PLASMODIUM-FALCIPARUM - EFFECT OF CHEMICAL-STRUCTURE ON EFFICACY AND SPECIFICITY OF ANTISENSE OLIGONUCLEOTIDES AGAINST MALARIA IN-VITRO

Antisense oligodeoxynucleotides (AS ODNs) have shown promise both as p otential anti-malarial chemotherapeutic agents and as a means for iden tifying genes critical for parasite survival. Because conventional ODN s containing phosphodiester (PO) groups are subject to rapid nuclease degradation, ODNs with phosphorothioate (PS) groups are commonly used. However, at high concentration, these lose target specificity, and in some animal models, they become toxic. We compared a variety of chemi cal modifications (PO, PS, PO-PS hybrids, 2'-O-methyl-7'-deoxy chimera s) and structural modifications (sequence alterations favoring self-st abilizing loop formation) for their ability to inhibit Plasmodium falc iparum malaria cultured in vitro. All modifications were done using an AS ODN sequence targeted against dihydrofolate reductase thymidylate synthase (DHFR). Inhibition by PO-PS hybrids containing as few as thre e PS groups at the 3'- and 5'-ends did not differ significantly from t hat obtained using compounds containing all-PS groups. Similarly, inhi bition by PS chimeric compounds containing 2'-O-methyl modifications d id not differ significantly from that of conventional PS compounds. In contrast, while inhibition by PO-PS hybrid chimeras did not differ si gnificantly from that of all-PS compounds at low concentrations, at 1 mu M they inhibited parasite growth 25% less (P < 0.001) than all-comp ounds or PS 2'-O-methyl-2'-deoxy chimeras. Extension of the nucleotide sequence to increase stem-loop formation yielded two compounds which inhibited parasite growth about 20% more than unmodified compounds, th ough this difference was not significant. Furthermore, most of this in crease appears to correlate with the greater number of PS groups assoc iated with the increased ODN length. We conclude that limiting the num ber of PS groups and inclusion of PO 2'-O-methyl groups may yield comp ounds with high antisense activity but low non-sequence-dependent effe cts. Such compounds are currently being tested in vivo. (C) 1998 Acade mic Press.