Molecular basis of inhibition of the amino acid transporter B0AT1 (SLC6A19)

Abstract

The epithelial neutral amino acid transporter B⁰AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B⁰AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B⁰AT1 inhibitors with IC₅₀-values of 31-90 nM. High-resolution cryo-EM structures of B⁰AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.

Fig. 1. Endogenous contributions to leucine transport in the B⁰AT1 flux assay.

L-leucine transport was characterized in CHO-BC cells using uptake of 150 µM L-[¹⁴C]leucine. a Uptake of L-leucine was measured in the presence (yellow) and absence (green) of Na⁺. An apparent increase of Na⁺-independent leucine transport is observed after application of increasing concentrations of B⁰AT1 inhibitor cinromide. L-Leucine transport is blocked incompletely due to endogenous transport activities. b The Na⁺-independent endogenous transport can be blocked by LAT1 inhibitor JPH203. The Na⁺-dependent component of L-leucine uptake that is sensitive to inhibition by GPNA (γ-glutamyl-p-nitroanilide) was assigned to ASCT2. The remaining transport activity was assigned to B⁰AT1. Transport components are indicated by colour. c In the presence of GPNA (3 mM) and JPH203 (3 µM), the remaining Na⁺-dependent transport activity is completely blocked by cinromide. (d) IC₅₀ of cinromide as determined with the optimized assay. All data shown as mean ± SD, individual datapoints were overlayed in all bar graphs (n = 3, n referring to individually seeded cell culture dishes used for each condition or concentration).

Fig. 2. Structure-activity relationships of SLC6A19 inhibitor JX98.

For each chemical modification the remaining transport activity (measured at 150 µM L-leucine) in the presence of 30 μM inhibitor is shown as a violin plot (n = 10, referring to individually seeded wells) with median (white dot), SD (whiskers), minimum and maximum (violin body) indicated. Better inhibitors result in lower residual activity. Green violins represent improvements, red violins undesirable modifications. Uninhibited cells and inhibition by 30 µM cinromide served as controls for the experiments. More details can be found in Table S1.

Fig. 3. Pharmacological properties of optimized B⁰AT1 inhibitors.

Structures of the optimized inhibitors are shown above. L-Leucine transport activity was determined by FLIPR assay (closed symbols) and by radioactive L-leucine uptake assay (open symbols) in CHO-BC cells at the indicated inhibitor concentrations (n = 6, n referring to individually seeded cell culture dishes or wells). Data are shown as mean ± SD. Uninhibited cells and inhibition by 30 µM cinromide served as controls for the experiments.

Fig. 4. Overall structure of the ACE2-B⁰AT1 bound with inhibitors.

a Cryo-EM map of the full length ACE2-B⁰AT1 heterodimer in complex with inhibitors. ACE2 and B⁰AT1 bound with JX225 (coral) are represented in sky blue and orange, respectively, while ACE2 and B⁰AT1 bound with JX98 (magenta) are shown in darker blue and pink, respectively. The structural comparison of JX225 and JX98 is presented in the box. b The inserts of the cryo-EM map of the human ACE2-B⁰AT1 complex show the density corresponding to JX98 and JX225 color-coded in magenta and coral, respectively. c Positioning of JX98 and JX225 in the ACE2-B⁰AT1 complex. Both JX98 and JX225 are centrally located within the presumed transport pathway. d, e The binding mode of JX225 in B⁰AT1 is shown, highlighting hydrogen, halogen and hydrophobic interactions. f, g The binding mode of JX98 in B⁰AT1 is presented, with schematic diagrams illustrating the interaction environment, following the same representation as in (d, e).

Fig. 5. The movement of TM1 and TM6 and the role Trp56.

A comparison is drawn between the apo structure of B⁰AT1 (PDB: 6M18) when bound with JX225 (a) and JX98 (b), respectively. The apo structure of B⁰AT1 is shown in green, while the ligand-bound structures are shown in orange (JX225) and pink (JX98). The alterations in the positions of TM1 and TM6 are depicted in the middle and right panels. JX225 (a) and JX98 (b) are represented in coral and magenta, respectively.