Abbreviations: AZA, 25-azalanosterol; BSF, bloodstream form; DOX, doxycycline; ED, effective dose; FGM, full-growth medium; ITC, itraconazole; LDM, lipid-depleted medium; PCF, procyclic form; RRTc, relative retention time with cholesterol used as standard; SAM, S-adenosyl-lmethionine; SDM, sterol C14-demethylase; SMT, sterol C24-methyltransferase; Tb SDM, Trypanosoma brucei analyzed by RNAi silencing and inhibition of sterol C14-demethylase; Tb SMT, Trypanosoma brucei analyzed by RNAi silencing and inhibition of sterol C24-methyltransferase; TetR, tetracycline repressor .

SUMMARY Trypanosoma brucei, the causal agent for sleeping sickness, depends on ergosterol for growth. Herein, we describe the effects of a mechanism-based inhibitor, 26-fluorolanosterol (26FL), which converts in vivo to a fluorinated substrate of the sterol C24-methyltransferase essential for sterol methylation and function of ergosterol and missing from the human host. 26FL showed potent inhibition of ergosterol biosynthesis and growth of procyclic and bloodstream forms while it has no effect on cholesterol biosynthesis or growth of human epithelial kidney cells. During exposure of cloned TbSMT to 26-fluorocholesta-5,7,24-trienol, the enzyme is gradually killed as a consequence of the covalent binding of the intermediate C25 cation to the active site (kcat/kinact = 0.26 min-1/0.24 min-1; partition ratio of 1.08) whereas 26FL is non-productively bound. These results demonstrate that poisoning of ergosterol biosynthesis by a 26-fluorinated Δ24-sterol is a promising strategy for developing new treatment for trypanosomiasis.

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Objectives: Dieting often fails because weight loss triggers strong counter-regulatory biological responses such as increased hunger and hypometabolism that are thought to be critically dependent on the master fuel sensor in the mediobasal hypothalamus (MBH). Because prolonged starvation has been shown to increase AgRP and NPY, the expression level of these two orexigenic genes has been taken as an experimental readout for the presence or absence of hunger. Roux-en-Y gastric bypass (RYGB) surgery leads to a significant weight loss without inducing the associated hunger, indicating possible changes in hypothalamic neuropeptides and/or signaling. Our goal was to assess key genes in the MBH involved in regulating body weight, appetite, and inflammation/oxidative stress after RYGB surgery in mice. Methods: Obese mice on a high-fat diet were subjected to either sham or RYGB surgery, or caloric restriction to match the weight of RYGB group. Chow-fed mice without surgery served as an additional control group. After 2 or 12 weeks post-surgery, hypothalamic genes were analyzed by real-time qPCR. Results: During the rapid weight loss phase at 2 weeks after RYGB surgery, hypothalamic AgRP and NPY gene expression was not increased compared to mice with sham surgery, indicating that the mice are not hungry. In contrast, the same weight loss induced by caloric restriction promptly triggered increased AgRP and NPY expression. This differential effect of RYGB and caloric restriction was no longer observed during the weight maintenance phase at 12 weeks after surgery. A similar differential effect was observed for ObRb, but not for POMC and CART expression. Furthermore, RAGE and IBA-1, two markers for inflammation/oxidative stress, were significantly suppressed after RYGB compared to caloric restriction at 2 weeks post-surgery. Conclusions: These findings suggest that RYGB prevents the biologically adaptive hunger response triggered by undernutrition and weight loss, and suppresses weight loss-induced hypothalamic inflammation markers.

The tightly coupled nature of the electrophilic alkylation reaction sequence catalysed by 24-SMT (sterol C-24-methyltransferase) of land plants and algae can be distinguished by the formation of cationic intermediates that yield phyla-specific product profiles. C-24-methylation of the cycloartenol substrate by the recombinant Glycine max (soybean) 24-SMT proceeds to a single product 24(28)-methylenecycloartanol, whereas the 24-SMT from green algae converts cycloartenol into two products cyclolaudenol [∆(25(27))-olefin] and 24(28)-methylenecycloartanol [(∆24(28))-olefin]. Substrate analogues that differed in the steric-electronic features at either end of the molecule, 26-homocycloartenol or 3β-fluorolanostadiene, were converted by G. max SMT into a single 24(28)-methylene product. Alternatively, incubation of the allylic 26-fluoro cyclosteroid with G. max SMT afforded a bound intermediate that converted in favour of the ∆(25(27))-olefin product via the cyclolaudenol cation formed initially during the C-24-methylation reaction. A portion of the 26-fluorocycloartenol substrate was also intercepted by the enzyme and the corresponding hydrolysis product identified by GC-MS as 26-fluoro-25-hydroxy-24-methylcycloartanol. Finally, the 26-fluorocycloartenols are competitive inhibitors for the methylation of cycloartenol and 26-monofluorocycloartenol generated timedependent inactivation kinetics exhibiting a kinact value of 0.12 min(-1). The ability of soybean 24-SMT to generate a 25-hydroxy alkylated sterol and fluorinated ∆(25(27))-olefins is consistent with our hypothesis that (i) achieving the cyclolaudenyl cation intermediate by electrophilic alkylation of cycloartenol is significant to the overall reaction rate, and (ii) the evolution of variant sterol C-24-methylation patterns is driven by competing reaction channels that have switched in algae from formation of primarily ∆(25(27)) products that convert into ergosterol to, in land plants, formation of ∆(24(28)) products that convert into sitosterol.