A. A. Poloznikov

Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode.

Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 μM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 μM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions.

Bioactive Flavonoids and Catechols as Hif1 and Nrf2 Protein Stabilizers - Implications for Parkinson’s Disease

Flavonoids are known to trigger the intrinsic genetic adaptive programs to hypoxic or oxidative stress via estrogen receptor engagement or upstream kinase activation. To reveal specific structural requirements for direct stabilization of the transcription factors responsible for triggering the antihypoxic and antioxidant programs, we studied flavones, isoflavones and catechols including dihydroxybenzoate, didox, levodopa, and nordihydroguaiaretic acid (NDGA), using novel luciferase-based reporters specific for the first step in HIF1 or Nrf2 protein stabilization. Distinct structural requirements for either transcription factor stabilization have been found: as expected, these requirements for activation of HIF ODD-luc reporter correlate with in silico binding to HIF prolyl hydroxylase. By contrast, stabilization of Nrf2 requires the presence of 3,4-dihydroxy- (catechol) groups. Thus, only some but not all flavonoids are direct activators of the hypoxic and antioxidant genetic programs. NDGA from the Creosote bush resembles the best flavonoids in their ability to directly stabilize HIF1 and Nrf2 and is superior with respect to LOX inhibition thus favoring this compound over others. Given much higher bioavailability and stability of NDGA than any flavonoid, NDGA has been tested in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-animal model of Parkinson’s Disease and demonstrated neuroprotective effects.

Enzyme–substrate reporters for evaluation of substrate specificity of HIF prolyl hydroxylase isoforms

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.

Antioxidant and antihypoxic properties of neuroprotective drugs

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.

Reactivation of HIF prolyl hydroxylase 2 from E.coli inclusion bodies

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).

HIF2 ODD-luciferase reporter: the most sensitive assay for HIF prolyl hydroxylase inhibitors

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.

Features of Construction of the Fluorescent Microscope for the Study of Epithelial-Mesenchymal Transition of Cells in Vitro

An optical luminescent microscope has been developed to monitor the behavior of tumor cells that were modified by GFP and RFP fluorescent proteins and to monitor the formation of metastases in biochips. Images of cells in the light of GFP and RFP fluorescence proteins, as well as in the photography mode, are recorded in the microscope by an noncooled CMOS matrix at the excitation of luminescence by blue and green LEDs and illumination of cells with white LEDs, respectively. The obtained fluorescent images and photos in white light are recorded by the matrix in the same coordinate system and do not require additional processing for combination. The power of irradiation of cells with LEDs does not exceed 50 μW/mm2 at an exposure time of 1 s, which is an order of magnitude lower than the radiation power causing photodestruction and phototoxicity of cells during long-term studies. Relative parameters of the optical channel of the luminescent microscope have been introduced that allow comparing the sensitivity of the recording system and the minimum power level of the radiation that excites luminescence for optical elements with different spectral characteristics.

Metal Ions as Activators of Hypoxia Inducible Factor

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 Au3+, Pb2+, Sn2+, Hg2+ 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–Mn2+, Zn2+, Cu2+ и Ni2+–activate at high submillimolar concentrations, which indicates low permeability of the cell membrane for transition metals. The highest activation is observed for Co2+ and Cd2+, however, Cd2+ is highly toxic even at 10 μM, in contrast to Co2+, which activates both reporters without toxicity signs up to 25 μM for 24 h. A significant activation by Co2+ is observed already in low micromolar range of concentrations, which can be recommended for use in hypoxia mimicking.

Which cytochrome P450 metabolizes phenazepam? Step by step in silico, in vitro, and in vivo studies

Abstract Background: Phenazepam (bromdihydrochlorphenylbenzodiazepine) is the original Russian benzodiazepine tranquilizer belonging to 1,4-benzodiazepines. There is still limited knowledge about phenazepam’s metabolic liver pathways and other pharmacokinetic features. Methods: To determine phenazepam’s metabolic pathways, the study was divided into three stages: in silico modeling, in vitro experiment (cell culture study), and in vivo confirmation. In silico modeling was performed on the specialized software PASS and GUSAR to evaluate phenazepam molecule affinity to different cytochromes. The in vitro study was performed using a hepatocytes’ cell culture, cultivated in a microbioreactor to produce cytochrome P450 isoenzymes. The culture medium contained specific cytochrome P450 isoforms inhibitors and substrates (for CYP2C9, CYP3A4, CYP2C19, and CYP2B6) to determine the cytochrome that was responsible for phenazepam’s metabolism. We also measured CYP3A activity using the 6-betahydroxycortisol/cortisol ratio in patients. Results: According to in silico and in vitro analysis results, the most probable metabolizer of phenazepam is CYP3A4. By the in vivo study results, CYP3A activity decreased sufficiently (from 3.8 [95% CI: 2.94–4.65] to 2.79 [95% CI: 2.02–3.55], p=0.017) between the start and finish of treatment in patients who were prescribed just phenazepam. Conclusions: Experimental in silico and in vivo studies confirmed that the original Russian benzodiazepine phenazepam was the substrate of CYP3A4 isoenzyme.

Role of IGFBP6 Protein in the Regulation of Epithelial-Mesenchymal Transition Genes

Protein IGFBP6 plays an important role in the pathogenesis of many malignant tumors, including breast cancer. The relationship between IGFBP6 protein and the expression of genes associated with the epithelial-mesenchymal transition is studied. Gene IGFBP6 knockdown does not trigger the epithelial-mesenchymal transition in MDA-MB-231 cells, but modifies significantly the expression of many genes involved in this process. A decrease of IGFBP6 expression can involve a decrease in the expression of N-cadherin and transcription factor Slug.