Impact of HIV-1 Tat protein on cocaine-dopamine transporter interaction

描述（由申请人提供）：在有效抗逆转录病毒治疗的时代，HIV-1相关神经认知障碍（HAND）仍然非常普遍。中枢神经系统内的HIV-1感染在HAND的发生发展中起着重要作用。滥用药物，如可卡因，已被证明会增加HAND的发病率，并通过增强病毒复制加剧HAND的严重程度。然而，可卡因和HAND进展之间的机械联系仍然不明确。虽然许多神经递质系统的变化可能有助于HAND，但中枢多巴胺（DA）系统在HAND患者神经认知功能障碍的发展和可卡因的精神兴奋作用的控制中起着至关重要的作用。HIV-1达特蛋白与可卡因的相互作用增加多巴胺能脑区内突触DA水平和达特释放。长期暴露于升高的DA和达特最终导致DA缺乏，从而加重HAND的严重程度并加速其进展。抗逆转录病毒药物不能阻止HIV-1病毒蛋白，如达特蛋白在含前病毒的脑细胞中的产生。目前还不清楚HIV-1阳性可卡因滥用者的DA系统是如何改变的。因此，迫切需要确定HIV-1感染导致的DA系统受损影响可卡因滥用者HAND进展的分子机制。突触前多巴胺转运体（DAT）是达特和可卡因作用于多巴胺系统的主要分子靶点，对神经认知功能具有重要作用。在本申请中，我们假设达特通过DAT中的变构结合位点，增强了 可卡因对DA转运的影响是HAND患者发生DA系统功能障碍的关键。我们的实验将研究达特和可卡因如何通过它们在人DAT上的识别结合位点与人DAT相互作用，从而导致DA系统功能障碍。我们的策略包括创建一个动态的三维计算模型来预测潜在的达特和可卡因结合口袋残基的人DAT，验证这些残基通过定点诱变，并分析随之而来的功能变化的达特和DAT的相互作用在神经元细胞和原代神经元。该申请的完成将确定DAT上的分子靶标，用于开发特异性阻断DAT中的达特结合位点并稳定生理多巴胺能张力的化合物，这应该有利于保留同时滥用可卡因的HAND患者的神经认知功能。

DESCRIPTION (provided by applicant): HIV-1 associated neurocognitive disorders (HAND) remain highly prevalent in the era of effective antiretroviral therapy. HIV-1 infection within CNS system plays a central role in the development of HAND. Drugs of abuse, such as cocaine, have been shown to increase the incidence and exacerbate the severity of HAND by enhancing viral replication. However, the mechanistic links between cocaine and HAND progression remain undefined. Although changes in many neurotransmitter systems may contribute to HAND, the central dopamine (DA) system plays a crucial role in the development of neurocognitive dysfunction in HAND patients and in the control of psychostimulant action of cocaine. The interplay of HIV-1 Tat protein with cocaine augments synaptic DA level and Tat release within dopaminergic brain regions. Long lasting exposure to elevated DA and Tat eventually lead to DA deficit that potentiates severity and accelerates progression of HAND. Antiretroviral agents cannot prevent the production of HIV-1 viral proteins, such as Tat protein, in proviral-containing brain cells. It is unclear how the DA system is altered in HIV-1 positive cocaine abusers. Therefore, there is a pressing need to define the molecular mechanism(s) by which the impaired DA system by HIV-1 infection affects the progression of HAND in concurrent cocaine abusers. Presynaptic DA transporter (DAT), which is critical for neurocognitive function, is a major molecular target for both Tat and cocaine to impact the DA system. In this application, we hypothesize that Tat, via allosteric binding sites in the DAT, potentiates inhibitory effects of cocaine on DA transport, which is the key to DA system dysfunction occurred in HAND patients. Our proposed experiments will investigate how Tat and cocaine interact with the human DAT through their recognition binding sites on human DAT, thereby leading to dysfunction of the DA system. Our strategy encompasses creating a dynamic 3D computational model to predict potential Tat and cocaine binding pocket residues of human DAT, validating these residues via site-directed mutagenesis, and analyzing the consequent functional changes of the Tat and DAT interaction in neuronal cells and primary neurons. The completion of this application will identify molecular targets on the DAT for developing compounds that specifically block Tat binding site(s) in DAT and stabilize physiological dopaminergic tone, which should be beneficial to the preservation of neurocognitive function in patients with HAND in concurrent cocaine abusers.