Journal of Medicinal Chemistry pubs.acs.org/jmc Article 3 (2 × Cq-Ar), 127.5, 127.0 (2 × CH-Ar), 126.4 (CH-Ar), 125.2, 125.1 Radiotracer Flux Assays. N-acetyl [6- H]neuraminic acid (20 (CH-Ar), 120.0 (2 × CH-Ar), 116.7 (Cq-Ar), 111.6 (CH-Ar), 110.9 Ci/mmol) and [4,5-3H] Fmoc-L-leucine (50 Ci/mmol) were (Cq-Ar), 103.4 (CH-Ar), 65.7 (OCH CH), 53.6 (C-α), 46.6 14 2 purchased from American Radiolabeled Chemicals. L-[1,2,1′,2′- C]- 3 (OCHCH), 32.7 (C-β), 31.2 (CH -Coum); HPLC (method A-2): cystine (200 mCi/mmol) and L-[2,3,4,5- H]-proline (75 Ci/mmol) 2 2 3 t = 16.8 min; HPLC-MS (method B): t = 17.6 min; HPLC-MS were from PerkinElmer. [ H]Neu5Ac uptake into whole HEK293 R + R + 6 (method C): t = 8.9 min; MS: (ESI ), m/z (%): [M + H] = 552.1 cells was measured 2 days after transfection as described with minor R − − (100%); MS: (ESI ), m/z (%): [M − H] = 550.1 (100%). changes. Cells were briefly washed and incubated for 15 min at room Modeling and Docking Computation. Homology Modeling. 3 temperature with [ H]Neu5Ac (12.5 nM; 0.05 μCi/well) in a The human sequence of sialin was retrieved from the UniProt + medium buffered with MES-Na pH 5.0. After two brief ice-cold database59 under the code Q9NRA2. The DgoT template structures washes, the cellular radioactivity in the cells was counted by liquid 6E9N and 6E9051 were retrieved form the Protein Data Bank. scintillation with a Tri-Carb 4910TR counter (PerkinElmer). For 60 Sequence alignment was carried out using CLUSTAL W as classical experiments, inhibitors were added simultaneously with ̀ 3 implemented in Discovery Studio (Dassault Systemes BIOVIA, [ H]Neu5Ac. However, for some experiments (Figure 6), inhibitors Discovery Studio, 2019) and further refined manually. The model were pre-incubated for 15 min at pH 5.0 followed by 15 or 30 min 61 and the best out of 100 models + was generated using MODELER, washes in a medium buffered at pH 7.0 with MOPS-Na before was selected according to the PDF total energy. 3 measuring [ H]Neu5Ac transport. For saturation kinetics, incubation Molecular Docking. Flexible docking of 45 to the inward-facing was shortened to 10 min to keep measurements within the linear 53 A set of nine residues with 3 model was carried out using GOLD. phase of uptake at all [ H]Neu5Ac concentrations. flexible side chains was used to define the binding site: F50, Y54, R57, 3 [ H]-Fmoc-Leu-OH uptake was measured similarly using 2 μM F115, F116, Y119, F179, Y306, and Y335. The Goldscore was used to (1/10 of IC ) and 0.05 μCi/well of the tracer. To compare Fmoc- 50 3 keep the best 10 out of 100 poses. Then, the pose that showed the Leu-OH and Neu5Ac in Figure S1,[H]Neu5Ac transport was best orientation according to the hypothesis made from the biological measured at a similar transporter occupancy (100 μM; 0.05 μCi/ data was selected and further refined through molecular dynamic well). simulation. Human cystinosin and rat LYAAT1 were assayed at the plasma Molecular Dynamics Simulations. The system with proteins and membrane of HEK293 cells in MES-Na+ pH 5.0 buffer one day after ligands was prepared in the CHARMM-GUI web server62 in order to 14 3 lipofection using [ C]cystine (20 μM; 0.08 μCi/well) and [ H]- generate a membrane around the protein and solvate with water and proline (100 μM; 0.1 μCi/well) as substrates, respectively, as ions. A heterogeneous membrane made of 90% POPG and 10% 46,47 previously described. cholesterol was chosen, and a TIP3 water model with NaCl (0.15 M) Immunofluorescence Analysis. Sialin distribution was analyzed counter ions was chosen for the solvation. The system was typed with 6 2 days after transfection as described. Cells were fixed with 4% a CHARMM36mforce field, and NAMD 2.13 was used. The system paraformaldehyde. After quenching with 50 mM NH Cl and several 4 was equilibrated through six constrained simulations for a total of 690 washes, cells were permeabilized and blocked with 0.05% saponin and ps by gradually diminishing the force constraints at each steps. The 2+ 2+ 0.2% BSA in PBS buffer containing Ca and Mg . Coverslips were following constraints were applied (each value represents an then incubated for ≥1 h with mouse anti-LAMP1 antibodies (H4A3; equilibration step): protein backbone (10/5/2.5/1/0.5/0.1 kcal/ Developmental Studies Hybridoma Bank) at 0.75 μg/mL in blocking mol), protein side chains (5/2.5/1.25/0.5/0.25/0.05 kcal/mol), lipid buffer, washed, and incubated with Cy3-conjugated donkey anti- heads (5/5/2/1/0.2/0 kcal/mol), and dihedral bonds (500/200/ mouse antibodies (Jackson ImmunoResearch) at 1.4 μg/mL in the 100/100/50/0 kcal/mol). Then, a production dynamics of 20 ns was samebuffer. Coverslips were then washed and mounted on glass slides carried out in NPT conditions at 303.15 K without any constraints. with Fluoromount-G (SouternBiotech). Epifluorescence micrographs Cell Culture. HeLa and HEK293 cells were grown at 37 °C under were acquired under a 100× objective lens with a Nikon Eclipse TE- 5% CO2 in glucose-rich, Glutamax-I-containing Dulbecco’s modified 2000 microscope equipped with a CCD camera (Coolsnap). The Eagle medium (DMEM) supplemented with 10% fetal bovine serum intracellular distribution of recombinant sialin was classified into three (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. categories (see Figure 8B) by an independent observer in a blind Fibroblasts from Salla patients were obtained from the Finnish manner. In other experiments, sialin/LAMP1 colocalization was Institute for Health and Welfare (THL), Helsinki, Finland, and used quantitated by assessing the spatial correlation between pixel in this study with ethical permission no. 78/13/03/00/16 (22 March intensities of the green and red channels. Dual-color images were 2016), issued by the Ethics Committee of the Helsinki and Uusimaa imported into the Fiji version (http://fiji.sc) of ImageJ,63 and the Hospital District, Finland. The fibroblasts tested for sialic acid storage Substract Background and Coloc 2 plugins were used to calculate rescue carried compound-heterozygote mutations in the SLC17A5 Pearson’s correlation coefficients across 20 to 25 cells/condition. gene: 115c→t/1007-1008del, corresponding to p.Arg39Cys/ Statistical analysis was made using Kruskal−Wallis nonparametric p.Leu336Trpfs at the protein level. Fibroblasts were grown at 37 one-way ANOVA with post hoc Dunn’s test. To test the effect of 45 °C under 5% CO in glucose-rich, Glutamax-I-containing DMEM on sialin localization, the compound was added during the 2 supplemented with 20% FBS, 100 U/mL penicillin, and 100 μg/mL transfection step. The 45-supplemented culture medium was replaced streptomycin. by a fresh one every day until 4 h before cell fixation. Expression of Recombinant Sialin. HeLa cells were transfected Quantification of Sialic Acid Levels in Cells by Mass 6 Spectrometry. Two confluent 75 cm2 flasks of human fibroblasts either by electroporation or lipofection. For electroporation, 2 × 10 HeLacells in 50 μL of ice-cold phosphate-buffer saline (PBS; pH 7.4) were used for each measurement. Cells were cultured for 2 days in the were mixed with 5 μg of wild-type or R39C pEGFP-C2-sialin presence of 30 or 168 μM 45 in 0.3% DMSO or with DMSO alone plasmid6 and immediately subjected to 10 square pulses (200 V, 3 for control at 37 °C under 5% CO . The 45-supplemented culture 2 ms) delivered at 1 Hz by a GHT 1287 electropulsator (Jouan) with 4 mediumwasreplaced by a fresh one twice. After the 2 days, cells were mm-spaced electrodes. Cells were then diluted with 7 mL of culture detached by trypsinization, washed with ice-cold PBS, and medium and distributed into 14 wells from a 24-well culture plate centrifuged. The resulting cell pellets were flash-frozen and kept at containing glass coverslips. For lipofection, HeLa were plated −20 °C until measurement. (100,000 cells/well) into 24-well plates containing glass coverslips Sialic acid measurements were done with some differences in two and transfected on the following day with Lipofectamine 2000 laboratories. In one protocol, pellets were submitted to osmolysis by (Invitrogen) according to the manufacturer’s protocol. addition of 100 μL of ultrapure water (1 h, 4 °C) and subsequently HEK293cells were plated (250,000 cells/well) into poly-D-lysine- sonicated (3 × 20 s with 10 s resting intervals). At this stage, the coated 24-well plates and lipofected similarly with a construct carrying protein concentration was determined for further normalization using 6 a sorting motif mutation (pEGFP-C2-sialin L22G/L23G) to express Micro BCA Protein Assay Kit (ThermoFisher). After centrifugation human sialin at the plasma membrane. (3000 rpm, 10 min, 4 °C), three volumes of ice-cold EtOH were 8245 https://dx.doi.org/10.1021/acs.jmedchem.9b02119 J. Med. Chem. 2020, 63, 8231−8249