a-high-throughput-compatible-assay-to-evaluate-drug-efficacy-against

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged <em>Mycobacterium tuberculosis</em>

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

University of Alabama at Birmingham

The continuous rise of multi-drug resistant pathogenic bacteria has become a significant challenge for the health care system. In particular, novel drugs to treat infections of methicillin-resistant Staphylococcus aureus strains (MRSA) are needed, but traditional drug discovery campaigns have largely failed to deliver clinically suitable antibiotics. More than simply new drugs, new drug discovery approaches are needed to combat bacterial resistance. The recently described phenomenon of copper-dependent inhibitors has galvanized research exploring the use of metal-coordinating molecules to harness copper's natural antibacterial properties for therapeutic purposes. Here, we describe the results of the first concerted screening effort to identify copper-dependent inhibitors of Staphylococcus aureus. A standard library of 10 000 compounds was assayed for anti-staphylococcal activity, with hits defined as those compounds with a strict copper-dependent inhibitory activity. A total of 53 copper-dependent hit molecules were uncovered, similar to the copper independent hit rate of a traditionally executed campaign conducted in parallel on the same library. Most prominent was a hit family with an extended thiourea core structure, termed the NNSN motif. This motif resulted in copper-dependent and copper-specific S. aureus inhibition, while simultaneously being well tolerated by eukaryotic cells. Importantly, we could demonstrate that copper binding by the NNSN motif is highly unusual and likely responsible for the promising biological qualities of these compounds. A subsequent chemoinformatic meta-analysis of the ChEMBL chemical database confirmed the NNSNs as an unrecognized staphylococcal inhibitor, despite the family's presence in many chemical screening libraries. Thus, our copper-biased screen has proven able to discover inhibitors within previously screened libraries, offering a mechanism to reinvigorate exhausted molecular collections.

Combinatorial phenotypic screen uncovers unrecognized family of extended thiourea inhibitors with copper-dependent anti-staphylococcal activity.

Co-infection with HIV-1 and Mycobacterium tuberculosis (Mtb) is a major public health issue. While some research has described how each pathogen accelerates the course of infection of the other pathogen by compromising the immune system, very little is known about the molecular biology of HIV-1/Mtb co-infection at the host cell level. This is somewhat surprising, as both pathogens are known to replicate and persist in macrophages. We here identify Protein Phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) as a molecular link between Mtb infection and increased HIV-1 susceptibility of macrophages. We demonstrate that both Mtb and HIV-1 infection induce the expression of PPM1A in primary human monocyte/macrophages and THP-1 cells. Genetic manipulation studies revealed that increased PPMA1 expression rendered THP-1 cells highly susceptible to HIV-1 infection, while depletion of PPM1A rendered them relatively resistant to HIV-1 infection. At the same time, increased PPM1A expression abrogated the ability of THP-1 cells to respond to relevant bacterial stimuli with a proper cytokine/chemokine secretion response, blocked their chemotactic response and impaired their ability to phagocytose bacteria. These data suggest that PPM1A, which had previously been shown to play a role in the antiviral response to Herpes Simplex virus infection, also governs the antibacterial response of macrophages to bacteria, or at least to Mtb infection. PPM1A thus seems to play a central role in the innate immune response of macrophages, implying that host directed therapies targeting PPM1A could be highly beneficial, in particular for HIV/Mtb co-infected patients.

Protein Phosphatase, Mg2+/Mn2+-dependent 1A controls the innate antiviral and antibacterial response of macrophages during HIV-1 and Mycobacterium tuberculosis infection.

In the last 40 years, only a single new antituberculosis drug was FDA approved. New tools that improve the drug development process will be essential to accelerate the development of next-generation antituberculosis drugs. The drug development process seems to be hampered by the inefficient transition of initially promising hits to candidate compounds that are effective in vivo. In this study, we introduce an inexpensive, rapid, and BSL-2 compatible infection model using macrophage-passaged Mycobacterium tuberculosis (Mtb) that forms densely packed Mtb/macrophage aggregate structures suitable for drug efficacy testing. Susceptibility to antituberculosis drugs determined with this Mtb/macrophage aggregate model differed from commonly used in vitro broth-grown single-cell Mtb cultures. Importantly, altered drug susceptibility correlated well with the reported ability of the respective drugs to generate high tissue and cerebrospinal fluid concentrations relative to their serum concentrations, which seems to be the best predictors of in vivo efficacy. Production of these Mtb/macrophage aggregates could be easily scaled up to support throughput efforts. Overall, its simplicity and scalability should make this Mtb/macrophage aggregate model a valuable addition to the currently available Mtb drug discovery tools.

A Macrophage Infection Model to Predict Drug Efficacy Against Mycobacterium Tuberculosis.

The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to replicate intracellularly while protecting it from antibiotic treatment. We recently described that Mtb infection upregulated expression of the host phosphatase PPM1A, which impairs the antibacterial response of macrophages. Here we establish PPM1A as a checkpoint target used by Mtb to suppress macrophage apoptosis. Overproduction of PPM1A suppressed apoptosis of Mtb-infected macrophages by a mechanism that involves inactivation of the c-Jun N-terminal kinase (JNK). Targeted depletion of PPM1A by shRNA or inhibition of PPM1A activity by sanguinarine restored JNK activation, resulting in increased apoptosis of Mtb-infected macrophages. We also demonstrate that activation of JNK by subtoxic concentrations of anisomycin induced selective apoptotic killing of Mtb-infected human macrophages, which was completely blocked in the presence of a specific JNK inhibitor. Finally, selective killing of Mtb-infected macrophages and subsequent bacterial release enabled rifampicin to effectively kill Mtb at concentrations that were insufficient to act against intracellular Mtb, providing proof of principle for the efficacy of a "release and kill" strategy. Taken together, these findings suggest that drug-induced selective apoptosis of Mtb-infected macrophages is achievable.

Kaitlyn Schaaf

Department of Medicine

University of Alabama at Birmingham

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

Kaitlyn Schaaf

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medicine

University of Alabama at Birmingham

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

Department of Medicine