26,27-Dehydrolanosterol and Cholesta-5,7,22,24-tetraenol are suicide substrates of sterol methyl Transferase in Acanthamoeba castellanii
Acanthamoeba castellanii (AC) is a pathogenic protozoan that causes blinding keratitis in humans. AC synthesizes C28 ergosterol while humans synthesize C27 cholesterol. The one carbon difference at C24 in the sterol structures of parasite and host is generated by sterol methyl transferase (SMT). In an effort to understand the mechanism of action of suicide inhibitors designed to inhibit sterol methylation that thereby prevents ergosterol biosynthesis and growth, 26,27-dehydrolanosterol (DHL) and cholesta-5,7,22,24-tetraenol (T4) were evaluated against cloned AcSMTs. During the course of the investigation, two AcSMTs were identified through bioinformatic and chemical analyses of the ergosterol biosynthetic pathway, one of them (24AcSMT) was shown to covert cycloartenol to 24(28)-methylenecycloartanol while the other one (28AcSMT) was shown to convert 24(28)-methylenelophenol to 4α-methyl 24β-ethylstigmasta-7,25(27)-dienol and other minor products. To determine the specificity of DHL and T4 as suicide inhibitors of 24AcSMT or 28AcSMT, a series of kinetic and chemical experiments were carried out against the cloned enzymes. Based on GC-MS analysis of enzyme generated methyl sterol diol formation, DHL inactivated 24AcSMT while T4 inactivated 28AcSMT. Based on steady-state kinetic analysis, DHL generated Ki values of 16µM against 24AcSMT and 40µM against 28AcSMT, while T4 generated Ki values of 18µM and 8µM respectively. Consistent with suicide behavior, DHL showed time-dependent inactivation properties against 24SMT exhibiting kinact of 0.04min-1 and the 3H-DHL intermediate-24SMT complex co-migrated with wild type Ac-24SMT on SDS-PAGE gel. The results show that DHL and T4 are potential therapeutic leads in the design of next generation drugs to treat amoeba infections.