Date of Award
Doctor of Philosophy (PhD)
The disease tuberculosis caused by Mycobacterium tuberculosis (Mtb) remains a global health threat, and the antibiotic resistance crisis challenges our ability to treat Mtb infections. For example, out of the approximately 10 million cases of tuberculosis each year, an estimated 10.7% of new cases and 27.2% of previously treated cases are resistant to the frontline antibiotic isoniazid (INH). There is a clear need for new therapies that enhance our current regimen and target these genetically resistant strains.
Identifying ways to enhance the antimicrobial activity of INH has the potential to significantly improve the efficacy of frontline treatment regimens. To this end, our lab identified a small molecule potentiator of INH, C10. While C10 on its own has poor growth-inhibitory activity, this compound enhances the bactericidal activity of INH against Mtb. Remarkably, we discovered that C10 suppresses the emergence of INH-resistant mutants in vitro and can even re-sensitize some INH-resistant mutants to this antibiotic. INH is a cell-wall targeting antibiotic that inhibits the biosynthesis of mycolic acid, an essential lipid in the outer layer of the mycobacterial cell envelope. However, INH is a pro-drug that must first be converted to its active form INH-NAD through the activity of the bacterial enzyme KatG. Activated INH-NAD can then bind and inhibit its target, the essential mycolic acid biosynthesis enzyme InhA. The vast majority of INH resistant isolates harbor mutations that decrease the activity of the activating enzyme KatG. Our work demonstrated that C10 could re-sensitize a set of INH-resistant katG mutants to INH, revealing that INH resistance is not absolute and can be reversed.
In pursuit of the mechanism of action of C10, I used transcriptional profiling, forward genetics, and assays of bacterial physiology to demonstrate that C10 disrupts Mtb energy metabolism, leading to decreased respiration and depletion of bacterial ATP. We found that at very high concentrations (>50μM), C10 exhibited bacterial toxicity that was mediated through disruption of Mtb energy metabolism. However, using a combination of genetic and chemical biology approaches, we discovered that the ability of C10 to disrupt Mtb energy metabolism was neither required nor sufficient to potentiate the bactericidal activity of INH. C10 must impart some other effect on Mtb that elicits the increased INH sensitivity.
To more specifically understand how C10 potentiates INH, we selected for mutants resistant to both INH and C10. The INH/C10-resistant mutants harbored katG-null mutations that abolished synthesis of INH-NAD, rendering the strains resistant to INH even in the presence of C10. In contrast, only INH-resistant strains that retained residual KatG activity and accumulated a low level of INH-NAD could be re-sensitized to inhibition by C10. Therefore, some low level of KatG activity is required for C10 to potentiate INH.
The best studied mechanism to enhance INH activity involves increasing the amount of INH-NAD synthesis in the bacteria. However, through a series of assays, we found that C10 sensitized Mtb to killing by INH without increasing KatG activity or INH-NAD levels. C10 therefore sensitizes Mtb to INH-NAD after it is synthesized without changing its levels, such that resistant mutants that accumulate sub-inhibitory levels of INH-NAD become inhibited by this concentration of INH-NAD in the presence of C10. In support of this model, we discovered that C10 sensitizes the bacteria to killing by the direct InhA inhibitor NITD-916. Notably, decreasing InhA expression or activity could sensitize Mtb to InhA inhibitors by titrating the concentration of antibiotic required to inhibit this target. However, we showed that C10 on its own does not decrease InhA expression or activity. Therefore, C10 enhances the bacterial vulnerability to InhA inhibitors through an un-precedented mechanism. These findings have implications for mycolic acid targeting antibiotics currently in use in the clinic and in clinical development.
The target and precise mechanism of action of C10 remains unknown. However, by using chemical genetic approaches to interrogate the pleiotropic effects of C10 against Mtb, my thesis work has enhanced our understanding of Mtb physiology and uncovered a novel mechanism to circumvent Mtb drug resistance.
In an independent approach to identify compounds that inhibit Mtb through a novel mechanism of action, our lab screened a small library of nucleotide mimetics to identify inhibitors of Mtb growth. We discovered a new series of antimycobacterial compounds, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit the growth of Mtb. Using forward genetics and assays of Mtb bioenergetics, we found that 4-amino-thieno[2,3-d]pyrimidines inhibit the QcrB subunit of the electron transport chain enzyme cytochrome bc1, a validated drug target.
Collectively this work highlights that Mtb energy metabolism is a promising target for the development of novel small molecule Mtb inhibitors. In attempts to address the Mtb drug resistance crisis, I have contributed to our understanding of how C10 sensitizes Mtb to clinically relevant antibiotics, and I have described a new chemical class of respiration inhibitors, 4-amino-thieno[2,3-d]pyrimidines, which inhibit a known Mtb drug target.
Chair and Committee
Christina L. Stallings
Jennifer A. Philips, Michael G. Caparon, Daniel E. Goldberg, Scott J. Hultgren,
Harrison, Gregory Alexander, "Circumventing Drug Resistance with Small Molecule Inhibitors of Mycobacterium tuberculosis" (2023). Arts & Sciences Electronic Theses and Dissertations. 2854.