S -Adenosyl- l -Methionine Inhibitors Δ ²⁴ -Sterol Methyltransferase and Δ ²⁴⁽²⁸⁾ -Sterol Methylreductase as Possible Agents against Paracoccidioides brasiliensis

Abstract

We studied the antiproliferative effects of three azasterol analogs [piperidyl-2-yl-5␣-pregnan-3␤,20(R)-diol (AZA-1), 22-piperidin-2-yl-pregnan-22(S),3␤-diol (AZA-2), and 22-piperidin-3-yl-pregnan-22(S),3␤-diol (AZA-3)] and their effects on the lipid composition of the pathogenic yeastlike phase of the dimorphic fungus Paracoccidioides brasiliensis. Inhibition was 100% for AZA-1 at 5 M, 62% for AZA-2 at 10 M, and 100% for AZA-3 at 0.5 M. The analogs inhibited different stages of the sterol biosynthesis pathway.Membrane sterol biosynthesis is one of the few areas of difference in primary metabolism between mammals and other eukaryotes, such as plants, fungi, and protozoa. While mammals synthesize C 27 cholestane-based members of the steroid family, pathogenic fungi, protozoa, and plants require the presence of endogenous sterols (typically ergosterol and 24-alkyl analogs) which act as essential growth factors for these organisms (4, 12). The enzyme responsible for the addition of these alkyl groups to carbon C-24 and for the regulation of carbon flow in the sterol pathway is the ⌬ 24 -sterol methyltransferase (SMT) (6). The critical role of this enzyme has stimulated considerable interest in the rational design of SMT inhibitors for potential clinical or agrochemical use as antifungal agents (1, 2, 12). Several sterol analogues behave as antiproliferative agents against fungi, yeast, protozoa, and plants in vitro (1,2,8,11,12). One sterol analogue, 20-piperidyl-2-yl-5␣-pregnan-3␤,20(R)-diol (AZA-1) has been reported to be an SMT inhibitor in the protozoan species Trypanosoma cruzi, Leishmania donovani, and the fungus Pneumocystis carinii (5,11,12). The antiproliferative effect of AZA-1 against these organisms coincided with the depletion of 24-alkyl-sterols and their complete replacement by ⌬ 24 -cholesta-type sterols. Recently, Atencio et al. (1) studied the molecular parameters of AZA-1 and its N-methyl derivative and established some structureactivity correlation. The information obtained from these compounds and the steric-electric plug model proposed by Nes (6) has allowed the design and synthesis of two new azasterols, 22-piperidin-2-yl-pregnan-22(S),3␤-diol (AZA-2) and 22-piperidin-3-yl-pregnan-22(S),3␤-diol (AZA-3) (Fig. 1) (Visbal et al., submitted).To explore the potential use of these new drugs, we studied the effects of azasterol analogs AZA-1, AZA-2, and AZA-3 on growth and sterol profile in the pathogenic yeastlike (Y) phase of Paracoccidioides brasiliensis. This fungus is a thermally dimorphic fungus, the causative agent of paracoccidioidomycosis, a prevalent human systemic mycosis in Latin America where it is geographically constrained. P. brasiliensis is sensitive to sterol biosynthesis inhibitors, such as ketoconazole, itraconazole, and saperconazole (9), and is also affected by ajoene, a derivative of allicin, extracted from garlic (8). In the search for new antifungal agents, we chose to test AZA-1, AZA-2, and AZA-3 in P. brasiliensis Y phase.Experiments were performed as previously reported...

Paracoccidioides brasiliensis: chemical and molecular tools for research on cell walls, antifungals, diagnosis, taxonomy

“…AZA-1 (0.1-5 lM) inhibited P. brasiliensis growth in a dose-dependent fashion, reaching 100% growth arrest at the latter concentration and above [60], a result that is similar to those previously reported for parasites (Trypanosoma cruzi, Leishmania donovani) [61] and fungi (Pneumocystis carinii) [62]. AZA-2, instead, was only able to inhibit 60% growth at the highest concentration used in these experiments (10 lM), while AZA-3 was the most powerful drug, since a concentration of 0.5 lM was able to completely inhibit fungal growth in a fungicydal manner [60].…”

“…While mammals synthesize C 27 cholestane-based members of the steroid family, pathogenic fungi, protozoa and plants require the presence of endogenous sterols C 28 -C 29 (typically ergosterol and 24-alkyl analogs) which act as essential growth factors for these organisms. The enzyme responsible for the addition of these alkyl groups to carbon C-24 and for the regulation of carbon flow in the sterol pathway is the D (24) -sterol methyl transferase (SMT) [59,60]. With this in mind, new compounds were synthesized.…”

“…Three of them were azasterol derivatives, i.e., 20-piperidyl-2-yl5a-pregnan-3b, 20(R)-diol (22,26-azasterol or AZA-1), 22-piperidin-2-yl-pregnan-22(S),3b-diol (AZA2) and 22-piperidin-3-yl-pregnan-22(S),3b-diol (AZA3) (Fig. 3a) [60]; additionally four sterol hydrazones, namely, 20-hydrazone-imidazolin-2-yl-5a-pregnan3b-ol (H1), 20-hydrazone-pyridin-2-yl-5a-pregnan-3b-ol (H2), 22-hydrazone-imidazolin-2-yl-chol-5-ene-3b-ol (H3) and 22-hydrazone-pyridin-2-yl-chol-5-ene3b-ol (H4) were also tested [Visbal et al manuscript under preparation]. AZA-1 (0.1-5 lM) inhibited P. brasiliensis growth in a dose-dependent fashion, reaching 100% growth arrest at the latter concentration and above [60], a result that is similar to those previously reported for parasites (Trypanosoma cruzi, Leishmania donovani) [61] and fungi (Pneumocystis carinii) [62].…”

“…3a) [60]; additionally four sterol hydrazones, namely, 20-hydrazone-imidazolin-2-yl-5a-pregnan3b-ol (H1), 20-hydrazone-pyridin-2-yl-5a-pregnan-3b-ol (H2), 22-hydrazone-imidazolin-2-yl-chol-5-ene-3b-ol (H3) and 22-hydrazone-pyridin-2-yl-chol-5-ene3b-ol (H4) were also tested [Visbal et al manuscript under preparation]. AZA-1 (0.1-5 lM) inhibited P. brasiliensis growth in a dose-dependent fashion, reaching 100% growth arrest at the latter concentration and above [60], a result that is similar to those previously reported for parasites (Trypanosoma cruzi, Leishmania donovani) [61] and fungi (Pneumocystis carinii) [62]. AZA-2, instead, was only able to inhibit 60% growth at the highest concentration used in these experiments (10 lM), while AZA-3 was the most powerful drug, since a concentration of 0.5 lM was able to completely inhibit fungal growth in a fungicydal manner [60].…”

“…In order to explore their possible mechanism(s) of action, a detailed analysis of the sterol composition of P. brasiliensis (Y phase) grown in the absence or presence of subinhibitory concentrations of the three azasterols analogs (1 lM (AZA-1), 2 lM (AZA-2), and 0.3 lM (AZA-3)), was carried out (Table 1) [60]. In control cells, main sterols were brassicasterol (G) (69.1%), ergosterol (F) (26.8%) and lanosterol (A) (4.1%), as reported before [63].…”