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 potential anti-malarial chemotherapeutic agents and as a means for identifying genes critical for parasite survival. Because conventional ODNs 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 chemical modifications (PO, PS, PO-PS hybrids, 2'-O-methyl-2'-deoxy chimeras) and structural modifications (sequence alterations favoring self-stabilizing loop formation) for their ability to inhibit Plasmodium falciparum 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 three PS groups at the 3'- and 5'-ends did not differ significantly from that obtained using compounds containing all-PS groups. Similarly, inhibition by PS chimeric compounds containing 2'-O-methyl modifications did not differ significantly from that of conventional PS compounds. In contrast, while inhibition by PO-PS hybrid chimeras did not differ significantly from that of all-PS compounds at low concentrations, at 1 microM they inhibited parasite growth 25% less (P < 0.001) than all-compounds 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, though this difference was not significant. Furthermore, most of this increase appears to correlate with the greater number of PS groups associated with the increased ODN length. We conclude that limiting the number of PS groups and inclusion of PO 2'-O-methyl groups may yield compounds with high antisense activity but low non-sequence-dependent effects. Such compounds are currently being tested in vivo.
Barker
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RH
Hybridon, Inc., Worcester, Massachusetts 01605, USA.
Metelev
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V
Coakley
A
A
Zamecnik
P
P
D000818:Animals; D002478:Cells, Cultured; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics; D014316:Tritium
D016376:Oligonucleotides, Antisense; D014316:Tritium; D019271:Hypoxanthine; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase