The sterol substrate analog 25-thialanosterol and its corresponding sulfonium salt were evaluated for their ability to serve as antifungal agents and to inhibit sterol methyltransferase (SMT) activity in Candida albicans. Both compounds inhibited cell proliferation, were fungistatic, interrupted the yeast-like-form to germ-tube-form transition, and resulted in the accumulation of zymosterol and related delta24-sterols concurrent with a decrease in ergosterol, as was expected for the specific inhibition of SMT activity. Feedback on sterol synthesis was evidenced by elevated levels of cellular sterols in treated vs. control cultures. However, neither farnesol nor squalene accumulated in significant amounts in treated cultures, suggesting that carbon flux is channeled from the isoprenoid pathway to the sterol pathway with minor interruption or redirection until blockage at the C-methylation step. Activity assays using solubilized C. albicans SMT confirmed the inhibitors impair SMT action. Kinetic analysis indicated that 25-thialanosterol inhibited SMT with the properties of a time-dependent mechanism-based inactivator Ki of 5 microM and apparent kinact of 0.013 min(-1), whereas the corresponding sulfonium salt was a reversible-type transition state analog exhibiting a Ki of 20 nM. The results are interpreted to imply changes in ergosterol homeostasis as influenced by SMT activity can control growth and the morphological transition in C. albicans, possibly affecting disease development.

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The sterol composition of Pneumocystis carinii, an opportunistic pathogen responsible for life-threatening pneumonia in immunocompromised patients, was determined. Our purpose was to identify pathway-specific enzymes to impair using sterol biosynthesis inhibitors. Prior to this study, cholesterol 15 (ca. 80% of total sterols), lanosterol 1, and several phytosterols common to plants (sitosterol 31, 24alpha-ethyl and campesterol, 24alpha-methyl 30) were demonstrated in the fungus. In this investigation, we isolated all the previous sterols and many new compounds from P. carinii by culturing the microorganism in steroid-immunosuppressed rats. Thirty-one sterols were identified from the fungus (total sterol = 100 fg/cell), and seven sterols were identified from rat chow. Unusual sterols in the fungus not present in the diet included, 24(28)-methylenelanosterol 2; 24(28)E-ethylidene lanosterol 3; 24(28)Z-ethylidene lanosterol 4; 24beta-ethyllanosta-25(27)-dienol 5; 24beta-ethylcholest-7-enol 6; 24beta-ethylcholesterol 7; 24beta,-ethylcholesta-5,25(27)-dienol 8; 24-methyllanosta-7-enol 9; 24-methyldesmosterol 10; 24(28)-methylenecholest-7-enol 11; 24beta-methylcholest-7-enol 12; and 24beta-methylcholesterol 13. The structural relationships of the 24-alkyl groups in the sterol side chain were demonstrated chromatographically relative to authentic specimens, by MS and high-resolution 1H NMR. The hypothetical order of these compounds poses multiple phytosterol pathways that diverge from a common intermediate to generate 24beta-methyl sterols: route 1, 1 --> 2 --> 11 --> 12 --> 13; route 2, 1 --> 2 --> 9 --> 10 --> 13; or 24beta-ethyl sterols: route 3, 1 --> 2 --> 4 --> 6 --> 7; route 4, 1 --> 2 --> 5 --> 8 --> 7. Formation of 3 is considered to form an interrupted sterol pathway. Taken together, operation of distinct sterol methyl transferase (SMT) pathways that generate 24beta-alkyl sterols in P. carinii with no counterpart in human biochemistry suggests a close taxonomic affinity with fungi and provides a basis for mechanism-based inactivation of SMT enzyme to treat Pneumocystis pneumonia.

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