Azasterol inhibition of .DELTA.24-sterol methyltransferase in Saccharomyces cerevisiae

Abstract

The inhibition of the delta 24-sterol methyltransferase (24-SMT) of Saccharomyces cerevisiae by side-chain azasterols is related to their nuclear skeleton and side chain nitrogen position. Inhibitory power [I50 (microM)] was found to be in the order of 25-azacholesterol hydrochloride salt (0.05) greater than 25-aza-24,25-dihydrozymosterol (0.08) greater than 25-azacholesterol approximately equal to 25-azacholestanol (0.14) greater than (20R)- and (20S)-22,25-diazacholesterol (0.18) greater than 24-azacholesterol (0.22) greater than 25-aza-24,25-dihydrolanosterol (1.14) greater than 23-azacholesterol (4.8). In the presence of azasterols, S. cerevisiae produces increased amounts of zymosterol, decreased amounts of ergosterol and ergostatetraenol, and the new metabolites cholesta-7,24-dienol, cholesta-5,7,24-trienol, and cholesta-5,7,22,24-tetraenol. Kinetic inhibition studies with partially purified 24-SMT and several azasterols suggest the azasterols act uncompetitively with respect to zymosterol and are competitive inhibitors with respect to S-adenosyl-L-methionine (SAM). These results are consistent with at least two kinetic mechanisms. One excludes competition of azasterol and zymosterol for the same site, whereas a second could involve a ping-pong mechanism in which 24-SMT is methylated by SAM and the methylated enzyme reacts with sterol substrate.

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth

“…6) ( 4, 44 ). Cell growth of wild-type PCF in FGM at day 6 was inhibited in a dose-dependent manner of ED 50 values from 1 to 3 M ; there was no signifi cant difference in the ED 50 …”

“…Tb SMT inhibitors and ITC were added to PCF or BSF cells from stock solutions in dimethyl sulphoxide, such that the organic solvent was less than 1% (v/v) of total culture volume. Five different concentrations of each compound were tested in triplicate between 0 (control) and 10 M; the 50% growth inhibition reported as the IC 50 relative to the growth of control. Cell densities were determined using a Neubauer hemocytometer counter.…”

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

“…In work with the yeast SMT, 25-azalanosterol was discovered to bind to the active center with a K d value of 4 µM similar to either the K d values of zymosterol or AdoMet at 4 µM [20]. The expectation for tight binding was borne out by the results of the 24-and 25-heteroatom substituted sterols assayed with SMT1 and SMT2, the carbocation mimic was bound to the Michaelis complex many orders more tightly than the sterol (or AdoMet) substrate generating a K m /K i ratio of approximately 1000 [16,23,38,[40][41][42][43][44][45][46]. The efficacy of 25-azacycloartenol, a substrate mimic, and solasodine have been compared in vitro and in vivo with the pathogenic yeastlike microbe P. wickerhamii which synthesizes ergosterol by the cycloartenol-cyclolaudenol route shown in Scheme 1.…”

“…The overall methyl transfer reaction catalyzed by SMT is proposed to proceed through a nucleophilic attack by the π-electrons of the Although the catalysis involves a conformationally flexible sterol substrate and very reactive ionic intermediates, the reaction progress is efficiently channeled through a coupled methylationdeprotonation step to produce single or sets of products with complete structural and stereochemical control (Scheme 3). The kinetics of the SMT catalyzed reaction involving two substrates have been hypothesized to proceed by one of two types of limiting mechanisms in which either a ternary or binary (ping pong) complex is formed [28,38]. These two mechanisms can be readily distinguished by steady-state kinetic velocities since they produce different kinetic patterns.…”

“…The compounds shown in Scheme 4 have been tested and found to be noncompetitive inhibitors against the native substrate of the SMT [22,23,28,39]. In spite of numerous side chain modifications [38,[40][41][42][43][44][45][46][47], only a limited number of structural variants exhibit binding affinities to the enzyme active site that are significantly greater than those of the native substrate.…”