1. Modeling bacterial phenotypes using conditional knockdown mutants
Dirk Schnappinger, PhD
Weill Cornell Medical College

2. Target-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.

3. Target-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.

4. Evaluating 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).

5. The biotin biosynthesis pathway
Amiclenomycin prevents mycobacterial growth through inhibition of BioA.
Crystal structure of BioA has been solved (Sacchettini).

6. Mtb DbioA

7. Mtb DbioA

8. Mtb DbioA

9. Mtb DbioA Biotin concentration in human serum:
0.1 to 3.3 nM (Hansen & Holm, Clin. Chem. 35/8, 1989)
0.01 to 0.2 nM (Hayakawa & Oizumi J Chrom )

10. Mtb DbioA

11. Mtb bioA TetON

12. Impact of silencing bioA during infections

13. Impact of silencing bioA during infections
Plus doxy
Doxy day 1 to 10
Doxy day 1 to 56
Day 56
Dav 112
Day 168
Day 224

14. Conclusions 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.

15. How efficient do BioA inhibitors have to be?
Mtb bioA TetON-1 grows without inducer in mice.

16. Alanine 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)

17. Engineer Mtb strains that are hypersusceptible to inhibition of a specific enzyme or pathway

18. Engineer Mtb strains that are hypersusceptible to inhibition of a specific enzyme or pathway
M. tuberculosis
bioA TetON-1

19. Summary
BioA 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.

20. Thanks to Weill Cornell Ehrt, Sabine Ferraras, Julian
Klotzsche, Marcus
Monteleone, Mercedes
Odaira, Toshiko
Park, Sae Woong
University of Minnesota
Aldrich, Courtney
Finzel, Barry
Duckworth, Benjamin
Shi, Ce
Wilson, Daniel
Novartis, Singapore
Camacho, Luis
Dartois, Veronique
Dick, Thomas
Manjunatha, Ujjini
Pethe, Kevin
Rao, Srini
NIH/NIAID
Barry, Clifton
Boshoff, Helena
University of Pittsburgh
Flynn, JoAnne
Lin, Philana
Imperial College
Robertson, Brian
Williams, Kerstin
Robert Wilkinson
Young, Douglas
Harvard University
Rubin, Eric
Wei, Jun-Rong
Stanford
Dolganov, Gregory
Schoolnik, Gary
Yonsei/Masan, Korea
Cho, Ray
Taek-Sun Song
SBRI
Rustad, Tige
Sherman, David