Systems-based screen of compounds that target nitrogen metabolism of Mycobacterium tuberculosis.

M. tuberculosis (Mtb), the etiological agent of TB, is presently the most devastating infectious agent of mortality worldwide, responsible for about 8 million cases of TB each year resulting in more than one million deaths. Co-infection with the human immunodeficiency virus (HIV), along with the emergence of multi- and extensively- drug resistant (MDR and XDR) strains of TB, has reaffirmed Mtb as a primary public health threat throughout the world. The limited number of drugs available that have activity against Mtb, and the prolonged multi-drug regimen needed to eradicate the infection, are the fundamental problems of TB treatment. New drugs active against Mtb are urgently needed. Intracellular metabolism of Mtb is an attractive target for development of novel anti-tuberculosis drugs; however most studies have focussed on carbon metabolism. Nitrogen is also an essential nutrient of Mtb but few studies have attempted to elucidate fundamental questions such as the nature of the nitrogen source of the pathogen when it grows inside the host. Our previous studies have identified the principle amino acids as sources of nitrogen for Mtb when growing inside host cells. In this study we aim to take this study forward to test the hypothesis that nitrogen metabolism represents an unexplored and potentially fruitful drug target for TB drug development.

We will use serine metabolism as a test case of this hypothesis. In our previous study we demonstrated that the enzyme SerC that performs the last step in the biosynthesis of serine is essential for intracellular replication of Mtb. This suggest that it is also essential for growth of the pathogen inside the host and thereby a good target for the development of novel drugs. To confirm this hypothesis, we will first measure the virulence of the SerC mutant of Mtb in a mouse model of infection. The next step will be to screen a 'library' of chemical compounds library to identify any that target SerC. Conventional drug screens identify compounds that are active against particularly enzymes, such as SerC. However, the identified compounds often fail to work in the natural host because of poor penetration or inactivation of the drug in host cells. To overcome this problem, we propose to screen compounds against whole live cells of Mtb. This is more difficult as Mtb makes hundreds of different enzymes, each of which might be inhibited by the test compounds. To identify compounds that specifically target SerC, we will search for those that are active against wild-type Mtb but not a SerC mutant strain supplemented with serine so that it no longer needs SerC to grow. We will then extend the study to other key genes involved in nitrogen metabolism and uptake of amino acids from host cells. Finally, we will develop a computer model of nitrogen and carbon metabolism in Mtb that can be used to design combinations of drugs that target nitrogen metabolism that will work effectively together and test predictions of the model experimentally.

New drugs active against Mycobacterium tuberculosis (Mtb) are urgently needed but conventional screens have developed only a handful in the last fifty years. Metabolism is central to cell replication and a source of many enzymes that might be targeted with novel drugs. A lot of interest has recently focussed on carbon metabolism but nitrogen is also an essential nutrient of Mtb that is likely to provide many novel drug targets. In our previous BBSRC-supported systems-based study of nitrogen metabolism of Mtb, we identified the major amino acids captured from host cells that are used as sources of nitrogen in Mtb and showed that the SerC transaminase is essential for intracellular replication. In this study will first measure the virulence of we will first measure the virulence of a SerC mutant of Mtb in the BALB/c mouse model of infection. The next step will be to screen a chemical compound library to identify any that target SerC. To identify compounds that specifically target SerC, we will search for those that are active against wild-type Mtb but not an auxotrophic SerC mutant strain supplemented with serine. We will then extend the study to other key genes involved in nitrogen metabolism and also genes involved in uptake of amino acids from host cells and screen auxotrophic mutants of these gene against the compound library. Finally, we will develop a computer model of nitrogen and carbon metabolism in Mtb that can be used to design synergistic combinations of drugs that target nitrogen metabolism and test predictions of the model experimentally.