Memoria CD.indd - ISHAM

and Cryptococcus neoformans. MICs for caspofungin in the pathogenic yeastlike phases of severaldimorphic fungi, fluctuate according to species and strains, e.g., Blastomyces dermatitidis (0.5-8µg/ml) or Histoplasma capsulatum ( Pb300 (35%) > Pb381 (27%) > Pb444 (21%) > Pb377 (17%) (values at 1.0 µgcaspofungin/ml). The mycelial phase, as expected, was highly susceptible to caspofungin, inhibitionfluctuating between 74% (Pb381) and 81% (Pb73). In no case, a 100% inhibition was achieved, evenat concentrations as high as 16 µM caspofungin, when 91% inhibition was the observed value forstrain Pb73, M phase. Scanning electron microscopic observations indicated structural modificationsin the cell walls of both phases as a consequence of caspofungin action. Such results may indicatevariability in P. brasiliensis cell wall composition, particularly with regards to the participation of β-1,3-glucan and the related activity of its synthesizing enzyme (GS). Experiments to solve this biologicalquestion are currently under way. But from a clinical point of view, caspofungin does not appear tobe a promising antifungal for the treatment of paracoccidioidomycosis.2. Azasterols and sterol hydrazones, experimental drugsThe sterol biosynthetic pathway has been largely studied for the search of antifungals. Azoles andallilamines act on differents steps of this pathway. However, they may interfere with similar steps inthe host. Hence, we have undertaken the search for drugs that may act on more specific steps in thesterol pathway. While mammals synthesize C27 cholestane-based members of the steroid family,pathogenic fungi, protozoa and plants require the presence of sterols (typically ergosterol and 24-alkyl analogs) which act as irreplaceable essential growth factors for these organisms. The enzymeresponsible for the addition of these alkyl groups to carbon C-24 and for the regulation of carbonflow in the sterol pathway is the ∆ (24) -sterol methyl transferase (SMT). There is no such enzyme inthe pathway of cholesterol biosynthesis, and thus SMT inhibition should selectively block ergosterolbiosynthesis, affecting only fungal cells, and theoretically bypassing any undesirable blockage in thesynthesis of the host’s cholesterol. The crucial and specific role of SMT has stimulated interest on thebiorational design of SMT inhibitors for potential clinical or agrochemical use as antifungal agents.SMT inhibitors such as azasterols and sterol hydrazones (AZA1-AZA3; H1-H3) have proven highlyeffective as antiproliferative agents against protozoa and some fungi, among them, Paracoccidioidesbrasiliensis. In the presence of AZA-1, a dose-dependent inhibition of P. brasiliensis growth (Yphase) was observed from 0.1 to 5 µM, to reach 100% growth arrest at the latter concentration andabove. AZA-2, instead, was only able to inhibit growth by 60% at the highest concentration used inthese experiments (10 µM). AZA-3 was the most powerful drug, as a concentration of 0.5 µM wasable to completely inhibit fungal growth. Lipid analyses indicated that in control cells, main sterolswere brassicasterol (69.1 %), ergosterol (26.8 %) and lanosterol (4.1 %). On exposure to AZA-1,ergosta-5,7,24(28)-trien-3β-ol (17.1%) and lanosterol (11%) accumulated, while AZA-2 led to animportant accumulation of ergosta-5,7,22,24(28)-tetraen-3β−ol (50.5 %). With AZA-3, instead, animportant accumulation of lanosterol (34.5 %) was observed, as a result of SMT inhibition. Concurrentwith the accumulation of these intermediates, final products of the sterol pathway (ergosteroland brassicasterol) decreased substantially. Similarly, sterol hydrazones (H1, H2, and H3) generateda dose-dependent effect in fungal growth, which were active at nanomolar concentrations, alsorelated to SMT inhibition. These drugs were active in the following sequence: AZA3 > AZA1 > H3≥ H1 >> H2 >> AZA2.Acknowledgement: Merck, Sharp & Dohme (Caracas, Venezuela), for partial support in the caspofunginexperiments.122