Utilization of an in vivo reporter for high throughput identification of branched small molecule regulators of hypoxic adaptation

Abstract

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-1 alpha 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 HIF1 alpha, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.

Figure 1. Mechanism of Reporter Activation and Response to Canonical HIF PHD Inhibitors

(A) Schematic presentation of reporter performance showing key steps/potential sites of inhibition. (B) Reporter response to canonical HIF PHD inhibitors: ciclopirox, DFO, DMOG, and DHB. All values are presented as mean ± SEM. Calculation of activation parameters from the titration curve in shown in Figure S1.

Figure 2. Effect of Mutations Adjacent to Pro564 on the Reporter Response to 10 μM Ciclopirox upon 3 hr Incubation

PHD1 and PHD2 are supposed to be the major HIF PHD isoforms present in neuroblastoma cell line as judged by PCR (Figure S2). All values are presented as mean ± SEM.

Figure 3. Determination of Apparent HIF PHD Inhibition Constants from Time Course of Reporter Activation

(A and B) Original kinetic curves for DHB (A; all values are presented as mean ± SEM) and ciclopirox (B). (C) Linear plot to calculate the apparent inhibition constants using Equation 3.

Figure 4. New Hit Groups Identified in HTS

(A) Chemical structures of hits: (I), Edaravon-type hit: (Z)-1, 3-diphenyl-4-(thiazol-2-yl-hydrazono)-1H-pyrazol-5(4H)-one (86%); (II), Hydralazine type hit: 2-hydrazinyl-4,6-diphenylpyrimidine (64%); (III), Dibenzoylmethane group hit: 1-phenyl-3-(1,3-thiazol-2-yl)thiourea (41%); (IV), Thiadiazole group hit: N-[5-(3-bromophenyl)-[1,3,4]thiadiazol-2-yl]pyrrolidine-2-carboxamide (35%); (V), Triazole group hit: 4-isopropyl-5-isoquinolin-1-yl-4H-[1,2,4]triazole-3-thiol (90%); (VI), Catechol group hit: (E)-5-(3,4-dihydroxybenzylidene)-3-phenyl-2-thioxothiazolidin-4-one (38%); docking of branched catechol hits is shown in Figure S3. (B) Schematic presentation of docking mode of hydroxylated HIF peptide into PHD2. (C) Docking of best hits, compounds 7 and 8, into PHD2.

Figure 5. Upregulation of HIF1α and HIF-Regulated Human Genes

Upregulation of HIF1α (A) and HIF-regulated human genes (e.g. EPO, VEGF, PGK1, LDHA) (B) upon 3 hr treatment of neuroblastoma cells with 5 μM inhibitor (7 and 8 as positive hits, oxyquinoline and 10 as negative hits, control with no inhibitor added). All values are presented as mean ± SEM. All compounds used have comparable cell permeability as judged by kinetics of reporter activation shown in Figure S4.

Figure 6. Neuroprotection Effects of Best Branched Oxyquinoline Hits (7,8) in Comparison with a Poor Hit from the Same Group (10) in Oxidative Stress (HCA) Model

(A) Concentration titration for (7,8 and 10). (B) Photographs of viability test for 0.6 μM for (8) and (10). All values are presented as mean ± SEM. See Figure S5 for discussion of specificity of compounds effects.