Targeting M. tuberculosis alanine ligase for drug design

DESCRIPTION (Provided by the applicant): Mycobacterium tuberculosis causes a
serious chronic infection in human beings. M. tuberculosis, along with
Mycobacterium avium,, is a major opportunistic pathogen of AIDS patients.
Although generally susceptible to antimycobacterial agents, multi-drug
resistant strains of M. tuberculosis have emerged, underlying the need for new
therapeutic agents. Peptidoglycan is the backbone of the mycobacterial cell
wall, and drugs that inhibit its biosynthesis cause a bactericidal effect due
to cell lysis. D-alanine is a required component of the mycobacteriai
peptidoglycan. Thus, those biosynthetic enzymes involved in the synthesis and
incorporation of D-alanine are attractive targets for new drug development,
especially because these enzymes are not found in mammalian hosts. The terminal
D-alanyl-D-alanine dipeptide of the peptidoglycan side chain is an essential
component for this process and its synthesis is catalyzed by the enzyme
D-alanyl-D- alanine synthetase, usually denominated D-alanine ligase (Ddl).
Unfortunately, the specific characteristics of the M. tuberculosis enzyme have
not been fully characterized, nor the essentiality of the gene has been
elucidated. In this context, our hypothesis for the proposed project is that
D-alanine ligase plays an essential role in M. tuberculosis physiology and is a
useful target for drug design. To test this hypothesis, we plan to: 1)
Overexpress, purify, and characterize biochemically the M. tuberculosis Ddl
enzyme; and 2) Test the essential role of Ddl enzyme in M. tuberculosis
physiology. These studies are expected to provide basic knowledge on key
enzymes involved in the pathway of peptidoglycan biosynthesis in mycobacteria.
Most importantly, we will obtain information on the physiological essentiality
and biochemical parameters of the Ddl enzyme necessary to develop assays for
the screening and testing of candidate compounds.