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The body’s natural response to hypoxia is elicited by stabilization of Hypoxia Inducible Factor (HIFs). HIF is a transcriptional factor that has the ability to induce the expression of genes providing hypoxic adaptation by controlling mitogenesis, apoptosis, erythropoiesis, angiogenesis, etc. The major role in regulation of HIF stability is played by HIF prolyl hydroxylases (PHDs), which belong to the group of alpha-ketoglutarate dependent non-heme iron dioxygenases. Hydroxylated proline 564 in HIF is recognized by von Hippel-Lindau protein, and subsequently by ubiquitinylation machinery, targeting it for proteasomal degradation. Under low oxygen, the enzyme is inhibited and HIF is stabilized. The same effect can be achieved under normal oxygen by using specific inhibitors of the enzyme activity. The development of enzyme inhibitors is usually done by a traditional approach based on the inhibition assay of homogeneous recombinant HIF prolyl hydroxylase. In this laboratory, the novel approach to drug discovery, based on the application of novel luciferase fusion reporters to drug screening, yielded novel HIF prolyl hydroxylase inhibitors that exert neuroprotection in various in vivo and in vitro scenarios of neuronal failure. In addition to drug discovery purposes, this type of reporters can be used for studying HIF prolyl hydroxylase substrate specificity with respect to various HIF isoforms – HIF1, HIF2, HIF3; amino acid sequence surrounding the hydroxylated proline, as well as the differences in substrate specificity between the three isoenzymes of HIF prolyl hydrohylase – PHD1, PHD2, and PHD3. In frames of ongoing RSF project (16-14-10226), a series of novel luciferase fusion reporters has been designed and validated for research and drug discovery purposes. Five single-point mutations have been introduced in the HIF1 ODD-luc reporter around the hydroxylated Pro564 (PYIP), namely PYIA, PAIP, PPIP, PGIP, PYLP. 7 stable cell lines including the original PYIP, its five mutants, and the negative control – mutant AYIA - have been generated using the neuroblastoma host strain. Based on the inhibitor analysis of the mutant reporter stable cell lines, the enzyme specificity towards amino acids neighboring Pro564 has been elucidated. In particular, Tyr565Pro and Tyr565Pro mutations are almost unrecognized by the enzyme, and Ile566Leu mutation is recognized very poorly. The Tyr565Ala (PAIP) and Pro567Ala (PYIA) mutations result in a 3-4-fold decrease in the affinity and could be used for screening purposes. The PAIP reporter line is mainly recognized by PHD2, as judged by RNA silencing. Hence, this mutant reporter can be used to develop PHD2 specific inhibitors. The optimization of the structure of the branched oxyquinoline series done with this mutant reporter yielded a more potent inhibitor than previously identified Compound 8 (the so called adaptaquin). In the case of the PYIA reporter line, in contrast to the PAIP reporter line, the sensitivity to PHD2 silencing was negligible, pointing to the possible engagement of PHD3 into the preferred recognition of this mutant. This particular reporter was used to optimize the structure of Compound 7 analogs. This reporter favored the presence of para-nitro-group in the phenyl ring of the branched tail resulted in the improvement of the activation constant, which is in agreement with the docking predictions. Double reporters expressing HIF1 ODD-luc/PHD1; HIF1 ODD-luc/PHD2; HIF1 ODD-luc/PHD3; HIF2 ODD-luc/PHD1; HIF2 ODD-luc/PHD2; HIF2 ODD-luc/PHD3; HIF3 ODD-luc/PHD1; HIF3 ODD-luc/PHD2; HIF3 ODD-luc/PHD3 fusions have been constructed as well as two novel single constructs - HIF2 ODD-luc and HIF3 ODD-luc. In accord with the results of all 9 double construct transient expression, the following conclusions had been made on the individual HIF prolyl hydroxylase isoforms preference for individual HIF isoforms. In particular, HIF1 and not HIF2, is the best isoform for PHD1, PHD2 is active towards all three HIF isforms, whereas PHD3 is almost inactive towards HIF3. This conclusion has been supported by the development and validation of stable cell lines expressing the double constructs. The work was supported by Russian Science Foundation (grant 16-14-10226).