Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

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.

Keywords: FPMINT; equilibrative nucleoside transporter; inhibitor; mechanism of action; structure-activity relationship.

FIGURE 1

Chemical structure of FPMINT.

FIGURE 2

Effects of FPMINT analogues (listed in Supplementary Table S1) on [³H]uridine uptake by ENT1 and ENT2 [³H]uridine uptake (1 μM, 2 μCi/ml) in (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells was measured in the presence of various concentrations of FPMINT analogues listed in Supplementary Table S1 (10 nM–100 μM).

FIGURE 3

Effects of FPMINT analogues (listed in Supplementary Table S2) on [³H]uridine uptake by ENT1 and ENT2 [³H]uridine uptake (1 μM, 2 μCi/ml) in (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells was measured in the presence of various concentrations of FPMINT analogues listed in Supplementary Table S2 (10 nM–100 μM).

FIGURE 4

Competitive inhibition of ENT1 and ENT2 by compound 3c. Kinetic study of [³H]uridine uptake (0.1 μM–1 mM) was measured in (A) PK15NTD/ENT1 and (D) PK15NTD/ENT2 cells in the presence of various concentrations of compound 3c (0, 0.01, 0.1, 1 and 10 µM) (B,E) 1/V versus 1/[S] plots of each curve in (A) and (D), respectively (C,F) The plot of slopes of each line in (B) and (E), respectively, versus the concentration of compound 3c. Data are presented as mean ± SE of three experiments.

FIGURE 5

Effects of compound 3c on cytotoxicity and protein expressions of ENT1 and ENT2. Cytotoxicity of compound 3c was measured after 24 h or 48 h treatment of PK15NTD/ENT1 and PK15NTD/ENT2 cells with various concentrations of compound 3c (0.5, 5 and 50 μM) or a vehicle (0.5% DMSO, as a control) (A) The cell viability and (B) cell membrane integrity were determined by the MTT assay and LDH release, respectively (C) PK15NTD/ENT1 and (D) PK15NTD/ENT2 cells were incubated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for 24 or 48 h. Western blotting assay was performed to determine the protein expression levels of ENT1 and ENT2, with β-actin as an internal reference. Cell surface proteins of (E) PK15NTD/ENT1 and (F) PK15NTD/ENT2 cells were biotinylated and treated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for 24 or 48 h. After cleavage of extracellular biotin, internalized biotinylated proteins were precipitated with immobilized streptavidin beads and detected with an anti-ENT1 and anti-ENT2 antibodies by western blotting. Total ENT1 and ENT2 from whole-cell lysates were also detected for a comparison. β-actin served as an internal reference. Representative blots are from three independent experiments. Data are presented as mean ± SE of three experiments.

FIGURE 6

Reversibility of the inhibitory effects of compound 3c on ENT1 and ENT2 (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells were incubated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for different time (0–60 min) The cells were then washed five times. After wash-out for every single time [³H]uridine uptake (1 μM, 2 μCi/ml) was measured. Data are presented as mean ± SE of three experiments. ^* p < 0.05 compared with the fifth wash.

FIGURE 7

Molecular docking analysis of compound 3c against ENT1. 3D structural overview of ENT1 with (A,B) compound 3c and (D,E) draflazine docked complexes. The black box showed the location of compound 3c. A close-up of the docked protein displaying the amino acid residues of ENT1 involved in the binding with (C) compound 3c and (F) draflazine.