29 research outputs found
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Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer
Many anticancer and
antiviral drugs are purine or pyrimidine analogues,
which use membrane transporters to cross cellular membranes. Concentrative
nucleoside transporters (CNTs) mediate the salvage of nucleosides
and the transport of therapeutic nucleoside analogues across plasma
membranes by coupling the transport of ligands to the sodium gradient.
Of the three members of the human CNT family, CNT3 has the broadest
selectivity and the widest expression profile. However, the molecular
mechanisms of the transporter, including how it interacts with and
translocates structurally diverse nucleosides and nucleoside analogues,
are unclear. Recently, the crystal structure of vcCNT showed that
the prokaryotic homologue of CNT3 forms a homotrimer. In this study,
we successfully expressed and purified the wild type human homologue,
hCNT3, demonstrating the homotrimer by size exclusion profiles and
glutaraldehyde cross-linking. Further, by creating a series of cysteine
mutants at highly conserved positions guided by comparative structure
models, we cross-linked hCNT3 protomers in a cell-based assay, thus
showing the existence of hCNT3 homotrimers in human cells. The presence
and absence of cross-links at specific locations along TM9 informs
us of important structural differences between vcCNT and hCNT3. Comparative
modeling of the trimerization domain and sequence coevolution analysis
both indicate that oligomerization is critical to the stability and
function of hCNT3. In particular, trimerization appears to shorten
the translocation path for nucleosides across the plasma membrane
and may allow modulation of the transport function via allostery
Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer
Many anticancer and
antiviral drugs are purine or pyrimidine analogues,
which use membrane transporters to cross cellular membranes. Concentrative
nucleoside transporters (CNTs) mediate the salvage of nucleosides
and the transport of therapeutic nucleoside analogues across plasma
membranes by coupling the transport of ligands to the sodium gradient.
Of the three members of the human CNT family, CNT3 has the broadest
selectivity and the widest expression profile. However, the molecular
mechanisms of the transporter, including how it interacts with and
translocates structurally diverse nucleosides and nucleoside analogues,
are unclear. Recently, the crystal structure of vcCNT showed that
the prokaryotic homologue of CNT3 forms a homotrimer. In this study,
we successfully expressed and purified the wild type human homologue,
hCNT3, demonstrating the homotrimer by size exclusion profiles and
glutaraldehyde cross-linking. Further, by creating a series of cysteine
mutants at highly conserved positions guided by comparative structure
models, we cross-linked hCNT3 protomers in a cell-based assay, thus
showing the existence of hCNT3 homotrimers in human cells. The presence
and absence of cross-links at specific locations along TM9 informs
us of important structural differences between vcCNT and hCNT3. Comparative
modeling of the trimerization domain and sequence coevolution analysis
both indicate that oligomerization is critical to the stability and
function of hCNT3. In particular, trimerization appears to shorten
the translocation path for nucleosides across the plasma membrane
and may allow modulation of the transport function via allostery
Identification and Quantitative Assessment of Uremic Solutes as Inhibitors of Renal Organic Anion Transporters, OAT1 and OAT3
One
of the characteristics of chronic kidney disease (CKD) is the
accumulation of uremic solutes in the plasma. Less is known about
the effects of uremic solutes on transporters that may play critical
roles in pharmacokinetics. We evaluated the effect of 72 uremic solutes
on organic anion transporter 1 and 3 (OAT1 and OAT3) using a fluorescent
probe substrate, 6-carboxyfluorescein. A total of 12 and 13 solutes
were identified as inhibitors of OAT1 and OAT3, respectively. Several
of them inhibited OAT1 or OAT3 at clinically relevant concentrations
and reduced the transport of other OAT1/3 substrates <i>in vitro.</i> Review of clinical studies showed that the active secretion of most
drugs that are known substrates of OAT1/3 deteriorated faster than
the renal filtration in CKD. Collectively, these data suggest that
through inhibition of OAT1 and OAT3, uremic solutes contribute to
the decline in renal drug clearance in patients with CKD
Computational Discovery and Experimental Validation of Inhibitors of the Human Intestinal Transporter OATP2B1
Human organic anion
transporters (OATPs) are vital for the uptake
and efflux of drugs and endogenous compounds. Current identification
of inhibitors of these transporters is based on experimental screening.
Virtual screening remains a challenge due to a lack of experimental
three-dimensional protein structures. Here, we describe a workflow
to identify inhibitors of the OATP2B1 transporter in the DrugBank
library of over 5,000 drugs and druglike molecules. OATP member 2B1
transporter is highly expressed in the intestine, where it participates
in oral absorption of drugs. Predictions from a Random forest classifier,
prioritized by docking against multiple comparative protein structure
models of OATP2B1, indicated that 33 of the 5,000 compounds were putative
inhibitors of OATP2B1. Ten predicted inhibitors that are prescription
drugs were tested experimentally in cells overexpressing the OATP2B1
transporter. Three of these ten were validated as potent inhibitors
of estrone-3-sulfate uptake (defined as more than 50% inhibition at
20 ÎĽM) and tested in multiple concentrations to determine exact
IC<sub>50</sub>. The IC<sub>50</sub> values of bicalutamide, ticagrelor,
and meloxicam suggest that they might inhibit intestinal OATP2B1 at
clinically relevant concentrations and therefore modulate the absorption
of other concomitantly administered drugs
Cis eQTL analysis of SLC2A2 in human liver tissue
A three-stage genome wide association study (GWAS) reports the finding of the C-allele of rs8192675 in the intron of SLC2A2, which encodes the facilitated glucose transporter GLUT2, was associated with a 0.17% (p=6.6x10<sup>-14</sup>) greater metformin induced HbA1c reduction in 10,577 participants of European ancestry (doi:10.1038/ng.3632). Â The SNP, rs8192675 is the top cis-eQTL for SLC2A2 in 1,226 human liver samples, suggesting a key role for hepatic GLUT2 in regulation of metformin action. Here, we deposit the SLC2A2 transcript levels (see Supplemental Table A) in 583 human liver samples and their genotypes within the SLC2A2 region (see Supplemental Table B). The data here are referred to as data set 4 in the published GWAS (doi:10.1038/ng.3632)
Metformin Is a Substrate and Inhibitor of the Human Thiamine Transporter, THTR‑2 (SLC19A3)
The
biguanide metformin is widely used as first-line therapy for
the treatment of type 2 diabetes. Predominately a cation at physiological
pH’s, metformin is transported by membrane transporters, which
play major roles in its absorption and disposition. Recently, our
laboratory demonstrated that organic cation transporter 1, OCT1, the
major hepatic uptake transporter for metformin, was also the primary
hepatic uptake transporter for thiamine, vitamin B1. In this study,
we tested the reverse, i.e., that metformin is a substrate of thiamine
transporters (THTR-1, SLC19A2, and THTR-2, SLC19A3). Our study demonstrated
that human THTR-2 (hTHTR-2), SLC19A3, which is highly expressed in
the small intestine, but not hTHTR-1, transports metformin (<i>K</i><sub>m</sub> = 1.15 ± 0.2 mM) and other cationic compounds
(MPP<sup>+</sup> and famotidine). The uptake mechanism for hTHTR-2
was pH and electrochemical gradient sensitive. Furthermore, metformin
as well as other drugs including phenformin, chloroquine, verapamil,
famotidine, and amprolium inhibited hTHTR-2 mediated uptake of both
thiamine and metformin. Species differences in the substrate specificity
of THTR-2 between human and mouse orthologues were observed. Taken
together, our data suggest that hTHTR-2 may play a role in the intestinal
absorption and tissue distribution of metformin and other organic
cations and that the transporter may be a target for drug–drug
and drug–nutrient interactions
Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1)
Organic cation transporter
1 (OCT1) plays a critical role in the
hepatocellular uptake of structurally diverse endogenous compounds
and xenobiotics. Here we identified competitive and noncompetitive
OCT1-interacting ligands in a library of 1780 prescription drugs by
combining in silico and in vitro methods. Ligands were predicted by
docking against a comparative model based on a eukaryotic homologue.
In parallel, high-throughput screening (HTS) was conducted using the
fluorescent probe substrate ASP<sup>+</sup> in cells overexpressing
human OCT1. Thirty competitive OCT1 ligands, defined as ligands predicted
in silico as well as found by HTS, were identified. Of the 167 ligands
identified by HTS, five were predicted to potentially cause clinical
drug interactions. Finally, virtual screening of 29 332 metabolites
predicted 146 competitive OCT1 ligands, of which an endogenous neurotoxin,
1-benzyl-1,2,3,4-tetrahydroisoquinoline, was experimentally validated.
In conclusion, by combining docking and in vitro HTS, competitive
and noncompetitive ligands of OCT1 can be predicted
Discovery of Potent, Selective Multidrug and Toxin Extrusion Transporter 1 (MATE1, SLC47A1) Inhibitors Through Prescription Drug Profiling and Computational Modeling
The human multidrug and toxin extrusion (MATE) transporter
1 contributes to the tissue distribution and excretion of many drugs.
Inhibition of MATE1 may result in potential drug–drug interactions
(DDIs) and alterations in drug exposure and accumulation in various
tissues. The primary goals of this project were to identify MATE1
inhibitors with clinical importance or in vitro utility and to elucidate
the physicochemical properties that differ between MATE1 and OCT2
inhibitors. Using a fluorescence assay of ASP<sup>+</sup> uptake in
cells stably expressing MATE1, over 900 prescription drugs were screened
and 84 potential MATE1 inhibitors were found. We identified several
MATE1 selective inhibitors including four FDA-approved medications
that may be clinically relevant MATE1 inhibitors and could cause a
clinical DDI. In parallel, a QSAR model identified distinct molecular
properties of MATE1 versus OCT2 inhibitors and was used to screen
the DrugBank in silico library for new hits in a larger chemical space
Discovery of Potent, Selective Multidrug and Toxin Extrusion Transporter 1 (MATE1, SLC47A1) Inhibitors Through Prescription Drug Profiling and Computational Modeling
The human multidrug and toxin extrusion (MATE) transporter
1 contributes to the tissue distribution and excretion of many drugs.
Inhibition of MATE1 may result in potential drug–drug interactions
(DDIs) and alterations in drug exposure and accumulation in various
tissues. The primary goals of this project were to identify MATE1
inhibitors with clinical importance or in vitro utility and to elucidate
the physicochemical properties that differ between MATE1 and OCT2
inhibitors. Using a fluorescence assay of ASP<sup>+</sup> uptake in
cells stably expressing MATE1, over 900 prescription drugs were screened
and 84 potential MATE1 inhibitors were found. We identified several
MATE1 selective inhibitors including four FDA-approved medications
that may be clinically relevant MATE1 inhibitors and could cause a
clinical DDI. In parallel, a QSAR model identified distinct molecular
properties of MATE1 versus OCT2 inhibitors and was used to screen
the DrugBank in silico library for new hits in a larger chemical space
Gene Expression Profiling of Transporters in the Solute Carrier and ATP-Binding Cassette Superfamilies in Human Eye Substructures
The barrier epithelia of the cornea and retina control
drug and
nutrient access to various compartments of the human eye. While ocular
transporters are likely to play a critical role in homeostasis and
drug delivery, little is known about their expression, localization
and function. In this study, the mRNA expression levels of 445 transporters,
metabolic enzymes, transcription factors and nuclear receptors were
profiled in five regions of the human eye: cornea, iris, ciliary body,
choroid and retina. Through RNA expression profiling and immunohistochemistry,
several transporters were identified as putative targets for drug
transport in ocular tissues. Our analysis identified <i>SLC22A7</i> (OAT2), a carrier for the antiviral drug acyclovir, in the corneal
epithelium, in addition to <i>ABCG2</i> (BCRP), an important
xenobiotic efflux pump, in retinal nerve fibers and the retinal pigment
epithelium. Collectively, our results provide an understanding of
the transporters that serve to maintain ocular homeostasis and which
may be potential targets for drug delivery to deep compartments of
the eye