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

Day 56

Dav 112

Day 168

Plus doxy

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.

Weill Cornell

Ehrt, Sabine

Ferraras, Julian

Klotzsche, Marcus

Monteleone, Mercedes

Odaira, Toshiko

Park, Sae Woong

Thanks toUniversity 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