Mycobacterium tuberculosis Zinc Metalloprotease-1 Assists Mycobacterial Dissemination in Zebrafish.

Zinc metalloprotease-1 (Zmp1) from Mycobacterium tuberculosis (M.tb), the tuberculosis (TB) causing bacillus, is a virulence factor involved in inflammasome inactivation and phagosome maturation arrest. We earlier reported that Zmp1 was secreted under granuloma-like stress conditions, induced Th2 cytokine microenvironment and was highly immunogenic in TB patients as evident from high anti-Zmp1 antibody titers in their sera. In this study, we deciphered a new physiological role of Zmp1 in mycobacterial dissemination. Exogenous treatment of THP-1 cells with 500 nM and 1 µM of recombinant Zmp1 (rZmp1) resulted in necrotic cell death. Apart from inducing secretion of necrotic cytokines, TNFα, IL-6, and IL-1ß, it also induced the release of chemotactic chemokines, MCP-1, MIP-1ß, and IL-8, suggesting its likely function in cell migration and mycobacterial dissemination. This was confirmed by Gap closure and Boyden chamber assays, where Zmp1 treated CHO or THP-1 cells showed ∼2 fold increased cell migration compared to the untreated cells. Additionally, Zebrafish-M. marinum based host-pathogen model was used to study mycobacterial dissemination in vivo. Td-Tomato labeled M. marinum (TdM. marinum) when injected with rZmp1 showed increased dissemination to tail region from the site of injection as compared to the untreated control fish in a dose-dependent manner. Summing up these observations along with the earlier reports, we propose that Zmp1, a multi-faceted protein, when released by mycobacteria in granuloma, may lead to necrotic cell damage and release of chemotactic chemokines by surrounding infected macrophages, attracting new immune cells, which in turn may lead to fresh cellular infections, thus assisting mycobacterial dissemination.

Ligand-free Pd-catalyzed C-N cross-coupling/cyclization strategy: an unprecedented access to 1-thienyl pyrroloquinoxalines for the new approach towards apoptosis.

The link between PDE4 and apoptosis prompted us to design and synthesize for the first time a series of novel 1-thienyl pyrroloquinoxalines as potential PDE4 inhibitors/apoptotic agents. A ligand-free Pd-catalyzed C-N cross-coupling/cyclization strategy has been developed for the rapid and milder access to this class of compounds some of which showed interesting pharmacological properties when tested in vitro and in zebrafish embryos.

A Pd-based regioselective strategy to indole-1,2-fused 8- and 9-membered rings: their evaluation as potential scaffolds for apoptosis in zebrafish.

A strategy based on Pd-mediated ring closure of 1,2-disubstituted indoles containing an unactivated olefin leading to indole-1,2-fused 8- and 9-membered rings has been developed for the identification of new and potential scaffolds for apoptosis. A large number of fused indole derivatives containing an endocyclic double bond were synthesized using this robust methodology. A representative compound showed promising apoptotic properties in zebrafish embryos.

Ligand/PTC-free intramolecular Heck reaction: synthesis of pyrroloquinoxalines and their evaluation against PDE4/luciferase/oral cancer cell growth in vitro and zebrafish in vivo.

A series of 1,3-disubstituted pyrrolo[2,3-b]quinoxalines has been designed for the potential inhibition of PDE4 without inhibiting luciferase. A ligand/PTC (phase transfer catalyst) free intramolecular Heck cyclization strategy was used to prepare these compounds, some of which showed significant inhibition of PDE4B (IC50≈ 5-14 µM) and growth inhibition of oral cancer cells (CAL 27) but not inhibition of luciferase in vitro. They also showed acceptable safety profiles but no apoptosis in zebrafish embryos.