Discovery and Investigation of Small Molecule Inhibitors of Sulfate Assimilation Pathway as Novel Anti-Latent Tuberculosis Therapy

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the single most deadly pathogen. An estimated 2 billion people are currently infected and 2 million die each year as a result of the disease. However, only 10% of the cases are symptomatic. Majority of the infections are latent, which is characterized by non-replicative, slow metabolic, and dormant Mtb surrounded by a plethora of immune called the granuloma in the lungs. The nonreplicating persistent state of infection poses a great challenge to the efforts to eradicate TB. The frontline drugs, which were discovered almost half a century ago, are primarily efficacious against actively dividing or metabolically active bacilli. To ensure the latent infection would not transition into active infection, in a process called reactivation, the drugs are to be administered up to 9 months. The long regimen leads to high non-compliance rates and directly fuels the appearance of multiple drug-resistant (MDR), extensively drug-resistant (XDR), and just recently, the discovery of totally drug-resistant (TDR) TB. To complicate matters, the co-infection with HIV promotes the progression to AIDS and animate dormancy to active infections. Unfortunately, due to the lack of financial incentive, TB research in pharmaceutical industry has largely been absent. The responsibility falls in the hands of academic labs to discover new drugs that can target Mtb in the latent phase to eliminate the episode of reactivation, shorten the regimen, and increase compliancy. This thesis presents the utilization of high-throughput screening in an effort to identify small molecule inhibitors that target the sulfate assimilation pathway, which is believed to be critical during the latent phase of TB infection. Chapter 1 reviews the molecular pathways that govern the sulfate assimilation pathway. Chapter 2 discusses the efforts of devising an assay amenable to high throughput screening, target production, library selection, and quality control. Chapter 3 presents follow-up studies that encompass the confirmation of hit compounds, the elimination of aggregate formers, and elucidation of the binding mechanism. In addition, structure activity relationship was investigated by testing compounds similar in structure to the most potent compounds. Finally, chapter 4 explores the in vivo activity of the lead compounds in growth inhibition of Mycobacterium tuberculosis, Escherichia coli, and Bacillus subtilis, as well as toxicity profile against mammalian cell line