INHIBITION OF 2,3-OXIDOSQUALENE CYCLASE AND STEROL BIOSYNTHESIS BY 10-AZASQUALENE AND 19-AZASQUALENE DERIVATIVES

The inhibition of 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7) (OSC) by new azasqualene derivatives, mimicking the proC-8 and proC-20 carbocationic high-energy intermediates of the cyclization of 2,3-oxidosqualene to lanosterol, was studied using pig liver microsomes, partially purified preparations of OSC, and yeast microsomes. The azasqualene derivatives tested were: 6E- and 6Z-10aza-10,11-dihydrosqualene-2,3-epoxide 17 and 18, 19-aza-18,19,22,23-tetrahydrosqualene-2,3-epoxide 19 and its corresponding N-oxide 20, and 19-aza-18,19,22,23-tetrahydrosqualene 21. The compounds 17 and 19 (i.e. the derivatives bearing the 2,3-epoxide ring and the same geometrical configuration as the OSC substrate) were effective inhibitors, as shown by the K-i obtained using partially purified OSC: 2.67 mu M and 2.14 mu M, respectively. Compound 18, having an incorrect configuration and the 19-aza derivative 21, lacking the 2,3-epoxide ring, were poor inhibitors, with IC50 of 44 mu M and 70 mu M, respectively. Compound 21 was a competitive inhibitor of OSC, whereas 17 and 19 were noncompetitive inhibitors, and showed a biphasic time-dependent inactivation of OSC, their apparent binding constants being 250 mu M and 213 mu M, respectively. The inhibition of sterol biosynthesis was studied using human hepatoma HepG2 cells. The incorporation of [C-14] acetate in the C-27 sterols was reduced by 50% by 0.55 mu M 17, 0.22 mu M 19, and 0.45 mu M 21, whereas 2 mu M 18 did not affect sterol biosynthesis. In the presence of 17, 19 and 21, only the intermediate metabolites 2,3-oxidosqualene and 2,3,22,23-dioxidosqualene accumulated, demonstrating a very specific inhibition of OSC.