3. Dangerous Trends – Not a Laughing Matter Source: Clatworthy et al , Nature Chemical Biology, 3 , 541 - 548 (2007)
4. Classic Approaches to Antibiotic Development ***In all cases, a heritable change to a bacterium’s DNA must be made*** Molecular Basis for Resistance Drug Targets
9. RecA and Recombination Paired homologous DNA can come from a sister chromatid (high-fidelity repair) or from another organism (horizontal gene transfer)
10. RecA and the Antibiotic Response Antibiotic Tolerance Antibiotic Resistance **Bactericidal antibiotics stimulate the production of DNA-damaging hydroxyl radicals** Concept: Kohanski et al. Cell . 2007 Sep 7;130(5):781-3.
11. recA Knockouts are More Sensitive to Bactericidal Antibiotics Kohanski et al. Cell . 2007 Sep 7;130(5):781-3.
15. Where we looked for inhibitors Directed vs. random screening
16. Conclusions Regarding the Metal Inhibitors Bismuth-dithiol inhibitors target two storage forms of RecA Wigle, Lee, Zeng and Singleton, submitted (2008)
17. Conclusions Regarding the Nucleotide Inhibitors Nucleotide Analogs are Conformationally Selective Competitive Inhibitors: N 6 –modifications target inactive monomers 2' and 3' modifications target the active conformation competitively Wigle , Gromova & Singleton, submitted (2008) Wigle, Lee & Singleton, Biochemistry (2006) K i ( μ M) % inhibition ATPase
18. Directed Screening: Intuitive Discovery of Natural Product Inhibitors of RecA - The Ames and umu tests are quick and easy methods to determine if compounds are carcinogenic without the use of live animals - Thinking backwards, looked for compounds that inhibited mutation/SOS activation in these tests AMES TEST (Ames et al. Mutat. Res. 1975 , 31, 347 – 363) Bacteria incapable of His synthesis due to point mutation in His synthesis genes Expose to potential mutagen Plate on medium lacking His Mutagen (carcinogenic) Not a Mutagen (non-carcinogenic) umu TEST (Oda et al. Mutat. Res. 1985 , 147, 219 – 229) umuC promoter (LexA regulated) lacZ gene (encodes β -Gal ) umu test plasmid Expose to potential mutagen If compound is a mutagen (carcinogen) this Rx proceeds
19. Directed Screening: Intuitive Discovery of Natural Product Inhibitors of RecA - Two major classes of natural products present in food sources were identified through a literature search for compounds/extracts that inhibited mutation (Ames) or SOS induction ( umu) Flavonoids Triterpenoids Quercetin Ursolic acid Betulinic acid Biflavonoids Hinokiflavone A total of 13 natural product phytoterpenoids and 53 synthetically modified phytoterpenoids (KH Lee, UNC) were subsequently screened and one modified phytoterpenoid has emerged as a potential lead.
23. Library Mining ♦ Initial screens effectively mined various libraries and returned hits belonging to chemotype clades as defined by molecular scaffold and shared pharmacophores L
24. Lead Discovery: Confirming Leads With IC 50 CLADE A (Biogen) IC 50 = 8 – 22 μ M A1 = Most potent CLADE B (Biogen) IC 50 = 22 – 104 μ M B1 = Most potent CLADE C (Biogen) IC 50 = 19 μ M CLADE L (NCI) IC 50 = 4 – 6 μ M CLADE M (NP) IC 50 = 38 – 61 μ M
27. Confirming Biological Activity Use of Oxygen Sensor Microplates to Screen All Drug-Like Small Molecules IRA to Date for Cipro & Amp Synergism in E. coli ( & B. subtilis) synergistic toxic No effect Allows for a rapid Yes/No decision on whether to pursue hit as a lead A screen of 28 compounds +ve Ctrl Cipro Amp Cipro Amp Cipro Amp Cipro Amp
31. HTS-Compatible Continuous ATPase Assays Wigle and Singleton Bioorg. Med. Chem. Lett . 2007 Jun 15;17(12):3249-53 Using in end-point mode Z' = 0.87
32. 15 μ M-nts 1 μ M-nts 10 μ M-nts 1 μ M-nts 15 μ M-nts increase ssDNA to 15 μ M-nts Biochemical Characterization of the Leads Quick test shows A1 appears to be competitive with ssDNA Add ssDNA to a fully inhibited reaction and it resumes RecA + ATP + ssDNA ADP + Pi
33. Biochemical Mechanism of Action Studies Comprehensive Competition Titrations of A1 & ATP or ssDNA Michaelis-Menten Kinetics modified for cooperativity R = [RecA] S 0.5 ≈ K m R = [RecA] n = DNA/RecA stoichiometry (nts/monomer) D = [DNA] in nts K d = RecA-DNA binding dissociation constant
34. Biochemical Mechanism of Action Studies Comprehensive Competition Titrations of A1 & ATP or ssDNA S 0.5 ATP ( μ M) K d ssDNA ( μ M) V max ( μ M/min) V max ( μ M/min) A1 ( μ M) A1 ( μ M)
35. Biochemical Mechanism of Action Studies RecA-Inhibitor Co-Crystallization with Charles Bell (Ohio State)
36. Biological Mechanism of Action Studies GFP Reporter Gene Assay Measured by High-Content Screening McCool JD, Long E, Petrosino JF, Sandler HA, Rosenberg SM, Sandler SJ. Mol Microbiol . 2004 Sep;53(5):1343-57
37. Biological Mechanism of Action Studies A1 Inhibits GFP-SOS Expression Induced by Cipro as Measured by Fluorescence Microscopy (HCS)
39. Biological Mechanism of Action Studies GFP Reporter Gene Assay Measured by Flow Cytometry Induced with Cipro Uninduced Cipro + A1 (inhibition of SOS)
40. Biological Mechanism of Action Studies A1 Potentiates Several Antibiotics Including Cipro We generally observe a 2 to 4-fold decrease in MIC for Ciprofloxacin, Ampicillin, Mitomycin C, Kanamycin, and Chloramphenicol when A1 is present at 50 μ M
41. Biological Characterization of Hits The ED 50 of A1 Corresponds to it’s Biochemical IC 50 (8 μ M) All antibiotics administered at half their IC 50 and A1 titrated
44. Selectivity & Universality Panel of 11 purified RecAs Almost all RecA inhibitors are universal across species
45. Current State of RecA-Antibacterial Development SFS to discuss with CICBDD Systems biology & Transcription profiling Additional Screening ↑ chemical space (Harvard ICCB) Initiate Mouse Studies