CNT3

CNT3 (concentrative nucleoside transporter 3)

Aliases: cib
Gene name: Solute carrier family 28 member 3 (SLC28A3)

Summary

Concentrative Nucleoside Transporter 3 (SLC28A3, CNT3) is a sodium-dependent transporter of naturally occurring nucleosides. CNT3 has a broad substrate specificity, accepting both purine and pyrimidine nucleosides, as well as anticancer and antiviral nucleoside derivative drugs. Current FDA and EMA recommendations do not include testing for CNT3 transporter liabilities

Localization and expression

CNT3 protein is expressed on the apical membrane of cells and can be found in the pancreas, trachea, bone marrow, mammary gland, and intestine. CNT3 is also present, although with modest expression levels, in the liver, lung, placenta, prostate, testis, as well as in some regions of the brain and heart. Also expressed in nasal mucosa, CNT3 is thought to contribute to nasal absorption of substrate drugs [1]. In HL 60 promyelocytic leukemia cells CNT3 expression increases with cell differentiation [2]. Although Ritzel et al. were originally unable to detect CNT3 in the kidney, it was later confirmed by others in this location [3]. Monocytes and monocyte-derived macrophages were also shown to express CNT3 [4]. A truncated but functional splice variant was reported by Errasti-Murugarren et al. to be expressed in the endoplasmic reticulum (ER) in many cell types [5]. Intracellular localization is a unique characteristic among CNTs. 

Function, physiology and clinically significant polymorphisms

CNT3, cloned by Ritzel et al. in 2001 [2], is a 72-kDa protein with a putative structure of 13 transmembrane domains [6]. Human CNT3 was shown to form a homotrimer [7]. As CNT3 is widely expressed and accepts both purine and pyrimidine-based substrates, it has the theoretical capacity for functionally substituting CNT1 and CNT2 wherever it is co-expressed with either of these. CNT3-mediated transport is electrogenic, and a sigmoidal dependence of uridine influx on Na+ concentration indicated a Na+/uridine coupling ratio of at least 2:1 [8]. A rare variant, Cys602Arg, was identified with a 1:1 ratio of cation/nucleoside transport [9]. A distinguishing feature of CNT3 among CNTs is the acceptance of protons in its ion-binding pocket, making it the only H+-coupled nucleoside transporter in mammals [5, 10]. Proton-dependent transport is most relevant to the ER-related function of the protein. 
The physiological role of this transporter has not been extensively investigated. Our general understanding that CNTs are mainly associated with cell differentiation and stress response derives from the studies of CNT1 and CNT2 (e. g. [11-13]). Notably, a recent study has shown that CNT3 transports deoxyadenosine monophosphate (dAMP), a nucleotide, albeit with about 500-fold less efficiently than it transports deoxyadenosine (dAdo) [14]. As dAMP affects intestinal cell growth, this interaction may be of physiological significance. In addition, CNT3 transports adenosine, and thus contributes to purinergic regulation [15]. Lactation has been shown to increase CNT3 mRNA levels 4-fold in lactating mammary epithelial cells, indicating a role for CNT3 in nucleoside accumulation in milk [16].
Important drug substrates of these proteins are listed comprehensively in [17], and include gemcitabine, fludarabine, cladribine, ddI, ddC, ribavirin, and entecavir [18].
CNT3 demonstrates low inter-individual diversity as only 10 nonsynonymous SNPs and 52 haplotypes were found in a study by Badagnani et al. Commonly occurring variants were tested for fludarabine and cladribine transport, but no significant changes were reported. Although the rare Gly367Arg SNP caused reduced transport of inosine and thymidine, interactions between the variant and clinically relevant drugs were not determined due to its low allele frequency. The glycine at position 367 is conserved not only in the SLC28A3 genes across mammalian species, but in SLC28A1 and SLC28A2 as well, indicating the significance of this residue [19]. 
A study from 2010 identified a haplotype with two separate nonsynonymous SNPs in the coding sequence, leading to decreased risk of hemolytic anemia in patients receiving ribavirin therapy, where the mutant haplotype is thought to take up less drug into the red blood cells, allowing them to survive [20]. 

Clinical significance

Considering its wide substrate specificity covering both purine and pyrimidine analogue drugs, CNT3 is an obvious choice to be tested against a broad range of anticancer and antiviral agents. The analysis of thiopurine-resistant cell lines showed lower mRNA levels of CNT3 [21], while ribavirin was shown to be less effective after CNT3 overexpression in ribavirin-resistant cells [22]. High expression of CNT3 in patients with chronic lymphocytic leukemia correlates with poor prognosis and, interestingly, immunostaining of the protein showed intracellular localization [23]. 

Regulatory requirements

Currently, there is no recommendation by the FDA or EMA for the evaluation of CNT2. However, in vitro evaluations may be required on a case-by-case basis.

References

1.    Al-Ghabeish, M., et al., Microarray Determination of the Expression of Drug Transporters in Humans and Animal Species Used for the Investigation of Nasal Absorption. Mol Pharm, 2015. 12(8): p. 2742-54.
2.    Ritzel, M.W., et al., Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). J Biol Chem, 2001. 276(4): p. 2914-27.
3.    Errasti-Murugarren, E., M. Pastor-Anglada, and F.J. Casado, Role of CNT3 in the transepithelial flux of nucleosides and nucleoside-derived drugs. J Physiol, 2007. 582(Pt 3): p. 1249-60.
4.    Minuesa, G., et al., Expression and functionality of anti-human immunodeficiency virus and anticancer drug uptake transporters in immune cells. J Pharmacol Exp Ther, 2008. 324(2): p. 558-67.
5.    Errasti-Murugarren, E., et al., A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside transporter-3 (hCNT3) protein localized in the endoplasmic reticulum. FASEB J, 2009. 23(1): p. 172-82.
6.    Podgorska, M., K. Kocbuch, and T. Pawelczyk, Recent advances in studies on biochemical and structural properties of equilibrative and concentrative nucleoside transporters. Acta Biochim Pol, 2005. 52(4): p. 749-58.
7.    Stecula, A., et al., Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer. Biochemistry, 2017. 56(27): p. 3475-3483.
8.    Ritzel, M.W., et al., Recent molecular advances in studies of the concentrative Na+-dependent nucleoside transporter (CNT) family: identification and characterization of novel human and mouse proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). Mol Membr Biol, 2001. 18(1): p. 65-72.
9.    Errasti-Murugarren, E., et al., Functional characterization of a nucleoside-derived drug transporter variant (hCNT3C602R) showing altered sodium-binding capacity. Mol Pharmacol, 2008. 73(2): p. 379-86.
10.    Smith, K.M., et al., The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics. J Biol Chem, 2005. 280(27): p. 25436-49.
11.    del Santo, B., et al., Developmental regulation of the concentrative nucleoside transporters CNT1 and CNT2 in rat liver. J Hepatol, 2001. 34(6): p. 873-80.
12.    Duflot, S., et al., ATP-sensitive K(+) channels regulate the concentrative adenosine transporter CNT2 following activation by A(1) adenosine receptors. Mol Cell Biol, 2004. 24(7): p. 2710-9.
13.    Aymerich, I., et al., Extracellular adenosine activates AMP-dependent protein kinase (AMPK). J Cell Sci, 2006. 119(Pt 8): p. 1612-21.
14.    Narumi, K., et al., Mutual role of ecto-5'-nucleotidase/CD73 and concentrative nucleoside transporter 3 in the intestinal uptake of dAMP. PLoS One, 2019. 14(10): p. e0223892.
15.    Pastor-Anglada, M. and S. Perez-Torras, Who Is Who in Adenosine Transport. Front Pharmacol, 2018. 9: p. 627.
16.    Alcorn, J., et al., Transporter gene expression in lactating and nonlactating human mammary epithelial cells using real-time reverse transcription-polymerase chain reaction. J Pharmacol Exp Ther, 2002. 303(2): p. 487-96.
17.    Molina-Arcas, M., F.J. Casado, and M. Pastor-Anglada, Nucleoside transporter proteins. Curr Vasc Pharmacol, 2009. 7(4): p. 426-34.
18.    Ma, Z., et al., Multiple SLC and ABC Transporters Contribute to the Placental Transfer of Entecavir. Drug Metab Dispos, 2017. 45(3): p. 269-278.
19.    Badagnani, I., et al., Functional analysis of genetic variants in the human concentrative nucleoside transporter 3 (CNT3; SLC28A3). Pharmacogenomics J, 2005. 5(3): p. 157-65.
20.    Doehring, A., et al., Role of nucleoside transporters SLC28A2/3 and SLC29A1/2 genetics in ribavirin therapy: protection against anemia in patients with chronic hepatitis C. Pharmacogenet Genomics, 2011. 21(5): p. 289-96.
21.    Fotoohi, A.K., et al., Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines. Biochem Biophys Res Commun, 2006. 343(1): p. 208-15.
22.    Ibarra, K.D. and J.K. Pfeiffer, Reduced ribavirin antiviral efficacy via nucleoside transporter-mediated drug resistance. J Virol, 2009. 83(9): p. 4538-47.
23.    Mackey, J.R., et al., Quantitative analysis of nucleoside transporter and metabolism gene expression in chronic lymphocytic leukemia (CLL): identification of fludarabine-sensitive and -insensitive populations. Blood, 2005. 105(2): p. 767-74.
24.    Vasko, B., et al., Inhibitor selectivity of CNTs and ENTs. Xenobiotica, 2019. 49(7): p. 840-851.