THE EFFECT OF (S)-9-(3-HYDROXY-2-PHOSPHONYLMETHOXYPROPYL) ADENINE ON NUCLEAR AND ORGANELLAR DNA-SYNTHESIS IN ERYTHROCYTIC SCHIZOGONY IN MALARIA

The very effective (ID50 = 47 nM) and selective antimalarial compound (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) abruptly arrests Plasmodium falciparum-cultured schizonts at concentrations between 1 and 10 x ID50 as soon as their DNA content reaches 8 times that of the haploid ringform stage. Even very high HPMPA concentrations do not inhibit the first 2-3 rounds of schizogonic DNA replication. Also, in the presence of HPMPA, replication of the 6-kb mitochondrial and 35-kb chloroplast-like DNA proceeds normally and in close concert with each other, both to a 16-fold amount within 5 h during the trophozoite stage. Hence the in in vitro assays HPMPApp-sensitive plasmodial DNA polymerase gamma-like enzyme (IC50 = 1 mu M) - assumed to be involved in mitochondrial DNA replication - is not the target of HPMPA in vivo (living parasites), nor seems to be the DNA polymerization activities of the - in vitro also HPMPA-sensitive (IC50 = 38 mu M) - DNA polymerase alpha or of any other nuclear DNA polymerase of Plasmodium. In vitro assays demonstrated that HPMPApp does not act as an alternative substrate for plasmodial polymerases, contradicting the suggestion that the observed delayed inhibition of plasmodial schizogony might be the result of DNA strand breakage caused by HPMPApp incorporation. Neither do results support the idea that the HPMPA-induced arrest of DNA replication might be due to chain termination as a result of such incorporation. We investigated whether arrest of DNA replication by HPMPA in schizonts could be explained by inhibition of the DNA synthesis rate limiting ribonucleotide reductase enzyme. For the first time its in vitro activity was demonstrated in Plasmodium, converting - in an allosterically controlled way - rNDPs into dNDPs. However, this conversion is not inhibited by HPMPA or HPMPApp, excluding this enzyme as well as the target of HPMPA. Putative alternative modes of action of HPMPA on Plasmodium are discussed.