— Reporter vectors expressing of amino acid residues 16–96 and 16–85 from Neh2-domain of Nrf2 transcription factor fused to firefly luciferase have been constructed. Cell lines stably expressing the above reporter constructs as well as an earlier developed Neh2-luc fusion reporter have been generated using HeLa cells. Comparative analysis of all three reporter lines has been performed using a well-known Nrf2 activator, nordihydroguaiaretic acid. The reporter expressing the minimal fragment of Neh2-domain containing 16–85 aa sequence exhibits a 2-fold higher amplitude of activation compared to Neh2-luc reporter, and thus can be recommended for high throughput screening for Nrf2 activators. A likely reason for the improved response of the above reporter could be ubiquitination of N-terminal lysine residues (7, 10, 11) of luciferase protein adjacent to the ubiquitinated Neh2 sequence.

Cell-based reporters expressing luciferase fusions with transcription factors or their protein stability domains can be considered as microbioreactors providing a way to directly monitor the stability of the luciferase labeled transcription factor or its domain in real-time. To understand principal advantages and/or limitations of these systems for the purposes of applied and fundamental research one needs to develop a quantitative description of their performance based on the determination of actual intracellular concentrations of the fusion proteins and rates of their production. In this work, the experimental data generated by means of luciferase activity calibration were used to calculate the steady-state intracellular concentrations of luciferase fusions in SH-SY5Y neuroblastoma cell lines stably expressing HIF1 ODD-luc and Neh2-luc proteins. For both reporters, the concentration of fusion proteins was determined as 60–80 nM, the values close to those for Michaelis constants for HIF prolyl hydroxylase (10–100 nM HIF) and the dissociation constant for Keap1-Nrf2 complex (50 nM), the parameters controlling rate-limiting steps of HIF1 ODD-luc and Neh2-luc reporter performance, respectively. New data allowed us to calculate the production rates and maximum concentrations for the fusion proteins under the conditions of irreversible activation and protein stabilization. The quantitative analysis of the Neh2-luc reporter performance employing the newly generated parameters explains the multi-order shift in the apparent activation constant versus the “real” dissociation constant determined for a known Nrf2 displacement activator using fluorescent polarization homogeneous assay with recombinant Keap1 and labeled Nrf2 peptide.

Activation of antihypoxic program under the action of a number of transition and heavy metals has been studied using cell-based HIF1 ODD-luc and HRE-luc reporters. It has been demonstrated that Au 3+ , Pb 2+ , Sn 2+ , Hg 2+ are weak HIF1 ODD-luc activators, likely reflecting their weak competition for the ironbinding site in the active center of HIF prolyl hydroxylase. Metals capable of replacing iron–Mn 2+ , Zn 2+ , Cu 2+ и Ni 2+ –activate at high submillimolar concentrations, which indicates low permeability of the cell membrane for transition metals. The highest activation is observed for Co 2+ and Cd 2+ , however, Cd 2+ is highly toxic even at 10 μM, in contrast to Co 2+ , which activates both reporters without toxicity signs up to 25 μM for 24 h. A significant activation by Co 2+ is observed already in low micromolar range of concentrations, which can be recommended for use in hypoxia mimicking.

Reporters expressing fusion proteins of HIF2 and HIF3 C-terminal oxygen degradable domain (ODD) with the firefly luciferase, HIF2 ODD-luc and HIF3 ODD-luc, were constructed and briefly characterized. Stable neuroblastoma cell lines expressing either reporter were generated, and their response to the known HIF prolyl hydroxylase inhibitors: dimethyloxalylglycine, ciclopirox, and adaptaquin, was studied and compared with the HIF1 ODD-luc reporter. The HIF2 ODD-luc reporter exhibited the highest sensitivity: its response in absolute luminescence value was almost an order of magnitude higher than that of the HIF1 ODD-luc reporter. The new reporter can be used for a fine discrimination of enzyme inhibitors stabilizing HIF2, and further structural optimization of adaptaquin discovered earlier by using the HIF1 ODD-luc reporter. The higher sensitivity of HIF2 ODD-luc reporter could be most likely explained by the lower affinity of the endogenous enzyme for this HIF isoform in comparison with the two others, which also resulted in the increased efficiency of inhibitors under the reporter assay conditions.

An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD iso-form preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of “paired” enzyme–substrate reporters to evaluate the potency of “branched tail” oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.

HIF prolyl hydroxylase is a major regulator of HIF stability. Branched tail oxyquinolines have been identified as specific inhibitors of HIF prolyl hydroxylase and recently demonstrated clear benefits in various scenarios of neuronal failure. The structural optimization for branched tail oxyquinolines containing an acetamide bond has been performed in the present study using HIF1 ODD-luc reporter assay. The special attention has been paid to the length of a linker between acetamide group and phenyl ring, as well as substitutions in the phenyl ring in the other branch of the tail. The optimized version of branched tail oxyquinolines is 3-fold more potent than the original one identified before and shows a submicromolar EC50 in the reporter assay. The compounds have been studied in a "liver-on-a-chip" device to question their hepatotoxicity towards differentiated human HepaRG "hepatocytes": the absence of hepatotoxicity is observed up to 200 μM concentrations for all studied derivatives of branched tail oxyquinolines.

We developed a cytochrome P450 substrate—inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC—mass-spectroscopy. The selected specific substrate—inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.

Benzimidazoles are drugs which target tubulin and are widely used to treat intestinal parasites. Four benzimidazoles are tested with the well-characterized and commercially available bacterial p-hydroxybenzoate hydroxylase (PHBH), which belongs to the group of Class A FAD-monooxygenases, which also includes such enzymes as the FAD-monooxygenase domain of MICAL. PHBH is shown to be competitively inhibited by all four benzimidazoles (mebendazole, albendazole, fenbendazole, and oxibendazole) in the micromolar range in the hydroxylase reaction, but not in the non-physiological NADPH-dehydrogenase reaction of ferricyanide reduction. The inhibition pattern is consistent with benzimidazoles competing with p-hydroxybenzoate for the resting state of the enzyme, indirectly indicating the ordered mechanism of substrate binding. Modeling studies support the conclusions derived from steady-state kinetics.

The flavin adenine dinucleotide-containing domain of the protein MICAL (molecule interacting with CasL) is a monooxygenase. This protein plays an important role in the regulation of axonal guidance. However, the mechanism of this process has been unknown until recently. Only two years ago, F-actin was found to be the physiological substrate. The oxidation of methionine residues of this substrate is catalyzed by MICAL, resulting in the depolymerization of actin. A year ago it was shown that methionine sulfoxide reductase (MSR) catalyzes the reverse reaction of reduction of oxidized actin methionines. Therefore, this couple of proteins is currently a target for manipulation of the ability of neurons to regenerate their axons. The present study deals with different approaches to the regulation of the activity of MICAL and MSR and the design of inhibitors of the former enzyme.