Photoaffinity labeling of oxidosqualene cyclase and squalene cyclase by a benzophenone-containing inhibitor

A new orally active oxidosqualene:lanosterol cyclase (OSLC) inhibitor (Ro48-8071; Morand, O. H. et al. (1997) J. Lipid Res. 38, 373-390) showed potent noncompetitive inhibition of bacterial squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius (IC50 = 9.0 nM, K-I = 6.6 nM) and OSLC (IC50 = 40 nM, K-I = 22 nM for homogeneous rat liver OSLC). A tritium-labeled isotopomer (18.8 Ci/mmol) of this nonterpenoid inhibitor, which possesses a benzophenone (BP) photophore, was chemically synthesized as a photoaffinity label. Specific, efficient covalent modification of both OSLC and SHC enzymes was observed after UV irradiation at 360 nm. Labelling of both OSLC and SHC by [H-3]Ro48-8071 was competitively displaced by coincubation with a 1000-fold molar excess of 18-thia-2,3-oxidosqualene or the nonterpenoid inhibitor BIBX79. Displacement of labelling of OSLC was also achieved with the suicide substrate (3S)-29-methylidene-2,3-oxidosqualene. Thus, the nonsubstrate Ro48-8071 and both terpenoid and nonterpenoid inhibitors of these enzymes appear to share a common binding site.