Partial purification and characterization of DNA topoisomerase II from Plasmodium falciparum.

DNA topoisomerase II from Plasmodium falciparum was partially purified by FPLC using three columns: Econo-Pac Q, heparin-agarose and Mono Q. The enzyme showed ATP- and Mg2 +/- dependent activities in a decatenation assay, with optimum concentrations of 0.5 and 10 mM, respectively. Furthermore, highest activity was detected in the presence of 100 mM KCI. Enzyme decatenation activity was not inhibited by the DNA topoisomerase I inhibitor, camptothecin, but was sensitive to both prokaryotic and eukaryotic DNA topoisomerase II inhibitors.

Purification and characterization of DNA polymerases from Plasmodium falciparum.

Fractionation of Plasmodium falciparum cellular extracts by fast protein liquid chromatography (FPLC) identified at least two different DNA polymerases. An aphidicolin-sensitive activity co-purified with a primase activity. This, in combination with other characteristics (processivity, sensitivity to other inhibitors), most likely classifies this enzyme as an alpha-like DNA polymerase. It was, however, relatively resistant to N2-(p-n-butylphenyl)deoxyguanosine 5'-triphosphate (IC50 = 6.6 microM) and differs in this aspect from the host homologue, possibly indicating structural differences between host and parasite DNA polymerase alpha. The other DNA polymerase matched eukaryotic DNA polymerase gamma in all properties tested.

Structure-activity relationships and modes of action of 9-anilinoacridines against chloroquine-resistant Plasmodium falciparum in vitro.

An in vitro investigation of the structure-activity profiles for a range of 9-anilinoacridines on drug-resistant Plasmodium falciparum is reported. C-3, 6-diamino substitution, low lipophilicity, and high pKa values substantially increased the activities of the 9-anilinoacridines tested. There appeared to be no correlation between DNA binding and antimalarial activity. 3,6-Diamino-1'-amino-9-anilinoacridine (compound 13) was the most active compound tested; it had a 50% inhibitory concentration of 25 nM. In vitro mammalian cell growth assays showed compound 13 to be one of the least cytotoxic 9-anilinoacridines (50% inhibitory concentration, 15 microM). Both compound 13 and the antimalarial drug pyronaridine inhibited the decatenation activity of P. falciparum DNA topoisomerase II at concentrations of 10 and 11 microM, respectively.

In vitro study of anticancer acridines as potential antitrypanosomal and antimalarial agents.

The requirement for rational drug design in the search for new agents that are active against parasitic protozoa prompted our in vitro studies with a group of 9-anilinoacridines. In vitro growth assays with Trypanosoma lewisi identified a series of C-1' alkylaminoacridines which possess previously unreported potent growth-inhibitory activities against T. lewisi at a concentration range of 0.1 to 1 microM. In contrast, several 9-anilinoacridines that possess acridine ring NH2 substituents at C-3 and C-6 were inactive against T. lewisi, but they possessed strong activity against Plasmodium falciparum at a concentration range of 0.1 to 2.8 microM. In mammalian cells, amsacrine [4'-(9-acridinylamino)methanesulfon-m-anisidide] inhibits DNA topoisomerase II; however, amsacrine was only weakly active against T. lewisi. Such differences in the patterns of susceptibility of mammalian cells, T. lewisi, and P. falciparum to these 9-anilinoacridines may reflect enzyme differences between different parasites and mammalian cells that can be exploited by further improvements in drug design.

A simple technique for large scale in vitro culture of Plasmodium falciparum.

The large scale in vitro cultivation method of Fairlamb et al (1985) was modified to contain human plasma-supplemented medium in HEPES buffer. After 4 days with no change of medium nor agitation of the culture flask, 27- to 50-fold increase in the starting parasitemia of trophozoites were obtained.

Inhibition of the growth of Plasmodium falciparum and Plasmodium berghei by the DNA polymerase inhibitor HPMPA.

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [HPMPA] belongs to a class of nucleoside analogues originally described as having potent activity against a broad spectrum of DNA viruses. We examined the effects of this class of drugs on the growth of cultured Plasmodium falciparum. Strong inhibition was observed by HPMPA (ID50 = 47 nM) at concentrations more than 1000-fold less than the cytotoxic dose for human cells. 3-deaza-HPMPA was even more strongly inhibitory (ID50 = 8 nM), whereas several other acyclic nucleosides were not effective. In mice infected with Plasmodium berghei, increase of parasitaemia can be blocked for 4-6 days by a single injection of HPMPA. Repeated drug administration blocks parasite growth for prolonged periods at doses that are clinically feasible. We also determined the inhibition of several purified Plasmodium DNA polymerases by diphosphorylated HPMPA (HPMPApp). DNA polymerase alpha-like enzymes of P. falciparum and P. berghei are inhibited with an IC50 = 40 microM and a gamma-like DNA polymerase from P. falciparum is even 40-fold more sensitive to the drug. The inhibition by HPMPApp is competitive with dATP, strongly suggesting that Plasmodium DNA polymerases are targets for this class of nucleotide analogue.