Anti-HIV NRTIs and the lysosomal toxicity

项目总结 核苷逆转录酶抑制剂(NRTI)具有很强的抗HIV活性，但它们的治疗作用 接受NRTI治疗的患者因严重的药物不良反应(ADR)而受益有限，因此 患者依从性差，药物疗效受损。我们的团队最近描述了一个不可或缺的 溶酶体核苷转运体ENT3通过缺失ENT3在小鼠溶酶体内稳态中的作用。 有趣的是，ENT3KO小鼠表现出与NRTI adr非常相似的临床表型。总体目标 这一应用的目的是评估ENT3缺失驱动的溶酶体毒性，作为一种可能的参与 NRTIs的慢性不良后遗症。这一提议的中心假设是，不干预的国家RTI 使用ENT3支持的溶酶体动态平衡或包含溶酶体信号转导试剂将使 NRTI不良反应的发生。目标1将评估在不影响药物的情况下避免NRTI毒性的策略 功效。我们关于这一目标的工作假设是，ENT3和NRTI之间相互作用的中断 加入溶酶体-自噬途径的药物激动剂将减轻急性胰腺炎的发病和严重程度 NRTI ADR.证实细胞表面NRTI转运体参与的初步研究 (例如，ENT1、CNT)而不是溶酶体ENT3对NRTI的有效性、AMPK和mTOR的错误调节 Ent3-/-小鼠的信号转运轴，以及使用A/A对Ent3-/-小鼠多器官功能障碍的挽救 药理AMPK激动剂AICAR；所有人都支持这一目标。目标2将阐明(S)的作用机制 NRTI特异性ADR征象的出现。我们对这一目标的工作假设是，当NRTIs存在于 临床相关的血液浓度，会抑制ENT3调节的成体干细胞功能，导致 破坏组织修复和再生。此外，我们假设NRTIs将不同地影响 ENT3在成人组织中发挥作用，带来不同的炎症、代谢和退行性变化 干细胞的改变，将解释临床观察到的NRTI ADR征象。初步研究表明， 证明通过ENT3转运许多产生ADR的NRTI，即溶酶体腺苷的抑制 NRTIs转运和Ent3-/-小鼠成体后腺苷溶酶体循环的扰动 干细胞耗尽，组织炎症和退化，以及中胚层组织完整性的破坏， 这些因素加在一起支持了这一目标。该项目将利用生化和分子方法，新颖 ENT3探针、新产生的ENT3小鼠模型、代谢组学、组织工程、药效团 建模、综合和筛选程序以及PKPD以实现目标。圆满完成 将为NRTI ADR的发生机制提供新的见解，并可能 优化治疗的转化效益(如长期疗效、依从性、耐受性等)在……里面 接受NRTI治疗的患者。

PROJECT SUMMARY The nucleoside reverse transcriptase inhibitors (NRTIs) have potent activities against HIV, but their therapeutic benefit in patients undergoing NRTI therapies is limited by significant adverse drug reactions (ADR), resulting in poor patient compliance and compromised drug efficacy. Our group has recently described an indispensable role of a lysosomal nucleoside transporter ENT3 in lysosomal homeostasis via deletion of ENT3 in mice. Intriguingly, ENT3 KO mice manifest clinical phenotypes closely resembling NRTI ADR. The overall objective of this application is to evaluate ENT3-loss driven lysosomal toxicity as a putative mechanism involved in the chronic adverse sequelae of NRTIs. The central hypothesis of this proposal is that NRTIs that do not interfere with ENT3-supported lysosomal homeostasis or the inclusion of lysosomal signaling agents will minimize the occurrence of NRTI ADR. Aim 1 will evaluate the strategies to avoid NRTI toxicity without compromising drug efficacy. Our working hypothesis for this aim is that disruption of interaction between ENT3 and NRTIs or the inclusion of pharmacological agonists of lysosomal-autophagy pathway will mitigate the onset and severity of NRTI ADR. The preliminary studies that demonstrate the involvement of the cell surface NRTI transporters (e.g., ENT1, CNT) and not the lysosomal ENT3 for NRTI efficacy, the misregulation of the AMPK and mTOR signaling axis in the Ent3-/- mice, and the functional rescue of multi-organ dysfunction in Ent3-/- mice using a pharmacological AMPK activator AICAR; all support this aim. Aim 2 will elucidate the mechanism(s) of occurrence of NRTI-specific ADR signs. Our working hypothesis for this aim is that NRTIs, when present at clinically relevant blood concentrations, will inhibit the ENT3-regulated adult stem cell functions resulting in disruption of tissue repair and regeneration. In addition, we hypothesize that NRTIs will differentially impact the ENT3 function in adult tissues to bring distinct inflammatory, metabolic and degenerative changes that coupled with stem cell alterations, will explain the clinically observed NRTI ADR signs. The preliminary studies that demonstrate the transport of many ADR-producing NRTIs by ENT3, the inhibition of lysosomal adenosine transport by NRTIs and the perturbation of lysosomal recycling of adenosine in Ent3-/- mice leading to adult stem cell exhaustion, tissue inflammation and degeneration, and breaches of mesodermal tissue integrity, which taken together supports this aim. The project will utilize biochemical and molecular approaches, novel ENT3 probes, newly generated ENT3 mouse models, metabolomics, tissue engineering, pharmacophore modeling, synthetic and screening procedures and PKPD to accomplish the goals. The successful completion of the project will provide new insights into the mechanisms of occurrence of NRTI ADR and may have translational benefit for optimizing treatments (such as long-term efficacy, adherence, tolerability, etc.) in patients undergoing NRTI therapies.