Utilization of an In Vivo Reporter for High Throughput Identification of Branched Small Molecule Regulators of Hypoxic Adaptation

Abstract

Summary Small molecules inhibiting hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are the focus of drug development efforts directed toward the treatment of ischemia and metabolic imbalance. A cell-based reporter produced by fusing HIF-1α oxygen degradable domain (ODD) to luciferase was shown to work as a capture assay monitoring stability of the overexpressed luciferase-labeled HIF PHD substrate under conditions more physiological than in vitro test tubes. High throughput screening identified novel catechol and oxyquinoline pharmacophores with a “branching motif” immediately adjacent to a Fe-binding motif that fits selectively into the HIF PHD active site in in silico models. In accord with their structure-activity relationship in the primary screen, the best “hits” stabilize HIF1α, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.

Novel Approach to High Throughput Screening for Activators of Transcription Factors

“…For both reporters discussed, this new feature opens a way towards design of specific stabilizers of the corresponding transciption factor. We were first to predict the possibility of development of isoform-specific PHD inhibitors employing variations in the motif adjacent to the iron binding core based on the results of HTS (Smirnova et al 2010). This prediction was supported by the identified branched oxyquinolines, which were characterized as PHD specific inhibitors and were confirmed to exert the predicted biological effects.…”

“…In accord with validation studies, the HIF ODD-luciferase reporter system is controlled by the rate of PHD-catalyzed reaction: the response of the ODD-luc reporter to canonical HIF PHD inhibitors, and the increased stability of single-point mutant reporters in accord with the predictions (see Smirnova et al 2010), provided confidence that this system could be utilized for screening for novel small molecule HIF PHD inhibitors. From an enzyme kinetics point of view, the HIF ODD-luciferase reporter system is a "capture assay" monitored by the consumption of a substrate, the heterologously expressed HIF ODDluciferase fusion protein.…”

“…As shown in Fig.15, long or rigid branch, immediately attached to the iron-coordinating core, has a profound effect on HIF1 ODD-luc reporter activation. In the case of branched oxyquinolines, we were able to study structure-activity relationship in detail (Smirnova et al 2010), and to demonstrate the specific character of PHD inhibition by branched oxyquinolines (see compound 7 in Fig.12 & compounds 1,4,7,8 in Fig. 14).…”

“…However, in our hands, the cell line's response to positive controls decreases after 7 passages, making the system not suitable for a robotic HTS on 384-well plates. Taking into account the low specific activity of recombinant enzymes, and the inadequacy of interpretation of the inhibition constant generated using different types of enzyme in vitro assays, we developed a cell-based reporter system for HTS of ODD-luc stability, a variant of the cell-based "capture" assay, and accomplished a screen of 85,000 structurally diverse compounds (Smirnova et al 2010). Cell-based assay for HIF1 activators working via inhibition of HIF PHDs employs the same strategy as the one used for screening of Nrf2 activators.…”

“…11. Experimental kinetics of reporter activation for ciclopirox (complete iron deprivation, which permits determination of the promoter capacity, K 0 ), dimethyloxalylglycine (DMOG), ketoglutarate analog competing with the latter for the binding in the active center of PHD, and one of the best hits in HTS (Smirnova et al, 2010), compound 7 (formula in Fig.12). …”