Tan G T, Kinghorn A D, Hughes S H, Pezzuto J M
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago 60612.
J Biol Chem. 1991 Dec 15;266(35):23529-36.
Psychotrine dihydrogen oxalate and O-methylpsychotrine sulfate heptahydrate (MP), the salts of isoquinoline alkaloids from ipecac, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). We currently report the results of additional studies designed to characterize the mechanism of inhibition facilitated by MP. The inhibition was noncompetitive with respect to TTP and uncompetitive with respect to poly(rA) and oligo(dT)12-18 (4:1) at low template-primer concentrations but competitive at high concentrations (greater than 200 microM). Identical non-Michaelis-type kinetics were observed when activated DNA was used as the template. The biphasic nature of the double-reciprocal plots and Hill coefficients of less than 1 indicate that MP functions as an allosteric inhibitor of the enzyme which appears to possess multiple active sites that interact in a cooperative (negative) fashion in the presence of the inhibitor. MP was selective for the recombinant HIV-1 RT (p66) utilizing poly(rA) and oligo(dT)12-18 (4:1) as template-primer. Greater inhibition was observed with this template primer as compared with other natural and synthetic template-primers tested. MP had significantly less effect on avian myeloblastosis virus RT as well as mammalian or bacterial DNA and RNA polymerases. Other members of the ipecac class of alkaloids, e.g. emetine hydrochloride, were inactive against all of these enzymes, including HIV-1 RT. Conversely, MP did not inhibit in vitro protein synthesis, a property manifested by all the other ipecac alkaloids tested. Studies conducted with structural analogs revealed that the imine functionality at positions 1' and 2' of MP is the key structural requirement for HIV-1 RT inhibitory activity. Therefore, MP appears to possess unique structural properties that enable interaction with HIV-1 RT in a manner that can be differentiated from other polymerases. Use of these alkaloids for the definition of this viral enzyme-specific topology may lead to the development of therapeutically useful chemotherapeutic agents.
吐根中异喹啉生物碱的盐类——草酸氢吐根碱和七水合O-甲基吐根碱硫酸盐（MP），被发现是人类免疫缺陷病毒1型逆转录酶（HIV-1 RT）DNA聚合酶活性的有效抑制剂。我们目前报告了旨在表征MP促进抑制机制的其他研究结果。在低模板引物浓度下，该抑制作用对三磷酸胸苷（TTP）是非竞争性的，对聚（rA）和寡聚（dT）12 - 18（4:1）是反竞争性的，但在高浓度（大于200 microM）时是竞争性的。当使用活化的DNA作为模板时，观察到相同的非米氏型动力学。双倒数图的双相性质和小于1的希尔系数表明，MP作为该酶的变构抑制剂起作用，该酶似乎具有多个活性位点，在抑制剂存在下以协同（负）方式相互作用。MP对利用聚（rA）和寡聚（dT）12 - 18（4:1）作为模板引物的重组HIV-1 RT（p66）具有选择性。与测试的其他天然和合成模板引物相比，使用该模板引物时观察到更大的抑制作用。MP对禽成髓细胞瘤病毒RT以及哺乳动物或细菌的DNA和RNA聚合酶的影响明显较小。吐根类生物碱的其他成员，例如盐酸依米丁，对所有这些酶包括HIV-1 RT均无活性。相反，MP不抑制体外蛋白质合成，这是所有测试的其他吐根生物碱所具有的特性。对结构类似物进行的研究表明，MP 1'和2'位的亚胺官能团是HIV-1 RT抑制活性的关键结构要求。因此，MP似乎具有独特的结构特性，能够以一种可与其他聚合酶区分开的方式与HIV-1 RT相互作用。使用这些生物碱来定义这种病毒酶特异性拓扑结构可能会导致开发出具有治疗用途的化学治疗剂。
