Developing Selective Inhibitors and Probes for Concentrative Nucleoside Transporters

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 decrease NRTI reuptake and mitigate against mitochondrial toxicity. A
multidisciplinary approach combining computational and synthetic chemistry and cell-based biological testing will be
applied. The success of the project will make available highly anticipated probe compounds for studying the biology
of and pharmacology of this important transporters that are critical to the success of nucleoside drug therapies.

Grant Number: 5R21AI181894-02
NIH Institute/Center: NIH
Principal Investigator: John Buolamwini