Gruegelsiepe Heike, Willkomm Dagmar K, Goudinakis Olga, Hartmann Roland K
Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, 35037 Marburg, Germany.
Chembiochem. 2003 Oct 6;4(10):1049-56. doi: 10.1002/cbic.200300675.
The ribonucleoprotein enzyme RNase P catalyzes endonucleolytic 5'-maturation of tRNA primary transcripts in all domains of life. The indispensability of RNase P for bacterial cell growth and the large differences in structure and function between bacterial and eukaryotic RNase P enzymes comply with the basic requirements for a bacterial enzyme to be suitable as a potential novel drug target. We have identified RNA oligonucleotides that start to show an inhibitory effect on bacterial RNase P RNAs of the structural type A (for example, the Escherichia coli or Klebsiella pneumoniae enzymes) at subnanomolar concentrations in our in vitro precursor tRNA (ptRNA) processing assay. These oligonucleotides are directed against the so-called P15 loop region of RNase P RNA known to interact with the 3'-CCA portion of ptRNA substrates. Lead probing experiments demonstrate that a complementary RNA or DNA 14-mer fully invades the P15 loop region and thereby disrupts local structure in the catalytic core of RNase P RNA. Binding of the RNA 14-mer is essentially irreversible because of a very low dissociation rate. The association rate of this oligonucleotide is on the order of 10(4) M(-1) s(-1) and is thus comparable to those of many other artificial antisense oligonucleotides. The remarkable inhibition efficacy is attributable to the dual effect of direct interference with substrate binding to the RNase P RNA active site and induction of misfolding of the catalytic core of RNase P RNA. Based on our findings, the P15 loop region of bacterial RNase P RNAs of the structural type A can be considered the "Achilles' heel" of the ribozyme and therefore represents a promising target for combatting multiresistant bacterial pathogens.
核糖核蛋白酶RNase P催化所有生命域中tRNA初级转录本的内切核酸5'成熟。RNase P对细菌细胞生长不可或缺，且细菌和真核生物RNase P酶在结构和功能上存在巨大差异，这符合将细菌酶作为潜在新型药物靶点的基本要求。在我们的体外前体tRNA（ptRNA）加工试验中，我们已鉴定出在亚纳摩尔浓度下就开始对结构A型细菌RNase P RNA（例如大肠杆菌或肺炎克雷伯菌的酶）表现出抑制作用的RNA寡核苷酸。这些寡核苷酸针对已知与ptRNA底物的3'-CCA部分相互作用的RNase P RNA的所谓P15环区域。先导探测实验表明，互补的RNA或DNA 14聚体完全侵入P15环区域，从而破坏RNase P RNA催化核心中的局部结构。由于解离速率非常低，RNA 14聚体的结合基本上是不可逆的。该寡核苷酸的缔合速率约为10⁴ M⁻¹ s⁻¹，因此与许多其他人工反义寡核苷酸相当。显著的抑制效果归因于直接干扰底物与RNase P RNA活性位点结合以及诱导RNase P RNA催化核心错误折叠的双重作用。基于我们的发现，结构A型细菌RNase P RNA的P环区域可被视为核酶的“阿喀琉斯之踵”，因此是对抗多重耐药细菌病原体的一个有前景的靶点。
