Category: Preclinical Development
Purpose: Human concentrative nucleoside transporter 3 (hCNT3, SLC28A3) is a sodium-coupled nucleoside transporter. hCNT3 exhibits a broad substrate specificity, tolerating not only both purine and pyrimidine nucleosides, but also nucleoside analogues such as anticancer and antiviral nucleoside-mimicking drugs. hCNT3 is one of important nucleoside transporters involved in the disposition, pharmacological efficiency and toxicity of a variety of nucleoside drugs. The purpose of the present study was to elucidate whether hCNT3 can recognize and transport antiviral nucleoside drugs without sugar moiety, aciclovir (ACV) and ganciclovir (GCV), and also how tolerant the substrate recognition by hCNT3 shows.
Methods: hCNT3 was expressed heterologously in Xenopus oocytes by capped cRNA injections. Oocytes were injected with 50 ng hCNT3 cRNA in a 50 nl volume and incubated for 3 – 6 days. The oocytes were used for transport studies 3 - 6 days after cRNA injection. The nucleoside transport activity via hCNT3 was determined by the uptake of [3H]-adenosine or [3H]-uridine, or [3H]-GCV by the oocytes and by the substrate-induced current due to the transport via hCNT3 by electrophysiological studies with two-electrode voltage clamp technique.
Results: We determined the uptakes of nucleosides (uridine and adenosine) and GCV by hCNT3 heterologously expressed in Xenopus oocytes. The uptakes of 1 μM uridine, adenosine and GCV by hCNT3 cRNA injected oocytes were enhanced more than 50-fold, compared to control (H2O-injected oocytes), indicating that hCNT3 was expressed robustly in Xenopus oocytes and that hCNT3 can recognize and transport GCV irrespective of sugar moiety in a nucleoside. These enhanced uptakes showed Na+-dependency as the uptake was reduced substantially when measured in the absence of Na+. The transport activities of adenosine and uridine via hCNT3 were inhibited significantly by 2.5 mM ACV and GCV, indicating that hCNT3 could also recognize and transport these antiviral nucleoside drugs. Furthermore, we assessed the ability of hCNT3 to transport ACV and GCV by monitoring an inward current due to its transport via hCNT3 in the oocyte, because hCNT3 is an electrogenic Na+/nucleoside cotransporter. Superfusion of hCNT3-expressing oocytes with 200 μM various nucleosides elicited substantially inwards currents. One mM ACV and GCV also induced substantially inward currents in hCNT3-expressing oocytes, which were abolished completely by Na+ removal in the perfusate. Such currents were not detectable in H2O-injected oocytes. Furthermore, we conducted the homology modeling of hCNT3 using the crystal structure of Vibrio Cholerae, CNT (vcCNT) (3TIJ) and the docking simulation of GCV to the hCNT3 model. The structure model of hCNT3 showed a putative binding pocket to accommodate uridine and GCV in a similar manner. Taken collectively, it has been demonstrated that hCNT3 exhibits a broad substrate recognition and transport ACV as well as GCV in a concentrative manner.
Conclusion: We here demonstrated that hCNT3 can transport antiviral nucleoside analogue drugs without sugar moiety, ACV and GCV in a Na+-coupled electrogenic manner. The potential of this transporter to exhibit a broad recognition raises the possibility that it might be involved in the distribution of these antiviral drugs, ACV and GCV to a tissue. Since hCNT3 is expressed not only in the intestinal tract and kidney, but also in various tissues such as the lung, pancreas and skin, hCNT3 might have the potential to govern the disposition, pharmacological efficiency and toxicity of these antiviral nucleoside analogue drugs.
Masayuki Masuda– Toho University, Funabashi, Chiba, Japan
Kazuaki Sugio– Toho University, Funabashi, Chiba, Japan
Shotaro Sasaki– Lecturer, Toho University, Funabashi, Chiba, Japan
Kazumi Shimono– Professor, Sojo University, Kumamoto, Kumamoto, Japan
Ikuko Tsukamoto– Professor, Kagawa University, Kita-gun, Kagawa, Japan
Ryoji Konishi– Kita-gun, Kagawa, Japan