Target identification

Novel drug targets may be identified in several ways. The classical approach is to study a process, identify a protein target and purify sufficient material to obtain sequence information, then clone the gene and use reverse genetics to confirm its essentiality. It is known that disruption of cell wall synthesis is lethal to the bacterium, and several steps in this process are being analysed in this way to reveal targets. In some cases, for example arabinan biosynthesis (Mikusova et al 1995) and linkage region biosynthesis (Mikusova et al 1996), this has only progressed as far as developing a crude assay system. In other cases, enzyme function has been correlated with a single gene product, e.g. UDP-galacto-pyranose mutase (Weston et al 1997), or several gene products, e.g. mycolyl transferase (Belisle et al 1997).

Alternatively, determining the mode of action of today's drugs identifies targets. For example, the inhA gene product was identified as a target for isoniazid (Banerjee et al 1994) and subsequently shown to possess enoyl reductase activity (Quemard et al 1995), a step in fatty acid metabolism. Biochemical studies have indicated that ethambutol acts by inhibiting arabinosyl transferase (Lee et al 1995) and further genetic studies indicated that the emb genes encode the target (Belanger et al 1996, Telenti et al 1997).

Progress in identifying essential genes in M. tuberculosis has been hampered by the lack of well-characterized mutants. The recent description of a set of temperature-sensitive mutants of Mycobacterium smegmatis (A. Belanger, J.C. Porter, G. Hatfull, unpublished paper, ASM Conference on Tuberculosis: past, present and future, 8—12 July 1997) is a step towards defining specific targets in M. tuberculosis. Moreover, libraries of M. tuberculosis mutants have been generated by randomly inserting a transposon into the chromosome, using plasmid (Pelicic et al 1997) and mycobacteriophage (Bardarov et al 1997) delivery systems. Although it is not possible to recover mutants generated when the transposon interrupts a gene essential for replication, we may nevertheless deduce which gene products are essential for vegetative growth by sequencing the insertion points and by referring to the genome sequence (with the proviso that there may be polar or other effects that will complicate interpretation).

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