Modeling bacterial phenotypes using
conditional knockdown mutants
Dirk Schnappinger, PhD
Weill Cornell Medical College
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.
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.
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).
The biotin biosynthesis pathway
• Amiclenomycin prevents
mycobacterial growth through
inhibition of BioA.
• Crystal structure of BioA has been
solved (Sacchettini).
Mtb DbioA
Mtb DbioA
Mtb DbioA
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
1987)
Mtb DbioA
Mtb bioA TetON
Impact of silencing bioA during infections
Impact of silencing bioA during infections
Plus doxy
Day 56
Dav 112
Day 168
Day 224
Doxy day 1 to 10
Doxy day 1 to 56
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.
How efficient do BioA inhibitors have to be?
Mtb bioA TetON-1 grows without inducer in mice.
Engineer Mtb strains that are hypersusceptible to
inhibition of a specific enzyme or pathway
Engineer Mtb strains that are hypersusceptible to
inhibition of a specific enzyme or pathway
M. tuberculosis
bioA TetON-1
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.
Thanks to
Weill Cornell
University of Minnesota
NIH/NIAID
Harvard University
Ehrt, Sabine
Aldrich, Courtney
Barry, Clifton
Rubin, Eric
Ferraras, Julian
Finzel, Barry
Boshoff, Helena
Wei, Jun-Rong
Klotzsche, Marcus
Duckworth, Benjamin
Monteleone, Mercedes
Shi, Ce
University of Pittsburgh
Stanford
Odaira, Toshiko
Wilson, Daniel
Flynn, JoAnne
Dolganov, Gregory
Lin, Philana
Schoolnik, Gary
Imperial College
Yonsei/Masan, Korea
Robertson, Brian
Cho, Ray
Williams, Kerstin
Taek-Sun Song
Park, Sae Woong
Novartis, Singapore
Camacho, Luis
Dartois, Veronique
Dick, Thomas
Manjunatha, Ujjini
Pethe, Kevin
Rao, Srini
Robert Wilkinson
Young, Douglas
SBRI
Rustad, Tige
Sherman, David