Abstract
Equilibrative nucleoside transporters (ENTs) play a vital role in nucleotide synthesis, regulation of adenosine function and chemotherapy. Current inhibitors of ENTs are mostly ENT1-selective. Our previous study has demonstrated that 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) is a novel inhibitor of ENTs, which is more selective to ENT2 than to ENT1. The present study aimed to screen a series of FPMINT analogues and study their structure-activity relationship. Nucleoside transporter-deficient cells transfected with cloned human ENT1 and ENT2 were used as in vitro models. The results of the [3H]uridine uptake study showed that the replacement of the naphthalene moiety with the benzene moiety could abolish the inhibitory effects on ENT1 and ENT2. The addition of chloride to the meta position of this benzene moiety could restore only the inhibitory effect on ENT1 but had no effect on ENT2. However, the addition of the methyl group to the meta position or the ethyl or oxymethyl group to the para position of this benzene moiety could regain the inhibitory activity on both ENT1 and ENT2. The presence of a halogen substitute, regardless of the position, in the fluorophenyl moiety next to the piperazine ring was essential for the inhibitory effects on ENT1 and ENT2. Among the analogues tested, compound 3c was the most potent inhibitor. Compound 3c reduced V max of [3H]uridine uptake in ENT1 and ENT2 without affecting K m. The inhibitory effect of compound 3c could not be washed out. Compound 3c did not affect cell viability, protein expression and internalization of ENT1 and ENT2. Therefore, similar to FPMINT, compound 3c was an irreversible and non-competitive inhibitor. Molecular docking analysis also showed that the binding site of compound 3c in ENT1 may be different from that of other conventional inhibitors. It is expected that structural modification may further improve its potency and selectivity and lead to the development of useful pharmacological agents.
Highlights
Nucleoside transporters are transmembrane proteins responsible for transporting physiological nucleosides essential for the salvage pathways for the biosynthesis of nucleotides
PK15NTD/ENT1 and PK15NTD/ENT2 cells were used to screen a series of FPMINT analogues listed in Supplementary Tables S1, S2
The results showed that compound 3c could form hydrogen bonds with Gly198, Ser199and Glu200 of ENT1 (Figures 7A–C) but draflazine from hydrogen bonds with Pro384 and Phe452 (Figures 7D–F)
Summary
Nucleoside transporters are transmembrane proteins responsible for transporting physiological nucleosides essential for the salvage pathways for the biosynthesis of nucleotides They participate in modulating the extracellular and intracellular concentrations of physiological nucleosides such as adenosine (Molina-Arcas et al, 2009), which participates in numerous essential physiological functions, including anti-inflammatory, cardioprotective and vasodilatory effects (Reiss et al, 2019). ENT4 is mainly expressed in the brain, heart and skeletal muscles (Barnes et al, 2006) It transports only adenosine, and its activity is increased in acidic environment. ENT1 and ENT2 can be inhibited by other compounds, including benzodiazepines, cilostazol, KF24345, propentofylline and troglitazone in the nanomolar to micromolar range (Patel et al, 1982; Parkinson et al, 1993; Liu et al, 2000; Seubert et al, 2000; Hammond and Archer, 2004; Leung et al, 2005) These compounds are more selective to ENT1 than to other ENTs. There has been a slow progress in the development of ENT2selective inhibitors. A representative analogue was selected for investigating its mechanism of action
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