PROJECT SUMMARY Although acute viral replication has been well controlled with combination antiretroviral therapies (cART) in the early stage of HIV infection long-term HIV-1 viral protein exposure within the CNS causes dysregulation of monoamine transmission, which has been implicated as a key factor in HIV associated neurocognitive and depression-like behavior deficits with concurrent cocaine abuse. Cocaine abuse has been shown to enhance the incidence of HIV-1 associated cognitive depression and exacerbate its severity. Dopamine (DA) plays a significant role in the prefrontal cortex, which is an important brain region for higher cognitive function. Both DA transporter (DAT) and norepinephrine transporter (NET) are responsible for maintaining dopaminergic homeostasis. While serotonin transporter (SERT) can transport not only serotonin but also DA, DA release is increased by inhibition of SERT. Among the viral proteins, Tat protein plays a significant role in viral replication in the early stage of HIV infection and in the pathophysiological effects on the development of cognitive depression impairments. Our published work has demonstrated that in vivo expression of Tat protein dysregulates monoamine transmission by its allosteric interaction with DAT and NET in inducible Tat transgenic (iTat-tg) mice. Furthermore, our preliminary studies demonstrate that in vivo Tat expression increases serotonin release and decreases serotonin uptake through SERT in the midbrain of iTat-tg mice. We have identified SRI- 32743, a novel allosteric modulator, which attenuates Tat-inhibited DA transport via DAT or NET and alleviates Tat-potentiated cognitive impairments and cocaine reward in iTat-tg mice. Through further structure activity relationship studies, we identified four analogs of SRI-32743 with improved solubility and metabolic stability. The four analogs display partial inhibitory capability for monoamine transporters and attenuate Tat-induced inhibition of DAT-mediated DA uptake through an allosteric mechanism. This R01 renewal application proposes to expand our ongoing studies of allosteric modulators interacting with DAT by identifying the unique allosteric modulatory sites on monoamine transporters for Tat and cocaine binding. We hypothesize that attenuating Tat and cocaine binding to monoamine transporters through novel allosteric modulators with minimal effects on physiological changes in monoaminergic transmission preserve neurocognitive function in HIV-infected individuals with the concurrent use of cocaine. Three specific aims are proposed in investigating this hypothesis: (1) Design and synthesize novel allosteric ligands in recognition for Tat and cocaine binding sites on monoamine transporters as potential probes for in vivo pharmacological studies; (2) Characterize the in vitro pharmacological profile of the compounds to identify optimized probe molecules; and (3) Determine the therapeutic role of novel allosteric modulators in alleviation ...