Nd

a Parasites were cultured in the presence ofCAAX mimetics or vehicle for 3d(T. brucei bloodstream and procyclic forms) or 7 d (T. cruzi amastigotes in mouse 3T3 cells and epimastigotes). b ND, not determined.

a Parasites were cultured in the presence ofCAAX mimetics or vehicle for 3d(T. brucei bloodstream and procyclic forms) or 7 d (T. cruzi amastigotes in mouse 3T3 cells and epimastigotes). b ND, not determined.

FTI-277 and GGTI-298, and L-745,631 and SCH-44342 were tested for their effect on the growth of the bloodstream and insect (procyclic) forms of T. brucei. Cells were cultured for 3 din the presence ofvarious amounts ofinhibitors, at which time potencies of the inhibitors were determined. The concentrations of inhibitors required to reduce the cell number relative to control culture by twofold (EC50) are listed in Table 2. Both FTI-277 and GGTI-298 are highly potent anti-parasite agents (EC50 = 0.7 and 1.7 pM, respectively). Growth curves for bloodstream form T. brucei in the presence ofdifferent concentrations ofthese compounds are shown in Fig. 3. Growth ofbloodstream parasites was completely blocked with 1 pM FTI-277 and with 5 pM GGTI-298. GGTI-298 has been shown to be useful for selective inhibition of mammalian GGTase I in vivo and for studyingthe consequence ofthis inhibition (31). However, growth inhibition of T. brucei caused by GGTI-298 might be owing to inhibition of protein farnesylation but not gera-nylgeranylation, because both this compound and FTI-277 are potent inhibitors of T. brucei FTase in vitro (Table 1). The bloodstream form is more sensitive to these compounds than the procyclic form (Table 2), possibly because ofpoor penetration ofCAAX mimetic through the dense protein layer that covers the plasma membrane of procyclic parasites. L-745,631 and SCH-44342, which are considerably less potent at inhibiting T. brucei FTase in vitro, are also less potent at stunting parasite growth. For all tested inhibitors, cell shape deformation was observed within 24 h after adding inhibitor, and significant cell lysis occurs during the course ofthe treatment. Thus, these CAAX mime-tics seem to be cytocidal rather than cytostatic.

The effect ofCAAX mimetics on the growth of T. cruzi amastigotes inside of3T3 host cells was assayed using the Tulahuen strain of this parasite that is stably transfected with the Escherichia coli P-galactosidase gene (LacZ) (32). This procedure permits the number of amastigotes to be readily quantified using an enzyme-linked immunoassay (ELISA) plate reader and a colorimetric substrate for E. coli P-galactosidase. Figure 4 shows that FTI-277, GGTI-298, and L-745,631 block growth of T. cruzi amastigotes in a dose-dependent manner (measured after 7 d of culture). The EC50s are listed in Table 2. Ofthe three CAAX mimetics tested, only GGTI-298 was toxic at low micromolar amounts to noninfected 3T3 host cells. On the other hand, concentrations of L-745,631 and FTI-277 sufficient to block completely amastigote growth did not affect the growth of non-infected host cells. GGTI-298 but not the FTase inhibitor FTI-277 has been reported to block cell-cycle phase progression from G0/G1 to S phase in 3T3 cells (31). As shown in Table 2, the CAAX mimetics also inhibit the growth of insect form T. cruzi (epimastigote)

Fig. 3. Growth inhibition ofbloodstream form T. brucei by CAAX analogs. (A) Bloodstream form T. brucei (5 X 104 cells/mL) was cultured with 0 (O), 0.5 (•), 0.75 (A), 1 pM (□) FTI-277, and cells were counted daily. (B) Same as (A) except with GGTI-298, 0 (O), 1 (•), 2.5 (A), or 5 pM (□). Adapted with permission from ref. (23).

Fig. 3. Growth inhibition ofbloodstream form T. brucei by CAAX analogs. (A) Bloodstream form T. brucei (5 X 104 cells/mL) was cultured with 0 (O), 0.5 (•), 0.75 (A), 1 pM (□) FTI-277, and cells were counted daily. (B) Same as (A) except with GGTI-298, 0 (O), 1 (•), 2.5 (A), or 5 pM (□). Adapted with permission from ref. (23).

in a dose-dependent manner. The compounds are roughly an order of magnitude less potent against epimastigotes compared to amastigotes. Studies are underway to test the CAAX mimetics as inhibitors ofpartially purified FTases from T. cruzi, L. amazonensis, and L. braziliensis as well as to test these compounds for their ability to block the growth of various Leishmania species in vitro (preliminary results look encouraging).

Fig. 4. Growth inhibition of T. cruzi amastigotes and of murine 3T3 fibroblasts by CAAX mimetics. Amastigotes (■) in 3T3 host cells were cultured in the presence of the indicated inhibitors for 7 d, and growth was measured by the colorimetric assay of ß-galactosidase (17,32). Separately, 3T3 fibroblasts (▲) were cultured in the presence of the inhibitor, and growth was measured with Alamar-Blue after 5 d. Adapted with permission from ref. (23).

Fig. 4. Growth inhibition of T. cruzi amastigotes and of murine 3T3 fibroblasts by CAAX mimetics. Amastigotes (■) in 3T3 host cells were cultured in the presence of the indicated inhibitors for 7 d, and growth was measured by the colorimetric assay of ß-galactosidase (17,32). Separately, 3T3 fibroblasts (▲) were cultured in the presence of the inhibitor, and growth was measured with Alamar-Blue after 5 d. Adapted with permission from ref. (23).

FPP analogs, which are low- to sub-micromolar inhibitors of mammalian and T. brucei FTases, are also able to block the incorporation of 3H-mevalonic acid into bloodstream T. brucei proteins (17). Low micromolar concentrations of these compounds are also toxic to T. brucei bloodstream and procyclic forms (17). Although FPP analogs are not being developed as anti-cancer drugs, these results provide useful data showing that inhibitors of FTase that have structures very different from CAAX mimetics are also lethal to parasites.

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