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RecA foscused antibacterial screening
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RecA foscused antibacterial screening

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Work leading to and for the RecAEdo startup

Work leading to and for the RecAEdo startup

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    RecA foscused antibacterial screening RecA foscused antibacterial screening Presentation Transcript

    •  
    • The Reality of Antibiotics
      • 2 million U.S. nosocomial infections/year
      • 70% of these infections are resistant to at least one drug
      • 90,000 die from these infections (up from 13,300 in 1992 – nearly 600% increase)
      • MRSA kills more Americans than AIDS (18,000/year) and is 10 th leading cause of death in U.S.
      • Worldwide antibiotic sales expected to increase from $22 to $26 billion by 2011
      • Resistance costs the U.S. healthcare system $6 billion/year
      • Factors making this problem worse:
      • Antibiotic misuse and overuse
      • Lax antibiotic restrictions in some countries
      • 50% antibiotics made in the U.S. are used on livestock in a prophylactic manner
      • Dense populations
      • Sports & locker rooms (see St. Louis Rams, Cleveland Browns, Washington Redskins)
      • Bioterrorism
      • International travel
      Source: Infectious Diseases Society of America (IDSA.org) and U.S. Center for Disease Control (cdc.gov)
    • Dangerous Trends – Not a Laughing Matter Source: Clatworthy et al , Nature Chemical Biology, 3 , 541 - 548 (2007)
    • 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
    • New Pharmaceutical Approaches Needed New Targets Slow Antibiotic Resistance Potentiate Existing Antibiotics RecA
    • RecA and the Bacterial Stress Response DNA Damage RecA Repair DNA Damage Upregulate Mutation
    • RecA is Activated by DNA Damaging Stress… … But What Does it Do? ATP Signaling ATPase powered motor activity
    • RecA Initiates the SOS Response to DNA Damage
    • RecA and Recombination Paired homologous DNA can come from a sister chromatid (high-fidelity repair) or from another organism (horizontal gene transfer)
    • 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.
    • recA Knockouts are More Sensitive to Bactericidal Antibiotics Kohanski et al. Cell . 2007 Sep 7;130(5):781-3.
    • RecA: A Novel & Non-Classical Drug Target?
    • Possible Therapeutic Outcomes of RecA Inhibition ?
    •  
    • Where we looked for inhibitors Directed vs. random screening
    • Conclusions Regarding the Metal Inhibitors Bismuth-dithiol inhibitors target two storage forms of RecA Wigle, Lee, Zeng and Singleton, submitted (2008)
    • 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
    • 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
    • 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.
    • Target-Based Screening for RecA Inhibitors: Early Stage Development Lead Discovery Platform
    • HTS of 70,000 Compounds Used an Endpoint-Based P hospho m olybdate B lue ( PMB ) ATPase Assay for HTS A 650 Z′ = 0.73
    • Lead Discovery: Single-Point HTS Screen
    • Library Mining ♦ Initial screens effectively mined various libraries and returned hits belonging to chemotype clades as defined by molecular scaffold and shared pharmacophores L
    • 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
    • Time-consuming, low-throughput, laborius, tedious, antiquated, frustrating, error prone + Cipro Lead Discovery: Confirming Biological Activity Low-Throughput Viability Assay
    • Lead Discovery: Confirming Biological Activity Higher-Throughput Viability Assay ex/em 465/595
    • 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
    • Biochemical Mechanism of Action Studies A1 Inhibits RecA In Vitro Strand Exchange 25 μ M A1
    • RecA-ssDNA Direct Binding Assays A1 interferes with ssDNA Binding Lee, Wigle and Singleton Analytical Biochemistry . 2007 367(2):247
    • Continuous ATPase Assays
    • 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
    • 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
    • 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
    • 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)
    • Biochemical Mechanism of Action Studies RecA-Inhibitor Co-Crystallization with Charles Bell (Ohio State)
    • 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
    • Biological Mechanism of Action Studies A1 Inhibits GFP-SOS Expression Induced by Cipro as Measured by Fluorescence Microscopy (HCS)
    • Biological Mechanism of Action Studies HCS Dose-Dependent Inhibition of GFP-SOS by A1
    • Biological Mechanism of Action Studies GFP Reporter Gene Assay Measured by Flow Cytometry Induced with Cipro Uninduced Cipro + A1 (inhibition of SOS)
    • 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
    • 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
    • Biological Mechanism of Action Studies A1 Slows Antibiotic Resistance Development
    • Selectivity & Universality Assayed RecA inhibitors against a panel of relevant enzymes and others that were readily available
      • RecA family ATPases
      • Smooth Muscle Myosin
      • F 1 -ATPase
      • rho Transcription Termination Factor
      • RecBCD (coming soon)
      • Rad51 (coming soon)
      • Nucleotide-Dependent Enzymes
      • Pyruvate Kinase
      • Lactate Dehydrogenase
      • Purine Nucleoside Phosphorylase
      • Dihydrofolate Reductase
      • Creatine Kinase
      • Other Enzymes
      • single-stranded DNA binding protein
      • horseradish peroxidase
      • trypsin
      • pyruvate oxidase
      • DNA-dependent Enzymes
      • Endonucleases
      • Exonucleases
      • LexA (coming soon)
      Almost all RecA inhibitors were found not to cross-inhibit other enzymes
    • Selectivity & Universality Panel of 11 purified RecAs Almost all RecA inhibitors are universal across species
    • Current State of RecA-Antibacterial Development SFS to discuss with CICBDD Systems biology & Transcription profiling Additional Screening ↑ chemical space (Harvard ICCB) Initiate Mouse Studies
      • Howard Hughes Medical Institute
      • (Boston University)
      • Dr. James Collins
      • Collins Lab Members
      • University of Massachussets
      • Dr. Steven Sandler
      • Ohio State University
      • - Dr. Charles Bell