Small molecules to treat angiogenesis and inflammation in an in-vitro model of diabetic retinopathy

Abstract

Purpose : Diabetic retinopathy (DR) is an inflammatory and microvascular disease. The ELAVL-1 (HuR) RNA binding protein is a chaperone protein that stabilizes mRNA of TNFα and VEGFA, markers of retinal inflammation and angiogenesis, respectively. Inhibition of HuR represents a novel approach towards DR treatment. We discovered small molecules capable to interfere with the HuR-mRNA complex, and inhibited TNFα and VEGFA release in retinal endothelial cells.

Methods : Virtual screening was carried out on a compounds database searching for novel HuR inhibitors. Dihydrotanshinone (DHTS) was used as positive control for in-silico studies and in-vitro studies. Selected in-silico compounds were tested in the in-vitro model of diabetic retinopathy: human retinal endothelial cells (HRECs), exposed to high glucose levels (HG, 25 mM) for 48 h. MMT assays was carried out to test the cell tolerability to compounds exposure. HuR, TNFα, VEGF expression was analyzed through western blotting. Commercial ELISA kits were used for quantification of TNFα and VEGF release by HRECs. Molecular dynamics simulations of HuR/inhibitor complexes were carried out in order to confirm at structural level the mechanism of action of tested compounds.

Results : Two indole compounds VP12/14 and VP12/110 were identified in-silico as best predicted inhibitors of HuR. These two compounds, and DHTS (positive control) protected HRECs from high glucose damage. DHTS, VP12/14 and VP12/110 significantly reduced the HuR, TNFα and VEGFA expression in HRECs exposed to HG condition. HG condition induced a significant (p<0.05) higher release of TNFα and VEGFA levels by HRECs, compared to control cells. Tested compounds significantly (p<0.05) counteracted the TNFα and VEGFA release induced by HG condition. The compounds showed an efficacy profile DHTS»VP12/110>VP12/14 that was explained by molecular dynamics simulation analysis.

Conclusions : VP12/110, as well as the known HuR inhibitor DHTS, is not only a competitve inhibitor of HuR, but also interferes with HuR chaperone function by conformational destabilization of TNFα and VEGF mRNA structure. VP12/110, on the basis of pharmacolodynamic assays carried out, can be further developed as innovative anti-angiogenic and anti-inflammatory drug for treatment of diabetic retinopathy.

This is a 2020 ARVO Annual Meeting abstract.