against untargeted bacteria. These results demonstrate that a functional, dual-targeted molecule can be constructed from a wide-spectrum antimicrobial peptide precursor.
PMCID: PMC2696886  PMID: 19188046
Antimicrobial peptide; Targeted therapeutic; Streptococcus mutans; Pseudomonas aeruginosa; Peptide synthesis; Novel antibiotic; STAMP; Specifically-targeted antimicrobial peptide; MH-STAMP
6.  Specific Binding and Mineralization of Calcified Surfaces by Small Peptides 
Calcified Tissue International  2009;86(1):58-66.
Several small (<25aa) peptides have been designed based on the sequence of the dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)n repeats, and though similar repeated sequences—(NTT)n, (DTT)n, (ETT)n, (NSS)n, (ESS)n, (DAA)n, (ASS)n, and (NAA)n—also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)n peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed.
PMCID: PMC2798077  PMID: 19949943
Dentin phosphoprotein; Peptide; Mineralization
7.  Novel Synthetic Antimicrobial Peptides against Streptococcus mutans▿  
Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents.
PMCID: PMC1855471  PMID: 17296741
8.  Enhancement of Antimicrobial Activity against Pseudomonas aeruginosa by Coadministration of G10KHc and Tobramycin▿  
Antimicrobial Agents and Chemotherapy  2006;50(11):3833-3838.
Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway colonization during cystic fibrosis. Previously, we converted the wide-spectrum antimicrobial peptide novispirin G10 into a selectively-targeted antimicrobial peptide (STAMP), G10KHc. Compared to novispirin G10, the STAMP had an enhanced ability to kill Pseudomonas mendocina. In this study, we explored the activity of G10KHc against P. aeruginosa. G10KHc was found to be highly active (as active as tobramycin) against P. aeruginosa clinical isolates. Most interestingly, we observed a synergistic-like enhancement in killing activity when biofilms and planktonic cultures of P. aeruginosa were cotreated with G10KHc and tobramycin. The data indicate that the mechanism of enhanced activity may involve increased tobramycin uptake due to G10KHc-mediated cell membrane disruption. These results suggest that G10KHc may be useful against P. aeruginosa during acute and chronic infection states, especially when it is coadministered with tobramycin.
PMCID: PMC1635211  PMID: 16940063
9.  Adding Selectivity to Antimicrobial Peptides: Rational Design of a Multidomain Peptide against Pseudomonas spp. 
Currently available antimicrobials exhibit broad killing with regard to bacterial genera and species. Indiscriminate killing of microbes by these conventional antibiotics can disrupt the ecological balance of the indigenous microbial flora, often resulting in negative clinical consequences. Species-specific antimicrobials capable of precisely targeting pathogenic bacteria without damaging benign microorganisms provide a means of avoiding this problem. In this communication, we report the successful creation of the first synthetic, target-specific antimicrobial peptide, G10KHc, via addition of a rationally designed Pseudomonas-specific targeting moiety (KH) to a generally killing peptide (novispirin G10). The resulting chimeric peptide showed enhanced bactericidal activity and faster killing kinetics against Pseudomonas spp. than G10 alone. The enhanced killing activities are due to increased binding and penetration of the outer membrane of Pseudomonas sp. cells. These properties were not observed in tests of untargeted bacterial species, and this specificity allowed G10KHc to selectively eliminate Pseudomonas spp. from mixed cultures. This work lays a foundation for generating target-specific “smart” antimicrobials to complement currently available conventional antibiotics.
PMCID: PMC1426969  PMID: 16569868

Results 1-9 (9)