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...

Two phaeoid strains of the fungus Cladosporium carrionii (SR3 from a xerophyte species and PP8201 from a patient), and one strain of Hormoconis resinae (Cladosporium resinae), isolated from oil-impregnated soil, were analyzed for their cell wall composition by colorimetric methods, X-ray diffraction, infrared spectroscopy, and solid-state 13C-nuclear magnetic resonance. Results suggested that the cell walls were composed mainly of hexoses (34%-47%) as beta-1,3-glucan (some galactose and mannose were also present) and melanin, chitin being absent. Electron microscopic observations suggested that melanin was found not only in the cell wall but also in intracellular bodies resembling melanosomes.

The water-soluble polysaccharide fraction of the cell wall alkali extract (F1SS) from the mycelial phase of the dimorphic fungus Paracoccidioides brasiliensis is compared with F1SS polysaccharides obtained from the Onygenalean mycelial fungi Ascocalvatia alveolata, Onygena equina and Aphanoascus terreus. These polymers were exclusively composed of mannose and galactose. Data from methylation and NMR analyses reveal that F1SS polysaccharides from the four fungi contain the same residues although in different proportions: [-->2,6)-alpha-D-Manp-(1 -->]; [2)-alpha-D-Manp-(1 -->]; [ -->6)-alpha-D-Manp-(1 -->]; and [alpha-D-Galf-(1 -->]. In P. brasiliensis, the repeating unit of the polysaccharide consists of a backbone of [(1 -->6)-alpha-D-Manp] substituted at the 0-2 position by the disaccharide [alpha-D-Galf-(1 -->6)-alpha-D-Manp-(1 -->], while the remaining 0-2 positions are substituted by single residues of mannose or short chains of (1 -->2)-mannose. The other species had a lower proportion of galactofuranose-containing side chains and higher proportion of mannose-containing side chains. The similarities found among the F1SS polysaccharides from P. brasiliensis and the Onygenalean A. alveolata, A. terreus and O. equina, reveal the close relatedness of all these fungi, show differences with polysaccharides from other fungal genera and agree with the molecular evidence provided in the scientific literature for the placement of P. brasiliensis within the Onygenales.

Putrescine and spermidine were the only polyamines found in Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans. Free polyamines (putrescine > spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine > putrescine). Conjugated and bound polyamines were also quantified. 1, 4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase. The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition of P. brasiliensis.

Zygosporium geminatum, isolated as a contaminant in a culture of the mycelial phase of Paracoccidioides brasiliensis, was lethal to the latter organism. Its lytic action was due to exocellular alpha-1,3- and beta-1,3-glucanases which degraded the P. brasiliensis cell wall. The alpha-1,3-glucanase was more active at 30 degrees C and the beta-1,3-glucanase at 23 degrees C, each having pH 6.0 as its optimum.