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Bernard Weisblum
Awards: Inducible Antibiotic Resistance and Drug Discovery We are using combinatorial methods (phage surface peptide display and bead peptide display) to identify peptide sequences which selectively bind to proteins responsible for clinical resistance to antibiotics. Two forms of resistance on which we are concentrating are resistance to vancomycin, and to erythromycin, respectively. We have discovered peptide sequences which bind to VanR, a positive regulator of vancomycin resistance, and will attempt to use these peptide sequences to devise mechanism-based high throughput assays for new drug lead discovery. Similarly, we are attempting to find peptides which will bind to and inactivate the enzyme ErmC methylase whose activity is responsible for erythromycin resistance. The present direction of our research is an outgrowth of our earlier studies of the action of antibiotics as regulators of gene expression. These previous studies led to the discovery that clinical isolates of erythromycin resistant strains modified their ribosomes by methylation of a single adenine residue in 23S ribosomal RNA. Further studies led to the discovery that methylase synthesis was induced by a translational control mechanism. According to this mechanism, methylase message responsible for conferring resistance becomes activated by undergoing a conformational change leading to unmasking of the sequestered translation initiation site in the ermC message. ONGOING PROJECTS (1) Regulation of vancomycin resistance by the vanRS gene cluster (with A. Ullijasz) VanR is an essential positive-acting transcriptional factor for the synthesis of the proteins which confer vancomycin resistance in Enterococcus faecium. We discovered a peptide ligand, by combinatorial phage display technology, which inhibits binding of VarR~P to its target, the vanH promoter, PVanH. The peptide sequence also resembles the catalytic center of VanS with thich VanR reacts in vivo. Both the VanS-like peptide sequence and VanS show a similar activity signature in a set of two respective parallel alanine-replacement constructs. These studies identify structural requirements for the regulation of vancomycin resistance. With additional development, the reactions and reagents used in these studies can be configured to craft an assay for lead discovery of inhibitors of VanR function. (2) Combinatorially synthesized toxic intracellular peptides - a new paradigm for drug lead discovery (with R. Giannattasio) We have constructed an intracellularly expressed dodecamer combinatorial peptide library displayed at the C-terminus of Escherichia coli thioredoxin; its expression is under control of the arabinose promoter P araBAD. Cells carrying determinants of inhibitory peptides form small colonies when grown in the presence of arabinose, and normal size (larger) colonies when grown in glucose-containing medium. By the use of pathways whose activity can be monitored with reporters, it is possible to know which enzymatic step is inhibited by the displayed peptide. Although inhibitory peptides discovered in this way cannot per se be considered to be drugs, they can be useful in identifying vulnerable antibiotics traces, or finding inhibitors of specific pathways. Using this approach, we have discovered a peptide which inhibits the expression of induced B-galactosidase. If the target of a peptide is known, a peptide-target interaction can be configured as an assay for drug lead discovery based on ligand displacement by chemical libraries.
(3) Genomic effects of combinatorially synthesized toxic intracellular peptides tested with DNA microarrays (with F. Blattner (Genetics)) Patterns of inhibition by combinatorial toxic peptides will be studied on a genome-wide basis. cDNA probes will be synthesized with cellular RNA templates from inhibited cells, followed by quantification of their hybridization to E coli DNA microarrays. The information from these studies will help us to determine the kinds of transcriptional signatures which are associated bacteriostatic versus bactericidal action of antibiotics. The use of an internally expressed peptide library allows us to screen for an unlimited number of prospective inhibitors. To obtain inhibitory peptides requires addition of arabinose, as inducer, to growing cells carrying the combinatorial library, and picking small colonies whose growth is retarded by an expressed inhibitory peptide.
(4) Genomic effects of antibiotics, tested wit DNA microarrays (with H. Jin and F. Blattner (Genetics)) Antibiotics with known inhibitory mechanisms will be used to study genome-wide responses to inhibitors of known pathways using DNA microarrays of the entire E.coli genome. Very little is known how to predict whether an antibiotics will be bacteriostatic or bactericidal, nor has the basis for the post-antibiotic effect been generalized. Genomic arrays will be used to determine the transcriptional signatures of known bactericidal and bacteriostatic antibiotics as a mean to predict the activity of newly discovered inhibitors whose activity is unknown. (5)DNA microarrays to study gene expression in response to antibiotics in Candida alibans (with D. Andes (Medicine)) We are constructing DNA microarrays that will enable us to determine transcriptional signatures of Candida albicans responding to azole antibiotics, including resistant mutants. The target of the azole antibiotics is lanosterol-14- demethylase, a key enzyme on the pathway from acetate to ergosterol. A DNA microarray containing sample targets which correspond to all 19 enzymes which form the pathway will be fabricated. At this time we already have 6 of the 19 needed samples as PCR amplimer products. The information from these studies will be correlated with antibiotics resistance levels seen in parallel animal model studies with susceptible and resistant strains of C. albicans.
Selected Publications:
For a complete list back to 1966 see <http://www.ncbi.nlm.nih.gov/PubMed/>
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