The disruption of dopamine (DA) neurotransmission by the HIV-1 transactivator of transcription (Tat) during HIV-1 infection has been linked to the development of neurocognitive disorders, even under combined antiretroviral therapy treatment. We have demonstrated that Southern Research Institute (SRI) 32742, a novel allosteric modulator of DA transporter (DAT), attenuates cocaine- and Tat-binding to DAT, alleviates Tat-induced cognitive deficits and potentiation of cocaine reward in inducible Tat transgenic mice. The current study determined the in vitro pharmacological profile of SRI-32743 and its optimized second-generation analogs and their effects as allosteric modulators. Through structure-activity relationship studies of SRI-32743, 170 compounds were synthesized and evaluated for their ability to modulate DAT function. We identified 21 analogs as atypical competitors of DAT (maximum attributable drug effect, ≤60%). Four compounds, SRI-46564, SRI-47056, SRI-46286, and SRI-47867, displayed IC₅₀ values for [³H]DA uptake inhibition from 9.33 ± 0.50 to 0.96 ± 0.05 μM and from 3.96 ± 1.36 to 1.29 ± 0.19 for DAT binding, respectively. The 4 analogs also displayed high potency at 2 different concentrations (0.5 nM and 0.05 nM) to attenuate Tat-induced inhibition of [³H]DA uptake and cocaine-mediated dissociation of [³H]WIN35,428 binding in Chinese hamster ovary cells expressing human DAT, suggesting that the effects occur through an allosteric mechanism. In further ex vivo studies using fast scan cyclic voltammetry, we demonstrated that the analogs do not disrupt the baseline phasic-like DA release. These findings provide a new insight into the potential for development of novel therapeutic agents to attenuate DAT-Tat interactions to normalize DA neurotransmission in NeuroHIV. SIGNIFICANCE STATEMENT: The allosteric inhibition of the dopamine (DA) transporter by the HIV-1 transactivator of transcription (Tat) disrupts DA homeostasis, leading to HIV-associated neurocognitive disorders. Analogs of Southern Research Institute 32743, a novel allosteric modulator of the Tat-DA transporter (DAT) interaction, were evaluated in the current study and characterized as atypical ligands of DA uptake. Four novel lead compounds demonstrated high potency to attenuate Tat-induced inhibition of human DAT-mediated DA uptake in an allosteric modulatory manner with no effects on the dynamics of DA uptake-release in DAT.

Prolonged exposure to HIV-1 transactivator of transcription (Tat) protein dysregulates monoamine transmission, a physiological change implicated as a key factor in promoting neurocognitive disorders among people living with HIV. We have demonstrated that in vivo expression of Tat in Tat transgenic mice decreases dopamine uptake through both dopamine transporter (DAT) and norepinephrine transporter (NET) in the prefrontal cortex. Further, our novel allosteric inhibitor of monoamine transporters, SRI-32743, has been shown to attenuate Tat-inhibited dopamine transport through DAT and alleviates Tat-potentiated cognitive impairments. The current study reports the pharmacological profiles of SRI-32743 in basal and Tat-induced inhibition of human NET (hNET) function. SRI-32743 exhibited less affinity for hNET binding than desipramine, a classical NET inhibitor, but displayed similar potency for inhibiting hDAT and hNET activity. SRI-32743 concentration-dependently increased hNET affinity for [³H]DA uptake but preserved the V_max of dopamine transport. SRI-32743 slowed the cocaine-mediated dissociation of [³H]Nisoxetine binding and reduced both [³H]DA and [³H]MPP+ efflux but did not affect d-amphetamine-mediated [³H]DA release through hNET. Finally, we determined that SRI-32743 attenuated a recombinant Tat_1–86-induced decrease in [³H]DA uptake via hNET. Our findings demonstrated that SRI-32743 allosterically disrupts the recombinant Tat_1–86–hNET interaction, suggesting a potential treatment for HIV-infected individuals with concurrent cocaine abuse.