Theses and Dissertations at Montana State University (MSU)
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Item Drug development for tuberculosis: design and synthesis of cytochrome bd oxidase inhibitors(Montana State University - Bozeman, College of Letters & Science, 2022) Hopfner, Sarah Marie; Chairperson, Graduate Committee: Mary J. Cloninger; This is a manuscript style paper that includes co-authored chapters.In 2019, more than 10 million people worldwide became ill with Tuberculosis (TB) according to the World Health Organization 2020 Global Tuberculosis Report. More disturbing is the continued rise in cases of drug resistant TB. Thus, there is an immediate need for the development of new antimicrobials and treatment options that can quickly eliminate resistant variants of Mycobacterium tuberculosis (Mtb) infection. The oxidative phosphorylation pathway in Mtb is an attractive target for drug development because only two terminal oxidases are present: cytochrome bcc : aa 3 (cyt-bcc:aa3) and cytochrome bd (cyt-bd). Q203, a small-molecule inhibitor, targets cyt-bcc:aa3 in the oxidative phosphorylation pathway. However, Q203 is bacteriostatic and does not inhibit respiration in Mtb. Thus, innovative drugs and effective drug combinations which target the oxidative phosphorylation pathway are still needed. In this dissertation, I report the synthesis and characterization of new cytochrome bd inhibitors that can be used in conjunction with a cytochrome bcc : aa 3 inhibitor such as Q203 to rapidly kill mycobacterium tuberculosis. This combination of drugs is expected to shut down oxidative phosphorylation in Mtb, thereby removing both the primary (cyt-bcc : aa 3) and the backup (cyt-bd) power sources of energy for Mtb. Additionally, the syntheses of molecules with a thieno[3,2-d]pyrimidine-4-amine core and substituted phenylethyl substituents are described. IC 50 values of these compounds against three mycobacterial strains are presented using M. bovis BCG, M. tuberculosis H37Rv, and M. tuberculosis clinical isolate N0145 strains. Since the structure of the Mtb cyt-bd oxidase has only very recently been reported, these molecules are important targets not only to study the efficacy of a dual drug therapy but also to study membrane association of thienopyrimidine molecules. Therefore, the membrane partitioning of thienopyrmidine molecules into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was studied using time-correlated single photon counting and correlated with activity against Mtb. Finally, the syntheses of molecules with substitutions on a quinazoline core and substituted phenylethyl substituents are described. Through focused structure-activity relationships, activity against all 3 bacterial strains was improved with 2 compounds showing greater activity than the naturally derived cyt-bd inhibitor aurachin D.Item Expression and purification of two CRISPR-CAS proteins, Csm3 and Csm5 from Mycobacterium tuberculosis(Montana State University - Bozeman, College of Letters & Science, 2015) Hashimi, Marziah; Chairperson, Graduate Committee: C. Martin LawrenceOne third of the World's population is infected with tuberculosis (TB). TB disease is caused by bacterium called Mycobacterim tuberculosis, which is a facultative intracellular parasite that is transferred through the air from one person to another in close contact. A six month course of four antimicrobial drugs is the only current treatment for drug-sensitive TB. However, multi-drug resistance TB is difficult to treat. Phage therapy might be one answer as a treatment for multi-drug resistance TB. In order for phage therapy to have a chance against TB, the immune system of bacteria, known as CRISPR/Cas needs to be inhibited. Our lab has taken a structural and biochemical approach to try to understand the CRISPR/Cas system in M. tuberculosis. We have cloned, expressed, and purified individual Csm proteins from the H37Rv M. tuberculosis strain. Two Csm protein, Csm3 and Csm5 were successfully purified to homogeneity in yields suitable for structure and biochemical studies. While to date, each has failed to produce crystals, the ability to the express and purify each of these proteins will allow further biochemical characterization of Csm3 and Csm5.