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High-Throughput In Situ Parametric Studies of Cytochrome P450 Catalysis

  • Author(s): Traylor, Matthew John
  • Advisor(s): Clark, Douglas S
  • et al.
Abstract

Cytochrome P450 enzymes are the main route of first pass oxidative metabolism of prescribed drugs in humans. As such, they are of paramount importance to the pharmaceutical industry in the metabolic and toxicity screening of lead compounds. P450 enzymes function by generating a highly reactive ferryl-oxo species with two electrons supplied via NADPH. This reactive species is capable of a wide range of oxidative transformations, often on unreactive carbon centers. Consequently, P450 enzymes are of general interest to the biotechnology industry due to the variety of enantioselective oxidation reactions that they catalyze.

The effective utilization of P450 enzymes within both industries has yet to be realized. In the biotech industry, P450 transformations have not been adopted, except in whole cell systems, due to slow reaction rates and an inefficient use of the expensive cofactor, NADPH. The P450 reaction mechanism often yields a broad array of coupled and uncoupled products from a single substrate. While it is well known that reaction conditions can drastically affect the rate of P450 catalysis, their effects on regioselectivity and coupling are not well characterized. To investigate such effects, the CYP1A2 oxidation of 7-ethoxymethoxy-3-cyanocoumarin (EOMCC) was examined as a function of buffer type, buffer concentration, pH, and temperature. A high-throughput, optical method was developed to simultaneously measure the rate of substrate depletion, NADPH depletion, and generation of the O-dealkylated product. Increasing the phosphate buffer concentration and temperature increased both the NADPH and EOMCC depletion rates by 6-fold, whereas coupling was constant at 7.9% and the regioselectivity of O-dealkylation to other coupled pathways was constant at 21.7%. Varying the buffer type and pH increased NADPH depletion by 2.5-fold and EOMCC depletion by 3.5-fold; however, neither coupling nor regioselectivity was constant, with variations of 14.4% and 21.6%, respectively. Because the enzyme-substrate binding interaction is a primary determinant of both coupling and regioselectivity, it is reasonable to conclude that ionic strength, as varied by the buffer concentration, and temperature alter the rate without affecting binding while buffer type and pH alter both.

Although the large effect of ion type and concentration on cytochrome P450 catalysis is well documented, the full kinetic consequence of this effect has yet to be explored. We measured the CYP1A2 and CYP3A4 mediated O-dealkylation of alkoxycoumarins as a function of ion type and concentration for twelve salts. We found that greatest rate enhancement for both isoforms with potassium fluoride, yielding increases of 19 and 2.5 fold for CYP3A4 and CYP1A2, respectively. Moreover, we measured the kinetic parameters and noncompetitive intermolecular isotope effect for the O-dealkylation of 7-methoxy-3-cyanocoumarin by CYP1A2 as a function of potassium fluoride and P450-reductase content. We found the rate increases seen with potassium fluoride were primarily due to increases in kcat. Furthermore, the noncompetitive intermolecular isotope effect was large (~6) and constant for all conditions assayed. A simplified P450 mechanism was used to support the possibility that the large isotope effects seen in this and other P450 systems are due primarily to the large rate of uncoupling, which serves to unmask the intrinsic isotope effect.

In vitro metabolic stability measurements are a critical component of pre-clinical drug development in the pharmaceutical industry. Available measurement strategies rely on chromatography and mass spectrometry, which are expensive, labor-intensive, and low-throughput. Using a reaction engineering approach, we have developed a general method to measure metabolic stability by fluorescently quantifying cofactors in the mechanisms of cytochrome P450 enzymes. This method combines the accuracy and generality of chromatography with the ease and throughput of fluorescence.

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