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Journal of Medicinal Chemistry Article system. We reasoned that 45 binding to the cytosolic side of Two ligands with this scaffold, Fmoc-Leu-OH 13 and 45, sialin might stabilize nascent R39C polypeptides in the were characterized in more detail. They inhibited Neu5Ac endoplasmic reticulum and rescue their delivery to lysosomes. transport with IC of 23 and 2.4 μM as compared to an IC 50 50 45 (12 μM) efficiently inhibited the residual transport of ∼1 mM for Neu5Ac. Neither compound was translocated activity of sialin R39C in our whole-cell assay (Figure S4), by sialin (Figures S1 and S2), and somewhat surprisingly, both showing that 45 binding is not impaired by the pathogenic inhibited Neu5Ac transport in a non-competitive, rather than a mutation. We thus transiently expressed wild-type (WT) or competitive, manner (Figure 5), notwithstanding the substrate- 33 R39C human sialin fused to EGFP (with an intact sorting binding pocket focus of our virtual screening. In contrast, motif) in HeLa cells in the absence or presence of 45 (70 μM) another sialin inhibitor identified in this screen, the endothelin- and examined their intracellular distribution by fluorescence A receptor antagonist FR139317, inhibited Neu5Ac transport 33 microscopy. As this distribution varies across cells in a given in a competitive manner. condition (Figure 8A), sialin was compared to a lysosomal This apparent paradox could be explained in a model where marker (LAMP1) and the dual staining in individual cells was the new compounds permeate biological membranes and bind classified into three categories: lysosomal (≥70% overlap to sialin in a cytosol-facing (inward-facing) rather than lumen- between sialin and LAMP1 puncta), non-lysosomal (≤30% facing (outward-facing) conformation. The protonation of the overlap), and mixed. In agreement with earlier studies, the carboxylate group at pH 5.0 and the high partition coefficient lysosomal category predominated in untreated cells expressing of these compounds facilitate their passive diffusion into the WTsialin (83%) whereas this category dropped to 11% for the cells in our assay of sialin transport activity. As structural R39C mutant with a predominance of the mixed (55%) and transition equilibriums are slower than ligand binding non-lysosomal (34%) categories. However, when sialin R39C equilibriums, this mechanism should trap sialin in an inward- was expressed in the presence of 45 (72 μM), the lysosomal facing state (Figure 5C) and prevent extracellular Neu5Ac category increased to 32% at the expense of the non-lysosomal binding to the lumen-facing state, thus decreasing the transport category (15%), while this treatment did not alter the capacity. A similar non-competitive inhibition of a transporter distribution of WT sialin (Figure 8B). Similar results were by ligands of the substrate pocket has been reported for the observed when sialin was expressed by lipofection instead of interaction between the glucose transporter GLUT1 and electroporation (Figure S4). 45 thus partially rescues the cytochalasin B or forskolin.55 An analogous mechanism also trafficking defect of the R39C mutant. To confirm this effect, occurs in enzymes undergoing a conformational change when we repeated these experiments and quantitated the colocaliza- inhibitors selectively bind to the product-favoring conforma- tion between EGFP-sialin and LAMP1 using scatter plots and tion.56 Selective or predominant binding of Fmoc-Leu-OH and Pearson’s correlation coefficient analysis of the pixel 45toaninward-facing state of sialin may thus account for their fluorescence intensities. This analysis showed a much lower non-competitive inhibition. level of sialin R39C/LAMP1 colocalization relative to the wild- We provide two pieces of evidence in support of this type. When sialin R39C-expressing cells were treated with a hypothesis. First, inhibition of sialin by Fmoc-Leu-OH and 45 high dose (300 μM) of 45, colocalization was rescued to a level in our whole-cell assay persisted after a 15 min wash (Figure close to that of the wild-type (Figure 8C). 6), in agreement with an action in the cytosolic rather than Next, we tested whether this increased delivery of sialin extracellular compartment. Second, we built a more accurate R39Ctolysosomes could decrease sialic acid storage in patient inward-facing homology model of human sialin based on the + cells. We used Salla fibroblasts from a compound-heterozygote recent crystallographic structures of the H /D-galactonate patient (R39C and L336W + frameshift alleles) because these symporter DgoT.51 Molecular docking of 45 to this model cells accumulate sialic acid to higher levels than homozygous showed that it binds well to the substrate pocket of the inward- R39C cells.54 Compound-heterozygote Salla fibroblasts and facing state, in agreement with our model. However, other control fibroblasts were thus treated or not with 45 (30 to 170 potential mechanisms for the non-competitive inhibition such μM)for2days,andtheir level of free sialic acid was assayed by as the existence of another 45-binding site cannot be excluded. mass spectrometry. Salla fibroblasts cultured in standard Further studies are needed to distinguish between these medium accumulated free sialic acid by ∼8-fold as compared possibilities. to control fibroblasts. The 45 treatment did not reduce but The best docked pose of 45 showed several interesting instead slightly increased this accumulation (Figure 8C), features, including an interaction of its carboxylate group with probably reflecting an inhibition of sialin R39C by 45 at the a conserved arginine in TM1 (R57 in human sialin) that is 51 lysosomal membrane. To circumvent this effect, we used a required for D-galactonate binding in DgoT. 45 may thus pulse-chase protocol in which the drug was applied for 2 days share common interactions with the anionic substrates of to correct the trafficking defect followed by a 6 h chase in drug- sialin. Another notable feature is the interaction of 45 with free medium to remove lysosomal inhibition. However, this residues in TM4 and TM10 that act as hinge regions for 51 treatment did not reverse sialic acid storage (data not shown). alternating-access structural transitions in DgoT. 45 binding to human sialin should thus impair its structural transitions, ■ DISCUSSION explaining why this compound blocks Neu5Ac transport. The identification of 45 as a cell-permeant ligand of the In this study, we exploited our previous virtual screening of “active” site (sialic acid pocket) of sialin prompted us to test 33 human sialin and report chemical substitutions of validated whether it might act as a pharmacological chaperone proof-of- virtual hits. This led to the identification of a novel ligand principle for the treatment of Salla disease. Pharmacological scaffold unrelated to sugar substrates, which is characterized by chaperones are selective ligands that bind to and stabilize an amino acid backbone, a free carboxylate, a N-linked misfolded mutant proteins to rescue their retention by the aromatic or heteroaromatic substituent, and a hydrophobic 57,58 protein quality control system. This approach is a side chain. promising option for Salla disease as this condition is almost 8240 J. Med. Chem. 2020, 63, 8231−8249

Amino Acids Bearing Aromatic or Heteroaromatic Substituents as a New Class of Ligands for the Lysosomal Sialic Acid Transporter Sialin - Page 10 Amino Acids Bearing Aromatic or Heteroaromatic Substituents as a New Class of Ligands for the Lysosomal Sialic Acid Transporter Sialin Page 9 Page 11