Date of Award

Summer 2019

Document Type

Open Access Dissertation

Department

College of Pharmacy

First Advisor

Jun Zhu

Abstract

More than 37 million people are living with HIV worldwide. Despite the widespread use of antiretroviral therapy (ART), up to 70% of HIV-positive individuals suffer from cognitive and behavioral deficits collectively known as HIV-associated neurocognitive disorders (HAND). HIV-mediated damage to the dopaminergic system is a mediating factor in HAND. Dopamine (DA) transporter (DAT)-mediated reuptake is essential for maintaining DA homeostasis. Because most ART cannot efficiently cross the blood-brain barrier (BBB), the brain serves as a viral reservoir that facilitates the spread of infection to microglia and astrocytes. Infected cells shed viral proteins such as Tat, which plays a critical role in HIV infection-induced dysregulation of the dopaminergic system by its allosteric inhibition of DAT. Because Tat does not compete with the DA uptake site, blocking the Tat-DAT interaction will have minimal effects on normal transporter function. In addition, cocaine, a DAT inhibitor, magnifies the effects of Tat on neurotoxicity, which contributes to HAND severity in cocaine-abusing HIV-infected patients. We hypothesize that Tat protein inhibits dopamine (DA) uptake by interacting with specific recognition binding residues on DAT thereby leading to dopaminergic dysregulation observed in HAND. Targeting the Tat-DAT interaction during the early stages of HIV-1 infection is therefore a potential therapeutic strategy to prevent HAND.

By utilizing integrated computational homology modeling and pharmacological validation, key recognition binding residues on human DAT that are critical to Tat-DAT interaction were identified. Mutations on these residues attenuated Tat-induced inhibition of DAT uptake while preserving (Y88F, D203L) or significantly enhancing (H547A) basal function of the transporter. H547A was found to enhance DAT function through alterations in phosphorylation, palmitoylation, and transporter conformation. Additionally, double (D206L/H547A) and triple (Y88F/D206L/H547A) combinations of these mutations blocked Tat inhibition. Introducing mutations on Tat1-86 (K19A, C22G) reversed the inhibitory effects of the protein, indicative of disrupted Tat-DAT binding.

Finally, to probe the therapeutic feasibility of developing allosteric modulators to block Tat and cocaine binding without interfering with normal DAT function, the SRI compounds, novel quinazoline-based allosteric modulators, were utilized. From the initial screening, SRI-32743 was selected due to its effective potency. SRI-32743 pharmacologically demonstrated allosteric properties and attenuated Tat inhibition in wild-type human DAT-expressing cells. When administered systemically to inducible HIV-1 Tat transgenic mice after 14 days of Tat induction via doxycycline, SRI-32743 dose-dependently ameliorated conditioned place preference for cocaine. These results demonstrate that developing allosteric modulatory molecules which attenuate cocaine and Tat binding to DAT will aid drug discovery efforts in the search for therapeutic interventions for HIV-infected patients who are concurrent abusers of cocaine.

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