Pathogen Box screening for hit identification against <i>Mycobacterium abscessus</i>

<div><p><i>Mycobacterium abscessus</i> is a rapidly growing life-threatening mycobacterium with multiple drug-resistance mechanisms. However, there is no official regimen for <i>M</i>. <i>abscessus</i> therapy. In this study, we screened the Pathogen Box, which contains 400 drug-like molecules active against neglected diseases, to identify active molecules targeting <i>Mycobacterium abscessus</i> using resazurin live/dead assays. In this screening assay, the Z-factor was 0.7, as an indicator of the statistical confidence of the assay. A cut-off of 80% growth inhibition in the screening resulted in the identification of four different compounds at a single concentration (20 μM). Dose-response curves identified three different hit candidates, i.e., MMV688508, MMV688844, and MMV688845, which generated good inhibitory curves. All hit candidates were expected to have different molecular targets. Among them, MMV688844 showed the best minimum inhibitory concentration value for not only wild-type <i>M</i>. <i>abscessus</i> but also for nine different R and S morphotype clinical isolates. Thus, we found that MMV688844, identified from the Pathogen Box screen, may be a promising candidate in the <i>M</i>. <i>abscessus</i> drug discovery pipeline.</p></div

Screening validation.

<p>The Z-factor of 0.7 demonstrates excellent assay.</p

Pathogen Box screening for hit identification against <i>Mycobacterium abscessus</i> - Fig 5

<p>Time–kill curves of (A) cefoxitin and (B) MMV688844 against <i>Mycobacterium abscessus</i>. The bacteria were grown in liquid culture (Middlebrook 7H9 medium) in the presence of the indicated concentrations of cefoxitin or MMV688844.</p

Screening of the Pathogen Box library for <i>Mycobacterium abscessus</i>.

<p>Scatter plot distribution showing the results of the <i>M</i>. <i>abscessus</i> screening of the Pathogen Box library using resazurin reduction assay. A total of 400 compounds from the Pathogen Box were screened at 20 μM against <i>M</i>. <i>abscessus</i>. Growth inhibition of at least 80% was defined as cut-off which resulted in 4 hits (1.0% hit rate) from the 400 compounds in the Pathogen Box library.</p

In vitro activity of MMV688844.

<p>Activity of MMV688844 against <i>Mycobacterium abscessus</i> clinical isolates. Dose-response curves were plotted from REMA data for <i>M</i>. <i>abscessus</i> strains treated with MMV688844. Data are expressed as the mean ± standard deviation (SD) of triplicates for each concentration. RFU, relative fluorescence units.</p

Mycobacterial strains inhibit apoptotic cell death induced by the chemical agents doxorubicin and cyclohexamide.

<p>Macrophages infected with mycobacterial strains for 2.5 hours (MOI 10) were induced to undergo apoptosis with doxorubicin (2.5 µM) (a,b) or cyclohexamide (9 µM) (a,c). After 20 hours, live cells were enumerated using Calcein-AM. (a) A representative comparison of cell death induced by doxorubicin and cyclohexamide in uninfected and H37Rv-infected macrophages. Survival is expressed as a percentage of control untreated cells. (b,c) A comparison of the ability of mycobacterial strains to inhibit doxorubicin- (b) and cyclohexamide- (c) induced apoptotic cell death. Survival of drug treated infected cells is expressed as a percentage of untreated, infected cells. (Bar graphs are Mean+SEM, n = 10, and are representative of two independent experiments. *p = <0.05, **p = <0.01, ***p = <0.001 by unpaired 2-tailed T-test.</p

Dose response curve (DRC) and chemical structure of hits.

<p>Dose response curve of MMV687146 (A), MMV688508 (B), MMV688844 (C), and MMV688845 (D) and reference compound (Amikacin; E) were plotted and chemical structure of each compounds were described.</p

Live mycobacteria inhibit doxorubicin-induced apoptosis in a dose-dependent manner.

<p>Macrophages were induced to undergo apoptosis with different doses of doxorubicin, and cell survival enumerated at 3,6 and 20 hours using a calcein-AM (a). Macrophages infected with mycobacterial strains at MOI 1–10 (b) or MOI 10 (c–d) for 2.5 hours were induced to undergo apoptosis with doxorubicin (2.5 µM). After 20 hours, live cells were enumerated using Calcein-AM (b–c). (d) ApoBrdU TUNEL staining was performed on uninfected (grey fill) and GC1237-infected (black line) macrophages after 20 hrs treatment with 2.5 µM doxorubicin, and cells analysed by flow cytometry (representative plots of quadruplicate samples are shown). Bar graphs are Mean+SEM, n = 10, and are representative of two independent experiments. *p = <0.05, **p = <0.01, ***p = <0.001 by unpaired 2-tailed T-test.</p

Polycaspase activation in <i>M. tuberculosis</i> infected RAW macrophages over 72 hours.

<p>RAW macrophages were infected with <i>M. tuberculosis</i> GC1237 wild-type and trafficking mutant strains Tn::<i>moaC1</i> and Tn::<i>Rv1503c</i>. After 24, 48 and 72 hours, nuclei were stained with Hoechst stain (blue), and active caspases stained with fluorescent polycaspase inhibitor (FLICA; red), and the cells analysed by automated confocal microscopy. Caspase staining (A) (surface of caspase/number of nuclei) and number of nuclei (B) were measured using Image Mining Software developed at the Institut Pasteur, Korea. Bar graphs are Mean+SEM of triplicate samples. *p = <0.05, **p = <0.01, ***p = <0.001 by 2-way ANOVA, with the upper and lower p values for GC1237 vs Tn::<i>moaC1</i> and Tn::<i>Rv1503c</i> respectively. (C) Representative microscopy at 72 hours is shown. Results are representative of 3 independent experiments with similar results.</p

Retinestatin, a Polyol Polyketide from a Termite Nest-Derived <i>Streptomyces</i> sp.

A new polyol polyketide, named retinestatin (1), was obtained and characterized from the culture of a Streptomyces strain, which was isolated from a subterranean nest of the termite Reticulitermes speratus kyushuensis Morimoto. The planar structure of 1 was elucidated on the basis of the cumulative analysis of ultraviolet, infrared, mass spectrometry, and nuclear magnetic resonance spectroscopic data. The absolute configuration of 1 at 12 chiral centers was successfully assigned by employing a J-based configuration analysis in combination with ROESY correlations, a quantum mechanics-based computational approach to calculate NMR chemical shifts, and a 3 min flash esterification by Mosher’s reagents followed by NMR analysis. Biological evaluation of retinestatin (1) using an in vitro model of Parkinson’s disease revealed that 1 protected SH-SY5Y dopaminergic cells from MPP+-induced cytotoxicity, indicating its neuroprotective effects