Mechanism of polyoxometalate-mediated inactivation of DNA polymerases: an analysis with HIV-1 reverse transcriptase indicates specificity for the DNA-binding cleft.
Mechanism of polyoxometalate-mediated inactivation of DNA polymerases: an analysis with HIV-1 reverse transcriptase indicates specificity for the DNA-binding cleft.
复制标题
DOI：
10.1042/bj3190619
复制
发表时间：
1996-10
期刊：
影响因子：
--
通讯作者：
S. Sarafianos;U. Kortz;M. T. Pope;M. Modak
中科院分区：
文献类型：
--
作者：
S. Sarafianos;U. Kortz;M. T. Pope;M. Modak
测定了一个多金属氧酸盐结构家族的抗DNA聚合酶活性。该家族中具有代表性的两个化合物[(O_3POPO_3)_4W_(12)O_(36)]16-(多金属氧酸盐I)和[(O_3PCH_2PO_3)_4W_(12)O_(36)]16-(多金属氧酸盐II)对所有被测DNA聚合酶都有抑制作用，IC50值在2~10微米之间。以HIV-1逆转录酶(RT)和Klenow聚合酶为代表的DNA聚合酶的比较研究表明，DNA的包涵体可以防止DNA失活，但不能通过脱氧核苷酸三磷酸(DNTPs)来实现。动力学分析表明，HIV-1RT抑制模式相对于DNA是竞争性的，而对于dNTP结合是非竞争性的。交联实验证实，这些抑制剂干扰了HIV-1逆转录酶的DNA结合功能。有趣的是，HIV-1RT的一些耐药突变株对多金属氧酸盐的敏感性与野生型HIV-1RT相当，这表明这些多金属氧酸盐在一个新的位点上相互作用。因为不同的聚合酶含有不同尺寸的DNA结合裂隙，所以应该有可能对多金属氧酸盐进行修饰，或者在酶特异性药物上添加一个链接，以便开发出更有效的抑制剂。利用HIV-1 RT的计算机模型，我们在一个二元复合体(酶-多金属氧酸盐I)中进行了对接研究，初步提出了HIV-1 RT中可能的相互作用部位，与现有的生化结果以及两个分子的几何和电荷限制一致。
The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12O36]16- (polyoxometalate I) and [(O3PCH2PO3)4W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 microM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme-polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules.