Genomics and Tuberculosis Research

The completion of the genome of Mycobacterium tuberculosis presents an enormous opportunity for understanding its pathogenesis, exploring drug resistance mechanisms identifying drug targets, and developing more effective new drugs. This scenario is further reinforced by high throughput gene expression analysis based on a microarray, a chip on which tens of thousands of genes are implanted.

Drug Resistance

How the tubercle bacillus develops resistance to INH (isoniazid) is a classical example to illustrate a common mechanism of drug resistance. As INH is activated by an enzyme called catalase-peroxidase of the bacillus, lack or alteration of this enzyme leads to drug resistance. An estimated 50% or more of INH resistance is caused by deletion or mutation in the katG gene, which encodes the sole catalase-peroxidase activity in the cell. It is thus possible to demonstrate the altered expression level of the katG gene for at least some INH-resistant strains in the microarray experiment. The hope here is that we can discover more drug-resistance associated genes much quicker than before.

Drug Discovery based on Genomics

Of the estimated 4000 proteins in M. tuberculosis genome, about 40% have known biochemical functions, another 44% have some sequence homology, and 16% are completely unknown. The focus of mycobacterial research now has shifted from gene hunting to understanding of genome behavior in an attempt to find clues for new drugs.

Potential drug targets can be selected using a variety of bioinformatic methods such as protein phylogenetic profiling, and screened for certain physical and chemical properties. Since Mycobacteria are prokaryotic organisms (without cell nucleus membrane) and the human host is an eukaryote (with cell nucleus membrane), the basic idea here is to select genes which are linked to essential prokaryotic genes but non-essential or absent in eukaryotes. The traditional approach focuses on rate-limiting steps in metabolic pathways unique to prokaryotes. Many such targets are obvious given the genome sequence. Protein targets which are specific to M. tuberculosis can be recognized by bioinformatic analysis and confirmed by molecular biology methods. Criteria based on protein solubility, domain size, and so on are also required for target selection.

Differential gene expression as monitored by DNA microarrays coupled with the discipline of combinatorial chemistry (ref. 1) is the emerging fundamental principle for high throughput drug discovery. The metabolism of the tubercle bacillus in the in vivo environment can be studied by taking the specimen from infected cells by bronchial lavage, for example. This will provide additional information on top of that from the in vitro bacterial culture study.

Many current antimycobacterials require some form of cellular activation such as hydrolysis, oxidation, or reduction to convert an inactive prodrug to active drug. Understanding the processes involved in drug activation will facilitate the development of analogs with alternative activation mechanisms or even none.