Comparison of the substrate specificities of human liver cytochrome p450s 2C9 and 2C18: Application to the design of a specific substrate of CYP2C18

A series of 2-aroylthiophenes derived from tienilic acid by replacement of its OCH2COOH substituent with groups bearing various functions have been sy nthesized and studied as possible substrates of recombinant human liver cyt ochrome P450s 2C9 and 2C18 expressed in yeast. Whereas only compounds beari ng a negative charge acted as substrates of CYP 2C9 and were hydroxylated a t position 5 of their thiophene ring at a significant rate, many neutral 2- aroylthiophenes were 5-hydroxylated by CYP 2C18 with k(cat) values of >2 mi n(-1). Among the various compounds that were studied, those bearing an alco hol function were the best CYP 2C18 substrates. One of them, compound 3, wh ich bears a terminal O(CH2)(3)OH function, appeared to be a particularly go od substrate of CYP 2C18. It was regioselectively hydroxylated by CYP 2C18 at position 5 of its thiophene ring with a K-M value of 9 +/- 1 mu M and a k(cat) value of 125 +/- 25 min(-1), which are the highest described so far for a CYP 2C. A comparison of the oxidations of 3, by yeast-expressed CYP 1 A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5, showed that only CYP 2C8, 2C18, and 2C19 were able to catalyze the 5-hydroxylation of 3. Howeve r, the catalytic efficiency of CYP 2C18 for that reaction was considerably higher (k(cat)/K-M value being 3-4 orders of magnitude larger than those fo und for CYP 2C8 and 2C19). Several human P450s exhibited small activities f ur the oxidative O-dealkylation of 3. The four recombinant CYP 2Cs were the best catalysts for that reaction (k(cat) between 1 and 5 min(-1)) when com pared to all the P450s that were tested, even though it is a minor reaction in the case of CYP 2C18. All these results show that compound 3 is a new, selective, and highly efficient substrate for CYP 2C18 that should be usefu l for the study of this P450 in various organs and tissues. They also sugge st some key differences between the active sites of CYP 2C9 and CYP 2C18 fu r substrate recognition.