Glucosylceramide synthase as target for new antiparasitic drugs. Effect of PPMP on different developmental stages of Trypanosoma cruzi

DUSCHAK, V.G.;; LANDONI, M;; GARAVAGLIA, P;; ESTEVA M. I.; COUTO, A.S.

Mendoza, Argentina.

Congreso; XXV Reunión de Protozoología y Enfermedades Parasitarias.; 2005

Instituto Nacional de Parasitología, Dr. Mario Fatala Chabén, Ministerio de Salud y Ambiente; CIHIDECAR, Depto de Química Orgánica, F.C.E. y N., UBA, Argentina. Biosynthesis of glycosphingolipids involves the sequential action of glycosyltransferases in mammals. The key step involves the transfer of glucose from UDP-glucose to ceramide catalyzed by a UDP-Glucose:glucosylceramide transferase (glucosylceramide synthase (GCS)) to form glucosylceramide, the precursor of most higher order glycosphingolipids. In Trypanosoma cruzi, little is known about the enzymes involved in their biosynthesis. It is known that PPMP (D,L-threo-phenyl-2-palmitoylamino-3-morpholine-1-propanol) at low concentrations inhibits the activity of GCS in mammals whereas at higher concentrations it is also effective on sphingomyelin synthase. In order to address the effect of the GCS inhibition on parasite growth, bloodstream trypomastigotes (Tul 2 strain) from infected mice were treated with different concentrations of PPMP (10-50mM), obtaining from 79 to 95.5 % of parasite lysis. Similarly, epimastigote forms in the presence of 10 mM PPMP, showed a 63% of parasite lysis. In order to confirm whether PPMP was affecting GCS, epimastigote cultures were incubated with 10 mM PPMP. After 24 h, parasites were labeled with NBD-ceramide for 24 h in the presence of the drug. Glycosphingolipids were extracted from the labeled parasites and purified by DEAE-Sephadex and TLC. When neutral glycosphingolipids obtained from the control parasites were compared with the analogous fraction from the PPMP-treated parasites by TLC and HPLC, a significant reduction of the synthesized glucosylceramide (60%) was shown. The results obtained reinforce the relationship between the enzymes involved in the glycosphingolipid biosynthesis and parasite viability, indicating that these enzymes might constitute new interesting targets for antiparasitic drugs.