Biomedical Chemistry: Research and Methods 20120, 3(3), e00134

Biosensor Selection of Small Compounds, Modulating a Complex Formation between Cytochrome P450s and Nadph-Dependent P450 Oxidoreductase

P.V. Ershov1, E.O. Yablokov1*, Y.V. Mezentsev1, L.A. Kaluzhskiy1, A.Ya. Luschik2, A.M. Tumilovich2, A.Yu. Karputs2, A.A. Gilep2, А.S. Ivanov1

1Institute of Biomedical Chemistry", Pogodinskaya str. 10, bldg 8, Moscow, 119121Russia; *e-mail: evgeyablokov1988@mail.ru

2Institute of Bioorganic Chemistry of National Academy of Science of Belarus, Kuprevicha str. 5/2, Minsk, 220141, Republic of Belarus.

Keywords:affinity modulators; kinetics modulators; low molecular weight compounds; NADPH; cytochrome P450; NADPH-dependent cytochrome P450 oxidoreductase

DOI:10.18097/BMCRM00134

The whole version of this paper is available in Russian.

The study of the effect of low-molecular-weight compounds (substrates, endogenous metabolites, drugs and xenobiotics) on the kinetic and equilibrium parameters of functionally significant binary protein-protein interactions (PPIs) is of both fundamental and clinical importance. The surface plasmon resonance (SPR) is the method of the first choice for studying PPIs. Earlier, SPR analysis revealed the modulating effect of steroidal substrates on the affinity of interactions between steroidogenic microsomal cytochromes P450 (CYP) and their redox partner (cytochrome b5). In this work, we have shown the suitability of the experimental approach for assessing the selective effect of the cofactor NADPH on the interaction between cytochromes CYP3A4 or CYP2E1 with NADPH-dependent P450 oxidoreductase (CPR).

Experiments have shown that the CYP3A4/CPR complex is not modulated by NADPH, while the dissociation rate of the CYP2E1/CPR complex in the presence of NADPH significantly decreased: the koff values in the absence and presence of NADPH were (3.6 ± 0.2) • 10-3 s-1 and (3.8 ± 0.2) • 10-4 s-1, respectively. Thus, in the presence of NADPH, an increase in the affinity of CYP2E1/CPR complex formation by approximately one order of magnitude was observed, while NADPH did not affect the kon value of this complex. Co-injection of NADPH at the CYP2E1/CPR complex preformed in the absence of NADPH had minor influence on the koff values (<10%). This suggests a stabilizing role of NADPH for the CYP2E1/CPR complex formation. Thus, the use of our approach made it possible to assess the effect of the main electron supplier for the microsomal cytochrome P450 monooxygenase system on the kinetic rate constants of CYP/CPR complexes.
Figure 1. SPR assessment of the NADPH effect on the kinetic parameters of protein-protein interaction using CYP2E1/CPR as an example: (A) - injection of 250 nM CYP2E1 via immobilized on the chip CPR protein in the absence (solid line) or presence of 100 µМ NADPH (dotted line). (B) - injection of the working buffer (solid line) or 100 µM of NADPH (dotted line) after the formation of CYP2E1/CPR complex in the absence of NADPH. All samples were injected for 5 min at a flow rate of 10 µl/min.
Figure 1. A scheme of the SPR analysis for assessment of the modulating effect of low-molecular weight compounds on the kinetics and affinity parameters of protein-protein interaction. Abbreviations: small compound (SC); protein-protein interaction (PPI); reference panel (different concentration of an analyte); control panel (different concentrations of a SC); target panel #1 (different concentrations of an analyte + first concentration of a SC) etc.

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Table 1. Association (kon) and dissociation (koff) rate constants and equilibrium dissociation constant (Kd) characterizing complex formation of cytochromes P450 with their redox partners in the absence/presence of the NADPH cofactor.

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Table 2. A typical experiment aimed at the detection of the modulating effect of a small compound (SC) on kinetics (affinity) of binary BBI.

FUNDING

This work was supported by the Russian Foundation for Basic Research (RFBR) (project № 18-04-00071).

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