HTS for Protein Splicing Inhibitors as TB Drugs

DESCRIPTION (provided by applicant): One-third of the world' s population (2 billion people) are infected with Mycobacterium tuberculosis and 5-10% of these suffer active tuberculosis, leading to nearly 3 million deaths annually. An alarmingly growing number of patients in developed country are suffering from multidrug-resistant TB, which is essentially refractory to therapy by existing drugs. The mainline anti-tuberculosis drugs need thus be supplemented by additional drugs against new targets, until those, too, succumb to resistance mechanisms. The aim of the proposed research is to discover drugs that inhibit the post-translational processing of the M. tuberculosis DnaB and RecA proteins by protein splicing. The DnaB and the RecA proteins play essential roles in DNA replication and DNA repair, respectively. Because in virulent strains of M. tuberculosis these proteins are synthesized as inactive precursors that need to be activated by protein splicing, the inteins that interrupt these proteins and promote the protein splicing reaction constitute anti-tuberculosis targets. This application focuses on developing in vitro fluorescent assays for DnaB and RecA protein splicing based on the activation of Green Fluorescent Protein (GFP) from inactive fusions with the DnaB or RecA intein, which can be adapted as a high-throughput screening system for protein splicing inhibitors. Inhibitors found with these screens will be for specificity and antibacterial activity using both in vitro and in vivo assay systems. Preference will be given to inhibitors which inhibit splicing of the DnaB as well as the RecA intein so as to interfere both with DNA replication and DNA repair. Because protein splicing occurs only in unicellular organism and in mycobacteria as the only pathogens, such inhibitors should be highly specific antimycobacterial agents with no effect on other bacteria associated with humans nor on the human host.