Keppler Brian R, Jarstfer Michael B
School of Pharmacy, Division of Medicinal Chemistry and Natural Products, University of North Carolina, Chapel Hill, North Carolina 27599-7360, USA.
Biochemistry. 2004 Jan 20;43(2):334-43. doi: 10.1021/bi035583e.
Telomerase is a ribonucleoprotein complex that acts as a reverse transcriptase in the maintenance of chromosome ends. Because the vast majority of cancer cells require telomerase activity, telomerase has become a target for anticancer drug discovery. Here, we describe a new approach for targeting telomerase by blocking the association between the telomerase catalytic subunit, hTERT, and key elements of the human telomerase RNA subunit, hTR. By examining the effects of oligonucleotides that hybridize to various regions of hTR, we identified two regions of the RNA subunit that are sensitive to molecular interactions leading to telomerase inhibition. Oligonucleotides that hybridize to either the P3/P1 pairing region or to the CR4-CR5 domain of hTR, hTRas009, and hTRas010, respectively, inhibit telomerase activity when added to recombinant hTERT and hTR prior to assemblage. However, addition of hTRas009 or hTRas010 to preassembled telomerase resulted in little or no inhibition. We also examined the ability of hTRas009 and hTRas010 to inhibit binding of hTR and hTR fragments to hTERT. We found that hTRas009 inhibited approximately 50% of the maximum binding between the pseudoknot fragment of hTR (nucleotides 46-209) and hTERT, whereas hTRas010 inhibited over 90% of the maximum binding between the CR4-CR5 fragment of hTR (nucleotides 243-328) and hTERT. In addition, neither oligonucleotide was able to appreciably inhibit the binding of full-length hTR to hTERT, although both oligonucleotides used in conjunction decreased binding by approximately 50%. We propose that the P3/P1 pairing region and CR4-CR5 domain represent viable targets to inhibit telomerase by perturbing proper assemblage of the active complex.
端粒酶是一种核糖核蛋白复合体，在维持染色体末端时作为逆转录酶发挥作用。由于绝大多数癌细胞需要端粒酶活性，端粒酶已成为抗癌药物研发的靶点。在此，我们描述了一种通过阻断端粒酶催化亚基hTERT与人端粒酶RNA亚基hTR的关键元件之间的结合来靶向端粒酶的新方法。通过检测与hTR不同区域杂交的寡核苷酸的作用，我们确定了RNA亚基中对导致端粒酶抑制的分子相互作用敏感的两个区域。分别与hTR的P3/P1配对区域或CR4-CR5结构域杂交的寡核苷酸hTRas009和hTRas010，在组装前添加到重组hTERT和hTR中时可抑制端粒酶活性。然而，将hTRas009或hTRas010添加到预先组装好的端粒酶中几乎没有抑制作用。我们还检测了hTRas009和hTRas010抑制hTR及其片段与hTERT结合的能力。我们发现，hTRas009抑制了hTR假结片段（核苷酸46 - 209）与hTERT之间最大结合的约50%，而hTRas010抑制了hTR的CR4-CR5片段（核苷酸243 - 328）与hTERT之间最大结合的90%以上。此外，尽管两种寡核苷酸联合使用可使结合减少约50%，但两种寡核苷酸均不能明显抑制全长hTR与hTERT的结合。我们认为，P3/P1配对区域和CR4-CR5结构域是通过干扰活性复合体的正确组装来抑制端粒酶的可行靶点。
