Molecular Basis of 1-Deoxygalactonojirimycin ... - ACS Publications

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Molecular Basis of 1‑Deoxygalactonojirimycin Arylthiourea Binding to Human α‑Galactosidase A: Pharmacological Chaperoning Efficacy on Fabry Disease Mutants Yi Yu,†,‡,¶ Teresa Mena-Barragán,§,¶ Katsumi Higaki,*,† Jennifer L. Johnson,∥ Jason E. Drury,∥ Raquel L. Lieberman,∥ Naoe Nakasone,⊥ Haruaki Ninomiya,⊥ Takahiro Tsukimura,# Hitoshi Sakuraba,∇ Yoshiyuki Suzuki,○ Eiji Nanba,† Carmen Ortiz Mellet,*,§ José M. García Fernández,◆ and Kousaku Ohno‡,⊕ †

Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Yonago 683-8503, Japan Division of Child Neurology, Institute of Neurological Sciences, Tottori University Faculty of Medicine, Yonago 683-8504, Japan § Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Sevilla 41012, Spain ∥ School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, Georgia United States ⊥ Department of Biomedical Regulation, School of Health Science, Tottori University Faculty of Medicine, Yonago 683-8503, Japan # Department of Functional Bioanalysis, Meiji Pharmaceutical University, Tokyo 204-8588, Japan ∇ Department of Clinical Genetics, Meiji Pharmaceutical University, Tokyo 204-8588, Japan ○ Tokyo Metropolitan Institute of Medical Science, Tokyo 156-0057, Japan ◆ Instituto de Investigaciones Químicas (IIQ), CSIC-Universidad de Sevilla, Sevilla 41092, Spain ‡

S Supporting Information *

ABSTRACT: Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene often leading to missense α-galactosidase A (α-Gal A) variants that undergo premature endoplasmic reticulum-associated degradation due to folding defects. We have synthesized and characterized a new family of neutral amphiphilic pharmacological chaperones, namely 1-deoxygalactonojirimycin-arylthioureas (DGJ-ArTs), capable of stabilizing α-Gal A and restoring trafficking. Binding to the enzyme is reinforced by a strong hydrogen bond involving the aryl-N′H thiourea proton and the catalytic aspartic acid acid D231 of α-Gal A, as confirmed by a 2.55 Å resolution cocrystal structure. Selected candidates enhanced α-Gal A activity and ameliorate globotriaosylceramide (Gb3) accumulation and autophagy impairments in FD cell cultures. Moreover, they acted synergistically with the proteostasis regulator 4-phenylbutyric acid, appearing to be promising leads as pharmacological chaperones for FD.

However, large-scale screenings of Italian and Taiwan male newborns revealed an occurrence of mutations in the GLA gene as high as 1:3100 and 1:1250, respectively (Hwu et al., 2009; Spada et al., 2006).5,6 The cocrystal structure of α-Gal A with galactose revealed 13 direct hydrogen bonding interactions with amino acid residues.7,8 The mutations affecting any of these amino acids result in the severe, classical phenotype of FD. To date, more than 500 mutations in the GLA gene have been identified including a variety of missense, nonsense, splicing, small deletion, or insertion mutations. About 60% of them were

Fabry disease (FD; OMIM 301500) is an X-linked lysosomal storage disorder (LSD) caused by mutations in the GLA gene that encodes lysosomal α-galactosidase A (α-Gal A; EC 3.2.1.22).1,2 α-Gal A cleaves α-linked galactosyl moieties of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3, also known as GL-3 and ceramide trihexoside). Deficiency in αGal A activity results in accumulation of Gb3, which gives rise to a variety of clinical manifestations such as cardiomyopathy, renal dysfunction, stroke, and, in some cases, neurological symptoms.2 Although FD follows X-linked inheritance, heterozygous females can be symptomatic.3 In FD cells, lysosomal storage and cellular dysfunction lead to compromised energy metabolism, impaired autophagosome maturation, and eventual cell death.4 The incidence of FD, according to the literature, varies from 1:476 000 to 1:117 000 live births.2 © 2014 American Chemical Society

Received: February 24, 2014 Accepted: April 28, 2014 Published: April 30, 2014 1460

dx.doi.org/10.1021/cb500143h | ACS Chem. Biol. 2014, 9, 1460−1469

ACS Chemical Biology

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Figure 1. Design criteria for DGJ-ArTs as PCs for FD. Key hydrogen-bonding interactions involving the catalytic aspartate (D170) and aspartic acid (D231) residues in the complexes of α-Gal A with DGJ (A) and D-galactose (B) and expected scenario for DGJ-ArT (C) pharmacological chaperones.

catalytic residue D231 stabilizes the corresponding complex by hydrogen bonding with the α-face-oriented anomeric OH1, which is not possible for a C1 carbon substituent (Figure 1B). We hypothesized that the later interaction could be maximized instead after transformation of the basic amino group of DGJ into a neutral arylthiourea functionality, with strong hydrogenbond donor capabilities (Figure 1C). As a proof of concept, we have now prepared a series of DGJ-arylthioureas (DGJ-ArTs) and assessed the molecular basis for α-Gal A binding by X-ray crystallography. Evaluation of the chaperoning efficiency in FDassociated mutants let identified compounds with improved PC activities in cultured cells as compared with DGJ.

missense mutations, which give rise to unstable protein targeted to premature degradation in the endoplasmic reticulum (ER) (Human Gene Mutation Database, http://www.hgmd.cf.ac. uk).9 It has been observed that the higher the level of the residual enzyme activity, the milder the disease phenotype. Enzyme replacement therapy (ERT) is available for the treatment of FD.10,11 This therapy requires an intravenous infusion of purified human α-Gal A every other week. Although the efficacy of ERT has been confirmed in many FD patients, intrinsic problems include instability of the enzyme protein in blood and adverse side effects associated with immunological reactions against the recombinant α-Gal A protein.12 An alternative therapeutic approach consists in restoring the proper folding and lysosome delivery of degradation-prone mutant enzymes by using small molecules as pharmacological chaperones (PCs).13,14 Once in the lysosome, the high local concentration of the substrate displaces the chaperone from the active site and normal processing is re-established. Competitive inhibitors with strong affinity for the target glycosidase can actually act as PCs. Thus, the iminosugar 1deoxygalactonojirimycin (DGJ, migalastat hydrochloride or AT1001), a mimic of the terminal galactose unit of Gb3, has been shown to bind to the active site of α-Gal A and to enhance the residual activities of several α-Gal A mutants.15,16 DGJ is a low molecular weight compound that can be administrated orally and is free from adverse immunological reactions.17 It is currently under clinical trials to be tested for its safety and efficacy in FD patients.18 Promising basic and preclinical results of PC therapy have been obtained also for other LSDs, supporting its potential as a novel therapeutic paradigm.19,20 The potential benefit of PCs as a supplement to ERT has been also pointed out.21 DGJ, as all amine-type iminosugars, is largely protonated at physiological pH and strongly hydrophilic, which limits diffusion through biological membranes. Incorporation of Nand C1-substituents onto the DGJ piperidine ring has been proposed in order to impart amphiphilicity and improve druglike properties. However, the resulting alkyl-DGJ derivatives systematically exhibited a significant decrease in α-Gal A binding affinity that correlated with lower chaperoning potential,22 which probably results from a penalty in enzymechaperone complementarity after derivatization. The crystal structure of the DGJ:α-Gal A complex involves a hydrogenbonding interaction between the protonated endocyclic amino group of the iminosugar, and the carboxylate group of the catalytic aspartate nucleophile residue D1708,23 that is likely to be hampered by N-substitution (Figure 1A). In the case of a neutral ligand such as galactose, the opposite carboxylic acid

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RESULTS AND DISCUSSION Pharmacological chaperones (PCs) are molecular ligands with the ability to bind to mutant enzymes at the ER and promote their correct folding and trafficking.13,24 With few exceptions,25,26 most of the reported PCs for LSDs are active-site directed low molecular weight compounds that behave as competitive inhibitors of the target glycosidase.13 This is indeed the case of DGJ, an iminosugar-type glycomimetic with α-Gal A inhibitory properties currently in clinical trials as a PC for Fabry disease.15,16 The available crystal structures of α-Gal A and its complexes with different ligands suggests that the efficacy of DGJ as an inhibitor and chaperone stems largely from its ability to mimic the α-galactopyranoside moiety of the natural Gb3 substrate while establishing an additional hydrogen-bonding interaction between the protonated endocyclic amino group and the catalytic aspartate residue D170.8,23 We conceived that the enhanced hydrogen-bond donor abilities of the N′H proton in DGJ-arylthioureas would reinforce instead the interaction with the opposite catalytic aspartic acid residue D231 in the chaperone:α-Gal A complex, mimicking the situation encountered in the corresponding complex with D-galactose, leading to a new family of neutral PCs with amphiphilic properties better suited for drug optimization strategies. Synthesis of Novel DGJ-ArT Pharmacological Chaperones. The electron withdrawing character of the N′-aryl substituent in DGJ-arylthioureas is entitled to enhance the hydrogen-bond donating abilities of the corresponding N′H thiourea proton, which at its turn was expected to stabilize the chaperone:α-Gal A complex after interaction with the catalytic aspartic acid D231. To test this hypothesis, the N′-(1-naphthyl) (DGJ-NphT), N′-(p-methoxyphenyl) (DGJ-pMeOPhT), N′(p-methylthiophenyl) (DGJ-pMeSPhT) and N′-(p-fluorophenyl) (DGJ-pFPhT) derivatives were prepared as DGJ-ArT representatives. Their synthesis was accomplished in one step and with total chemoselectivity by reaction of fully unprotected 1461



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Figure 2. Crystal structure of α-Gal A in complex with DGJ-pFPhT. (A) Location of DGJ-pFPhT and key active site residues. Gray mesh is final 2Fo-Fc density contoured at 1σ. Green mesh is Fo−Fc difference density contoured at 3s (σ) after initial molecular replacement and first round of refinement, prior to modeling the inhibitor. (B) H-bonding network involving DGJ-pFPhT in the active site of human α-Gal A. Distances are averages of two copies in the asymmetric unit. (C) H-bonding network involving DGJ in the active site of human α-Gal A from PDB code 3GXT. Distances are in Ångstrom.

DGJ with 1-naphtyl, p-methoxyphenyl, p-methylthiophenyl, or p-fluorophenyl isothiocyanate, respectively (see Supporting Information). For comparative purposes, the conformationally constrained bicyclic isothiourea-type derivative 5N,6S-(pfluorophenylimino methylidene)-6-thio-1-deoxynojirimycin (pFPhIM-DGJ) and the DGJ N′-benzylthiourea (DGJ-BnT), an alkylthiourea representative, were also prepared. The structures of all compounds were confirmed by mass spectrometry, 1H and 13C NMR spectroscopy and microanalytical data (see Supporting Information). As expected from their chemical structures, the synthesized DGJ-thioureas behaved as nonionizable compounds in a pH range that largely surpassed the physiological window (pKa > 12 as determined by 1H NMR titration experiments), which is in contrast with the basic character of DGJ (pKa 7.1)27 or the imino derivative pFPhIM-DGJ (1H NMR determined pKa 5.32). DGJ-thioureas also exhibited significantly higher octanol/water partition coefficients. The logarithm of this value, log P, is the parameter that determines the lipophilicity of a molecule and is currently used to estimate, among other biological properties, the membrane crossing capabilities of drug candidates.28 Log P values between 0.63 and 1.13 were obtained for the four ArTs studied in this work as compared with −1.98 for DGJ, supporting their better druglikeness. Crystal Structure of the DGJ-pFPhT:α-Gal A Complex and Comparison with DGJ- and D-Galactose-bound Structures. The 2.55 Å resolution crystal structure of the DGJ-pFPhT:α-Gal A complex reveals the location of the glycomimetic in the catalytic site, with clear 2Fo-Fc density for the iminosugar and fluorobenzyl ring, but not the intervening thiourea linkage (Figure 2A). The structure of the α-Gal A polypeptide is unchanged upon ligand binding, as seen in prior structures of ligand-bound α-Gal A.7 The hydroxyl groups OH2, OH3, OH4, and OH-6 of DGJ-pFPhT are involved in extensive hydrogen-bonding network with the amino acid residues Asp 92, Asp 93, Asp 170, Lys 168, Arg 227, as well as the catalytic nucleophile Asp 231 (Figure 2B). The piperidine ring adopts an orientation that is similar to the six-membered ring of DGJ (Figure 2B,C) or D-galactose (not shown) in the corresponding complex structures,7,8 but with key differences that are in agreement with our molecular design hypothesis. Namely, the interactions of Asp 170 and Asp 231 have changed. The N′H proton in DGJ-pFPhT likely forms a hydrogen bond

with Asp 231, while this residue is H-bonded to OH2 in the case of DGJ. Conversely, Asp 170 contributes to the stabilization of DGJ-pFPhT by interacting with OH4 rather than the endocyclic nitrogen as in DGJ. Additional observations from the structure include the fact that the distances from the chaperone to the amino acid residues are somewhat longer in the case of DGJ-pFPhT compared to DGJ, which may be a result of the fact that the fluoro substituent is forming a hydrogen-bonding interaction with the carbonyl oxygen of a Arg 193 from a neighboring polypeptide chain in the lattice or because the DGJ-pFPhT:α-Gal A complex structure was solved at pH 4.5, thus changing the protonation state of some interacting residues. Another key feature revealed is the position of the aryl group, which is nestled among hydrophobic residues Leu 206, Ala 230, and Tyr 207. In summary, the cocrystal structure confirms that we achieved the desired orientation of our inhibitor within the active site of α-Gal A, and opens up the possibility for future analogues optimization for potency by further exploring chemical substituents on the aryl ring. Effects of DGJ-ArTs on Human α-Gal A In Vitro. The inhibitory activities of the prepared DGJ-ArTs against lysosomal hydrolases from normal human skin fibroblasts were assessed29,30 in comparison to that of DGJ and the bicyclic isothiourea pFPhIM-DGJ and alkylthiourea derivative DGJBnT. The activities against α-Gal A are summarized in Figure 3A and the IC50 values were calculated from these curves and shown in Table 1. The IC50 values (pH 5.0) for DGJ-ArTs were in the range 0.0083 to 1.6 μM, with the values for pmethylthiophenyl derivative DGJ-pMeSPhT (0.0083 μM) in the same order of magnitude as the corresponding value for DGJ (0.003 μM). The inhibitory potential dropped by three-tofour orders of magnitude for the conformationally constrained bicyclic derivative pFPhIM-DGJ and the alkylthiourea DGJBnT, confirming the superiority of DGJ-ArTs as active sitedirected α-Gal-A ligands. The inhibitory potency of DGJpMeOPhT increased by 4.6-fold on going from pH 5.0 (IC50 0.074 μM) to pH 7.0 (IC50 0.016 μM). Considering that a pharmacological chaperone must tightly bind to the enzyme in the endoplasmic reticulum (neutral pH) but being released at the lysosome (acid pH) this is a favorable feature. No inhibition was detected against β-galactosidase (β-Gal) or hexosaminidase (Supporting Information Figure S2). Similarly to DGJ, the four 1462



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Figure 3. Effects of DGJ-arylthioureas on α-Gal in vitro. (A) Inhibition activities of DGJ-ArTs on α-Gal in vitro. Enzyme activity in lysate from human normal fibroblasts was determined in the absence or presence of increasing concentrations of DGJ-ArTs compounds. Each point represents the mean ± SEM of three determinations each done in triplicate. (B) Stabilization activities of DGJ-ArTs on α-Gal in vitro. Cell lysate from human normal fibroblasts was incubated at pH7.0 and 48 °C for the indicated time and the α-Gal activity was measured. Each point represents means of triplicates obtained in three independent experiments. Values were expressed as relative to the activity in the absence of compound as 100%.

new DGJ-ArTs showed moderate inhibitory activities against αN-acetylgalactosamine (α-NAGAL) (Supporting Information Figure S2). We then evaluated the abilities of DGJ-ArTs to protect the enzyme from heat-induced inactivation as reported previously.29,30 The α-Gal A activity in the cell extracts decreased to 20% of the initial value after 40 min incubation at 48 °C at pH 7. DGJ-ArTs suppressed this activity reduction in a dosedependent manner much more efficiently than pFPhIM-DGJ and DGJ-BnT. The N′-(p-methoxyphenyl)- and N′-(p-

methylthiophenyl) derivatives DGJ-pMeOPhT and DGJpMeSPhT were the most efficient chaperones in this test, achieving 95 and 85% α-Gal A protection, respectively, after 1 h heating at 30 μM concentration (Figure 3B). α-Gal A Activity Enhancements in Cultured Normal and FD Fibroblasts after Treatment with DGJ-ArTs. To evaluate the enzyme activity enhancements induced by DGJArTs, normal and FD fibroblasts expressing Q279E mutant αGal A were cultured for 96 h in the absence or presence of each chaperone at 0, 0.3, 3, and 30 μM concentrations for 96 h 1463



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favor substrate rather than imino sugar, moving the kinetic balance toward Gb3 processing.31 It remains to be confirmed that enzyme activity is still able to balance hydrolysis at the expenses of inhibition when substrate becomes depleted in the lysosome to below Km. In any case, the neutral character of DGJ-ArTs must facilitate inhibitor displacement as compared with a protonated tight-binding inhibitor such as DGJ. Mutation Profiling of the α-Gal A Chaperoning Activity of DGJ-ArTs. To investigate the chaperone effect of DGJ-ArTs on a range of mutant α-Gal A, a wild-type and 17 types of missense mutants were transiently transfected into COS7 cells. The activities of at least 15 out of the 17 mutants as well as the wild-type were significantly enhanced by the four DGJ-ArTs. Overall, the arylthioureas were more efficient than the isothiourea and alkylthiourea DGJ derivatives pFPhIM-DGJ and DGJ-BnT, with the p-methoxyphenyl and the pfluorophenyl thioureas DGJ-pMeOPhT and DGJ-pFPhT being the most efficient representatives (Figure 4D and Supporting Information Figure S9). Nevertheless, some exceptions deserve to be noted. Thus, the bicyclic DGJ isothiourea derivative pFPhIM-DGJ, a weak inhibitor of α-Gal A, was significantly more efficient than the DGJ-ArTs as a chaperone in the A20P mutant, whereas the benzylthiourea DGJ-BnT was the most efficient chaperone for the M296I mutant. These data are in line with previous studies on the molecular mechanisms of PC functioning in several LSDs, which suggest that mutants susceptible of reacting to pharmacological chaperone therapy retain full or partial catalytic activities with trafficking and processing defects.20,29 Up-Regulation of Autophagic Proteins in FD Cells and Its Restoration by DGJ-pMeOPhT and DGJ-FPhT in Transformed FD Fibroblasts. The senescence of primary cultured skin fibroblasts is an obvious inconvenience for their use in any assay platform. On the other hand, excess level of gene product is a disadvantage of the transient expression of glycosidases in COS7 cells. To circumvent these problems, we established SV40-mediated transformed cell lines from normal and Q279E FD fibroblasts (FD-SV). We first assessed the chaperone effects of DGJ-pMeOPhT and DGJ-FPhT and found that both of these DGJ-ArTs increased the residual α-Gal A activities in FD-SV cells at 3 and 30 μM concentration, exhibiting a significantly higher chaperoning efficiency than DGJ at 30 μM (Figure 5A). With next used the FD-SV platform to assess the levels of autophagy-related proteins in the cells. Impairment of autophagy is well-established as a hallmark of cellular pathology in lysosomal storage diseases.4,32 We were thus interested in assaying the effect of the new pharmacological chaperones in this cellular pathophysiology condition. Immunoblot analyses revealed a marked increase in the level of LC3-II, a specific autophagosome marker, in FD-SV cells compared with that in control cells (Figure 5B). The level of p62, a protein that interacts with ubiquitinated proteins and LC3, was also increased in FD-SV cells. However, neither the levels of beclin-1, a regulator of autophagy, nor that of Bip, an ER stressrelated protein, were altered in FD-SV cells. When cells were incubated with serum-starvation medium, the levels of LC3-II and p62 increased in normal but not in FD-SV cells, supporting that autophagy is actually impaired in the later. Treatment of FD-SV cells with the arylthioureas DGJ-pMeOPhT and DGJpFPhT significantly reduced the levels of both LC3-II and p62 (Figure 5C), confirming their potential in new FD therapeutic strategies.

Table 1. Inhibition Activities of Compounds Against Human α-Gal A In Vitro pH 5 DGJ-NphT DGJ-pMe OPhT DGJ-pMe SPhT DGJ-pF PhT pF PhIM-DGJ DGJ-BnT DGJ

1.60 0.074 0.0083 0.34 9.67 20.26 0.0030

± ± ± ± ± ± ±

0.092 0.0064 0.00051 0.047 0.88 2.43 0.00048

pH 7 0.37 0.016 0.0014 0.043 2.18 4.41 0.00078

± ± ± ± ± ± ±

0.045 0.0020 0.00031 0.0062 0.39 0.65 0.00013

Inhibition activities was determined as described in Figure 3A. Each value represents IC50 (μM) of the mean ± SEM of three determinations done in triplicate.

before determination of α-Gal A activities in extracts. In normal cells, three of the four DGJ-ArTs assayed, namely DGJ-NphT, DGJ-pMeOPhT, and DGJ-pFPhT, caused 1.2- to 1.5-fold increases in α-Gal A activities (Figure 4A). In Q279E FD cells, all the four arylthioureas, including DGJ-pMeSPhT, significantly enhanced the activity of the mutant α-Gal A at concentrations of 3 and 30 μM (Figure 4B). In agreement with our starting hypothesis, the chaperoning efficiency was markedly higher for DGJ-ArTs as compared with pFPhIM-DGJ and DGJ-BnT. Notably, treatment with 30 μM DGJ-pMeOPhT led to a more than 7-fold α-Gal A activity increase as compared with untreated cells, tripling the effect of DGJ at its optimal concentration of 20 μM (Figure 4B). DGJ-pMeSPhT, but not DGJ-pMeOPhT or DGJ-pFPhT, appeared to be toxic to cells as evaluated by an LDH assay (Supporting Information Figure S3). Therefore, we selected the two later compounds for further analyses. The maximum chaperoning efficiency was reached at 30 μM. Immunofluorescence analyses revealed that treatment of Q279E fibroblasts with DGJ-ArTs significantly reduced accumulation of Gb3, indicating that the rescued enzyme is actively processing the substrate at the lysosome, (Figure 4C). Time course analysis found that chaperone activities on Q279E mutant α-Gal A activity was increased time dependently with 4 days treatment (Supporting Information Figure S4). When cells were washed out, the activity gradually decreased to the basal level within 4 days. Decrease of Gb3 levels was also detected within 4 days treatment, then Gb3 levels increased again after 3 days washout (Supporting Information Figure S5). At higher concentrations, the inhibitory activity overcame the chaperoning effect as confirmed in both normal and FD fibroblasts expressing α-Gal A with the Q279E mutation (Supporting Information Figure S6). High concentration of treatment yielded no obvious changes in normal cells and modest decreases in Gb3 levels in Q279E fibroblasts (Supporting Information Figure S7). The chaperoning activity was also confirmed in R301Q FD fibroblasts (Supporting Information Figure S8). It is someway shocking that an increase in the catalytic activity of the α-Gal A Fabry variants is observed with all DGJArTs albeit using concentrations far in excess of their IC50 values. Actually, the optimal concentration of 30 μM is about 3 orders of magnitude higher. This apparent paradox has previously been observed for DGJ and other active-site directed pharmacological chaperone imino sugars. It must be considered, however, that the Km of the enzyme for the natural Gb3 substrate in the lysosome is probably extremely low. Substrate concentrations are then in excess of Km, especially when storage becomes pronounced, and the competition for binding will 1464



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Figure 4. Chaperone activities of DGJ-pMe OPhT in cultured human fibroblasts. Chaperone activities of DGJ-pMe OPhT on normal (A) and FD patient’s (B) fibroblasts and COS7 cells with α-Gal A expression (D) were measured as described in Methods. Each bar represents the mean ± SEM of three determinations each done in triplicate. *p < 0.05, statistically different from the value of untreated samples. (C) Immunofluorescence of antiGb3 and the quantification of Gb3 intensity. Treatment of DGJ-pMe OPhT decreased the level of anti-Gb3 signal in FD fibroblasts. Scale bar, 20 μm.

Synergetic Effects of DGJ-pMeOPhT and Proteostasis Regulators. Recent investigations on the molecular basis of FD support that α-Gal A deficiency can be attributed to the interplay of defective biosynthesis, loss of kinetic capability, and excess degradation of the mutant enzyme.33 Aggregation of mutant α-Gal A, for instance, has been shown to negatively affect the correct trafficking of the enzyme, aggravating the FD phenotype.34 Moreover, aggregation-prone mutants are less susceptible to be responsive to PC treatment probably due to impaired access to the catalytic site in the aggregates, as demonstrated by the failure of response to DGJ.34 The ability of chemical agents that target ER-related protein homeostasis

(or proteostasis) pathways to rescue misfolded mutant proteins has been demonstrated.35 To explore the potential of a combined proteostasis regulator-pharmacological chaperone therapy for FD, we evaluated the ability of 4-phenylbutyric acid (4-PBA) and celastrol, two proteostasis regulators, to rescue Q279E mutant α-Gal A in the absence or presence of DGJ-pMeOPhT. 4-PBA, but not celastrol, enhanced the mutant enzyme activity. Notably, the combined treatment with 4-PBA and DGJ-pMeOPhT caused significant synergetic effects in SVFD cells (Figure 6). 4-PBA is a short-chain fatty acid approved for treatment of a urea cycle disorder.36 It also acts as a histone deacetylase inhibitor,37 which has been argued to be relevant 1465



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potential of a combined proteostasis regulator-PC therapy for the treatment of FD. In conclusion, we have synthesized a new family of PCs for FD, namely DGJ-arylthioureas (DGJ-ArTs), and elucidated the molecular basis for their efficient binding to α-Gal A. The neutral amphiphilic character of DGJ-ArTs makes them more suitable than the parent iminosugar DGJ for drug optimization and development. In vitro results support a very high efficacy of the new PCs to enhance the residual activity of FD-associated α-Gal A mutants, reducing the accumulation of the substrate Gb3 in FD cells. In addition PCs act in a synergetic manner with the proteostasis regulator 4-PBA. This suggests that approaches based in the use of DGJ-ArTs are worth exploring as FD therapeutic strategies. Moreover, our data suggest that similar structure-based, molecular diversity-oriented PC design tactics may be applicable to develop new therapies for other LSDs.

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METHODS

Synthesis of DGJ-arylthioureas and other DGJ Derivatives. 1-Deoxygalactonojirimycin (DGJ) was prepared in our laboratory from 3,4-O-isopropylidene-1-deoxygalactonojirimycin following a reported route.40 The new DGJ-ArT pharmacological chaperones DGJ-NphT, DGJ-pMeOPhT, DGJ-pMeSPhT, and DGJ-pFPhT (Figure 7) were synthesized from DGJ by reaction with 1-naphtyl, p-methoxyphenyl, p-methylthiophenyl, or p-fluorophenyl isothiocyanate, respectively. Two additional DGJ derivatives, namely the bicyclic p-fluorophenyl isothiourea pFPhIM-DGJ and the alkythiourea DGJBnT, were synthesized for comparative purposes (Figure 7). The first one was obtained by HCl-promoted cyclization of the monocyclic thiourea precursor DGJ-pFPhT, whereas DGJ-BnT was obtained by nucleophilic addition of DGJ to benzyl isothiocyanate. Detailed synthetic protocols and physicochemical characterization data are provided in the Supporting Information. For biological evaluation studies, solutions were prepared in DMSO and kept at −30 °C. Cell Culture, Transfection and Chaperone Test. COS7 cells and human skin fibroblasts were cultured in DMEM with 10% FBS. Immortalized human fibroblast lines were established by transfecting SV40 large T cDNA expression vector, pET321-T.41 For chaperone test, human fibroblasts were cultured in the medium with or without chaperone compounds for 96 h as described.29,30 When immortalized human fibroblasts cells were used, they were exposed to chaperone compounds for 48 h. Transfection of COS7 cells with wild-type and mutant α-Gal A cDNA42 was performed using Lipofectamine 2000

Figure 5. Effects of DGJ-pMe OPhT and DGJ-pF PhT in the impairment of autophagy of transformed FD fibroblasts. (A) Chaperone effects on transformed FD (SV-FD) fibroblasts. Each bar represents the mean ± SEM of three determinations each done in triplicate. *p < 0.05, statistically different from the value of untreated samples. (B) Immunoblot analyses of autophagy-related proteins (LC3, p62, and beclin-1) and Bip protein in the lysate from transformed normal and FD fibroblasts. β-Tubulin was used as a control. (C) Effects of DGJ-pMe OPhT and DGJ-pF PhT on the levels of expression of LC3-II and p62 protein in the lysates of transformed FD fibroblasts.

for eliciting cellular events in cells from patients suffering from LSDs such as Gaucher disease or Niemann−Pick C disease.38,39 To the best of our knowledge, this is the first evidence of the

Figure 6. Synergetic effects of proteostatis regulators with DGJ-pMe OPhT on α-Gal A activity. Transformed FD fibroblasts were treated with proteostasis regulators (4-PBA and celastrol) and/or DGJ-pMe OPhT. Each bar represents the mean ± SEM of three determinations each done in triplicate. *p < 0.05, statistically different from the value of in the absence of 4-PBA and celastrol controls. 1466



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Figure 7. Structures of the new DGJ-ArTs and other DGJ derivatives prepared and evaluated in this work. hyde in phosphate buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS, and incubated with primary antibodies for 1 h. Bound antibodies were detected with Alexa-Fluor-conjugated secondary antibodies. Fluorescence images were obtained using a confocal laser microscope (Leica TSC SP-2; Wetzler, Germany). Quantification of Gb3 intensity was measured using Leica confocal software, normalized with nuclear (DAPI) intensity. More than 100 cells in 10 randomly obtained images were evaluated in each experiment. Immunoblotting. All the procedures were carried out at 4 °C. Cultured cells were lysed by sonication in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and a protease inhibitor cocktail (Roche Diagnostics). Detergent-resistant membrane microdomains were obtained by centrifugation of the lysates in 1% Triton X100 at 100 000 g for 30 min. Immunoblotting was performed as described.20 Signals from horseradish peroxidase (HRP)−conjugated secondary antibodies were visualized by ECL detection kit (GE Healthcare Bioscience) and images were obtained using LAS-4000 lumino image analyzer (Fujifilm, Tokyo, Japan). Statistical Analyses. Statistical analyses were conducted by paired t-test using Excel Software.

reagent. After 5 h, the medium was replaced with fresh medium supplemented with or without chaperone compounds and incubated for 48 h. Cytotoxicity of the compounds in human fibroblasts was evaluated by the LDH assay. Measurement of Lysosomal Enzyme Activities. Lysosomal enzyme activities were measured by using 4-MU-conjugated substrates: 4-MU-conjugated α-D-galactopyranoside for α-Gal, β-Dgalactoside for β-Gal, N-acetyl-β-D-glucosaminide for total Hex and αN-acetyl-D-galactosaminide for α-NAGA. For α-Gal assay, 10 μL of cell lysates in 0.1% Triton X-100 in dH20 mixed with 20 μL of 4-MU substrate (5 mM 4-MU α-D-galactopyranoside and 0.1 M N-acetyl-Dgalactosamine in 0.1 M citrate buffer (pH 4.5)) was incubated at 37 °C for 60 min and the reaction was terminated by adding 0.2 M glycinNaOH (pH 10.7). The librated 4-MU was masured with a fluorescence plate reader (excitation 340 nm; emission 460 nm; Infinite F500, TECAN, Kawasaki, Japan). Enzyme activity was normalized by protein concentration (Protein Assay Rapid Kit; Wako, Tokyo, Japan). Inhibition and Stabilization of α-Gal A In Vitro. For inhibition assay, 0.1% Triton X-100 extracts from normal skin fibroblasts were mixed with 4-MU substrates in absence or presence of increasing concentrations of DGJ derivatives.29 For heat-induced degradation, extracts were incubated in 0.1 M citrate buffer (pH 7) at 48 °C for the time indicated. The incubation was terminated by adding 0.1 M citrate buffer (pH 4.5). The enzyme activities were measured as above. Crystallization and X-ray Data Collection. Crystals of apo αGal A were grown as described previously7 with 10 mg mL−1 α-Gal A at pH 4.5. To the crystallization drop, 0.15 μL of 1 mM DGJ-pFPhT encapsulated in β-cyclodextrin (βCD)43 was added and incubated for 4.5 h. Crystals were then harvested for data collection by cryocooling in mother liquor supplemented with 30% ethylene glycol. Diffraction data were collected at beamline 22-ID of the Southeast Regional Collaborative Access Team (SER-CAT) at the Advanced Photon Source, Argonne, Illinois. Data were indexed, integrated and scaled in HKL-200044 in space group P3221. Molecular replacement was performed in Phaser45 using a single chain of apo acid-α-galactosidase (PDB code 3GXN) as a model.7 The model was refined in Phenix,46 and Coot47 was used for model building. eLBOW48 within Phenix was used to generate restraints of DGJ-pFPhT from coordinates generated using PRDRG.49 The final occupancy of the drug is 0.6. Crystallographic statistics appear in Table S1 in the Supporting Information, and the structure has been deposited to the Protein Data Bank with accession code 4NXS. Immunofluorescence Staining. All the procedures were carried out at RT.14,20 Cells on coverslips were fixed with 4% paraformalde-

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ASSOCIATED CONTENT

S Supporting Information *

Eleven figures, one table, and supplemental experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

Coordinates and structure factors have been deposited in the Protein Data Bank under code 4NXS.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +81-859-38-6472. Fax: +81-859-38-6470. Email: [email protected] *Tel: +34-9559806. Fax: +34-954624960. Email: [email protected]. Present Address ⊕

Sanin Rosai Hospital, Yonago, Japan

Author Contributions ¶

Y.Y. and T.M.-B. contributed equally to this work

Notes

The authors declare no competing financial interest. 1467



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ACKNOWLEDGMENTS We thank Dr. S. Ishii and Dr. S. Sugano for kindly providing the human α-Gal A and SV40 cDNA expression vectors, respectively. This study was supported by the Ministry of Education, Culture, Science, Sports and Technology of Japan (22390207 and 23591498), the Ministry of Health, Labour and Welfare of Japan (H17-Kokoro-019, H20-Kokoro-022), the Spanish Ministerio de Ciencia e Innovación (Contract Nos. SAF2010-15670 and CTQ2010-15848), the Fundación Ramón Areces, the Junta de Andaluciá (Project P08-FQM-03711), the European Regional Development Funds (FEDER), and the European Social Funds (FSE). The Center for Research, Technology and Innovation of the University of Seville (CITIUS) is also acknowledged. K.H. was supported by Takeda Science Foundation. J.L.J. was supported by in part by US Department of Education GAANN grant P200A090307 and Georgia Tech Molecular Biophysics traineeship. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

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