Project description:The transcription factor Nrf2 coordinates a multifaceted response to various forms of stress and to inflammatory processes, maintaining a homeostatic intracellular environment. Nrf2 anti-inflammatory activity has been related to the crosstalk with the transcription factor NF-κB, a pivotal mediator of inflammatory responses and of multiple aspects of innate and adaptative immune functions. However, the underlying molecular basis has not been completely clarified. By combining into new chemical entities, the hydroxycinnamoyl motif from curcumin and the allyl mercaptan moiety of garlic organosulfur compounds, we tested a set of molecules, carrying (pro)electrophilic features responsible for the activation of the Nrf2 pathway, as valuable pharmacologic tools to dissect the mechanistic connection between Nrf2 and NF-κB. We investigated whether the activation of the Nrf2 pathway by (pro)electrophilic compounds may interfere with the secretion of pro-inflammatory cytokines, during immune stimulation, in a human immortalized monocyte-like cell line (THP-1). The capability of compounds to affect the NF-κB pathway was also evaluated. We assessed the compounds-mediated regulation of cytokine and chemokine release by using Luminex X-MAP® technology in human primary peripheral blood mononuclear cells (PBMCs) upon LPS stimulation. We found that all compounds, also in the absence of electrophilic moieties, significantly suppressed the LPS-evoked secretion of pro-inflammatory cytokines such as TNFα and IL-1β, but not of IL-8, in THP-1 cells. A reduction in the release of pro-inflammatory mediators similar to that induced by the compounds was also observed after siRNA mediated-Nrf2 knockdown, thus indicating that the attenuation of cytokine secretion cannot be directly ascribed to the activation of Nrf2 signaling pathway. Moreover, all compounds, with the exception of compound 1, attenuated the LPS-induced activation of the NF-κB pathway, by reducing the upstream phosphorylation of IκB, the NF-κB nuclear translocation, as well as the activation of NF-κB promoter. In human PBMCs, compound 4 and CURC attenuated TNFα release as observed in THP-1 cells, and all compounds acting as Nrf2 inducers significantly decreased the levels of MCP-1/CCL2, as well as the release of the pro-inflammatory cytokine IL-12. Altogether, the compounds induced a differential modulation of innate immune cytokine release, by differently regulating Nrf2 and NF-κB intracellular signaling pathways.

Project description:The inability of the adult human heart to regenerate lost or damaged myocardial tissue has created one of the most pressing public health dilemmas due to the devastating impact of heart failure. Our group and others have outlined several regulators of cardiomyocyte mitosis that may impact the regenerative capacity of the adult myocardium in mammals. Recently, we reported that the transcription factors Meis1 and Hoxb13 regulate postnatal cardiomyocyte cell cycle arrest, where concomitant deletion of both genes induced cardiomyocyte proliferation and myocardial regeneration following ischemic injury. These studies suggest that pharmacological targeting Meis1 and Hoxb13 transcriptional activity could be a viable path towards heart regeneration. Therefore, we performed an in-silico screen to identify FDA-approved drugs that can inhibit Meis1 and Hoxb13 transcriptional activity based on the published crystal structure of Meis1 and Hoxb13 bound to DNA. Our screen yielded several candidates based on binding profiles to either Meis1 DNA binding domain, Hoxb13 DNA binding domain, or the interface between Meis1 and Hoxb13, as well as safety and side effect profiles. Out of the shortlist of top hits, paromomycin and neomycin induced proliferation of neonatal rat ventricular myocytes in vitro and displayed dose-dependent inhibition of Meis1 and Hoxb13 transcriptional activity by luciferase assay, and disruption of DNA binding by EMSA. X-ray crystal structure revealed that both paromomycin and neomycin bind to Meis1 near the Hoxb13 interaction site where they interact with 3 out of the 5 Meis1 amino acids that participate in DNA-binding. The crystal structure also demonstrates that both drugs bind at the interface of Meis1 homodimer, therefore placing one of the Meis1 monomers in a position incompatible with DNA-binding. Importantly, administration of paromomycin-neomycin combination by intraperitoneal injection in mice induced cardiomyocyte proliferation, improved left ventricular (LV) systolic function and decreased scar formation in two models of ischemic reperfusion injury in mice. Finally, dual intravenous administration of the paromomycin-neomycin combination in Yorkshire pigs following cardiac ischemia/reperfusion injury induced cardiomyocyte proliferation, improved LV systolic function, and decreased scar formation. Collectively, we identified paromomycin and neomycin as FDA-approved drugs with therapeutic potential for induction of heart regeneration in humans.

Dataset Information

Identification of Non-Electrophilic Nrf2 Activators from Approved Drugs.

Publications

Identification of Non-Electrophilic Nrf2 Activators from Approved Drugs.

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