Nucleoside Transporters In HAART Mitochondrial Toxicity

描述（由申请人提供）：高效抗逆转录病毒疗法（HAART）的引入成功地延长了艾滋病毒/艾滋病患者的生命。然而，HAART是一种几乎总是含有核苷HIV逆转录酶抑制剂（NRTI）的联合治疗，长期使用HAART与主要毒性相关，包括肝损伤、神经病、胰腺炎、肌病、神经病、乳酸血症和脂肪代谢障碍，其中一些可能导致患者死亡。这些毒性的罪魁祸首被认为是HAART中的NRTI。这些核苷类药物，包括齐多夫定（AZT），司他夫定（d4 T），去羟肌苷（DDI），扎西他滨（DDC）和拉米夫定（3 TC）被认为是导致线粒体！不同程度的消耗，导致细胞死亡和组织毒性。这种线粒体消耗主要源于NRTI的三磷酸代谢物对线粒体（mt）DNA聚合酶γ的抑制。核苷进入线粒体和细胞通过称为核苷转运蛋白的专门的膜载体蛋白发生。一旦进入线粒体，核苷被线粒体激酶如线粒体特异性胸苷激酶（TK-2）等依次磷酸化，产生活性三磷酸代谢物，然后抑制mtDNA合成。最近已经表明，哺乳动物细胞中的线粒体在其膜中表达平衡型核苷转运蛋白1（ENT 1），并且这种表达增强了抗病毒核苷药物的线粒体毒性。根据这一观察结果，我们假设选择性抑制线粒体核苷转运蛋白可以阻止NRTI进入接受HAART的患者的线粒体，可以用作降低这些抗HIV药物的线粒体毒性的方法。在探索降低NTRI线粒体毒性的策略时，将追求以下具体目标。1)合成并表征核苷转运体抑制剂双嘧达莫的新型酯前药，包括ENT转运体抑制、细胞渗透和酶促酯水解。2)确定适当的双嘧达莫酯前药对抗抗HIV核苷的线粒体毒性的能力。方法将包括合成和分析化学，核苷转运蛋白结合和核苷摄取测定，流式细胞术和实时PCR。如果成功，这项研究将增加我们对核苷转运蛋白在线粒体毒性中作用的理解，并可能导致一种新的策略，用于减少或预防HIV/AIDS患者长期使用HAART的NRTI毒性，并提供一种新的药理学方法来解决核苷类似物抗病毒或抗癌药物的线粒体毒性，并为抑制线粒体核苷转运的作用提供了新的认识。

DESCRIPTION (provided by applicant): The introduction of highly active antiretroviral therapy (HAART) has been successful in prolonging the lives of HIV/AIDS patients. However, long-term use of HAART, which is a combination therapy that almost invariably contains nucleoside HIV reverse transcriptase inhibitors (NRTIs), is associated with major toxicities including liver damage, neuoropathy, pancreatitis, myopathy, neuropathy, lactic acidemia and lipodystrophy, some of which could result in patient fatalities. The culprits for these toxicities are believed to be the NRTIs in HAART. These nucleoside drugs, including zidovudine (AZT), stavudine (d4T), didanosine (DDI), zalcitabine (DDC) and lamivudine (3TC) are believed to cause mitochondria! depletion to varying extents, leading to cell death and tissue toxicity. This mitochondrial depletion stems mainly from the inhibition of mitochondrial (mt) DNA polymerase gamma by the triphosphate metabolites of NRTIs. The entry of nucleosides into mitochondria and cells occurs through specialized membrane carrier proteins termed nucleoside transporters. Upon entry into mitochondria, nucleosides are sequentially phosphorylated by mitochondrial kinases such as the mitochondrial-specific thymidine kinase (TK-2) and others to yield the active triphosphate metabolites, which then inhibit mtDNA synthesis. It has recently been shown that mitochondria in mammalian cells express the equilibrative nucleoside transporter 1 (ENT1) in their membranes and that this expression enhances mitochondrial toxicity of antiviral nucleoside drugs. In light of this observation, we hypothesize that selective inhibition of mitochondrial nucleoside transporters can prevent entry of NRTIs into mitochondria of patients undergoing HAART, can be used as an approach to reduce the mitochondrial toxicity of these anti-HIV drugs. The following specific aims will be pursued in exploring this strategy for reducing NTRI mitochondrial toxicity. 1) Synthesize and characterize novel ester prodrugs of the nucleoside transporter inhibitor dipyridamole in terms of ENT transporter inhibition, cellular permeation and enzymatic ester hydrolysis. 2) Determine the ability of the appropriate dipyridamole ester prodrug to protect against mitochondrial toxicity of anti-HIV nucleosides. Methods will include synthetic and analytical chemistry, nucleoside transporter binding and nucleoside uptake assays, flow cytometry and real-time PCR. If successful, this research will increase our understanding of the role of nucleoside transporters in mitochondrial toxicity and could lead to a new strategy for reducing or preventing NRTI toxicities of long-term use of HAART in HIV/AIDS patients, and provide a novel pharmacological approach to addressing mitochondrial toxicity of nucleoside analog antiviral or anticancer agents, as well as furnish new knowledge on the effects of inhibiting mitochondrial nucleoside transport in situ.