Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

Sheila J. Sadeghi et al
Preliminary electrochemical experiments of the fld-BMP fusion protein, free in solution
were carried out using a glassy carbon electrode [125]. The cyclic voltammograms of
both the fld-BMP fusion protein and BMP are shown in Figure 9. While no current was
observed for P450 BM3 enzyme on the bare glassy carbon electrode, both BMP and
fld-BMP show measurable redox activities. Furthermore, the fld-BMP fusion protein
interacts better with the electrode as measured by the larger current compared to that of
BMP. This current is further enhanced in the presence of neomycin, a positively
charged aminoglycoside which is believed to overcome the electrostatic repulsion
between the negatively charged fld and the negatively charged electrode surface [126].
3. Screening methods for P450 activity
After the crucial role of P450s in the determination of the pharmacological/
toxicological properties of drugs was known, the need to develop efficient systems for
the in vitro screening of drug-P450 interactions emerged. Thus in the recent years a
great amount of effort has been put into the generation of model systems for studying in
vitro the metabolism of pharmaceutical drugs in humans. Such model systems are
aiming at obtaining valid information for the metabolism of the drug candidates in vivo.
Additionally, it has been widely recognised that inter-individual differences in P450
content are capable of causing significant variation in pharmacokinetics, thus leading to
inaccurate estimation of the toxicological and pharmaceutical action of drug candidates.
As a result, the future of pharmacokinetics seems to be getting directed towards
individualised drug treatment [127], where the drug therapy to be followed will be
based on the genotype of the specific patient.
On the other hand the recent development of efficient non-rational protein engineering
techniques [ 13-14] generating large combinatorial protein libraries has provided us with
the capability to evolve protein function towards directions of choice. Strategies for
screening protein libraries have been reviewed by Zhao and Arnold [128]. We tried to
address both issues in our laboratory by developing a general assay for screening for
turnover of compounds of interest by NAD(P)H-dependent oxidoreductases.
3.1. ASSAY METHODS FOR P450-LINKED ACTIVITY
A rapid investigation of the in vitro drug metabolism has been facilitated in the recent
years by the development of high-throughput screening assays together with the
advances in the creation of automated miniaturised systems for liquid handling and
detection. The screens available can be categorised into
Assays aiming at the detection of P450 inhibitors using non-specific P450
substrates which are turned over into detectable metabolites (for example
fluorescent or radiolabelled) by the P450. Inhibition by a compound can thus
be detected by reduction (or abolishment) of the production of the detectable
metabolites [129-133].
Assays, using an instrumental chemical analysis method, able to distinguish
between the parent compound and its metabolites after turnover by the P450
[134-136].
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