Developments in Structure Based Drug Design: A Promising Method for Targeting Rifampicin Resistant Mycobacterium tuberculosis RNA Polymerase
Open Access
- Author:
- Sutherland, Catherine
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- November 21, 2019
- Committee Members:
- Katsuhiko Murakami, Thesis Advisor/Co-Advisor
Timothy Iwao Miyashiro, Committee Member
Jean-Paul Armache, Committee Member
Wendy Hanna-Rose, Program Head/Chair
Joyce Jose, Committee Member - Keywords:
- Tuberculosis
Mycobacterium tuberculosis
Rifampicin
Rifampin
Resistance - Abstract:
- Tuberculosis (TB) is the world’s most deadly infectious disease. Though there is a treatment regimen in place, treatment of TB is becoming increasingly difficult due to antibiotic resistance. Resistance to the first-line antibiotic rifampicin is particularly problematic as rifampicin greatly reduces treatment time and is effective against both actively growing and latent TB infections. Mycobacterium tuberculosis (MTB), the causative agent of TB, acquires rifampicin resistance through mutations of the rifampicin-binding pocket found in the RNA polymerase (RNAP) β subunit. An initiative to develop rifamycin derivatives that are effective against rifampicin resistant MTB and reduce drug-drug interactions has yielded candidate compound CCG-262723. An x-ray crystal structure of CCG-262723 bound to RNAP reveals that the two substituent side chains of CCG-262723 make unique contacts with the template DNA and σ factor. With knowledge of the binding mode of CCG-262723, further structure-based modification of CCG-262723 to improve efficacy can be undertaken. High-resolution crystallization systems allow for structure-based investigations of new therapeutic compounds. Until recently, there were no such systems available for MTB RNAP. Newly published crystallization conditions for MTB σH holoenzyme can be used to fill this void. An expression and purification system that yields crystals under the published conditions has been established. This will allow for further study of new RNAP inhibitory compounds in the therapeutic context of the MTB RNAP. Rifampicin resistance confers an advantage to MTB under selective pressure. However resistance mutations can cause changes in the activity of RNAP, thus incurring a fitness cost to the bacteria. In order to compensate for this cost, secondary mutations known as compensatory mutations may arise. The most commonly encountered rifampicin resistance mutation in the clinic is rpoB-S450L. The compensatory mutation rpoC-V483G occurs with rifampicin resistance mutation rpoB-S450L with high frequency across many different MTB lineages. The preferred concurrence of compensatory mutation rpoC-V483G with rifampicin resistance mutation rpoB-S450L is not understood. To probe this notable relationship, rifampicin resistant mutants isolated from Mycobacterium smegmatis with wild-type genetic backgrounds and those isolated with an rpoC-V483G genetic background were compared. The difference in distribution and frequency of rifampicin resistance mutations between the two genetic backgrounds provides insight into the driving force of the apparent concomitant relationship between rpoB-S450L and rpoC-V483G.