Pharmacological and structural insights into nanvuranlat, a selective LAT1 (SLC7A5) inhibitor, and its N-acetyl metabolite with implications for cancer therapy

Abstract

L-type amino acid transporter 1 (LAT1, SLC7A5), overexpressed in various cancers, mediates the uptake of essential amino acids crucial for tumor growth. It has emerged as a promising target for cancer therapy. Nanvuranlat (JPH203/KYT-0353), a LAT1 inhibitor, has shown antitumor activity in preclinical studies and efficacy in biliary tract cancer during clinical trials. This study provides a comprehensive pharmacological characterization of nanvuranlat and its N-acetyl metabolite, including structural insights into their LAT1 interactions. Both compounds demonstrated high selectivity for LAT1 over LAT2 and other amino acid transporters. Nanvuranlat acts as a competitive, non-transportable LAT1 inhibitor (K_i = 38.7 nM), while its N-acetyl metabolite retains selectivity but with reduced affinity (K_i = 1.68 µM). Nanvuranlat exhibited a sustained inhibitory effect on LAT1 even after its removal, indicating the potential for prolonged therapeutic effects. Both compounds showed comparable dissociation rates, suggesting that N-acetylation does not affect the interaction responsible for slow dissociation. The U-shaped conformation adopted by nanvuranlat when bound to LAT1 likely contributes to its high affinity, selectivity, sustained inhibitory effect, and non-transportable nature observed in this study. These insights into nanvuranlat's mechanism and metabolic impact provide essential information for understanding its clinical efficacy and advancing LAT1-targeted cancer therapies.

Keywords: Amino acid transporter; Cancer therapy; Drug metabolism & efficacy; LAT1 inhibitor; Pharmacological selectivity.

Fig. 1

Chemical structures of nanvuranlat and N-acetyl-nanvuranlat.

Fig. 2

The inhibitory effect of nanvuranlat and N-acetyl-nanvuranlat. (a) and (b), inhibition of l-[¹⁴C]leucine uptake in HEK293-hLAT1 cells (a) and l-[¹⁴C]alanine uptake in HEK293-hLAT2 cells (b). Nanvuranlat and N-acetyl-nanvuranlat at 0.1, 1 and 10 µM inhibited LAT1-mediated uptake of l-[¹⁴C]leucine (1 µM), whereas they did not inhibit LAT2-mediated uptake of l-[¹⁴C]alanine (1 µM). (c) and (d), growth inhibition of HEK293-hLAT1 and HEK293-hLAT2 cells by nanvuranlat and N-acetyl-nanvuranlat. Nanvuranlat and N-acetyl-nanvuranlat suppressed the proliferation of HEK293-hLAT1 cells in a concentration-dependent manner with GI₅₀ values of 1.83 ± 0.31 µM for nanvuranlat (c). For N-acetyl-nanvuranlat, the GI₅₀ value could not be determined, as the inhibition did not reach 50% at the maximum concentration tested (30 µM). Neither nanvuranlat nor N-acetyl-nanvuranlat inhibited the proliferation of HEK293-hLAT2 cells (d). Data are presented as mean ± S.D., n = 4. Nanv.: nanvuranlat; N-Ac-Nanv.: N-acetyl-nanvuranlat.

Fig. 3

Inhibition kinetics of nanvuranlat and N-acetyl-nanvuranlat on LAT1. The uptake of l-[¹⁴C]leucine (1–400 µM) was measured in the absence or presence of nanvuranlat (0.1 and 0.5 µM) and N-acetyl-nanvuranlat (1 and 5 µM) in HEK293-hLAT1 cells. Uptake data were plotted against leucine concentration and fitted to Michaelis–Menten curves (a and c). Lineweaver–Burk plot analysis was performed to evaluate the inhibition kinetics of nanvuranlat and N-acetyl-nanvuranlat (b and d), revealing competitive inhibition for both compounds. The calculated K_i values were 38.7 ± 5.1 nM for nanvuranlat and 1.68 ± 0.23 µM for N-acetyl-nanvuranlat. Data are presented as mean ± S.D., n = 4. Nanv.: nanvuranlat; N-Ac-Nanv.: N-acetyl-nanvuranlat.

Fig. 4

Sustained inhibition of LAT1-mediated leucine transport by nanvuranlat and N-acetyl-nanvuranlat. HEK293-hLAT1 cells were treated with nanvuranlat, N-acetyl-nanvuranlat (20 µM), or 0.6% DMSO (control) for 30 min. After washing out the compounds, cells were incubated in a fresh culture medium and assessed for leucine uptake at 0, 10, 20, 30, and 60 min (a) and 24 h (b). The leucine uptake values were normalized to the control at each time point, with the control uptake value set at 100%. Experiments were performed independently three times, each with four replicates. Data are presented as mean ± S.D. Nanv.: nanvuranlat; N-Ac-Nanv.: N-acetyl-nanvuranlat. The inhibition reduction rate was similar for both compounds: 29.8% for nanvuranlat and 30.8% for N-acetyl-nanvuranlat. This rate represents the percentage reduction in inhibition over the first 10 min, relative to the inhibition value at 0 min, calculated as follows: Inhibition reduction rate = (% Leu uptake at 10 min – % Leu uptake at 0 min)/(100 – % Leu uptake at 0 min) × 100.

Fig. 5

Absence of ¹⁴C-nanvuranlat uptake in HEK293-hLAT1 cells. Uptake measurements of 1 µM l-[¹⁴C]leucine (¹⁴C-Leu) and 1 µM ¹⁴C-nanvuranlat (¹⁴C-Nanv.) were conducted at 37 °C and on ice in HEK293-hLAT1 cells. The cell-associated counts of l-[¹⁴C]leucine were temperature-dependent, with significantly lower uptake observed on ice than 37 °C. In contrast, the cell-associated counts of ¹⁴C-nanvuranlat were unaffected by temperature, indicating that LAT1 does not transport ¹⁴C-nanvuranlat. Data are presented as mean ± S.D., n = 4.

Fig. 6

Inhibitory effect of nanvuranlat and N-acetyl-nanvuranlat on system L transporters. Uptakes of typical substrates (50 µM l-[¹⁴C]leucine for LAT1 (a), LAT3 (c), and LAT4 (d); 50 µM l-[¹⁴C]lalanine for LAT2 (b)) were measured in the presence of 1 µM and 10 µM of nanvuranlat and N-acetyl-nanvuranlat for 30 min in Xenopus oocyte expression system. Measurements were conducted in Na⁺-free uptake solution using control Xenopus oocytes injected with water instead of cRNA (“Water”) and the oocytes injected with cRNA for expressing each transporter. Uptake rates are expressed as mean ± S.D. (n = 8–12). Nanv.: nanvuranlat; N-Ac-Nanv.: N-acetyl-nanvuranlat. The data shown in (a), (c), and (d) were obtained using the same batch of oocytes. The l-[¹⁴C]leucine uptake data from control oocytes are shared across (a), (c), and (d), but are presented in each panel (a, c, and d) to allow comparison on the same scale as the l-[¹⁴C]leucine uptake in oocytes expressing each respective transporter.

Fig. 7

Effect of nanvuranlat and N-acetyl-nanvuranlat on neutral amino acid transporters other than system L that transport branched chain or aromatic amino acids. Uptakes of 50 µM l-[¹⁴C]leucine (Leu) by y⁺LAT1 (a), y⁺LAT2 (b), ATB^0,+ (c), B⁰AT1 (d); the uptake of 50 µM l-[¹⁴C]tyrosine (Tyr) by TAT1 (e); and the uptake of 50 µM l-[¹⁴C]cystine by b^0,+ (f) were measured in the presence of 1 µM and 10 µM of nanvuranlat and N-acetyl-nanvuranlat for 30 min in Xenopus oocyte expression system. Measurements were conducted using control Xenopus oocytes injected with water instead of cRNA (“Water”) and the oocytes injected with cRNA for expressing each transporter. Uptake rates are presented as mean ± S.D. (n = 8–12). For the functional expression of b^0,+ in (f), rBAT, an auxiliary subunit of system b^0,+ transporter that strongly induces system b^0,+ activity in Xenopus oocytes, was expressed. Thus, the induced system b^0,+ activity was of Xenopus origin, although human rBAT was expressed. Na⁺-free uptake solution was used for TAT1 and b^0,+, whereas ND96 solution was used for the other transporters. Nanv.: nanvuranlat; N-Ac-Nanv.: N-acetyl-nanvuranlat. The data shown in panels (a) and (b) and those shown in panels (c) and (d) were obtained using the same respective batches of oocytes. The l-[¹⁴C]leucine uptake data from control oocytes are shared between panels (a) and (b) and separately between panels (c) and (d), but they are presented in each panel (a and b, and c and d) to allow for comparison on the same scale as the l-[¹⁴C]leucine uptake in oocytes expressing each respective transporter.