Trypanocidal furamidine analogues: influence of pyridine nitrogens on trypanocidal activity, transport kinetics, and resistance patterns

Abstract

Current therapies for human African trypanosomiasis (HAT) are unsatisfactory and under threat from emerging drug resistance linked to the loss of transporters, e.g., the P2 aminopurine transporter (TbAT1). Here we compare the uptake and trypanocidal properties of furamidine (DB75), recently evaluated in clinical trials against stage 1 (haemolymphatic) HAT, and two aza analogues, DB820 and CPD0801 (DB829), which are candidate compounds for treatment of stage 2 (neurological) disease. Values of 50% inhibitory concentrations (IC50s) determined in vitro against both wild-type and transporter mutant parasites were submicromolar, with DB75 trypanotoxicity shown to be better than and DB820 trypanotoxicity similar to that of the widely used veterinary trypanocide diminazene, while CPD0801 was less active. Activity correlated with uptake and with the minimum drug exposure time necessary to kill trypanosomes: DB75 accumulated at double and 10-fold the rates of DB820 and CPD0801, respectively. All three compounds inhibited P2-mediated adenosine transport with similar Ki values, indicating affinity values for this permease in the low to submicromolar range. Uptake of DB75, DB820, and CPD0801 was significantly reduced in tbat1-/- parasites and was sensitive to inhibition by adenine, showing that all three compounds are substrates for the P2 transporter. Uptake in vitro was significantly less than that seen with parasites freshly isolated from infected rats, correlating with a downregulation of P2 activity in vitro. We conclude that DB75, DB820, and CPD0801 are actively accumulated by Trypanosoma brucei brucei, with P2 as the main transport route. The aza analogues of DB75 accumulate more slowly than furamidine itself and reveal less trypanocidal activity in standard in vitro drug sensitivity assays.

Fig. 1.

Structure of DB75 (furamidine) and its aza analogues DB820 and CPD0801 (DB829) compared to those of pentamidine and diminazene.

Fig. 2.

Uptake of diamidines by T. b. brucei strain s427. Trypanosomes isolated from infected female Wistar rats were immediately exposed to 10 μM permeant in complete HMI-9 media for up to 1 h at RT. Intracellular diamidine concentrations were measured using a fluorescence-based assay. The data are representative of the results of at least three similar experiments, each performed in at least triplicate. ○, DB75; ■, DB820; ▴, CPD0801. Bars represent standard errors of the means (SEM) of quadruplicate determinations of this particular experiment. When not shown, error bars fall within the symbol.

Fig. 3.

Inhibition of P2-mediated adenosine transport by furamidine analogues. Transport of 0.050 μM [³H]adenosine was assessed in the presence and absence of various concentrations of DB075 (▾), DB820 (○), and CPD0801 (Δ) over a period of 30 s. Uptake was determined in the presence of 1 mM inosine, which does not inhibit P2-mediated [³H]adenosine transport but does competitively inhibit P1-mediated uptake (15). The data are representative of the results of at least three similar experiments, each performed in triplicate. Bars represent SEM.

Fig. 4.

Inhibition of diamidine uptake by the P2 substrate adenine. T. b. brucei s427 parasites were isolated from infected rats and immediately exposed to 10 μM permeant in complete HMI-9 media for up to 1 h at RT in the presence (■) or absence (○) of 100 μM adenine. DB75 (a), DB820 (b), and CPD0801 (c) accumulation levels are plotted as percentages of intracellular concentration at 60 min. The data shown are representative of the results of three similar experiments, each performed in at least triplicate. Bars represent SEM of triplicate determinations of the experiment displayed. When not shown, error bars fall within the symbol. Averaged over three independent experiments, permeant concentrations after 60 min ± SEM were 541 ± 169 μM (DB75), 112 ± 43 μM (DB820), and 31 ± 3.3 μM (CPD0801).

Fig. 5.

Uptake of diamidines by T. b. brucei strain tbat1^−/−. Trypanosomes isolated from infected rats were immediately exposed to 10 μM permeant in complete HMI-9 media for up to 1 h at RT. Levels of accumulation of DB75 (a), DB820 (b), and CPD0801 (c) by tbat1^−/− (○) and wild-type s427 (■) are shown as percentages of the permeant concentration in s427 at 60 min. The data are representative of the results of at least three similar experiments, each performed in at least triplicate. Bars represent SEM of the triplicate determinations in this particular experiment; when not shown, error bars fall within the symbol.

Fig. 6.

Apparent kinetic parameters of diamidine uptake by trypanosomes. DB75 (a), DB820 (b), and CPD0801 (c) uptake rates were measured by immediately incubating s427 cells isolated from female Wistar rats for 10 min with 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, or 30 μM permeant in complete HMI-9 media. The data are representative of the results of at least three similar experiments, each performed in at least triplicate. Bars represent SEM of the triplicate determinants in this particular experiment. When not shown, bars fall within the symbol. Apparent kinetic constants (averages ± SEM of the results of 3 or 4 independent experiments), determined by nonlinear regression using the Michaelis-Menten equation, are shown in Table 5.

Fig. 7.

P2-mediated [³H]adenosine uptake by s427 cells brought into culture immediately after isolation from rat blood and cultured for the indicated times. P1 transporter activity was inhibited by the presence of 1 mM inosine. Incubation time was 30 s. Error bars represent SEM of the results of triplicate determinations.

Fig. 8.

Uptake of diamidines by trypanosomes cultured in vitro. Trypanosomes of strains 427 (■), tbat1^−/− (○), and B48 (▴) were exposed to 7.5 μM DB75 (a), DB820 (b), or CPD0801 (c) in complete HMI-9 media under standard culture conditions, and intracellular diamidine concentrations were measured after 1, 4, 8, and 24 h of exposure. The data are representative of the results of at least three similar experiments, each performed in at least triplicate. Bars represent SEM.