CNT2

CNT2 (concentrative nucleoside transporter 2)

Aliases: HCNT2, HsT17153, SPNT1
Gene name:  Solute carrier family 28 member 2 (SLC28A2)

Summary

Concentrative Nucleotide Transporter 2 (SLC28A1, CNT2), also referred as SPNT (sodium-dependent purine nucleoside transporter), is a sodium-dependent transporter mainly for purine nucleosides and uridine. Besides naturally occurring nucleosides, CNT2 is capable of transporting nucleoside-based anticancer and antiviral drugs. Current FDA and EMA recommendations do not include testing for CNT2 transporter liabilities.

Localization and expression

CNT2, cloned in 1998 by Ritzel et al. [1], is expressed in various tissues including the kidney, liver, heart, placenta, pancreas, spleen, skeletal muscle, colon, rectum and small intestine [2, 3]. Expression of CNT2 was quantifiable by LC-MS/MS-based proteomics in microsomal fractions of intestinal tissue [4].
CNT2 was also found ubiquitously in the immune system [3, 5]. CNT2 is widespread in the brain, being most abundant in the amygdala, hippocampus, cerebellum and certain neocortical regions [6]. CNT2 is the only concentrative nucleoside transporter that is present in myocytes [7]. The expression levels of CNT2 in rat lymphocytes were shown to be modulated by insulin [8], while experiments showed that the mRNA levels of rCnt2 were significantly altered in the diabetic heart, liver, and kidney [9]. Studies conducted on hepatocytes have indicated that hepatocarcinogenesis leads to a selective loss of CNT2 [10]. Histone hypoacetylation in colorectal cancer due to increased expression of histone deacetylase (HDAC) 7 results in chromatin condensation and reduced expression of CNT2, leading to resistance to CNT2 substrate nucleoside analogs; consistently, HDAC inhibitors were shown to counter resistance [11].

Function, physiology, and clinically significant polymorphisms

CNT2 is a 72-kDa protein with a putative structure of 13 transmembrane domains. CNT2 preferably transports purine nucleosides, but uridine is also a substrate of the transporter [12]. The stoichiometry of nucleoside:sodium transport appears to be 1:1 [13]. 
Extracellular level of adenosine, a substrate of several nucleoside transporters with CNT2 among them [14] is responsible for the regulation of anti-inflammatory signaling [15] and energy pathways across the body [16, 17]. Extracellular adenosine also offers a cardioprotective effect during ischemic stress or high myocardial workload [18], and counteracts neuronal damage caused by metabolic or physiological stress [19]. Adenosine uptake through the BBB is also modulated by CNT2 [20]. Important drug substrates of the transporter include anticancer and antiviral nucleoside analogues, and have been neatly summarized in a couple of reviews (e.g. [12, 21]).
CNT2 expression, similar to that of CNT1, is coupled with cell differentiation, as differentiation inducing glucocorticoids increase CNT2 protein and mRNA levels in rats [22]. In contrast to CNT1, CNT2 has a lower overall genetic variation at the amino acid level. Only six non-synonymous variants and 17 haplotypes were described by Owen et al [23]. No change in ribavirin transport by these variants was observed, although uridine transport by variant Phe355Ser was decreased. The study of Li et al. investigating CNT2 mutations from Asian populations found variant Glu385Lys to be less sensitive to uridine, inosine and ribavirin [24].

Clinical significance

Transport of naturally occurring purines contributes to the development of hyperuricemia. Inhibiting CNT2-mediated absorption of these nucleosides in the intestine can lower the urinary excretion of uric acid, the end product of purine degradation [25]. CNT2 is also considered to play an important role in the absorption of purine nucleoside analogs [25].
Purine analog nucleoside drugs are highly effective against blood cancers. For example, drug sensitivity of resistant leukemia cells against fluoropyrimidine nucleosides can be increased by CNT2 transgenic expression [13]. CNT2 transports antiviral drugs, such as didanosine (ddI) and ribavirin, which are used in HIV and Hepatitis C therapy. It also transports entecavir that has high efficacy against Hepatitis B Virus (HBV) and used as a first line treatment in chronic hepatitis B infections [26].

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.    Ritzel, M.W., et al., Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine. Mol Membr Biol, 1998. 15(4): p. 203-11.
2.    Che, M., D.F. Ortiz, and I.M. Arias, Primary structure and functional expression of a cDNA encoding the bile canalicular, purine-specific Na(+)-nucleoside cotransporter. J Biol Chem, 1995. 270(23): p. 13596-9.
3.    Pennycooke, M., et al., Differential expression of human nucleoside transporters in normal and tumor tissue. Biochem Biophys Res Commun, 2001. 280(3): p. 951-9.
4.    Nakamura, K., et al., Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM. Proteomics, 2016. 16(15-16): p. 2106-17.
5.    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.
6.    Guillen-Gomez, E., et al., Distribution of CNT2 and ENT1 transcripts in rat brain: selective decrease of CNT2 mRNA in the cerebral cortex of sleep-deprived rats. J Neurochem, 2004. 90(4): p. 883-93.
7.    Barnes, K., et al., Distribution and functional characterization of equilibrative nucleoside transporter-4, a novel cardiac adenosine transporter activated at acidic pH. Circ Res, 2006. 99(5): p. 510-9.
8.    Sakowicz, M., A. Szutowicz, and T. Pawelczyk, Differential effect of insulin and elevated glucose level on adenosine transport in rat B lymphocytes. Int Immunol, 2005. 17(2): p. 145-54.
9.    Pawelczyk, T., M. Podgorska, and M. Sakowicz, The effect of insulin on expression level of nucleoside transporters in diabetic rats. Mol Pharmacol, 2003. 63(1): p. 81-8.
10.    Dragan, Y., et al., Selective loss of nucleoside carrier expression in rat hepatocarcinomas. Hepatology, 2000. 32(2): p. 239-46.
11.    Ye, C., et al., Inhibition of histone deacetylase 7 reverses concentrative nucleoside transporter 2 repression in colorectal cancer by up-regulating histone acetylation state. Br J Pharmacol, 2018. 175(22): p. 4209-4217.
12.    Pastor-Anglada, M., et al., SLC28 genes and concentrative nucleoside transporter (CNT) proteins. Xenobiotica, 2008. 38(7-8): p. 972-94.
13.    Lang, T.T., et al., Acquisition of human concentrative nucleoside transporter 2 (hcnt2) activity by gene transfer confers sensitivity to fluoropyrimidine nucleosides in drug-resistant leukemia cells. Mol Pharmacol, 2001. 60(5): p. 1143-52.
14.    Pastor-Anglada, M. and S. Perez-Torras, Who Is Who in Adenosine Transport. Front Pharmacol, 2018. 9: p. 627.
15.    Thiel, M., et al., Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury. PLoS Biol, 2005. 3(6): p. e174.
16.    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.
17.    Aymerich, I., et al., Extracellular adenosine activates AMP-dependent protein kinase (AMPK). J Cell Sci, 2006. 119(Pt 8): p. 1612-21.
18.    Shryock, J.C. and L. Belardinelli, Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology. Am J Cardiol, 1997. 79(12A): p. 2-10.
19.    Cunha, R.A., Adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles, different sources and different receptors. Neurochem Int, 2001. 38(2): p. 107-25.
20.    Pardridge, W.M., Blood-brain barrier drug targeting: the future of brain drug development. Mol Interv, 2003. 3(2): p. 90-105, 51.
21.    Errasti-Murugarren, E. and M. Pastor-Anglada, Drug transporter pharmacogenetics in nucleoside-based therapies. Pharmacogenomics, 2010. 11(6): p. 809-41.
22.    Aymerich, I., M. Pastor-Anglada, and F.J. Casado, Long term endocrine regulation of nucleoside transporters in rat intestinal epithelial cells. J Gen Physiol, 2004. 124(5): p. 505-12.
23.    Owen, R.P., et al., Genetic analysis and functional characterization of polymorphisms in the human concentrative nucleoside transporter, CNT2. Pharmacogenet Genomics, 2005. 15(2): p. 83-90.
24.    Li, L., et al., Identification and functional analysis of variants in the human concentrative nucleoside transporter 2, hCNT2 (SLC28A2) in Chinese, Malays and Indians. Pharmacogenet Genomics, 2007. 17(9): p. 783-6.
25.    Hiratochi, M., et al., Hypouricemic effects of novel concentrative nucleoside transporter 2 inhibitors through suppressing intestinal absorption of purine nucleosides. Eur J Pharmacol, 2012. 690(1-3): p. 183-91.
26.    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.
27.    Vasko, B., et al., Inhibitor selectivity of CNTs and ENTs. Xenobiotica, 2019. 49(7): p. 840-851.