Human Metabolites

An extraordinarily broad substrate range and very diverse reactions catalyzed by eukaryotic P450s make them attractive for pharmaceutical research. However, the low stability and activity of these enzymes hinder their application for drug synthesis. Implementation of more stable and active bacterial P450s, which are able to accept substrates of the mammalian counterparts, would open up a route to the production of new pharmaceuticals.

Wild-type P450BM-3 (fatty acid hydroxylase) does not metabolize any drug-like compounds, but it can be tuned by protein design and directed evolution into an enzyme which oxidizes compounds typical for human P450 monooxygenases. The R47L/F87V/L188Q mutant was shown to metabolize testosterone (Scheme 4.11), amodiaquine, dextromethorphan, acetaminophen, and 3,4-methylenedioxymethylamphetamine [87]. Reactions catalyzed by this mutant include not only hydroxylation, as in the case of testosterone, but also N-dealkylation of e.g. dextromethorphan, O-demethylenation of 3,4-methylenedioxymethylam-phetamine, and dehydrogenation of acetaminophen. In general the activity of R47L/F87V/L188Q for these compounds was very low, but in the case of 3,4-methylenedioxymethylamphetamine and acetaminophen it could be stimulated up to 70-fold by the addition of caffeine, a known activator of rat P450 3A2.

Testosterone Scheme 4.11

Several mutants of P450BM-3 obtained by directed evolution have been shown to oxidize propranolol (Scheme 4.12), a multi-function P-adrenergic blocker. A mutant "9C1," which has 14 amino acid substitutions, produced a very similar product pattern in a bioconversion of propranolol to the human CYP2D6 and CYP1A2 [88]. After further optimization by saturation mutagenesis several new mutants were identified, yielding different ratios of products. Among the produced metabolites, the ring-hydroxylated products are of particular importance as they have been demonstrated to be equipotent to propranolol as a P-receptor antagonist. Most of the substituted amino acids (R47, A74, A82, F87) of importance are located in the active site of the enzyme and had already been shown to affect the substrate range and selectivity.

Propanolol Scheme 4.12

The mutant 9-10A/F87A, constructed on the basis of the 9-10A evolved previously for alkane hydroxylation, was able to hydroxylate the a-position of the peptide group of buspirone. Buspirone (Scheme 4.13) as well as its human metabolite, 6-hydroxybuspirone, are anti-anxiety agents. Remarkable is the high enantio- and regioselectivity of the enzyme in this reaction. (R)-6-Hydroxybuspirone was the sole product formed with > 99.5% ee [89].

Testosterone Scheme 4.11

Propanolol Scheme 4.12

Scheme 4.13

Buspirone

Scheme 4.13

Eliminating Stress and Anxiety From Your Life

Eliminating Stress and Anxiety From Your Life

It seems like you hear it all the time from nearly every one you know I'm SO stressed out!? Pressures abound in this world today. Those pressures cause stress and anxiety, and often we are ill-equipped to deal with those stressors that trigger anxiety and other feelings that can make us sick. Literally, sick.

Get My Free Ebook


Post a comment