HIV combined with either latent or active tuberculosis poses devastating consequences both in the U.S. and overseas. With no new drug class approved for tuberculosis since 1967 and with the growing problem of multidrug-resistant tuberculosis (MDRTB), the development of new agents to treat active or latent TB in the setting of HIV is a high priority. Modern drug discovery is a pipeline, which initiates with the identification of an appropriate target and culminates in pre-clinical evaluation of drug candidates and clinical trials. In this application we propose basic research on the identification of drug targets plus the development of several novel approaches to preclinical antimicrobial characterization in vitro and in vivo. A central tenet of the first aim of the application is that the best drug targets are those which are strictly essential for the organism. Through the use of random transposon mutagenesis, precise characterization of 1425 mutants by DNA sequencing, and a Monte Carle biostatistical model of essential genes, we have identified 878 non-essential genes of M. tuberculosis and 22 genes with probabilities of being essential ranging from 93-75%. Several of the likely essential genes have known inhibitors which we have found to be active against M. tuberculosis in vitro. We propose to extend this work to identify and characterize additional essential gene targets. Secondly towards advancing the later-stages of the drug development pipeline, we have developed the use of diffusion bioreactors with flow rate control for assessing critical pharmacodynamic parameters of drugs active against mycobacteria. In a related vein we have developed traditional and novel animal models which may predict drug activity against human latent tuberculosis infection (LTBI). We propose to extend the use of these tools, and to evaluate novel preventive therapy regimens for LTBI in the setting of exposure to drug-resistant TB such as might occur in areas with high MDRTB prevalence or in a bioterrorism attack.
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