Characterization of simian malarial parasite (Plasmodium knowlesi)-induced putrescine transport in rhesus monkey erythrocytes. A novel putrescine conjugate arrests in vitro growth of simian malarial parasite (Plasmodium knowlesi) and cures multidrug resistant murine malaria (Plasmodium yoelii) infection in vivo

Abstract

A stage-dependent increase in the level of putrescine, spermidine, and spermine during intraerythrocytic growth of Plasmodium knowlesi in rhesus monkey erythrocytes was observed. Further, intraerythrocytic P. knowlesi-induced putrescine influx was found in trophozoite stage-infected erythrocytes and process was time- and temperature-dependent and showed saturable kinetics. Characteristics of induced putrescine influx appears in infected erythrocytes to be close to the normal erythrocytes in terms of affinity of putrescine to the putrescine transporter (Km 34.6 +/- 3.8 microM as normal erythrocytes and Km 37.2 +/- 5.2 microM in infected erythrocytes). However, the difference involves the significant increase in the putrescine influx rate after infection (Vmax = 4.21 nmol/min/10(10) normal erythrocytes, compared with 11.6 nmol/min/10(10) infected erythrocytes). Energy dependence, involvement of -SH group, and noninterference by amino acid, spermidine, and spermine in the putrescine influx process clearly demonstrate the presence of a distinct transporter for putrescine in infected erythrocytes. A putrescine conjugate N1,N4-bis(7-chloroquinoline-4-yl)butane-1, 4-diamine (BCBD) was synthesized, which inhibits the putrescine influx in the P. knowlesi infected erythrocytes (Ki of 43.2 microM) as well as in vitro growth of P. knowlesi (IC50 value, 7.64 +/- 0.97 ng/ml BCBD, 10.8 +/- 0.45 ng/ml chloroquine). Addition of exogenous polyamines failed to reverse the inhibitory effect of BCBD in vitro. Administration of BCBD (24 mg/kg body weight, intraperitoneal, twice a day for 4 days) cured the Swiss mice infected with multidrug-resistant infection of Plasmodium yoelii. Therefore, inhibition of putrescine transport in malaria-infected erythrocytes offers a lead in the search of a new class of chemotherapeutic molecules against malaria.