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New iodoacetamide derivatives, containing a dodecyl or a squalenyl moiety, were synthesized. The effect of these new thiol-reacting molecules was studied on two mutants of Alicyclobacillus acidocaldarius squalene-hopene cyclase constructed especially for this purpose. In the quintuple mutant, all five cysteine residues of the enzyme are substituted with serine; in the sextuple mutant, this quintuple substitution is accompanied by the substitution of aspartate D376, located at the enzyme's active site, with a cysteine. N-Dodecyliodoacetamide had little activity toward either mutant, whereas N-squalenyliodoacetamide showed a stronger effect on the sextuple than on the quintuple mutant, as expected.

Various (1E,3E)- and (1Z,3E)-conjugated methylthio derivatives of oxidosqualene (OS) and conjugated and non-conjugated phenylthio derivatives of OS were obtained. These compounds, designed as inhibitors of pig liver and Saccharomyces cerevisiae 2,3-oxidosqualene-lanosterol cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene-hopene cyclase (SHC) (EC 5.4.99.-), contain the reactive function adjacent to carbons involved in the formation of the third and the fourth cycle during OS cyclization. All the new compounds are inhibitors of OSC and SHC, with various degrees of selectivity. The conjugated methylthio derivatives behaved as potent inhibitors of S. cerevisiae OSC, whereas most of the phenylthio derivatives were especially active toward SHC.

Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed. These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-). They contain one or two sulfur atoms in alpha-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site. Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity. The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S. cerevisiae OSC, with a concentration inhibiting 500% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent. These results could offer new insight for the design of antifungal drugs.

The inhibition of squalene-hopene cyclase (SHC) (E.C. 5.4.99.-), an enzyme of bacterial membranes catalyzing the formation of pentacyclic sterol-like triterpenes, was studied by using different classes of compounds originally developed as inhibitors of oxidosqualene cyclase (OSC) (E.C. 5.4.99.7), the enzyme of eukaryotes responsible for the formation of tetracyclic precursors of sterols. The mechanism of cyclization of squalene by SHC, beginning with a protonation of the 2,3 double bond by an acidic residue of the enzyme, followed by a series of electrophilic additions of the carbocationic intermediates to the double bonds, is similar to the mechanism of cyclization of 2,3-oxidosqualene by OSC. The inhibitors studied included: (i) analogs of the carbocationic intermediates formed during cyclization, such as aza-analogs of squalene and 2,3-oxidosqualene; (ii) affinity-labeling inhibitors bearing a methylidene reactive group; and (iii) vinyldioxidosqualenes and vinylsulfide derivatives of the substrates. Comparison of the results obtained with the two enzymes, SHC and OSC, showed that many of the most effective inhibitors of OSC were also able to inhibit SHC, while some derivatives acted as specific inhibitors. Differences could be easily explained on the basis of the different substrate specificity of the two enzymes.

19-Azasqualene-2,3-epoxide was more inhibitory than the corresponding N-oxide against 2,3-oxidosqualene cyclase (OSC) solubilized from Saccharomyces cerevisiae (IC50 7+/-2 and 25+/-5 microM, respectively). Both compounds showed a reversible, noncompetitive-type inhibition on solubilized OSC. Different inhibitory properties between the compounds were especially evident when measuring [14C]acetate incorporation into nonsaponifiable lipids extracted from treated cells. In cells treated with 19-azasqualene-2,3-epoxide at 30 microM, the radioactivity associated with the oxidosqualene fraction, which was negligible in the controls, rose to over 40% of the nonsaponifiable lipids, whereas it remained at a slightly appreciable level in cells treated with the N-oxide derivative under the same conditions. 19-Azasqualene-2,3-epoxide was also more effective than the N-oxide as a cell growth inhibitor (minimal concentration of compound needed to inhibit yeast growth: 45 and100 microM, respectively). The two inhibitors underwent different metabolic fates in the yeast: while 19-azasqualene-2,3-epoxide did not undergo any transformation, its N-oxide was actively reduced to the corresponding amine in whole and in "ultrasonically stimulated" cells. The N-oxide reductases responsible for this transformation appear to be largely confined within the microsomal fractions and require NADPH for their activity. A possible relationship between the inhibitory properties of the two compounds and their metabolic fates is discussed.

2,3-Oxidosqualene cyclases (OSC) are enzymes which convert 2,3-oxidosqualene (OS) into polycyclic triterpenoids such as lanosterol, cycloartenol, and alpha- and beta-amyrin. Our interest in the study of OSC is the development of new OSC inhibitors for potential use as hypocholesterolemic, antifungal, or phytotoxic drugs. In particular, we describe the biological activity and the mechanism of a series of acyclic azasqualene derivatives mimicking the C-2, C-8, and C-20 carbonium ions formed during OS cyclization. Some of these carbonium ion analogues are very promising as specific hypocholesterolemic agents. The toxicity, the biodistribution, and the pharmacokinetics of different azasqualene derivatives in mice are also presented. In order to obtain new, site-directed irreversible inhibitors of OSC, a series of squalene derivatives containing functional groups that can link covalently to an active-site thiol group was designed. Among these compounds, squalene maleimide was the most active toward mammalian OSC, whereas squalene Ellman behaved as an irreversible inhibitor of OSC from yeast.