Specific inhibitory effect of 3-deazaneplanocin A against several Leishmania mexicana and L. braziliensis strains.

The growth inhibitory effect of 3-deazaneplanocin A (c3NpcA) was tested against some pathogenic members of the family of American Trypanosomatidae. Under our culture conditions, c3NpcA displayed a strongly and uniformly leishmanistatic effect on all 23 American Leishmania (L. mexicana and L. brasiliensis) strains in the study (mean dose producing 50% inhibition compared with control parasite growth [ID50] = 96 ng/ml, 0.32 microM), but showed no inhibition against the several T. cruzi and T. rangeli strains tested with concentrations up to 10,000 ng/ml. This compound also induced a substantial expansion of the intracellular pools of both S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet), as well as a significant diminution of the AdoMet:AdoHcy ratio. Strong AdoHcy hydrolase activity was detected in American Leishmania promastigotes. At at a dose of 200 ng/ml, c3NpcA inhibited S-adenosyl-L-3H-methylmethionine and 3-thymidine incorporation by promastigotes after four days incubation in the presence of the drug. At a dose of 100 ng/ml, c3NpcA eliminated approximately 56% of the L. mexicana and L. brasiliensis from infected human macrophages, compared with simultaneously cultivated controls. Two schedules of 10 consecutive intraperitoneal injections of c3NpcA, with doses ranging from 0.5 to 1.5 mg/kg/day, significantly reduced development of cutaneous leishmanial infection produced in inbred BALB/c mice by L. b. guyanensis inoculation, although a few parasites remained at the inoculation site.

Uptake and metabolism of S-adenosyl-L-methionine by Leishmania mexicana and Leishmania braziliensis promastigotes.

Promastigotes of Leishmania mexicana and Leishmania braziliensis incorporate S-adenosyl-L-[3H-methyl]methionine (AdoMet) against a concentration gradient through a saturable system. This concentrative uptake requires metabolic energy and is sensitive to temperature and sulfhydryl reagents such as N-ethyl maleimide. Intracellular AdoMet exchanges with external AdoMet. At steady state, unaltered ADoMet in the intracellular pool is at about a 1800-fold concentration in relation to that found in the external medium. Glucose, galactose and ribose did not stimulate uptake rates. Incorporated AdoMet goes into the soluble AdoMet pool, where a small fraction is metabolized, chiefly into methylthioadenosine, decarboxylated AdoMet and methanol. After a 60 min pulse the radioactivity associated with the [3H]AdoMet incorporated disappears with a half-time of 2 h. Transmethylation reactions were analyzed following [3H]AdoMet incorporation. Fractionation experiments indicate that 45-62% and 30-42% of the radioactivity is incorporated into lipids and protein methyl esters respectively, with 5-14% present in the soluble pool of parasites. Sinefungin or its cyclic derivative (1 and 10 micrograms ml-1) in the incubation medium produces 58% and 64% inhibition of AdoMet incorporation into Leishmania promastigotes. Most transmethylation reactions are inhibited, as there is a 50% decrease in the total radioactivity present in both the base-labile and lipidic fraction, with a parallel increase in the percentage of radioactivity in the soluble pool. Previous results give evidence of the importance of AdoMet in American Leishmania promastigote metabolism.

Inhibitory effects of sinefungin and its cyclic analog on the multiplication of Trypanosoma cruzi isolates.

Sinefungin and its cyclic analog were evaluated in vitro for activity against the multiplication of Trypanosoma cruzi. When either drug was tested for eight days on twelve T. cruzi epimastigote isolates, an 800-fold difference in drug sensitivity was found. Both drugs were trypanostatics at a concentration range from 0.1 micrograms/ml to 300 micrograms/ml. The 50% effective concentration (EC50) of sinefungin and its cyclic analog at which the growth of a given isolate was inhibited was 0.38 micrograms/ml for sinefungin and 0.31 micrograms/ml for the cyclic analog against the Ma, Marin, OPS-86, Y, and Ya isolates, and > 300 micrograms/ml for sinefungin and 217 micrograms/ml for the cyclic analog against the A-35, Bertoldo, DS, EP, ES, OPS-58, and FL isolates. Incubation of drug-sensitive isolates for more than 10 days in glucose-saline (GS) medium, but not in minimal essential medium, in the presence of a 30-fold EC50 concentration of the drug induced an increase in the drug-resistant population, which maintained this phenotype for several passages in drug-free culture medium. Growth curves were analyzed as a function of parasite inoculum; it was observed that with sinefungin-sensitive T. cruzi epimastigote isolates grown in GS medium in the presence of 10 micrograms/ml of the drug, the inhibitory effects of the drug were dependent on the initial inoculum: 1 x 10(3)-1 x 10(4) parasites/ml were strongly inhibited even after 16 days. Significant impairment of thymidine incorporation into the DNA of parasites by both drugs was observed only in drug-sensitive epimastigote isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

On the acid phosphatase isoenzymes existing in American Leishmania promastigotes.

1. Two partially purified acid phosphatase activities present in American Leishmania promastigote homogenates were characterized by biochemical methods. 2. One isoenzyme acts preferentially on p-nitrophenyl phosphate, is strongly inhibited by 30 mM alloxan, citrate, maleate, malonate and succinate, and strongly stimulated by 3 mM spermine. Its pI is 4.8. 3. The other isoenzyme acts preferentially on beta-glycerophosphate and is resistant to 30 mM alloxan, citrate, maleate, malonate and succinate and also to 3 mM spermine. Its pI is 5.7. 4. Both acid phosphatase isoenzymes have an optimum pH of 5.2, are tartrate-sensitive and strongly membrane-bound, as shown by differential centrifugation and density gradient equilibration. 5. Both isoenzymes were separated by using homogenates prepared in 2% Triton X-100, differential centrifugation, Sepharose 4B/CL-4B gel filtration, ion exchange chromatography and electrofocusing. After this procedure, they were still contaminated with several different proteins. 6. Purification was around 150-fold, with a 32% yield. 7. When these acid phosphatase activities were measured in total homogenates from 12 different Leishmania isolates, p-nitrophenyl phosphatase specific activity values were quite close; beta-glycerophosphatase-specific activity had around a 2-fold variation. 8. This variation was independent from taxonomic classification or infectivity of susceptible hosts.

Biological activity of analogs of guanine and guanosine against American Trypanosoma and Leishmania spp.

The growth inhibitory effects of six guanine and guanosine analogs, 3-deazaguanine (compound 1); 3-deazaguanosine (compound 2); 6-aminoallopurinol (compound 3); 9-beta-xylofuranosyl guanine (compound 4); a ribosylated derivative of compound 3, 6-aminopyrazolo(3,4-d)pyrimidin-4-one (compound 5); and 5-aminoformycin B (compound 6), were tested against some pathogenic members of the family of American Trypanosomatidae. Compounds 1 and 2 were highly active against Trypanosoma cruzi, Trypanosoma rangeli, and American Leishmania spp. in in vitro culture forms. Both compounds also showed antiprotozoal activity in T. cruzi-infected mice, with the optimal dose being about 30 mg/kg of body weight per day given as 10 consecutive doses. Compound 3 was the most active compound in vitro, inhibiting all of the American Trypanosomatidae culture forms tested. It was also highly inhibitory in mice that were acutely infected with T. cruzi, with the optimal dose being about 10 mg/kg of body weight per day. Ribosylation of compound 3 resulted in a derivative that showed decreased inhibitory activity on Trypanosomatidae multiplication. Compound 6 was highly inhibitory of in vitro multiplication of American Leishmania and T. rangeli but had no effect on T. cruzi epimastigotes and on mice that were acutely infected with T. cruzi. Compound 4 showed only a slight effect on T. cruzi epimastigotes.

Biological action of pyrazolopyrimidine derivatives against Trypanosoma cruzi. Studies in vitro and in vivo.

The capacity of 54 different pyrazolo(3,4-d) or (4,3-d)pyrimidine derivatives to inhibit Trypanosoma cruzi epimastigote and trypomastigote multiplication, and for some of them its chemotherapeutic activity, was evaluated. Six pyrazolo(3,4-d)pyrimidines showed inhibitory activity against epimastigote forms, 4-aminopyrazolo(3,4-d)pyrimidine being the most active, 5-fold more so than 4-hydroxypyrazolo(3,4-d)-pyrimidine. Neither compound was active against freshly isolated trypomastigotes, suggesting biochemical differences between culture and bloodstream forms of T. cruzi. On both epimastigote and trypomastigote forms, 7-amino-3-beta-D-ribofuranosylpyrazolo-(4,3-d)pyrimidine (FoA) was about 2-fold more active than 7-hydroxy-3-beta-D-ribofuranosylpyrazolo-(4,3-d)pyrimidine (FoB); however, when tested on T. cruzi-infected mice, only FoB exhibited significant chemotherapeutic activity. Previous results suggest that, except for FoB and FoA: (a) pyrazolopyrimidine insensitivity is trypomastigote-specific and (b) drug-insensitivity is lost when trypomastigotes transform into epimastigotes and vice versa.

Action of pyrazolopyrimidine derivatives on American Leishmania spp. promastigotes.

The capacity of 54 different pyrazolo-(3,4-d)- or -(4,3-d)-pyrimidine derivatives to inhibit American Leishmania promastigote multiplication was evaluated. Among pyrazolo-(3,4-d)-pyrimidines, eight derivatives showed leishmanistatic activity, 4-aminopyrazolo-(3,4-d)-pyrimidine (APP) being the most active, about eight-fold more than 4-hydroxy-pyrazolo-(3,4-d)-pyrimidine (HPP). 7-Hydroxy-3-beta-D-ribofuranosylpyrazolo-(4,3-d)-pyrimidine (FoB) was as active as 7-amino-3-beta-D-ribofuranosylpyrazolo-(4,3-d)-pyrimidine (FoA), a situation different to that found for pyrazolo-(3,4-d)-pyrimidines. Furthermore, different chemical modifications in formycin structure did not modify inhibitory effects. It can be concluded that regarding American Leishmania the chemical analogy to hypoxanthine or inosine of pyrazolo-(3,4-d)- and pyrazolo-(4,3-d)-pyrimidine, respectively, is not absolutely critical, as different modifications on the heterocyclic ring did not abolish the inhibitory activity of these compounds.

Action of pyrazolopyrimidine derivatives on Trypanosoma rangeli culture forms.

The capacity of 54 different pyrazolo(3,4-d)- or pyrazolo(4,3-d)pyrimidine derivatives to inhibit the multiplication of Trypanosoma rangeli culture forms was evaluated. Among pyrazolo(3,4-d)pyrimidines, 14 derivatives showed trypanostatic activity, 4-aminopyrazolo-(3,4-d)pyrimidine (APP) being the most active, with 4-hydroxypyrazolo(3,4-d)pyrimidine (HPP) lacking trypanostatic activity. 7-Hydroxy-3-beta-D-ribofuranosylpyrazolo(4,3-d)pyrimidine (FoB) was as active as 7-amino-3-beta-D-ribofuranosylpyrazolo(4,3-d)pyrimidine (FoA), both compounds being five-fold less inhibitory than APP. It can be concluded that, regarding T. rangeli, the chemical analogy to hypoxanthine or inosine of pyrazolo(3,4-d)- and pyrazolo(4,3-d)pyrimidine, respectively, is not absolutely critical, as different modifications on the heterocyclic ring did not abolish the inhibitory activity of these compounds.