With >1.4 million people dying every year from Mycobacterium tuberculosis (Mtb) infection, tuberculosis (TB) is considered a pandemic, which is increasingly difficult to control. The deadly synergy between TB and HIV infection, and the alarming number of drug-resistant Mtb strains threaten our ability to control TB, even in developed countries. To halt and reverse this development, novel therapeutics that dramatically shorten and simplify TB treatment and that use new drug targets are needed. We recently discovered copper homeostasis as an important virulence factor of Mtb. We can demonstrate that genetic perturbation of Mtb copper homeostasis induces copper hypersensitivity which is associated with a strong virulence defect. Copper ions are employed by macrophages to kill Mtb hence compounds that synergize with copper dependent innate immune functions create novel opportunities for therapeutic interference. In this project we will develop a robust combinatorial high-throughput screening (HTS) assay for the identification of copper-boosting drugs that either (i) inhibit individual components of (ii) or increase copper uptake and thereby overwhelm the intrinsic copper resistance pathways of Mtb.
In Aim 1, we will optimize and transfer an HTS screening assay for copper-boosting drugs to a robotic platform utilizing a BSL2 approved mutant of Mtb H37Rv.
In Aim 2, we will develop and validate novel and more rapid secondary assays, which will guide hit prioritization. In particular, we will validate and utilize a flow cytometry-based approach to evaluate viability of drug exposed avirulent Mtb cells. This assessment is 1,000 times faster than traditional CFU counts, is HTS compatible, supports kinetic read-outs and requires less than 1,000 individual cells per read and condition. The various assays are designed to test whether the identified hits act as bactericidal or bacteriostatic compounds, exhibit activity against latent (non-growing) Mtb or interact with antiretroviral drugs. In research leading to this application, we have already identified two proof of concept compounds that target Mtb in a copper dependent manner and exhibit an in vitro therapeutic index of >20.
In Aim 3, we will perform a pilot screen to evaluate overall assay performance. Upon completion of this pilot campaign, we intend to apply for Fast Track entry of our screening assay into the NIH Molecular Libraries Probe Production Centers Network (Aim 4).
To achieve control over the global tuberculosis (TB) epidemic, new drugs are needed that break the deadly synergy between TB and HIV and are highly effective against multidrug and extensively drug resistant Mycobacterium tuberculosis (Mtb). Copper resistance pathways of Mtb are a promising and previously unexplored candidate for being a drug target. In this project, we will develop an automated combinatorial high- throughput screening assay to identify copper-boosting drugs that act against Mtb in synergy with natural immune functions. Identified compounds will validate copper homeostasis as novel drug target and will serve as leads for the development of new anti-Mtb drugs.
|Schaaf, Kaitlyn; Smith, Samuel R; Duverger, Alexandra et al. (2017) Mycobacterium tuberculosis exploits the PPM1A signaling pathway to block host macrophage apoptosis. Sci Rep 7:42101|
|Shah, Santosh; Dalecki, Alex G; Malalasekera, Aruni P et al. (2016) 8-Hydroxyquinolines Are Boosting Agents of Copper-Related Toxicity in Mycobacterium tuberculosis. Antimicrob Agents Chemother 60:5765-76|
|Dalecki, Alex G; Malalasekera, Aruni P; Schaaf, Kaitlyn et al. (2016) Combinatorial phenotypic screen uncovers unrecognized family of extended thiourea inhibitors with copper-dependent anti-staphylococcal activity. Metallomics 8:412-21|
|Sun, Jim; Schaaf, Kaitlyn; Duverger, Alexandra et al. (2016) Protein phosphatase, Mg2+/Mn2+-dependent 1A controls the innate antiviral and antibacterial response of macrophages during HIV-1 and Mycobacterium tuberculosis infection. Oncotarget 7:15394-409|
|Dalecki, Alex G; Wolschendorf, Frank (2016) Development of a web-based tool for automated processing and cataloging of a unique combinatorial drug screen. J Microbiol Methods 126:30-4|
|Dalecki, Alex G; Crawford, Cameron L; Wolschendorf, Frank (2016) Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay. J Vis Exp :|
|Schaaf, Kaitlyn; Hayley, Virginia; Speer, Alexander et al. (2016) A Macrophage Infection Model to Predict Drug Efficacy Against Mycobacterium Tuberculosis. Assay Drug Dev Technol 14:345-54|
|Dalecki, Alex G; Haeili, Mehri; Shah, Santosh et al. (2015) Disulfiram and Copper Ions Kill Mycobacterium tuberculosis in a Synergistic Manner. Antimicrob Agents Chemother 59:4835-44|
|Neyrolles, Olivier; Wolschendorf, Frank; Mitra, Avishek et al. (2015) Mycobacteria, metals, and the macrophage. Immunol Rev 264:249-63|
|Haeili, Mehri; Moore, Casey; Davis, Christopher J C et al. (2014) Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 58:3727-36|
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