Creation of mutant corticosteroid binding globulin: One step closer to a diagnostic device

Abstract

Much is known about how traditional and alternative medicines interact separately. However, there is very little knowledge of how the usage of alternative medicine affects the metabolism of traditional medicines when they are used together. This is especially important because of the amount of people who take alternative medicine in addition to antiretroviral drugs. The practice of using traditional and alternative medicines may induce cytochrome p450 enzymes, which metabolize traditional medicines used to treat HIV and AIDS. Cortisol is a steroid hormone produced by the body at a fairly constant rate. It is metabolized to 6OH-cortisol through the P450 enzyme pathway. As such, the ratio of hydroxycortisol to cortisol is an indirect indicator of the P450 enzyme activity. The ultimate goal of this project is to create a simple diagnostic device that will aid in assessing whether the usage of traditional medicine in combination with alternative medicine induces CYP3A4, to dangerous levels. The diagnostic device will detect the levels of 6OH-cortisol with a mutant version of the protein corticosteroid binding globulin (CBG), that preferentially binds to the 6OH-cortisol complex. The first step in this process was to create the mutant version of CBG. The mutation was found to have the closest binding pocket with a change in the asparagine 17 site to a valine, modeled by several computer programs. PCR was then performed with primers that contained the mutation and the wild type CBG, thus creating the CBG containing the mutation. A fluorescence assay is to be performed to determine how well the mutant CBG actually binds to the 6OH-cortisol. The results of the assay are in progress.