témavezető: Ötvös László
helyszín (magyar oldal): Temple University, College of Science and Technology, Dept. of Biology helyszín rövidítés: TU
A kutatási téma leírása:
A kutatási téma leírása:
Resistance to current antibiotics reached a critical stage when hospitals and health care agencies call for innovative legislative measures to stimulate the development of new antimicrobial drugs. In March, 2006 officials of the Infectious Diseases Society of America called government panels to introduce legislation that would promote research and development of novel antimicrobials for highly dangerous, drug-resistant microbes. They identified a “hit list” of strains that are most in need of research — Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Aspergillus species, Enterococcus faecium and Pseudomonas aeruginosa. The situation is not helped by many large pharmaceutical companies, who recently abandoned antimicrobial research and by biotech investor attitude that neglects new molecules that have not reached the clinical trial stage yet. To fill the void throughout the pipeline, this program contains a lead optimization aim to provide a new peptide-based antimicrobial compound for immediate preclinical development as well as a long-term strategy to identify antimicrobial compounds, peptides and non-peptides alike, that kill microbes by a novel, proprietary mode of action.
In a series of papers between 2000 and now, Professor Otvos’ laboratory at The Wistar Institute identified the bacterial killing mechanism of the short, proline-rich native antimicrobial peptide family, designed in vivo active analogs, studied the synergy with small molecule antibiotics and looked for potential resistance induction. Remarkably, our newest combinatorial peptide kills clinical fluoroquinolone-resistant E. coli and K. pneumoniae urinary tract pathogens, and, when added in sequence with small molecule antibiotics, recovers full activity of the conventional drugs against resistant strains that express resistance-inducing enzymes. This is possible because the major mode of action is binding the 70 kDa bacterial heat shock protein DnaK with ensuing inhibition of protein folding in bacteria, including enzymes involved in antibacterial resistance. Since we located the exact peptide binding site to the C-terminal D-E helix (where only very little or no homology to mammalian Hsp70 is found), the road is paved for the de novo design or library selection of new antimicrobial molecules acting by a novel mechanism. To this end, in our program we will:
1) Optimize the current lead A3-APO prodrug, or better yet the proprietary single chain metabolite Chex1-Arg20 for activity against Enterobacteriaceae in vitro, for stability in mammalian serum, for lack of in vitro toxicity to human cells and for improved pharmacokinetic properties. After synthesis of about 40-50 of these proline-rich designer peptide analogs, a final compound will be selected for detailed in vivo efficacy measures in mouse infection models and for evaluating toxicity in mammals.
2) Study the synergy of proline-rich antimicrobial peptides with conventional antibiotics against bacteria resistant to these small molecules. We plan to identify the conditions in which A3-APO or the monomer best synergizes given antimicrobial compound families, characterize strain limitations as well as dose range and timing. When all these parameters are established, in vivo mouse toxicity and efficacy assays of the combination therapy, similar to those in Aim 1, will follow.
3) Identify novel antibiotics that bind the D-E helix of bacterial DnaK and fungal Hsp70 and kill the target organisms. This will be done in three independent phases:
a. A targeted one-bead-one-peptide proline-rich library will be made with the DnaK-binding residues degenerate and the membrane-penetrating unit constant. The resin-bound library will be interrogated with labeled synthetic DnaK (Hsp70) D-E helix fragments corresponding to series of bacterial strains, fungi, and control mammals.
b. Peptide-based antagonists to currently non-susceptible bacteria will be designed based on homology models of DnaK proteins taking into consideration the known E. coli coordinates and the D-E helix amino acid alterations in the non-responsive strains.
c. Small molecule inhibitors of the peptide binding site will be selected in silico from available virtual libraries. Similarly to Aim 3b, we will use the published crystal structure of E. coli DnaK, and homology modeled DnaK (Hsp70) analogs of other bacteria and fungi. Once again, after thorough in vitro efficacy and toxicity screening, the best peptide and non-peptide antibiotics will be submitted to in vivo efficacy and toxicity testing in appropriate mouse models.
4) Treat bacteria (preferably E. coli) with sublethal doses of peptide A3-APO or the single chain metabolite and look for variations in the dnaK gene or other loci to identify potential resistance induction. Based on our preliminary results (and the need for efficacious membrane penetration and peptide activity on membranes, the minor mode of proline-rich antibacterial peptide action), specific attention will be paid to the assembly of the membrane structure. These studies may provide directions for future design of even more efficacious DnaK inhibitor peptides without introduction of yet another layer of bacterial resistance.
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