Development and application of a cell-based assay to allow the study of selective inhibitors of Hypoxia Inducible Factor (HIF) Prolyl Hydroxylase 1 (PHD1)

Introduction Hypoxia Inducible Factor (HIF) is a heterodimeric transcription factor that drives the expression of genes involved in the hypoxic response. It is negatively regulated by a family of 2-oxoglutarate-, Iron- and Oxygen- dependent Prolyl Hydroxylases (PHD1, PHD2 and PHD3). Inhibition of PHDs by a non-selective inhibitor has a potential utility in the treatment of ischemic disease but has the unwanted side effect of increasing haematocrit through the stimulation of EPO expression. Clinical studies and mouse knock-out work suggests that a selective inhibitor of PHD1 may be a point of pharmacological intervention in both ischemic diseases (1,2) and also in Irritable Bowel Disease (IBD)(3,4).

However, interrogating the inhibition of PHD1 in a cell-based assay is complicated by the ubiquitous expression of PHD2. In this study, this was overcome by the generation of a knock-out PHD2 cell line using Zinc-finger nuclease technology. The choice of cell line was made after the relative expression of PHDs was measured in a range of cell lines and HepG2 cells were found to have high expression of PHD1 and PHD2 but very low levels of PHD3. The knock-out cell line could therefore be used to measure, more specifically, the inhibition of PHD1 by monitoring the compound concentration-dependent stabilization and accumulation of HIF1-alpha.

Methods The quantification of inhibition of PHD1 and PHD2 in a recombinant enzyme assay was performed as described previously (5). For the cell-based assay, the cells were seeded overnight in full medium in the presence of inhibitor (16hours). The activity of compound was quantified by measuring the concentration-dependent stabilization and accumulation of HIF1-alpha using an MSD plate-based assay (6)

Results

Conclusion

The use of the PHD2-/- cell-line removes the effect of PHD2 from ablating the assay readout and thus obscuring the apparent efficacy of PHD1-selective compounds in a cell-based assay.

(1) Schneider Met al Gastroenterology (2010) 138(3): 1143-54.

(2) Adluri RS et al Antioxid Redox Signal. (2011) 15(7): 1789-97.

(3) Van Welden S et al J Inflamm (Lond). (2013) 10(1): 36

(4) Tambuwala MM et aI Gastroenterology. (2010) 139(6): 2093-101.

(5) A.C.R. Epstein et al. Cell. (2001) 107: 43–54

(6) https://www.mesoscale.com/en/products/k150dkd-2/