Selective Activity of5-Fluoroorotic Acidagainst Plasmodium falciparum InVitrot

Unlike mammalian cells, malarial parasites arecompletely dependent on denovo pyrimidine metabolism. Eventhough these parasites donotuseexternal uracil or uridine, orotic acid, an intermediate ofpyrimidine biosynthesis, issuccessfully transported into theparasite andincorporated intoparasite nucleic acids. Onthis basis, itwas hypothesized that5-fluoroorotate, a cytotoxic derivative oforotic acid, may bea potentand selective antimalarial agent.Invitro, 5-fluoroorotate caused 50% inhibition ofthegrowthofPlasmodium falciparum ata concentration of6.0nM. Incontrast, 5-fluorouracil, 5-fluorouridine, and5-fluoro 2'deoxyuridine were muchless effective against malarial parasites. Chloroquine-susceptible andchloroquineresistant clones ofP.falciparum were equally susceptible to5-fluoroorotate. Thetoxicity of5-fluoroorotate was evaluated onfourhumancell lines (HT-1080, IMR-90, HeLaS3,andHL-60) andone mouse cell line (L-1210). Compared withmalarial parasites, themammalian cells were relatively tolerant of5-fluoroorotic acid(50% inhibitory concentration, 0.9to10,uM). Finally, inthepresenceof1mM uridine, allmammaliancells were partially protected from5-fluoroorotate cytotoxicity, buturidine offered no protection toP.falciparum. According toWorldHealth Organization estimates of 1982, morethan350million people live inareas oftheworld wheremalaria ishighly endemic andwherenospecial antimalarial measures arebeingapplied (43). About2.2 billion people live inareas wheremalaria isstill endemic but wherecontrol measures havedecreased thelevel ofendemicity. Plasmodium falciparum strains resistant tochloroquine areconfirmed inatleast 40tropical andsubtropical countries (27). Thefuture ofmalarial chemotherapy isparticularly alarming inviewofparasite strains thatdisplay cross-resistance toseveral structurally unrelated drugs (42; T.E.Hubbert, A.M.Oduola, D.L.Klayman, andW.K. Milhous, paper presented atthe35th Annu.Meet.Am.Soc. Trop. Med.Hyg.1986). Forthese reasons, itisimportant to develop novel antimalarial drugs, particularly those effective against strains ofP.falciparuim thatdisplay multiple drug resistance. Theerythrocytic phase ofthelife cycle ofP.falciparum is associated with theclinical symptoms ofmalaria. During this 48-hasexual cycle, eachparasite inside anerythrocyte generates 6to24offspring thatburst outandindividually invade fresh erythrocytes. Theexponential increase inparasite DNA andRNA requires asteady supply ofpurine and pyrimidine nucleotides. Malarial parasites usetherich pool ofadenine nucleotides inside theerythrocyte toobtain their supply ofpurines, buttheparasites havetosynthesize pyrimidines denovo(35). Two lines ofevidence suggested thatmalarial parasites wereunable toutilize exogenous preformed pyrimidines. First, early studies showedthatradioactive carbon dioxide wasincorporated intopyrimidine units ofmalarial nucleic acidswhileuracil, uridine, thymine, thymidine, cytidine, anddeoxycytidine werenot(3,13,35,39,40). Later, several