TB is the second leading cause of morbidity world wide caused by a single infectious organism. One third of the world's population are infected with the causative agent, Mycobacterium tuberculosis, and every year it kills 1.7 million people. There is an urgent need to find new Anti - TB that are efficacious, affordable and compatible with HIV and other Anti - TB used in developing countries, where the disease is prevalent. These drugs must also shorten treatment time and be active against resistant forms of the mycobacteria that will help increase patient compliance. One such lead compound which may be developed to meet these criteria is thiolactomycin (TLM). This naturally occurring antibiotic has been shown to be active against M tuberculosis by inhibiting the F AS 11 condensing enzymes mtFabH, KasA and KasB involved in mycolic acid biosynthesis. We thus embarked on a project to design and synthesize analogues of TLM, and related compounds, as new lead molecules for consideration as potential Anti - TB. A series of synthetic procedures, amenable for use in an iterative drug design process, were postulated and investigated to generate enantiomerically pure analogues of TLM derivatised at the third and fifth positions of the thiolactone ring. The first was using a method starting from D-alanine which generated a pair of diastereoisomers which could be separated to give both enantiomers as analogues. However, this route depended upon the production of enantiomerically pure (S)-thiolactic acid which could not be isolated. However, it was possible to obtain racemic analogues using this approach. A second strategy was attempted by the chiral resolution of racemic thiolactic acid to generate a series of organic salts.

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Tuberculosis is the world's largest cause of death from an infectious agent. Treatment is by an extended period of combination chemotherapy. Drug resistance is an increasing problem in tuberculosis therapy, particularly to the frontline anti-tubercular drug isoniazid (INH). Recombinant arylamine N-acetyltransferase (NAT) of Mycobacterium tuberculosis N-acetylates INH using the cofactor Acetyl Coenzyme A. NAT from M. tuberculosis is a polymorphic enzyme and also acetylates INH in vivo. Acetylated INH is inactive therapeutically against M. tuberculosis both in vivo and in vitro. The acetylation of isoniazid in the mycobacterial cell may compete with the activation of INH by the catalase-peroxidase, katG, and hence contribute to INH resistance in clinical isolates. Inhibition of NAT in M. tuberculosis may thus increase the efficacy of INH therapy. A novel assay based around the detection of free Coenzyme A released during the acetylation reaction was used to determine the substrate specificity of recombinant NAT from the related Mycobacterium M. smegmatis (MSNAT). A relationship was observed between the lipophilicity of simple arylamine substrates and the rate of acetylation by MSNAT. Several MSNAT substrates possess antibacterial activity. The assay could also be used to screen compound libraries for MSNAT inhibitors. Synthesis of seventeen thiazolidinedione sultams in collaboration with Dr.Vickers (Dyson Perrins), identified as weak inhibitors of MSNAT, gave a minimum competitive inhibitory constant of 14μM. Screening a library of 5,074 drug-like compounds for inhibition of MSNAT identified thirteen compounds with semi-maximal inhibition constants (IC₅₀) of below 10μM. Based on this, fifteen maleimides were synthesised and were irreversible inhibitors of MSNAT with submicromolar potency. Similarly, ninety-six aminothiazoles were synthesised by Dr. Vickers and were uncompetitive inhibitors of MSNAT with a minimum IC₅₀ of 1.5μM. The most potent aminothiazole showed no effect on the growth of M. smegmatis or M. bovis BCG or the sensitivity of the bacteria to isoniazid. However the aminothiazoles were shown not to penetrate the cells.

The current worldwide epidemic of Mycobacterium tuberculosis infection is a huge global health problem. Widespread BCG vaccination remains a useful tool in combating this epidemic; however, its variable efficacy requires urgent development of novel vaccines against Mycobacterium tuberculosis. Such a candidate vaccine is a serotype 5 adenovirus expressing antigen 85A from M. tuberculosis (Ad85A). In animal models Ad85A confers significant protection when administered intra-nasally. The work in this thesis demonstrates that intra-nasal immunisation with Ad85A results in inhibition of M. tuberculosis growth in the lung early after infection, in contrast to the late inhibition induced by parenterally administered vaccines. Early inhibition correlates with the presence in the lung of a highly activated population of antigen-specific CD8 T cells, maintained for at least 6 months post-immunisation by persistent antigen. For intra-nasal Ad85A to be effective, the vaccine must be delivered into the lower respiratory tract, as immunisation targeting only the nasal-associated lymphoid tissue (NALT) does not result in protection. Following a change of animal facility, the lung immune response to intra-dermal immunisation with Ad85A increased and this route of immunisation now induced protection, though growth of M. tuberculosis was inhibited only late after infection. However, this response and protection can be altered by exposure to environmental mycobacteria. Further experiments showed that simultaneous respiratory and parenteral immunisations (SIM) act additively, where local lung immunity inhibits the growth of M. tuberculosis early after infection and systemic immunity protects later. SIM regimes generate greatly improved protection over either immunisation alone and do not depend on priming and boosting.