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）的引入已经成功地延长了艾滋病毒/艾滋病患者的生命。然而，长期使用HAART（一种几乎总是含有核苷类HIV逆转录酶抑制剂（NRTIs）的联合疗法）与主要毒性相关，包括肝损伤、神经病变、胰腺炎、肌病、神经病变、乳酸血症和脂肪营养不良，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（d4T），二腺苷（DDI），齐西他滨（DDC）和拉米夫定（3TC），被认为会引起线粒体！不同程度的耗竭，导致细胞死亡和组织毒性。这种线粒体耗竭主要源于NRTIs的三磷酸代谢物对线粒体（mt） DNA聚合酶的抑制。核苷通过称为核苷转运蛋白的特殊膜载体蛋白进入线粒体和细胞。进入线粒体后，核苷依次被线粒体激酶磷酸化，如线粒体特异性胸苷激酶（TK-2）和其他激酶，产生活性三磷酸代谢物，然后抑制mtDNA的合成。最近有研究表明，哺乳动物细胞的线粒体在其膜中表达平衡核苷转运蛋白1 (ENT1)，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NRTIs进入HAART患者的线粒体，可以作为降低这些抗hiv药物线粒体毒性的一种方法。以下具体目标将在探索这一策略，以减少NTRI线粒体毒性。1)合成核苷转运体抑制剂双嘧达莫的新型酯前药，并从ENT转运体抑制、细胞渗透和酶促酯水解等方面进行表征。2)确定合适的双嘧达莫酯前药对抗hiv核苷线粒体毒性的保护能力。方法将包括合成和分析化学，核苷转运体结合和核苷摄取测定，流式细胞术和实时PCR。如果成功，本研究将增加我们对核苷转运体在线粒体毒性中的作用的理解，并可能导致减少或预防长期使用HAART的HIV/AIDS患者NRTI毒性的新策略，并为解决核苷类似物抗病毒或抗癌药物的线粒体毒性提供新的药理学方法，以及提供关于抑制线粒体核苷原位运输的新知识。

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.