Presentation on theme: "Modeling bacterial phenotypes using conditional knockdown mutants"— Presentation transcript:
1Modeling bacterial phenotypes using conditional knockdown mutants Dirk Schnappinger, PhDWeill Cornell Medical College
2Target-based antibacterial drug discovery has been difficult Payne et al (2007) Drugs for bad bugs: confronting challenges of antibacterial discovery. Nature Reviews Drug Discovery 6, 29.
3Target-based antibacterial drug discovery – some reasons for failure Selection of an inappropriate target→ Develop and apply a genetic approach to identify Mtb genes required for growth and survival during all phases of an infection.→ Measure vulnerability of Mtb to partial inactivation specific enzymes.Biochemical screens against purified enzymes do not select for compounds that are able to enter the bacterial cell→ Engineer Mtb strains that are hypersusceptible to inhibition of a specific enzyme or pathway.
4Evaluating Mtb proteins / pathways as new targets for drug development: biotin metabolism Biotin is required to synthesize several essential components of the mycobacterial cell envelope.Transposon mutants are strongly attenuated in mice (Sassetti and Rubin).
5The biotin biosynthesis pathway Amiclenomycin prevents mycobacterial growth through inhibition of BioA.Crystal structure of BioA has been solved (Sacchettini).
13Impact of silencing bioA during infections Plus doxyDoxy day 1 to 10Doxy day 1 to 56Day 56Dav 112Day 168Day 224
14Conclusions relevant to the evaluation of BioA (biotin metabolism) as a drug target Small molecules that efficiently and specifically inhibit BioA are predicted:to be inactive in the presence of >25 nM biotin,to be bactericidal in the absence of biotin,to be effective during acute and chronic infections (given sufficient bioavailability),to require months to eliminate Mtb during an infection.Correlation between chemistry and genetics will of course not be perfect.
15How efficient do BioA inhibitors have to be? Mtb bioA TetON-1 grows without inducer in mice.
16Alanine racemase (alr) / D-cycloserine (depletion was >97% effective) DHFR (trimethoprim) (depletion was >97% effective)GyrA (quinolones) (depletion was not that efficient, quinolones have a special mechanism of action which includes inhibition of DNA religation → double strand breaks)KasA (thiolactamycin)InhA (isoniazide) (depletion was >97% effective)RpoB (rifampicin) (depletion was ~80% effective)
17Engineer Mtb strains that are hypersusceptible to inhibition of a specific enzyme or pathway
18Engineer Mtb strains that are hypersusceptible to inhibition of a specific enzyme or pathway M. tuberculosisbioA TetON-1
19SummaryBioA is required for (i) growth and survival of Mtb in biotin-free liquid medium, and (ii) growth and persistence of Mtb in mice. BioA is a low vulnerability target but an otherwise “druggable” target and partial knockdown mutants might facilitate the development of BioA inhibitors with whole cell activity. Developed system with improved gene silencing activity and faster kinetics of inactivation.