Characterization of 6-Mercaptopurine Transport by the SLC43A3-Encoded Nucleobase Transporter

Abstract

6-Mercaptopurine (6-MP) is a nucleobase analog used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disorders. However, the mechanisms underlying its transport into target cells have remained elusive. The protein encoded by SLC43A3_1 [equilibrative nucleobase transporter 1 (ENBT1)] has recently been shown to transport endogenous nucleobases. A splice variant (SLC43A3_2), encoding a protein with 13 additional amino acids in the first extracellular loop, is also expressed but its function is unknown. We hypothesized that 6-MP is a substrate for both variants of ENBT1. Human embryonic kidney 293 (HEK293) cells (lacking endogenous ENBT1 activity) were transfected with each of the coding region variants of SLC43A3. ENBT1 function was assessed via the rate of flux of [ 3 H]adenine and [ 14 C]6-MP across the plasma membrane. Both SLC43A3 variants encoded proteins with similar functional properties. [ 14 C]6-MP and [ 3 H]adenine had K m values (6S.D.) of 163 6 126 and 37 6 26 mM, respectively, for this system. Decynium-22, 6-thioguanine, and 6-methylmercaptopurine inhibited 6-MP uptake with K i values of 1.0 6 0.4, 67 6 30, and 73 6 20 mM, respectively. ENBT1 also mediated adenine-sensitive efflux of 6-MP from the SLC43A3-HEK293 cells. MRP4 also contributed to the efflux of 6-MP in this model, but was less efficient than ENBT1 in this regard. Furthermore, transfection of HEK293 cells with SLC43A3 increased the sensitivity of the cells to the cytotoxic effects of 6-MP by more than 7-fold. Thus, both variants of ENBT1 are key players in the transfer of 6-MP into and out of cells, and changes in SLC43A3 expression impact 6-MP cytotoxicity.

Impact of SLC43A3/ENBT1 Expression and Function on 6-Mercaptopurine Transport and Cytotoxicity in Human Acute Lymphoblastic Leukemia Cells

“…In the SUP-B15 cells, we observed a significantly lower V max for ENBT1-mediated 6-MP uptake of 22 ± 5 pmol/µl/s and a K m of 133 ± 3E). These K m values for 6-MP transport by ENBT1 are similar to those determined previously for SLC43A3-transfected HEK293 cells (Ruel, Nguyen et al 2019).…”

“…However, the mechanism by which 6-MP gets into cells, especially lymphoblasts (the target in ALL treatment), remained elusive until the recent identification of the SLC43A3-encoded equilibrative nucleobase transporter 1 (ENBT1). There are two alternative splice variants of SLC43A3 that we have shown to encode transport proteins that are functionally similar with respect to their ability to transport 6-MP and adenine (Ruel, Nguyen et al 2019). Furthermore, we have established that ENBT1, heterologously expressed in HEK293 cells, can transport 6-MP at concentrations within the therapeutic range.…”

Current Understanding of the Intestinal Absorption of Nucleobases and Analogs

“…With regard to basolateral efflux for exiting the epithelial cells following brush border uptake (Fig. 1), the facilitative transporters ENT1 [14][15][16][17][18][19][20][21][22] and ENBT1 [23][24][25] could be in operation, as mentioned in the preceding section. On the other hand, OAT1, OAT2, and OAT3, which could potentially operate for 5-fluorouracil and/or 6-mercaptopurine, are not likely to be involved in the basolateral efflux of those drugs, as also mentioned above, since these OATs are expressed only poorly or at negligible levels in the human small intestine.…”

“…44) In addition to ENT1, ENBT1, a purine-specific nucleobase transporter identified more recently, could also potentially be involved in the basolateral efflux of purine nucleobases. [23][24][25] Although the ENT1 and ENBT1 of rat have not been evaluated for their nucleobase transport functions, they could be expected to be capable of transporting nucleobases, as demonstrated for their human counterparts. These transporters are facilitative transporters that can operate for both influx and efflux across the cellular membrane, depending on the concentration gradient of the substrate.…”

“…[16][17][18][19][20][21][22] Equilibrative nucleobase transporter 1 (ENBT1/SLC43A3), a nucleobase-specific facilitative transporter, could also be involved in such transport in many animal species, although its expression in the small intestine has not yet been fully proven. [23][24][25] Notably, ENT2/SLC29A2, 15,26,27) a close homolog of ENT1, can transport nucleobases as well as nucleosides, but its expression in the small intestine has not been evident in recent studies. [19][20][21][22] For the absorption of nucleosides as nutrients closely related to nucleobases, the Na + -dependent uptake transporters of concentrative nucleoside transporter 1 (CNT1/SLC28A1), CNT2/ SLC28A2, and CNT3/SLC28A3 are present at the brush border membrane to constitute the absorption pathway in combination with ENT1, but it is suggested that they do not operate for nucleobases.…”