Selective Killing of Dormant Mycobacterium tuberculosis by Marine Natural Products

Abstract

The dormant phenotype acquired by Mycobacterium tuberculosis during infection poses a major challenge in disease treatment, since these bacilli show tolerance to front-line drugs. Therefore, it is imperative to find novel compounds that effectively kill dormant bacteria. By screening 4,400 marine natural product samples against dual-fluorescent M. tuberculosis under both replicating and nonreplicating conditions, we have identified compounds that are selectively active against dormant M. tuberculosis. This validates our strategy of screening all compounds in both assays as opposed to using the dormancy model as a secondary screen. Bioassayguided deconvolution enabled the identification of unique pharmacophores active in each screening model. To confirm the activity of samples against dormant M. tuberculosis, we used a luciferase reporter assay and enumerated CFU. The structures of five purified active compounds were defined by nuclear magnetic resonance (NMR) and mass spectrometry. We identified two lipid compounds with potent activity toward dormant and actively growing M. tuberculosis strains. One of these was commercially obtained and showed similar activity against M. tuberculosis in both screening models. Furthermore, puupehenone-like molecules were purified with potent and selective activity against dormant M. tuberculosis. In conclusion, we have identified and characterized antimycobacterial compounds from marine organisms with novel activity profiles which appear to target M. tuberculosis pathways that are conditionally essential for dormancy survival.

Mycobacterium tuberculosis Reporter Strains as Tools for Drug Discovery and Development

“…Av‐Gay and coworkers conducted a screen for Mtb survival in THP‐1 macrophage‐like cell lines by conducting a primary screen with a constitutive luciferase based reporter and then confirmed hits using a GFP‐based reporter strain . Rohde and coworkers used a dual fluorescent Mtb reporter, constitutively expressing both mCherry and GFP to screen a marine natural product extract collection for inhibitors of replicating and non‐replicating Mtb . Inclusion of two distinct fluorescent signals was used to identify false‐positive extracts that are specific GFP or mCherry fluorescence quenchers, which may be common in natural product extracts.…”

Plakinamine P, A Steroidal Alkaloid with Bactericidal Activity against Mycobacterium tuberculosis

“…This differs from other reported plakinamines in which the H-3 proton is equatorial. A trans-ring-fusion of the A and B rings indicating a chair conformation was established based on NOESY correlations between the axial protons H-5 (δ H 1.37) and H-9 (δ H 1.64) along with 1,3-diaxial ROESY correlations between H 3 This differs from other reported plakinamines in which the H-3 proton is equatorial. A trans-ringfusion of the A and B rings indicating a chair conformation was established based on NOESY correlations between the axial protons H-5 (δH 1.37) and H-9 (δH 1.64) along with 1,3-diaxial ROESY correlations between H3-19 (δH 0.75), H-2b (δH 1.48), H-4b (δH 1.41), and H-11b (δH 1.42).…”

“…(HBOI.010.F07) inhibited actively growing M. tuberculosis CDC1551 by 97.4%, and 62% inhibition was detected against nonreplicating dormant bacteria. The data with nonreplicating bacteria were obtained using a multistress dormancy model which includes hypoxia, acidic pH, and starvation as the cues for Mtb to stop replicating [3]. Activity against dormant Mtb was absent when colony forming units (CFUs) were evaluated [16].…”

“…Further purification yielded 1 mg of pure compound which did not retain activity against dormant Mtb, therefore the dormancy assay was no longer used for this compound. All the microbiological results reported were obtained with auto-luminescent mycobacteria expressing the luxABCDE operon as previously described [3]. Dose-response curves were conducted against Mtb as well as a panel of opportunistic non-tuberculous mycobacterial (NTM) pathogens, including both rapid-growing NTMs (M. massilliense, M. abscessus, M. fortuitum) and slow-growing NTMs (M. avium, M. intracellulare, M. simiae).…”

“…Recently, a large-scale screen of a marine natural products (MNP) library containing 4400 prefractionated peak fraction samples yielded highly potent inhibitors of Mtb [3]. A sample from the marine invertebrate Plakina sp.…”