New To TB Award Recipients
Fred Buckner, MD (UW)
Utilizing the trehalose transporter pathway to delivery methionyl tRNA synthetase inhibitors against TB
Erkang Fan, Ph.D. (UW)
Utilizing the trehalose transporter pathway to delivery methionyl tRNA synthetase inhibitors against TB
In this project, we propose early-stage research to develop methionyl-tRNA synthetase (MetRS) inhibitors as novel anti-TB therapeutics. Our lab has developed highly potent inhibitors of MetRS enzymes that are efficacious in murine models of Staphylococcus and Streptococcus infections. We have screened over 60MetRS inhibitors against the MTB MetRS enzyme, successfully identifying several compounds with low nanomolar IC50 values. (MTB has a single copy of the MetRS of the Type 1 category similar to that found in Gram-positive bacteria). Unfortunately, the MetRS inhibitors have relatively weak activity on MTB cultures (in the 10-40 µM range) raising concern that the compounds are not efficiently penetrating inside MTB cells. It is well recognized that the thick cell envelope of MTB poses a permeability barrier that precludes entry of most substances, including drugs (1). To circumvent this problem, we propose a “Trojan Horse” strategy in which MetRS inhibitors will be conjugated to a substrate of an MTB membrane transporter for active uptake into the
cell. Specifically, we plan to exploit the trehalose uptake pathway which transports the disaccharide, trehalose, from the extra-cellular environment to the MTB cytoplasm.
Aim 1 Synthesize and test MetRS inhibitor-trehalose conjugates for MTB MetRS inhibitory activity
Aim 2 Conjugated inhibitors (and unconjugated parent MetRS inhibitors) will have MICs tested against MTB
cells for proof of principle that the conjugated compounds inhibit whole cell growth.
Aim 3 Conjugated inhibitors (and unconjugated parent MetRS inhibitors) will have MICs tested against MTB
cells for proof of principle that the conjugated compounds inhibit whole cell growth.
Alexis Kaushansky, Ph.D. (SCRI)
Kinase regulators of respiration in mTb-infected macrophage
Host-bacteria interactions control Mycobacterium tuberculosis (mTb) survival, persistence, dormancy, and host responses. The initial site of infection for the bacteria is the alveolar macrophage, whose metabolism has been linked to outcomes for the bacteria and immune responses in the host. While many phenotypes associated
with the host response to mTb are monitored on the scale of weeks, months or years, metabolic changes can be observed rapidly, providing a possible window into identifying short-term interventional strategies with longterm outcomes for mTb progression and immunity. We have recently developed a tool, Temporally REsolved
KInase Network Generation (TREKING), which uses kinetic data on the activity of a small panel of kinase inhibitors on a given phenotype, and their known biochemical activity against a panel of 300 human kinases, to build predicted phosphosignaling networks that mediate the phenotype of interest. We have previously
validated this tool for its capacity to build meaningful networks that control the brain endothelial barrier in response inflammatory stimuli. Here, we propose to apply TREKING to identify host phosphosignaling networks that control macrophage respiration during mTb infection. We hypothesize that these regulatory
networks differ when compared to uninfected cells; these differences might provide an opportunity to control mTb infection and/or tune the subsequent immune response.
Aim 1: Build phosphosignaling networks that control macrophage respiration in mTb-infected
macrophages.
Aim 2: Assess TREKING predictions by generating macrophage CRISPR/Cas9 knockouts.
Shao-En Ong, Ph.D. (UW)
Mapping protein-protein interaction changes in the kinome of THP-1 cells upon Mtb infection
Kinases are central nodes of signaling activity, controlling phosphorylation-dependent mechanisms, and recruiting other proteins to kinase signaling complexes in most cellular processes. Dynamic changes in the composition of kinase complexes reflect regulatory mechanisms and provide direct quantitative evidence of signaling activity in cells. We developed new mass spectrometry-based chemoproteomics methods to enrich kinases, their interacting proteins, and phosphorylation states to measure kinome activity in
cell lines and clinical tissue samples. In this proposal, we will use our kinobead affinity reagents to study a time course of Mycobacterium tuberculosis (Mtb) infection in THP-1 macrophages; identifying activated signaling pathways at different stages of infection can provide insights into new drug targets and host-pathogen interactions in the kinome. Developing these kinome-centric proteomics tools to study Mtb infection will provide new analytical capabilities and valuable datasets for the Mtb research community.
Aim 1: Characterize THP-1’s kinome interactome and its changes upon Mtb infection.
Aim 2: Study changes in THP-1 kinome activity across five time points of Mtb infection.