Barker R H, Metelev V, Coakley A, Zamecnik P
Hybridon, Inc., Worcester, Massachusetts 01605, USA.
Exp Parasitol. 1998 Jan;88(1):51-9. doi: 10.1006/expr.1998.4192.
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.
反义寡脱氧核苷酸（AS ODNs）已显示出有望成为潜在的抗疟化疗药物以及识别对疟原虫生存至关重要基因的手段。由于含有磷酸二酯（PO）基团的传统ODNs会迅速被核酸酶降解，因此通常使用含硫代磷酸酯（PS）基团的ODNs。然而，在高浓度下，这些ODNs会失去靶标特异性，并且在一些动物模型中，它们会变得有毒。我们比较了多种化学修饰（PO、PS、PO-PS杂合体、2'-O-甲基-2'-脱氧嵌合体）和结构修饰（有利于形成自稳定环的序列改变）对体外培养的恶性疟原虫疟疾的抑制能力。所有修饰均使用针对二氢叶酸还原酶胸苷酸合酶（DHFR）的AS ODN序列进行。在3'和5'端含有少至三个PS基团的PO-PS杂合体的抑制作用与使用全PS基团化合物所获得的抑制作用没有显著差异。同样，含有2'-O-甲基修饰的PS嵌合化合物的抑制作用与传统PS化合物的抑制作用没有显著差异。相比之下，虽然PO-PS杂合嵌合体在低浓度下的抑制作用与全PS化合物没有显著差异，但在1 microM时，它们对寄生虫生长的抑制作用比全PS化合物或PS 2'-O-甲基-2'-脱氧嵌合体少25%（P < 0.001）。延长核苷酸序列以增加茎环形成产生了两种化合物，它们对寄生虫生长的抑制作用比未修饰的化合物高约20%，尽管这种差异不显著。此外，这种增加的大部分似乎与与增加的ODN长度相关的更多PS基团有关。我们得出结论，限制PS基团的数量并包含PO 2'-O-甲基基团可能会产生具有高反义活性但低非序列依赖性效应的化合物。此类化合物目前正在进行体内测试。
