PIETER SWART, ETIENNE P. DE VILLIERS, AMANDA C. SWART, KIRSTEN J. VAN DER h4ERWE and PENELOPE C. TODRES. Department of Biochemistry ...
Biochemical SocietyTransactions ( 1 993) 21 41 3 s The interaction o f biogenic amines with adrenal cytochrome P450-dependent enzymes. PIETER SWART, ETIENNE P. DE VILLIERS, AMANDA C. SWART, KIRSTEN J. VAN DER h4ERWE and PENELOPE C. TODRES. Department of Biochemistry and Institute for Biotechnology, University of Stellenbosch, Stellenbosch, 7600, South Africa. The shrub Salsola tirberculatiformis Botsch contains labile compounds which interact with sheep adrenal cytochrome P45Ocll (P450cll) [ I ] Using P45Ocl1 as an assay for biological activity, we isolated an extremely labile active substance, S2, which interacts with P450cll and inhibits the binding of the natural substrates, cortisol and corticosterone, to the active site of the enzyme. In addition S2 binds to P450cll in the absence of substrate and elicits a type I1 spectral response. It was subsequently shown with fast atom bombardment mass spectrometry (FAB-MS) that S2 decomposes to synephrine (I) in an acidic medium (pH 1) [ 2 ] The structural relationship of synephrine to biogenic amines like adrenaline (11). noradrenaline (III), octopamine (IV) and
Table 1. Influence of 111, IV and aminonlutethimide on P45Oscc Compound
Concentration (pM)
%Inhibition
111
3.65
27
111
7.3
60
IV IV aminoglutethimide
3.65 7.3
13 31
107
50
Table 2. Influence of I. IV and V on the activitv of P 4 5 0 c 1 7 d P45Oc21 exuressed in COS 1 cells Each value represents the mean + S E.M. for three assays Inhibitor (% of control) P450c2 1 P45Ocl7 ComDound Proeesterone Proaesterone Premenolone (100 PM) I
(1I.w 45rt3
IV
25+2
V
3535
(1 PM) 55k8 60f9 40+7
(1 PM) 4535
35+7 0
(0 synephrine: R,=H; R;CH, (11) adrenaline: R,-OH; R,=CH, (Ill) noradirMUne: RI=OH; %=H
(V) mtanephrine: R,-OCH,; %-CH,
metanephrine (V) prompted an investigation of the interaction of these biogenic amines with P45Ocll and the other cytochrome P450-dependent enzymes in the adrenal cortex namely; P45Oscc, P450c21andP450c17 P450cll and P45Oscc were isolated from sheep adrenals as previously described [3]. The influence of compounds I-V, on the spectral properties of P450cl1, was investigated as described in [ Z ] . Deoxycorticosterone (3.25 pM) was used as substrate. P45Oscc activity was assayed as follows: P45Oscc (0.54 pM), adrenodoxin reductase/adrenodoxin (35 units), potassium phosphate buffer (25 mM, pH 7.2; Tween 20 0,45% v/v), DMF (1.88% v/v) and cholesterol and ''C-cholesterol, solubilized in Tween 20, were combined and preincubated for 5 minutes under O2before initiating the reaction with NADPH (0.4 mM). The total incubation volume was 200 PI and the final mixture contained 0,3% Tween 20. The reaction was terminated after 5 minutes by the addition of methanol (200~1)and kept at 0°C before extraction of the steroids with three 200 pI aliquots of CHzClz Steroid extracts were analysed as previously described [4] Inhibitors were added to the reaction mixture together with the cholesterol at concentrations indicated in table I . The results of these experiments are summarised in table 1 To determine the influence of the biogenic amines on the activities of bovine P450c17 and P450c21 the enzymes were expressed in COS 1 cells and assayed as described previously [5] These results are summarked in table 2 Spectral studies with P45Ocll and the test compounds I-V showed that 111 and IV were the most active inhibitors of the enzyme Deoxycorticosterone binding was inhibited by 50% at concentrations of 3 5 and 2 5 pM respectively. Both compounds interacted directly with the active site of P450cl1 eliciting a type I1 spectral response The Ks values (spectral binding constant) for 111 and IV were 0 80 mM and 0 29 mM respectively Compounds I, I1 and V were considerably less active than I11 and IV in inhibiting substrate binding to P45Ocll and could not induce any spectral changes in the enzyme. Compounds 111 and IV were the only two substances that significantly inhibited the activity of P45Oscc (table I), while the activity of the microsomal enzymes P45Oc17
and P45Oc21, expressed in COS 1 cells, was affected only by I, IV and V (table 2). It is interesting to note that only I1 and IV significantly affected the adrenal mitochondria1 hemoproteins, P45Ocll and P45Oscc Of the five substances investigated only I1 and IV have free amino groups and the type I1 spectral response elicited in P450cl1 indicates interaction of this functional group with the iron in the active site of the enzyme. From the concentrations needed for 50% inhibition and the Ks values it can be deduced that IV is bound by P450cll with greater affinity than 111. In the P450scc-assay, however, 111 was a significantly better inhibitor of pregnenolone biosynthesis than IV. The interpretation of this finding is difficult as both enzymes were not assayed in the same manner. Both 111 and IV were significantly stronger inhibitors of P450scc-activity than aminoglutethimide. These results show that, for the mitochondrial enzymes, biogenic amines with free amino groups appear to be better inhibitors than those with N-methylated amino groups. The activity of the expressed microsomal adrenal hemoproteins in COS 1 cells, were significantly affected only by I, IV and V Progesterone metabolism by P450c17 and P450c21, was affected by all three substances to more or less the same extent. The metabolism of pregnenolone by P45Ocl7 was, however, not influenced by V, indicating a substrate selective inhibition of P450c 17-activity The instability of isolated P450-dependent enzymes of the adrenal cortex makes it difficult to assay these enzymes under identical conditions and a direct comparison of the inhibitory effect of the different biogenic amines is therefor not possible. These results do, however, indicate that substances structurally related to I-V can be modified to produce more specific and selective inhibitors of adrenal cytochromes P450. 1 . Swart, P., van der Menve, K.J., Swart, A.C., Todres, P.C. & J-H S. Hofmeyr. (1993) Planta Medica (In press). 2. Fourie, L., van der Menve, K.J., Swart, P & de Kock, S S Analytical Chimica Acta 279, 163-166 (1993). 3 . Suhara, K., Gomi, T., Sato, H., Itagaki, E., Takemori, S. & Katagiri, M (1978) Arch. Biochem. Biophys. 190, 290-299. 4. Hanukogklu, I & Jefcoate, C.R (1980) J. Chromatogr. 190, 256-262. 5. Swart P., Estabrook R.W., Mason J.I.& Waterman M.R. (1989) Biochem.. SOC.Trans. 17, 1025-1026.
