Structure-activity characteristics of phenylalanine analogs selectively transported by L-type amino acid transporter 1 (LAT1)

Abstract

L-type amino acid transporter 1 (LAT1) is a transmembrane protein responsible for transporting large neutral amino acids. While numerous LAT1-targeted compound delivery for the brain and tumors have been investigated, their LAT1 selectivity often remains ambiguous despite high LAT1 affinity. This study assessed the LAT1 selectivity of phenylalanine (Phe) analogs, focusing on their structure-activity characteristics. We discovered that 2-iodo-L-phenylalanine (2-I-Phe), with an iodine substituent at position 2 in the benzene ring, markedly improves LAT1 affinity and selectivity compared to parent amino acid Phe, albeit at the cost of reduced transport velocity. L-Phenylglycine (Phg), one carbon shorter than Phe, was found to be a substrate for LAT1 with a lower affinity, exhibiting a low level of selectivity for LAT1 equivalent to Phe. Notably, (R)-2-amino-1,2,3,4-tetrahydro-2-naphthoic acid (bicyclic-Phe), with an α-methylene moiety akin to the α-methyl group in α-methyl-L-phenylalanine (α-methyl-Phe), a known LAT1-selective compound, showed similar LAT1 transport maximal velocity to α-methyl-Phe, but with higher LAT1 affinity and selectivity. In vivo studies revealed tumor-specific accumulation of bicyclic-Phe, underscoring the importance of LAT1-selectivity in targeted delivery. These findings emphasize the potential of bicyclic-Phe as a promising LAT1-selective component, providing a basis for the development of LAT1-targeting compounds based on its structural framework.

Figure 1

Chemical structures of Phe and its analogs used in this study. (A) The parent amino acid Phe, α-methyl-Phe, bicyclic-Phe (with its methylene group marked by an asterisk), and Phg. (B) Halogenated phenylalanines classified into three groups based on the position of the halogen in the benzene ring: Group 1 with halogens at position 2 (ortho-position), Group 2 with halogens at position 3 (meta-position), and Group 3 with halogens at position 4 (para-position).

Figure 2

LAT1 and LAT2 selectivity of halogenated phenylalanines. (A) Inhibition of LAT1-mediated l-[¹⁴C]leucine uptake by iodinated Phe isomers (positions 2, 3, and 4) in HEK293-hLAT1 cells. The uptake of l-[¹⁴C]leucine (1 μM) was measured for 1 min in the absence (control, Ctrl) or presence of Phe and iodinated Phe isomers at 10, 30, and 100 μM. (B) Inhibition of LAT2-mediated l-[¹⁴C]alanine uptake by iodinated Phe isomers in HEK293-hLAT2 cells. Similar to (A), uptake was measured in the absence (control, Ctrl) or presence of the Phe and iodinated Phe isomers at 10, 30, and 100 μM. (C and D) Inhibitory impact of ortho-position (position 2) halogenated phenylalanines on l-[¹⁴C]leucine uptake in HEK293-hLAT1 cells (C) and l-[¹⁴C]alanine uptake in HEK293-hLAT2 cells (D). (E and F) Examination of the inhibitory effects of meta-position (position 3) halogenated phenylalanines on l-[¹⁴C]leucine uptake in HEK293-hLAT1 cells (E) and l-[¹⁴C]alanine uptake in HEK293-hLAT2 cells (F). The uptake values are presented as the percentage of l-[¹⁴C]leucine or l-[¹⁴C]alanine uptake relative to control (Ctrl). Data are expressed as mean ± S.D., n = 3–4.

Figure 3

LAT1 and LAT2 selectivity of Phe and its analogs. Inhibition of LAT1-mediated l-[¹⁴C]leucine (1 μM) uptake (A) or LAT2-mediated l-[¹⁴C]alanine (1 μM) uptake (B) was measured in the absence (control, Ctrl) or presence of Phe and its analogs 2-I-Phe, α-methyl-Phe, bicyclic-Phe, and Phg at 10, 30, and 100 μM. The uptake values are expressed as the percentage of l-[¹⁴C]leucine or l-[¹⁴C]alanine uptake relative to control (Ctrl). Data are presented as mean ± S.D., n = 4.

Figure 4

Inhibition kinetics of Phe analogs on LAT1 and LAT2. This figure presents Lineweaver–Burk plots to analyze the inhibition kinetics of Phe and its analogs on l-[¹⁴C]leucine uptake in HEK293-hLAT1 cells (A-E) and l-[¹⁴C]alanine uptake in HEK293-hLAT2 cells (F-J). The uptake of l-[¹⁴C]leucine or l-[¹⁴C]alanine (50, 100, 200, and 400 μM) was measured in the absence (closed circles) or presence of Phe and its analogs (open circles). Data are expressed as mean ± S.D., n = 3–4.

Figure 5

LAT1-mediated efflux of l-[¹⁴C]leucine and LAT2-mediated efflux of l-[¹⁴C]alanine induced by Phe analogs. (A and B) LAT1-mediated efflux of pre-loaded l-[¹⁴C]leucine from HEK293-hLAT1 cells (A) and LAT2-mediated efflux of pre-loaded l-[¹⁴C]alanine from HEK293-hLAT2 cells (B) induced by Phe and its analogs were measured. The efflux assessment was conducted for 1 min in the absence (control, Ctrl) or presence of Phe, 2-I-Phe, α-methyl-Phe, and bicyclic-Phe at concentrations of 10, 30, and 100 μM. The efflux values are presented as the percentage of pre-loaded l-[¹⁴C]leucine or l-[¹⁴C]alanine. (C and D) The concentration dependence of l-[¹⁴C]leucine efflux from HEK293-hLAT1 cells (C) and l-[¹⁴C]alanine efflux from HEK293-hLAT2 cells (D) on the extracellularly applied Phe and its analogs is shown. The efflux was measured for 1 min in the absence (control, Ctrl) or presence of varying concentrations of Phe (closed circles), 2-I-Phe (closed triangles), α-methyl-Phe (open rhombus), and bicyclic-Phe (open circles) ranging from 3 to 1000 μM. Efflux values in the absence of test compound (Ctrl) were subtracted from those in the presence of test compounds and fitted to Michaelis–Menten curves. Data are presented as mean ± S.D., n = 3.

Figure 6

Tissue accumulation of 2-F-Tyr and bicyclic-Phe in tumor-bearing mice. (A and B) Biodistribution of 2-F-Tyr (A) and bicyclic-Phe (B) was measured in tumor-bearing mice at 30 and 60 min following intravenous administration. Plasma concentrations, as well as tumor and small intestinal accumulations, were quantified in mice bearing B16-F10 back tumors, 30 and 60 min post-intravenous injection of equimolar doses of 2-F-Tyr (15.6 mg/kg) and bicyclic-Phe (15 mg/kg). (C and D) Tissue-to-plasma concentration ratios in tumor-bearing mice at 30 (C) and 60 (D) min after intravenous administration of 2-F-Tyr or bicyclic-Phe. T/P refers to the tumor-to-plasma ratio; I/P indicates the small intestine-to-plasma ratio. Data are expressed as mean ± S.D. for n = 4. N.S. (not significant) indicates no statistically significant difference, represented by p > 0.05. Differences were considered significant at p < 0.05.