Its mode of CYP11A1 inhibition is currently difficult to explain

Its mode of CYP11A1 inhibition is currently difficult to explain. and tizanidine. In studies of CYP46A1, desogestrel had no effect, and pemirolast and tizanidine had a STAT3-IN-3 slight stimulatory effect on enzyme activity. These compounds increased CYP11A1 activity by 161%, 125%, and 170%, respectively. Open in a separate windows Fig. 1 Effect of CYP46A1 inhibitors on activity of CYP11A1. STAT3-IN-3 Dashed and open bars correspond to CYP11A1 and CYP46A1, respectively. Data on the activity of CYP46A1 are taken from Mast et al., 2012, and shown for comparison. Enzyme assay was carried out as described under Section 2. The assay mixture contained PLs. The results are the mean S.D of three independent measurements. Drugs mentioned in Sections 3 and 4 are shown in strong. 3.2. Spectral changes in CYP11A1 elicited by inhibitors and activators The identified strong CYP11A1 inhibitors and all of the enzyme activators were then evaluated in the spectral binding assay. Of them, only 4 elicited significant spectral shifts in CYP11A1 (Fig. 2C4). These were two inhibitors (ketoconazole and posaconazole) and two activators (clobenpropit and dexmedetomidine). At saturating concentrations, ketoconazole and posaconazole shifted max in the CYP11A1 absolute spectrum from 417 nm to 422 nm (Fig. 2A and C), the same wavelength as observed in previous studies with amine-containing steroids that bind to the CYP11A1 active site and serve as the enzyme inhibitors by coordinating the P450 heme iron with their nitrogen atom (Linens et al., 1982; Sheets et al., 1983). Clobenpropit and dexmedetomidine also red-shifted the STAT3-IN-3 max of CYP11A1 (Fig. 3A, ?,4A),4A), but the position of the Soret peak was at a shorter wavelength (420C421nm). The difference spectra of CYP11A1 in the presence of ketoconazole, posaconazole, clobenpropit and dexmedetomidine were all comparable and of type II (Remmer et al., 1966; Schenkman et al., 1967) with the troughs at 412 nm and the peaks at 433C434 nm (Fig. 2B and D, ?,3B,3B, ?,4B).4B). Equilibrium binding constants were determined from the difference spectra (Table 1). The spectral Kd values were 1.0 M and 1.5 M for the inhibitors, and 7.0 Ntf3 M and 18 M for the activators. Open in a separate windows Fig. 2 Spectral analysis of CYP11A conversation with inhibitory drugs. The concentration of CYP11A1 was 0.4 M, and the buffer was 40 mM KPi, pH, 7.2, containing 1 mM EDTA. Numbers above or below the spectra indicate the wavelengths of absorption maxima or minima. A and C, absolute spectra; solid and dashed lines represent CYP11A1 spectrum in the absence and presence of ketoconazole (A) and posaconazole (B), respectively. Inhibitors concentrations were 15 M (ketoconazole) and 10 M (posaconazole), equal to 10 Kd of for the studied drug (1.5 M and 1.0 M, respectively) for substrate-free CYP11A1. B and D, difference spectra. Open in a separate window Fig. 3 Spectral analysis of CYP11A interaction with clobenpropit. Numbers above or below the spectra indicate the wavelengths of absorption maxima or minima. A, absolute spectra; black and gray lines represent the spectra of cholesterol-free (black) and cholesterol-bound (gray) CYP11A1 in the absence (solid line) and presence (dashed line) of clobenpropit. The concentration of CYP11A1 was 0.4 M; the concentrations of cholesterol and clobenpropit were 4 M and 150 M, respectively. These ligand concentrations are equal to 20 Kd of cholesterol (0.2 M) and 8 Kd of clobenpropit (18 M) for cholesterol-free CYP11A1. BCE, difference spectra of 0.4 M.