Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design

Abstract

Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C₂₈- and C₂₉-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC₅₀ values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding ∼K_i 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.

Keywords: Acanthamoeba; anti-amoeba drugs; ergosterol biosynthesis inhibitors; phytosterol biosynthesis; sterol C24-methyltransferase.

Fig. 1.

Metabolic pathways for the conversion of phyla-specific protosterol to C₂₈-ergosterol, C₂₉-7-dehydroporiferasterol and C₂₇-cholesterol in pathogenic protozoa and human host; SMT, sterol methyltransferase.

Fig. 2.

Comparison of amino acid sequences of sterol methyltransferases of Acanthamoeba castellanii with related enzymes across kingdoms. Cr, Chlamydomonas reinhardtii (green alga); Ac, Acanthamoeba castellanii (amoeba protozoa); Gm, Glycine max (land plant); Tb, Trypanosoma brucei (kinetoplastid protozoa); Tc, Trypanosoma cruzi (kinetoplastid protozoa); and Sc, Saccharomyces cerevisiae (fungus). Sterol methylation carried out as the first (Δ²⁴⁽²⁵⁾-substrate) C₁-transfer reaction and second (Δ²⁴⁽²⁸⁾-substrate) C₁-transfer reaction is correlated to the corresponding 24-SMT (=SMT1) and 28-SMT (=SMT2) sequences detected in the Genebank. Substrate binding segments established in references , for sterol (Regions I, III and IV) and SAM (Region II) are highlighted by black and blue boxes, respectively. Conserved residues are in red.

Fig. 3.

An amino acid-based phylogenetic tree of selected SMT enzymes across kingdoms associated with their substrate preference for a Δ²⁴⁽25⁾ - sterol or Δ²⁴⁽28⁾ -sterol. Putative A. castellanii 24-SMT and 28-SMT are in green labeled SMT1 and SMT2, respectively, as they are identified in the Genebank. The tree was constructed as described in Materials and Methods; substitution per site is labeled on the branch.

Fig. 4.

Expression of A. castellani 24-SMT (SMT1) and 28-SMT (SMT2) in E. coli. AcSMT1 and AcSMT2 genes were cloned in pET11a expression plasmid as described in the Materials and Methods and transformed in competent E. coli BL21(DE3) cells. Gel was stained by Coomassie Brilliant Blue (0.2%) to visualize the protein bands. Molecular weight markers are shown. The position of the recombinant protein is indicated by an arrow. IPTG, isopropyl thio-galactoside.

Fig. 5.

GC/MS analysis of Ac24-SMT generation of single product and Ac28-SMT generation of multiple products. A: cycloartenol (pk-1, substrate) conversion to 24(28)-methylene cycloartanol or D₂-24(28)-methylenecyloartanol (pk-2) by 24-SMT. B: mass spectra of products for SAM (top) and [²H₃-methyl]SAM (bottom) incubations with cycloartenol yielding pk-2, top. C: lanosterol (pk-1, substrate) conversion to eburicol (pk-2) by 24-SMT. D: mass spectra of products for SAM (top) and [²H₃-methyl]SAM incubations with lanosterol yielding pk-2, (D). E: 24(28)-methylenelophenol (pk-1, substrate) conversion to 24β-ethyl 4α-methyl stigmast-7, 25(27)-dienol (pk-2), 4α-methyl stigmast-7, 24(28)E-ethylidene dienol (pk-3) and 4α-methyl stigmasta 7, 24(28)Z-ethylidine dienol (pk-4). F: mass spectrum of sterol in pk-2 (E); inset is the olefinic region in ¹HNMR of the sample recovered from HPLC and containing trace 24(28)Z-ethylidene isomer. G: mass spectrum of sterol in pk-2. H: mass spectrum of sterol in pk-4. Incubations were carried out overnight with sterol substrate and SAM at saturation as described in Materials and Methods.

Fig. 6.

Product ratios generated by Ac24-SMT and Ac28-SMT incubated with either a Δ²⁴⁽25⁾ - or Δ²⁴⁽28⁾ -sterol substrate. The product ratios are normalized to cycloartenol at ∼70% conversion rates. Sterols are: (A), cycloartenol, (B), lanosterol, (C), 31-norlanosterol, (D), 14α-methylzymosterol, (E), zymosterol, (F), cholesta-5,7,24-trienol, (G) 24(28)-methylenecycloartanol, (H) eburicol, (I), obtusifoliol, (J), cycloeucalenol, (K), 24(28)-methylenelophenol, (L), fecosterol and (M), episterol.

Fig. 7.

Sterol methylation pathways for the conversion of Δ²⁴⁽²⁵⁾ - or Δ²⁴⁽²⁸⁾ -substrate to Δ²⁴⁽²⁸⁾ - or Δ²⁵⁽27⁾ -olefin products, indicating possible hydride shift and deprotonation mechanisms along with proposed site of inhibition by 24(R,S),25-epiminolanosterol (I) and mechanism of enzyme inactivation by 26,27-dehydrolanosterol (II).

Fig. 8.

Inhibition of Ac24-SMT and Ac28-SMT by 24(R,S),25-epiminolanosterol yielding IC₅₀ values of 44 nM ± 4 nM and 20 nM ± 2 nM, respectively; K_i values of steroidal inhibitor were determined from the corresponding IC₅₀ value as detailed in Materials and Methods. Normalized activity is related to control incubations for 100% activity. Incubations were carried out under initial velocity conditions of 45 min with sterol (100 µM) and [³H₃-methyl]SAM (150 µM). For control, ³H-methyl product afforded ∼1 × 10⁶ dpm for substrate cycloartenol or 5 × 10⁵ dpm for substrate 24(28)methylene lophenol whereas assays without sterol yield ∼500 dpm. Error bars represent ± SEM of three independent experiments.

Fig. 9.

Kinetic and chemical analysis of Ac24-SMT activity from incubation with 26,27-dehydrolanosterol (DHL). A: K_m curve of DHL paired with saturating levels of 150 µM SAM assayed under initial velocity conditions described in Materials and Methods. B: GC/MS analysis of enzyme-generated products recovered from nonsaponifiable lipid fraction of assay with saturating cosubstrates at 2.4 mg total protein. C: Inactivation of 24-SMT with the mechanism-based inhibitor in the concentration range and scheduled time of incubation as shown; inset: plot of 1/k_inact plotted as T_1/2 (min) versus 1/inhibitor. Specific labeling of Ac24-SMT with DHL. D: The Coomassie-stained SDS-PAGE gel of the partially pure cloned enzyme referenced to Bio-Rad ladder 20 to 100 kDa aligned next to the gel of DHL C26-methyl metabolite-complexed with 24-SMT. The position of 24-SMT is shown by predicted molecular mass associated with the 40 kDa marker.

Fig. 10.

Differential effect of steroidal inhibitors and reference voriconazole on Ac trophozoite growth. DHL, 26,27-dehydrolanosteol, El, 24(R,S),25-epiminolanosterol, VOR, voriconazole. The SEMs are less than 5% of three independent experiments.

Scheme 1.

Kinetic schemes for AcSMT inhibition by substrate analogs. In 1, binding of enzyme (E) and Δ²⁴-sterol substrate (S) followed by enzyme catalysis with formation of methyl product (P). In 2, inhibitor (I) binds to enzyme followed by activation via methylation (E_ˑI*) and this intermediate can partition along path b and dissociate from the active site (E + P) or enter into path a and irreversibly bind to the enzyme (E−I*).