Abstract
Purpose: :
To expand the search for a pharmaceutical treatment of retinopathy of prematurity based on our recent observation of preventing oxygen induced vascular obliteration by activation of hypoxia inducible factor 1 (HIF-1). In this study we have conducted high throughput large scale screening of the Chembridge small molecule library for compounds capable of upregulating HIF-1.
Methods: :
A Chemical DiverSet library of 34,000 chemical compounds consisted of handcrafted drug-like organic molecules with molecular weight in a range of 250 - 550. Primary screening was performed using NIH3T3 cell line stably expressing reporter gene of firefly luciferase under a HIF-1-inducible promoter. Hierarchical multi-step validation of the reporter gene activators was performed by assessing their effect on endogenous HIF-1 levels and erythropoietin (Epo) secretion in cell cultures.
Results: :
We have identified and selected 23 potential HIF inducers that demonstrate ability to stimulate HIF-dependent transcription at list 3-fold. Six compounds increased Epo protein levels while only four compounds substantially induced HIF-1 levels in cultured cells. One compound lead to HIF-1 upregulation that did not lead to Epo expression and three other compounds caused Epo production, but only minimal induction of HIF-1. As a result of secondary screening we have selected 3 compounds that induce robust increase of intracellular HIF-1 and secreted Epo by cultures cells.
Conclusions: :
A striking number of compounds capable of HIF-1 upregulation were from a similar family of small molecules. Furthermore, newly identified HIF activators were potent at relatively low micromolar concentrations. Finally, their differential effects on HIF and Epo induction provide us with a molecular tool to dissect the pathways involved in the protection of the neurovascular unit.
Keywords: retinopathy of prematurity • hypoxia • transcription factors