Design, Synthesis, and Evaluation of Novel Hybrid Efflux Pump Inhibitors for Use against Mycobacterium tuberculosis

Efflux pumps are considered a major potential contributor to the development of various forms of resistance in Mycobacterium tuberculosis leading to the emergence of multidrug-resistant tuberculosis (TB). Verapamil (VER) and tricyclic chemosensitizers such as the phenothiazines are known to possess efflux pump inhibition properties and have demonstrated significant efficacy in various TB disease models. Novel hybrid molecules based on fusion of the VER substructure with various tricyclic, as well as nontricyclic, chemosensitizer cores or their structural motifs are described. These hybrid compounds were evaluated in vitro and ex vivo individually for their intrinsic activity and in combination for their potentiating potential with the frontline anti-TB drugs, rifampin and isoniazid. In addition, efflux pump inhibition was assessed in an ethidium bromide assay. This study led to the identification of novel compounds, termed hybrid efflux pump inhibitors, with intrinsic antimycobacterial activities (MIC₉₀ ≤ 3.17 μg/mL) and intracellular activity in macrophages at a low concentration (≤6.25 μg/mL)

Proposed model of granzyme A-mediated suppression of intracellular mycobacterial growth by γ₉δ₂ T cells.

<p>Mycobacteria-infected macrophages present antigens to γ₉δ₂ T cells (1) which secrete granzyme A upon activation (2). Granzyme A in turn induces TNF-α production by infected and/or bystander macrophages (3) apparently independent of perforin. TNF-α activates intracellular mechanisms which alone or in concert with other unknown granzyme A-induced intracellular mechanisms suppress mycobacterial growth (4).</p

TNF-α is critical for γ₉δ₂ T cell-mediated inhibition of intracellular mycobacterial growth.

<p><i>A</i>, γ₉δ₂ T cell lines significantly suppressed intracellular growth of mycobacteria (n = 5; *p<0.05 by Wilcoxon matched-pairs test). γ₉δ₂ T cells were co-cultured with BCG-infected macrophages for 3 d. Surviving bacteria were quantified by H³-uridine incorporation and compared with cultures absent of γ₉δ₂ T cells. <i>B</i>, The effector mechanism responsible for γ₉δ₂ T cell-mediated mycobacterial growth inhibition involves soluble factors. γ₉δ₂ T cells were stimulated in the top chambers of semi-permeable transwell membrane (0.4 µm pores) systems with BCG-infected macrophages and/or soluble HMB-PP. The levels of intracellular growth inhibition were assayed in the bottom chambers and compared to the levels of inhibition achieved by direct co-culture of γ₉δ₂ T cells with the bottom layer of BCG-infected macrophages (*p<0.05, n = 5 by Wilcoxon matched pairs tests compared with unstimulated γ₉δ₂ T cells separated by transwells; **p<0.02, n = 7 by Wilcoxon matched pairs tests compared with HMB-PP stimulated γδ T cells separated by transwells; ***p<0.003, n = 15 by Wilcoxon matched pairs tests compared with HMB-PP stimulated γδ T cells separated by transwells). <i>C</i>, Neutralization of TNF-α alone prevents γ₉δ₂ T cell-mediated inhibition while neutralization of IFN-γ alone did not (*p<0.02, n = 7 by Wilcoxon matched pairs tests compared with no antibody control; **p<0.02, n = 6 by Wilcoxon matched pairs tests compared with no antibody control; n.s. – not significantly different). γ₉δ₂ T cells were co-cultured with BCG-infected macrophages in the presence or absence of the indicated neutralizing antibody and the surviving bacteria quantified by H³-uridine incorporation. <i>D</i>, shRNA-mediated knockdown of TNF-α in macrophages, but not γ₉δ₂ T cells, eliminates γ₉δ₂ T cell-mediated mycobacterial growth inhibition. (*p<0.02, n = 7 by Wilcoxon matched pairs tests compared with adenovirus expressing the empty vector). γ₉δ₂ T cells were co-cultured with BCG-infected macrophages. Prior to co-culture, either the T cells or uninfected macrophages were infected with shRNA expressing Adenovirus. Surviving bacteria were quantified by H³-uridine incorporation.</p

Granzyme A secretion by γ₉δ₂ T cells mediates inhibition of intracellular mycobacteria by induction of inflammatory responses in mycobacteria-infected macrophages.

<p><i>A</i>, The levels of granzyme A produced by γ₉δ₂ T cells are directly and highly correlated with inhibition of intracellular mycobacterial growth (r = 0.7564; p<0.0001). Supernatant levels of granzyme A, measured by CBA, were correlated with the level of mycobacterial growth inhibition observed in γ₉δ₂ T cell co-cultures with BCG-infected macrophages. <i>B</i>, Purified granzyme A induces the production of pro-inflammatory cytokines TNF-α and IL-1β by BCG-infected macrophages (*p<0.05, n = 4; statistical comparisons performed using Friedman's test). The indicated concentrations of purified granzyme A were added to BCG-infected monocytes for 3 days and the levels of TNF-α and IL-1β in the culture supernatants determined by CBA. <i>C-D</i>, Purified granzyme A alone can induce inhibition of intracellular mycobacterial growth in the absence of perforin. The indicated concentrations of granzyme A were added to BCG-infected (<i>C</i>) or <i>M. tuberculosis</i> H37Rv-infected (<i>D</i>) co-cultures for 3 days and the surviving bacteria quantified by H³-uridine incorporation and CFU counting. (*p<0.03; n = 6–12 by Wilcoxon matched pairs test comparing levels of mycobacterial intracellular growth in cultures with and without added purified granzyme A). Purified granzyme A had no direct effects on extracellular mycobacterial growth (data not shown). <i>E</i>, siRNA knockdown of granzyme A in γ₉δ₂ T cells reduces the inhibitory activity of γ₉δ₂ T cells for mycobacterial growth (*p<0.05; n = 3 by one-way ANOVA comparing levels of intracellular mycobacterial growth inhibition in cultures with or without γ₉δ₂ T cells producing granzyme A). γ₉δ₂ T cells transduced with the indicated siRNA-lentivirus targeting GzmA or a negative control (NC) were co-cultured with BCG-infected macrophages for 3 days and surviving bacteria quantified by H³-uridine incorporation.</p

Classical effector mechanisms involving direct contact of γ₉δ₂ T cells with BCG-infected macrophages are not required for mycobacterial suppression.

<p>γ₉δ₂ T cells were co-cultured with BCG-infected macrophages in the presence or absence of the indicated treatment and surviving bacteria quantified by H³-uridine incorporation. Blocking of Fas/FasL interactions did not prevent γ₉δ₂ T cell-mediated inhibition (p = 0.23, n = 7). In contrast, γ₉δ₂ T cells pre-treated for 16 hours with 25 mM strontium chloride were unable to mediate mycobacterial inhibition (*p<0.05, n = 5 by Wilcoxon matched pairs test compared with untreated γ₉δ₂ T cells). Despite strontium depletion indicating the importance of γ₉δ₂ T cell cytolytic granules for mycobacterial inhibitory effects, antibody neutralization of perforin had no effect (p = 0.28, n = 3).</p