Proteins are the vital components of nearly every cellular process. The diversity of property and function achieved by combining just twenty amino acids is fascinating. I use biochemical and structural biology methods to understand how proteins function. My current work mainly involves studying M. tuberculosis and aligns with the drug discovery process. However, I am also fortunate to learn about proteins from muscle filaments, phages, fungi, and other bacteria through my collaboration projects. Figuring out how the structural and physicochemical properties of proteins are realized into their functions brings us closer to understanding the intricately tuned and regulated molecular activities of a living cell and enabling methods of manipulating them for therapeutic purposes.

Central carbon metabolism as a drug target for tuberculosis

As an obligatory intracellular human pathogen, Mycobacterium tuberculosis (Mtb) evolved to efficiently utilize carbon sources available in multiple niches it encounters during infection. While featuring all the specific bacteria enzymes and pathways, Mtb developed unique regulation mechanisms enabling it to co-utilize multiple carbon sources and tune the carbon/energy metabolism to survive challenging environments of macrophage or caseous lesions. One of the whole marks is Mtb’s ability to utilize fatty acids derived from carbon for growth and energy efficiency. While doing so, the bacteria reroutes the flux in the TCA cycle to rely heavily on glyoxylate shunt. Genetic experiments have shown that loss of the enzymes of this shunt, isocitrate lyase (ICL), and malate synthase (MS) cripple the ability of Mtb to establish infection and to persist in the host. There is no glyoxylate shunt homolog in human cells, making it an attractive drug target. Structure-based drug discovery efforts led us to three series of compounds potently inhibiting malate synthase, which is whole-cell active, and one of them has shown to reduce the bacterial load in the mouse model of the disease. Drug discovery work on ICL and MS are ongoing.

S. Puckett, C. Trujillo, Z. Wang, H. Eoh, T. R. Ioerger, I. Krieger, J. Sacchettini, D. Schnappinger, K. Y. Rhee, S. Ehrt, Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation in mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A.114, 2225–2232 (2017).

J. F. Ellenbarger, I. V. Krieger, H. L. Huang, S. Gómez-Coca, T. R. Ioerger, J. C. Sacchettini, S. E. Wheeler, K. R. Dunbar, Anion-π interactions in computer-aided drug design: Modeling the inhibition of malate synthase by phenyl-diketo acids. J. Chem. Inf. Model 58, 2085–2091 (2018).

Metabolic adaptations responsible for persistence of Mtb

In response to host defense and antibiotic treatment, Mtb is capable of adopting a non-replicative “quiescent” state. In this state, the bacteria are not sensitive to current antibiotic treatment and can persist in the host indefinitely. This ability is responsible for the limited success of the tuberculosis treatment leaving the chance of relapse contributing to the emergence of drug-resistant strains. We strive to understand the metabolic adaptations which enable Mtb to survive in this state and later successfully recover from it. Targeting those mechanisms would greatly potentiate current treatment strategies, shorten the burden of 6-9 months of treatment regiment, increase compliance, and curb the relapse and resistance rates. One of the factors driving Mtb into a non-replicating state is hypoxia. The consequences of a shortage of the terminal electron acceptor for respiration include failure to regenerate NAD(P) from NAD(P)H. The bacteria respond by a remodeling of the tricarboxylic acid (TCA) cycle from the oxidative reactions that occur during respiration to a reductive pathway that regenerates NAD+ and produces succinate to sustain membrane potential, ATP synthesis, and anaplerotic pathways. We study the mechanism of this adaptation and assess the potential of inhibiting the targets crucial for this remodeling for therapeutic use against persistent bacteria. To avoid toxicity, we employ structure-based drug design to develop inhibitors selective to Mtb enzyme over the human homolog.

E. S. C. Rittershaus, S. H. Baek, I. V. Krieger, S. J. Nelson, Y. S. Cheng, S. Nambi, R. E. Baker, J. D. Leszyk, S. A. Shaffer, J. C. Sacchettini, C. M. Sassetti, A lysine acetyltransferase contributes to the metabolic adaptation to hypoxia in mycobacterium tuberculosis. Cell Chem. Biol. 25, 1495–1505 (2018).