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The structural optimization of adaptaquin, viz., 7-(4-chlorophenyl)[(3-hydroxypyridin-2-yl)amino]methylquinolin-8-ol, a HIF (hypoxia inducible factor) prolyl hydroxylase inhibitor, was carried out. This was done by cell-based screening using cell lines stably expressing a reporter construct consisting of the fusion protein of HIF oxygen-dependent domain and firefly luciferase (HIF1 ODD-luc). More than 100 structural analogs of adaptaquin were screened and the structures active in the submicromolar range were selected. The highest activity was found for analogs containing isobutyl, isopropyl, trifluoromethyl, and nitro group or halogen atom in the para-position of the benzene ring, but not a dimethylamino group or more bulky substituents. The lack of dependence on the donor-acceptor properties of substituents attests to predominance of the steric effect. The presence of methyl group in positions 3 and 5 of the pyridine ring provides additional activation. The 2-methyl group present in the hydroxyquinoline scaffold (50 compounds in the set) prevents coordination of the inhibitor in the active site and significantly reduces the activation. According to the real-time PCR results, the newly improved adaptaquin analog induces HIF target genes 15-times more efficiently than the FG-4592 inhibitor developed by Fibrogen.

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.

Adaptaquin, 7-{(4-chlorophenyl)[(3-hydroxypyridin-2-yl)amino]methyl}quinolin-8-ol (1), was identified in our earlier study as a hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor by high-throughput screening of cell lines, which stably express the reporter construct encoding a fusion protein composed of the HIF oxygen degradable domain and firefly luciferase (HIF1 ODD-luc). Adaptaquin is a specific inhibitor of this enzyme and exhibits neuroprotective properties in cell models of oxidative stress and in vivo models of hemorrhagic stroke. The role of the chiral carbon atom in the inhibitor molecule was studied in order to further improve the adaptaquin structure. The separation into two enantiomers was performed. Both enantiomers were shown to equally activate the HIF1 ODD-luc reporter. The adaptaquin structure can be significantly modified and made more sophisticated in order to improve specificity toward this group of enzymes and subsequently toward individual isozymes.

The screening of the library containing 320 drugs and biologically active compounds was carried out. The library was created for testing potent agents on Rett syndrome models (SMART library) using new-generation luciferase reporters to identify stabilizers of transcription factors triggering genetic programs for defense against hypoxia and oxidative stress (HIF1 and Nrf2, respectively). Nine compounds proved to be activators of HIF1, and 18 compounds were shown to activate Nrf2. A histone deacetylase inhibitor (oxamflatin) and a carbonic anhydrase inhibitor (ethamide) were found to be the most powerful Nrf2 activators, which are equipotent or superior to sulforaphane and quercetin. Oxamflatin was also shown to activate HIF with potency comparable or superior to the commercial HIF activators developed by Fibrogen (USA) and GlaxoSmithKline (UK), but it was a significantly weaker activator than branched tail oxyquinolines, developed in our previous studies. The structural motif identified in oxamflatin can be used in the future design of branched oxyquinolines having higher activity and/or more specific against individual isoforms of HIF prolyl hydroxylase.

Hypoxia inducible factor (HIF) prolyl hydroxylases (PHD) belong to α-ketoglutarate-dependent non-heme iron dioxygenases catalyzing hydroxylation of HIF prolines. The catalytic domains of all three enzyme isoforms (PHD1–3) were expressed in E.coli with the highest expression level being observed for the PHD2 isoform. In all cases, the recombinant portion of protein was mainly expressed as insoluble inclusion bodies. PHD was reactivated by refolding from inclusion bodies. To optimize the refolding procedure, a novel continuous assay based on measurement of ferrocyanide oxidation activity in the presence of HIF peptide or protein was developed. The comparison between the purified soluble enzyme samples and the PHD2 samples reactivated from inclusion bodies showed a 4–5-fold higher specific activity of the latter (5 mol min−1 vol) in the α-ketoglutarate consumption determined by fluorescence detection of ketoglutarate—o-phenylene diamine adduct. The PHD2 isoform is highly hydrophobic, has to be handled in high-molarity buffer solutions to prevent aggregation and precipitation, and is inactivated rapidly in the absence of dithiothreitol (DTT).

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.

Radioenzymology was used to study the recombinant and mutant forms of horseradish peroxidase, namely, F221W, Q176E, Q176A, S35K, E64P, E64S, S35A, and S35KQ176E. Both removal of the Trp residue and introduction of an additional one result in a simpler dose response; the insertion of polar residues stabilizes the enzyme molecule through realization of a more closed conformation. The greatest oscillation changes were found for the replacement by Ala. It was assumed that the binding site of guaiacol as a substrate is located near the residue 64, which is structurally related to the residue 176 and the heme. A scheme of formation of the intermediate through rotation of the Trp aromatic ring was proposed.

The stability of recombinant wild-type horseradish peroxidase and its tryptophan-less mutant Trp117Phe toward γ-radiation was studied. The absence of tryptophan in the enzyme molecule results in a certain stabilization, which is manifested as the absence of the initial drop in activity and appearance of a lag period for doses of up to 45 Gy. Contrary to the wild-type enzyme, the dose response of the mutant is almost independent of the nature of the substrate used to measure the catalytic activity; this indirectly indicates that Trp117 participates in the oxidation of substrates. Pretreatment of the wild-type recombinant enzyme with hydrogen peroxide destabilizes the enzyme towards irradiation, while the same procedure for the mutant enzyme has virtually no effect on the dose response curve. This suggests the modification of Trp117 in the oxidation of the native enzyme with H2O2 in the absence of electron-donor substrates, which is the modification of Trp171 in the recombinant lignin peroxidase.

Radiation-induced inactivation of soybean, horseradish, peanut, and tobacco peroxidases in the presence and in the absence of calcium and magnesium salts involves two steps which differ in the character of the effect of the metal cation. The effect of calcium cations is determined by the total charge of the enzyme molecule under experimental conditions. In general, calcium stabilizes the peroxidases, although in the case of anionic soybean and tobacco peroxidases it destabilizes the enzymes at the first inactivation step, probably due to absorption on their surface and changes in the native conformation. The effect of magnesium ions is determined by the specific features of the enzyme structure, and the data obtained allow us to suggest the existence of additional metal-binding sites in tobacco and peanut peroxidases.

The inactivation of native and recombinant horseradish peroxidase in the presence of hydrogen peroxide and under ionizing radiation was studied. The types of peroxidase activity differ in sensitivity towards the inactivating effect of H2O2: the activity in relation to the iodide ion is more stable than the activity in relation to ammonium 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonate) (ABTS) ando-phenylenediamine. Similar inactivation was observed in the course of the radiolysis of peroxidase. It was assumed that the initial period of peroxidase inactivation in the presence of hydrogen peroxide has a radical nature and is related to the generation of Superoxide radicals, which modify the protein moiety, resulting in the destruction of heme. The R-670 compound was not formed under the conditions studied. However, the E → EI transition occurred, depending on the radiation dose and the enzyme concentration.

Comparative studies of the inactivation of native and recombinant horseradish peroxidase in the course of an enzymatic reaction, at elevated temperatures and in a wide range of radiation doses, have been performed. The protective effect of the carbohydrate component of the native peroxidase providing for stabilization of the enzyme against various inactivating factors was demonstrated. It was proposed that radioactive inactivation is related to dysfunction in heme interaction with the protein component and to an increase in the conformational mobility around the active site of the enzyme.

The homogeneous recombinant horseradish peroxidase preparation fromE. coli inclusion bodies exhibits higher specific activity towards ammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) than the native one. The differences in substrate specificity can be assigned to the native enzyme inactivation in the course of metabolic reactions in living plant cells, while the recombinant enzyme reconstructedin vitro completely realizes the original catalytic abilities. Application of the method of radiation-induced inactivation demonstrates the existence of different binding sites for the iodide anion. ABTS, phenol, and guaiacol and allows one to assume a common character of the binding sites of phenol ando-phenylenediamine.

The recombinant horseradish peroxidase and its single-point F41H mutant have been reactivated fromE. coli inclusion bodies. The influence of the mutation on the heme entrapment, stability and activity of the enzyme was demonstrated. The catalytic rate constants for H2O2 cleavage and ammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) oxidation decrease by two and one orders of magnitude, respectively. Unlike the wild-type recombinant horseradish peroxidase, the elimination of the ABTS oxidation product is not a rate-determining step for the mutant. The F41H replacement results in significant changes of kinetics of iodide ion oxidation. The reaction rate is linear to the concentrations of iodide, H2O2, and the enzyme. The results suggest the direct interaction of iodide with the porphyrin ring of the heme. The decrease in ABTS oxidation activity accompanied by retention of activity in iodide oxidation in the course of low-dosage radiolysis of the F41H mutant is additional evidence of the direct electron transfer from iodide to the heme, in contrast to ABTS oxidation, in which the electron transfer chain in the protein molecule is involved.