MECHANISM BASED INHIBITION OF M TUBERCULOSIS ENZYMES

Project: Research project

Project Details

Description

This proposal's long-range objectives are the design, synthesis and testing
of mechanism-based inhibitors which will be effective in vitro and in vivo
against M. tuberculosis, and causative agent of tuberculosis. The
increasing incidence of tuberculosis worldwide, particularly in the United
States, and the recent appearance of multi-drug resistant varieties of M.
tuberculosis, makes the development of effective, second generation drugs
critical. A mechanism-based inhibition approach has been chosen as most
appropriate due to the favorable combination of high specificity, low
toxicity and biological efficiency exhibited by such inhibitors. As the
most attractive target enzyme for these studies, we have selected
dihydrodipicolinate reductase (DHPR), due to its ease of assay, its
favorable chemistry and its cloning from BCG (bacille Calmette-Guerin).
This enzyme is responsible for a critical early step in the lysine
biosynthetic pathway, which has as one of its intermediates,
diaminopimelate, an essential component of the mycobacterial cell wall.
The products of both of both the asd (aspartate semialdehyde dehydrogenase)
and ask (aspartokinase) genes are essential for growth of mycobacteria, and
the dual biosynthetic importance of this pathway makes the selection of
DHPR as a target especially appropriate. The cloned DHPR gene from BCG
will be used to clone the corresponding gene from M. tuberculosis, and this
gene will be sequenced. Using this sequence, the gene will be
overexpressed in E. coli or M. smegmatis. The enzyme will be purified, and
its kinetic and chemical mechanism will be determined. The specificity of
the enzyme for substrates and competitive inhibitors will be determined.
Attempts will be made to crystallize the homogeneous enzyme, and the three-
dimensional structure of the enzyme, and its binary and ternary complexes
with substrates will be determined. Mechanism-based inhibitors will be
synthesized and tested against the purified enzyme to ensure that all
kinetic and mechanistic criteria for such inhibitors are met. Those
inhibitors meeting these criteria and exhibiting favorable in vitro
properties will be tested against cultures of M. tuberculosis, and other
pathogenic bacteria. These studies should provide potent new therapeutics,
using targets which have not been suggested or explored previously.
Additional enzyme targets for this mechanism-based inhibitor approach exist
in this same pathway (succinyldiaminopimelate aminotransferase and
diaminopimelate decarboxylase), and similar strategies could be applied to
these enzymes.
StatusFinished
Effective start/end date9/30/928/31/93

Funding

  • National Institute of Allergy and Infectious Diseases

ASJC

  • Drug Discovery
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biochemistry
  • Biotechnology

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