ABSTRACT: Nucleoside transporters (NTs) mediate the cellular transport of physiological nucleosides and many synthetic derivatives like the reverse transcriptase inhibitors (NRTIs) used in HIV/AIDS therapy. The emerging toxicity and adverse effects like potentially irreversible kidney toxicity, weight gain/metabolic syndrome and potential fetal abnormalities plaguing the most popular HIV/AIDS drugs tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF) and integrase inhibitor dolutegravir, makes relying on NRTIs, which constitute the backbones of many HIV/AID combination therapies, attractive. As such it is imperative to seek to optimize their therapeutic outcomes. Human (h) Concentrative Nucleoside Transporters (hCNTs) family comprises three members, hCNT1, 2 and 3, that mediate sodium-dependent nucleoside transport. In contrast to their ubiquitously expressed human Equilibrative Nucleoside Transporters (hENTs) counterparts, hENT1, hENT2, hENT3 and hENT4, hCNTs are restricted in tissue distribution, being prevalent in absorptive tissues. Unlike hENTs with broad substrate specificity, CNTs have limited substrate specificities, with hCNT1 and hCNT2 preferring pyrimidine and purine nucleoside substrates, respectively, while hCNT3 transports both nucleoside classes. They occur on the luminal side of kidneys and are principal drivers of reabsorption of nucleoside drugs like the NRTIs, which could result in toxicities. The time is now to optimize NRTI therapies, and one way is to address their potential mitochondrial toxicity (mitotoxicity), which is very troubling in early drugs like zalcitabine (ddC) and didanosine (ddI), limiting use. The current frequently used NRTIs, zidovudine (AZT), lamivudine (3TC) and emtricitabine (FTC) also harbor mitotoxicity, particularly AZT, that has not been addressed. The goals of this research are to develop hCNT subtype selective inhibitors for which there is a woeful lack, hampering the study of hCNTs’ biology and pharmacology, and for therapeutic applications like blocking drug reabsorption to mitigate against mitotoxicity. Unlike their hENT counterparts, for which there are specific inhibitors with IC50’s down to nM levels, hCNTs lack them. Of note, the standard hCNT inhibitor, phloridzin (PHZN), has hCNT1 inhibitory IC50 as high as 250 μM, with low subtype selectivity, and also inhibits sodium-glucose transporters (SGLTs) even more potently. We are applying a multidisciplinary approach comprising structure- and ligand-based design, synthesis, cell-based bioassays and ADMET to discover and optimize potent hCNT subtype selective/specific inhibitors. We have already identified lead compounds with IC50 values down to 2 μM, and up to 25-fold improvement over phloridzin against hCNT1. Our specific aims are: 1) to discover and optimize potent subtype selective hCNT inhibitors, to be used as biological tools, and 2) to use hCNT3-specific/selective inhibitor for proof-of-concept that targeting hCNT3 can decreas...