View Abstract

A5706 - Identification of Novel SIRT3 Activators as Therapeutic Prospects for PH-HFpEF
Author Block: A. Levine, T. Lear, B. Chen, Y. Lai, M. T. Gladwin; Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, United States.
Rationale
SIRT3 is a protein deacetylase which regulates mitochondrial metabolism and bioenergetics. In humans, a single nucleotide polymorphism which encodes for catalytic domain of SIRT3 is linked to the metabolic syndrome. SIRT3 deficiency has been identified in animal models of pulmonary hypertension and the metabolic syndrome. Our laboratory has demonstrated that rats with the metabolic syndrome and accompanying pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) have SIRT3 deficiency exclusively in the skeletal muscle. Nitrite and metformin clinically improve PH-HFpEF in animals in a skeletal muscle SIRT3 dependent manner. This suggests a casual role of skeletal muscle SIRT3 activation in the management of metabolic syndrome-associated PH-HFpEF and offers a potential therapeutic target for PH-HFpEF, which currently lacks any pharmacologic therapies. In this study, we attempt to identify more potent SIRT3 activators using high throughput screening (HTS) as potential novel therapies for PH-HFpEF.
Methods
1100 FDA-approved drugs were screened for SIRT3 activity via high throughput screen. Each drug was incubated (at final concentration 10uM) with human recombinant SIRT3, NAD+, and substrate which comprises the protein sequence Gln-Pro-Lys-Lys(e-acetyl)-AMC. The assay was carried out at 37 degrees for 45 minutes. Developer cleaves the substrate and results in sample fluorescence. Compounds identified in this cell free system were then assayed in vitro using C2C12 murine skeletal muscle cells. Western blots and cell morphology analysis were done to assess SIRT3 activation and downstream effects.
Results
Five novel SIRT3 activators were identified by HTS: Dolutegravir, Chlortetracycline, Prazosin, Terazosin, and Alfusozin. Preliminary Western blot analysis confirms that a subset of the compounds identified by HTS activate skeletal muscle SIRT3 and the downstream target AMPK in vitro in C2C12 skeletal muscle cells. Furthermore, we trialed and validated previously reported SIRT3 activators in our skeletal muscle cell culture system.
Conclusions
We identified five potential SIRT3 activators by HTS with a small library of FDA-approved drugs. Our data suggests that Dolutegravir, Chlortetracycline, Prazosin, Terazosin, and Alfusozin function as skeletal muscle SIRT3 activators, which may offer a new avenue for treating PH-HFpEF. We plan to replicate the SIRT3 enzyme assay and in vitro experiments at physiological doses. Efficacious drugs will be trialed in primary human skeletal muscle cell culture and our ZSF1 sugen-hypoxia rat model of PH-HFpEF with metabolic syndrome. This study indicates that repurposing of several FDA approved, well tolerated drugs may confer a previously unrecognized benefit to cardiovascular health and offer novel therapeutic agents to PH-HFpEF.