Glucosidase Inhibitors: Binding Orientation and Inﬂuence on Selectivity

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Article Strategy for Designing Selective Lysosomal Acid α-Glucosidase Inhibitors: Binding Orientation and Inﬂuence on Selectivity

Atsushi Kato 1,* , Izumi Nakagome 2, Mizuki Hata 1, Robert J. Nash 3, George W. J. Fleet 4 , Yoshihiro Natori 5, Yuichi Yoshimura 5 , Isao Adachi 1 and Shuichi Hirono 2 1 Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan; [email protected] (M.H.); [email protected] (I.A.) 2 School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan; [email protected] (I.N.); [email protected] (S.H.) 3 Institute of Biological, Environmental and Rural Sciences, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK; [email protected] 4 Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK; george.ﬂ[email protected] 5 Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai 981-8558, Japan; [email protected] (Y.N.); [email protected] (Y.Y.) * Correspondence: [email protected]

Academic Editor: Derek J. McPhee Received: 27 May 2020; Accepted: 18 June 2020; Published: 19 June 2020

Abstract: Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1-C-heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC50 value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1-C-alkyl chain. It is noteworthy that α-1-C-heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.

Keywords: iminosugars; glucosidase inhibitor; lysosomal acid α-glucosidase; ER α-glucosidase II; ligand docking; molecular dynamics; drug design

1. Introduction Glycosidases are involved in a wide range of important biological processes; these include carbohydrate catabolism in the intestines, lysosomal catabolism of glycolipids, biosynthesis of oligosaccharide chains, and quality control of N-linked glycoproteins in the endoplasmic reticulum (ER).

Molecules 2020, 25, 2843; doi:10.3390/molecules25122843 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 13 Molecules 2020, 25, 2843 2 of 13 oligosaccharide chains, and quality control of N-linked glycoproteins in the endoplasmic reticulum (ER). Accordingly, the inhibition of these glycosidases can have profound effects on the intestinal Accordingly, the inhibition of these glycosidases can have profound effects on the intestinal digestion, digestion, catabolism of glycolipids in lysosome, the maturation, transport, and secretion of catabolism of glycolipids in lysosome, the maturation, transport, and secretion of glycoproteins, glycoproteins, and can alter cell-cell or cell-virus recognition processes. This provides a basis for the and can alter cell-cell or cell-virus recognition processes. This provides a basis for the potential use of potential use of glycosidase inhibitors in diabetes, genetic lysosomal disorders, cancer, and viral glycosidase inhibitors in diabetes, genetic lysosomal disorders, cancer, and viral infection [1–3]. infection [1–3]. Iminosugars are sugar mimics with a nitrogen atom in place of the ring oxygen of monosaccharides. Iminosugars are sugar mimics with a nitrogen atom in place of the ring oxygen of They usually bind to the active sites of glycosidases in a reversible and competitive manner, as a result monosaccharides. They usually bind to the active sites of glycosidases in a reversible and of their structural resemblance to the terminal sugar moiety in the natural substrates. A large number competitive manner, as a result of their structural resemblance to the terminal sugar moiety in the of iminosugars have been discovered from natural sources. They are classified into five structural natural substrates. A large number of iminosugars have been discovered from natural sources. They classes: polyhydroxylated pyrrolidines, piperidines, indolizidines, pyrrolizidines and nor-tropanes [4,5]. are classified into five structural classes: polyhydroxylated pyrrolidines, piperidines, indolizidines, Among them, the inhibition specificity of piperidine iminosugars (Figure1) is usually easy to predict pyrrolizidines and nor-tropanes [4,5]. Among them, the inhibition specificity of piperidine from the configurations of hydroxy groups, which are identical to those of the corresponding pyranose. iminosugars (Figure 1) is usually easy to predict from the configurations of hydroxy groups, which For example, 1,5-dideoxy-1,5-imino-d-glucitol (deoxynojirimycin (DNJ)) corresponding to glucose are identical to those of the corresponding pyranose. For example, 1,5-dideoxy-1,5-imino-D-glucitol in the pyranose configuration has high inhibition specificity against α-glucosidases. Similarly the (deoxynojirimycin (DNJ)) corresponding to glucose in the pyranose configuration has high polyhydroxylated piperidine analogues of mannose and galactose, 1,5-dideoxy-1,5-imino-d-mannitol inhibition specificity against α-glucosidases. Similarly the polyhydroxylated piperidine analogues of (deoxymannojirimycin (DMJ)), and 1,5-dideoxy-1,5-imino-d-galactitol (deoxygalactonojirimycin (DGJ)), mannose and galactose, 1,5-dideoxy-1,5-imino-D-mannitol (deoxymannojirimycin (DMJ)), and show the expected inhibition of α-mannosidase and α-galactosidase, respectively [6]. The structural 1,5-dideoxy-1,5-imino-D-galactitol (deoxygalactonojirimycin (DGJ)), show the expected inhibition of basis of the inhibition of glycosidases by these compounds is clear, but this inhibition selectivity α-mannosidase and α-galactosidase, respectively [6]. The structural basis of the inhibition of based on high similarity with the substrate may cause side effects. The anti-diabetes drug, miglitol, glycosidases by these compounds is clear, but this inhibition selectivity based on high similarity was designed from DNJ to suppress postprandial hyperglycemia, by inhibiting the hydrolysis of with the substrate may cause side effects. The anti-diabetes drug, miglitol, was designed from DNJ disaccharides such as maltose, isomaltase, and sucrose in the brush border membrane, and delaying to suppress postprandial hyperglycemia, by inhibiting the hydrolysis of disaccharides such as the absorption of carbohydrates from the small intestine. However, miglitol is nearly completely maltose, isomaltase, and sucrose in the brush border membrane, and delaying the absorption of absorbed from the intestinal tract [7]. Therefore, it inhibited not only intestinal α-glycosidases, but carbohydrates from the small intestine. However, miglitol is nearly completely absorbed from the also the processing ER enzymes α-glucosidase I and II [8]. Therefore, closely mimicking iminosugars intestinal tract [7]. Therefore, it inhibited not only intestinal α-glycosidases, but also the processing can be expected to give potent inhibition, but may cause selectivity problems between glycosidase ER enzymes α-glucosidase I and II [8]. Therefore, closely mimicking iminosugars can be expected to isozyme species. give potent inhibition, but may cause selectivity problems between glycosidase isozyme species.

Figure 1. Cont.

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Figure 1. Structures and glycosidase inhibition of piperidine and pyrrolidine iminosugars, and acarbose. (FigureA): α-glucosidase 1. Structures inhibitors. and glycosidase (B): α-mannosidase inhibition inhibitor.of piperidine (C): α -galactosidaseand pyrrolidine inhibitor. iminosugars, and acarbose. (A): α-glucosidase inhibitors. (B): α-mannosidase inhibitor. (C): α-galactosidase inhibitor. This study is focused on lysosomal acid α-glucosidase (GAA; EC 3.2.1.20). GAA is a retaining exo-glucosidaseThis study responsibleis focused on for lysosomal the cleavage acid of α the-glucosidaseα-glycosidic (GAA; bond EC of 3.2.1.20). glycogen GAA to release is a retaining glucose. Pompeexo-glucosidase disease is responsible caused by mutationsfor the cleavage in the of gene the thatα-glycosidic encodes bond the GAA; of glycog theseen mutations to release resultglucose. in reducedPompe disease cellular is enzyme caused activity,by mutations and the in progressivethe gene that accumulation encodes the ofGAA; glycogen these inmutations the lysosomes result ofin heart,reduced skeletal cellular muscles, enzyme and activity, other and tissues the [progressiv9]. At present,e accumulation more than of 300 glycogen different in pointthe lysosomes mutations of haveheart, been skeletal identified muscles, within and the other GAA tissues gene [10[9].]. At These present, GAA more mutations than result300 different in a decrease point ofmutations enzyme stability,have been and identified a subsequent within decrease the GAA in gene the amount[10]. These of enzyme GAA mutations activity in result lysosome. in a decrease Pharmacological of enzyme chaperones,stability, and such a subsequent as some reversible decrease competitive in the amount inhibitors, of enzyme appear activity to be in able lysosome. to act as Pharmacological a template that stabilizeschaperones, the such native as folding some reversible state in the competitive ER by occupying inhibitors, the activeappear site to ofbe theable mutant to act enzyme,as a template thus allowingthat stabilizes its maturation the native and folding traffi ckingstate in to the the ER lysosome by occupying [11,12]. the It was active reported site of that the DNJmutant can enzyme, act as a pharmacologicalthus allowing its chaperone,maturation while and trafficking also showing to the inhibition lysosome of [11,12]. ER α-glucosidase It was reported I and that II, which DNJ can might act beas linkeda pharmacological to its side eff ectschaperone, [13,14]. while also showing inhibition of ER α-glucosidase I and II, which mightThus, be linked our strategy to its side has effects been to [13,14]. design effective pharmacological chaperones for Pompe disease that aThus,ffect theour stabilitystrategy has of GAA,been to which design not effectiv onlye showpharmacological a strong a ffichaperonesnity for GAA, for Pompe but also disease do notthat interfereaffect the with stability glycoprotein of GAA, processing.which not only We show herein a strong describe affinity that thefor GAA, glucosidase but also inhibitor do not 1,4-dideoxy-1,4-imino-interfere with glycoproteinl-arabinitol processing. (LAB) with Weα -1-hereinC-heptyl describe chain that is a morethe glucosidase selective inhibitor inhibitor of GAA1,4-dideoxy-1,4-imino- than DNJ. We alsoL-arabinitol investigated (LAB) the e ffwithect of αα-1--1-CC-heptyl-alkyl chainchain length is a more on the selective selectivity inhibitor for GAA of vs.GAA ER thanα-glucosidase DNJ. We also II. An investigated additional the aim effect was of to α demonstrate-1-C-alkyl chain the molecularlength on the docking selectivity properties for GAA of αvs.-1- ERC-alkyl-LAB α-glucosidase with II. GAA. An additional aim was to demonstrate the molecular docking properties of α-1-C-alkyl-LAB with GAA. 2. Results and Discussion

2.1.2. Results Structural and Similarity Discussion of Active Site between GAA and ER α-Glucosidase II

2.1. StructuralTo date, various Similarity DNJ of derivativesActive Site between and stereoisomers GAA and ER have α-Glucosidase been synthesized II and investigated for the inhibition activities toward GAA [15,16], and ER α-glucosidase II [17]; however, there are no reports To date, various DNJ derivatives and stereoisomers have been synthesized and investigated for of the selective inhibition of each enzyme. the inhibition activities toward GAA [15,16], and ER α-glucosidase II [17]; however, there are no Several crystal structures of human GAA and mouse ER α-glucosidase II have been deposited reports of the selective inhibition of each enzyme. in the Protein Data Bank. Among them, we selected the complex crystal structure of GAA with DNJ Several crystal structures of human GAA and mouse ER α-glucosidase II have been deposited (PDB ID: 5NN5) and the complex crystal structure of ER α-glucosidase II with DNJ (PDB ID: 5IEE); in the Protein Data Bank. Among them, we selected the complex crystal structure of GAA with DNJ these structures were superimposed on each other by using the Protein Structure Alignment tool in (PDB ID: 5NN5) and the complex crystal structure of ER α-glucosidase II with DNJ (PDB ID: 5IEE); Maestro. Our selected complex crystal structures used DNJ as a common ligand, suitable for clarifying these structures were superimposed on each other by using the Protein Structure Alignment tool in the diﬀerent interactions between both enzymes. The amino acids around 5Å of DNJ are shown in Maestro. Our selected complex crystal structures used DNJ as a common ligand, suitable for Figure2. The orientation and the conformation of DNJ are almost completely overlapped between clarifying the different interactions between both enzymes. The amino acids around 5Å of DNJ are GAA (light green) and ER α-glucosidase II (yellow). Moreover, as shown in Figure2, amino acid shown in Figure 2. The orientation and the conformation of DNJ are almost completely overlapped residues surrounding the DNJ bound to the sugar recognition site of both enzymes are almost identical; between GAA (light green) and ER α-glucosidase II (yellow). Moreover, as shown in Figure 2, amino the only two diﬀerences are that (i) Leu405 (GAA) has been replaced by Ile452 (ER α-glucosidase II) acid residues surrounding the DNJ bound to the sugar recognition site of both enzymes are almost identical; the only two differences are that (i) Leu405 (GAA) has been replaced by Ile452 (ER

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α-glucosidase II) and (ii) Ser676 (GAA) has been replaced by His700 (ER α-glucosidase II). andFurthermore, (ii) Ser676 the (GAA) hydrogen has been bonding, replaced ionic by His700and cation- (ER απ-glucosidase network between II). Furthermore, these enzymes the hydrogen and DNJ bonding,were very ionic similar and cation- (see πSupportingnetwork between Information these enzymes S1). These and DNJresults were suggested very similar that (see the Supporting lack of Informationspecificity of S1). DNJ These may results be attributed suggested to that the the high lack degree of speciﬁcity of structural of DNJ may and be sequence attributed similarity to the high of degreeglucose of recognition structural and site sequence of GAA with similarity ER α of-glucosidases. glucose recognition site of GAA with ER α-glucosidases.

Figure 2. Structural overlap of lysosomal acid α-glucosidase (GAA) (light green) and endoplasmic reticulumFigure 2. (ER)Structuralα-glucosidase overlap IIof (yellow) lysosomal at the acid deoxynojirimycin α-glucosidase (GAA) (DNJ) binding(light green) site. Upper and endoplasmic light green: aminoreticulum acid residues(ER) α-glucosidase of GAA. Lower II (yellow) yellow: at amino the deoxynojirimycin acid residues of ER (DNJ)α-glucosidase binding II.site. Amino Upper acid light in agreen: box: uncommonamino acid amino residues acid of residues. GAA. Lower yellow: amino acid residues of ER α-glucosidase II. Amino acid in a box: uncommon amino acid residues. 2.2. Influence of α-1-C-alkylation of LAB on Inhibition of GAA and ER α-Glucosidase II 2.2. InfluenceOn the basis of α-1-C-alkylation of these findings, of LAB we turnedon Inhibition our attention of GAA and to the ER designα-Glucosidase of selective II GAA inhibitors, with theOn goalthe basis of increasing of these the findings, stability we of GAA.turned Table our 1 attentionshows the to 50% the inhibitorydesign of concentrationsselective GAA (ICinhibitors,50) of LAB with and the its α goal-1-C -alkylof increasing derivatives the against stability GAA of andGAA. ER αTable-glucosidase 1 shows II. the DNJ 50% was inhibitory used as a positiveconcentrations control. (IC The50) parentof LAB compound, and its α-1- LAB,C-alkyl showed derivatives potent against inhibitory GAA activities and ER against α-glucosidase GAA and II. ERDNJα-glucosidase was used as a II, positive with an control. IC50 value The ofparent 0.52 andcompound, 2.8 µM respectively.LAB, showed Inhibition potent inhibitory potency activities of GAA wasagainst 3.5 timesGAA weakerand ER thanα-glucosidase DNJ, whereas II, with LAB an was IC50 2.9 value times of stronger0.52 and inhibitor2.8 µM respectively. than GAA toward Inhibition ER αpotency-glucosidase of GAA II. Consequently,was 3.5 times weaker selectivity than for DNJ, GAA whereas against LAB ER α was-glucosidase 2.9 times IIstronger of LAB inhibitor was 10 times than lowerGAA thantoward DNJ. ER We α-glucosidase next focused II. on Consequently, the anomeric selectivity position and for whether GAA against extending ER α the-glucosidase alkyl chain II atof thisLAB position was 10 might times cause lower diff erentialthan DNJ. inhibition We next by LAB.focused Figure on3 showsthe anomeric the logarithm position of theand reciprocal whether ofextending pIC50 for theα-1- alkylC-alkyl-LAB chain at this against position both might enzymes caus ase differential a bar graph. inhibition Of these by compounds, LAB. Figure with 3 shows the introductionthe logarithm of of a propyl the reciprocal instead ofof apIC methyl50 for group, α-1-C-alkyl-LAB the inhibitory against activity both toward enzymes both as enzymes a bar graph. tends toOf decrease,these compounds, due to alkyl with chain the extension.introduction Thereafter, of a propyl both instead inhibitory of a methyl activities group, were the gradually inhibitory recovered activity goingtoward from both propyl enzymes to heptyl tends due to todecrease, the elongation due to ofalkyl the alkylchain chain.extension. However, Thereafter, it is notable both thatinhibitory there isactivities a large di werefference gradually in the degreerecovered of reduction going from and prop recoveryyl to ofheptyl the inhibitory due to the activity elongation for both of enzymes.the alkyl Thechain. eff ectHowever, of inhibition it is notable of GAA that was there small; is evena large the difference most reduced in theα degree-1-C-ethyl-LAB of reduction was and only recovery 21-fold lowerof the thaninhibitory LAB (Table activity1). for Furthermore, both enzymes. the inhibition The effect of of GAA inhibition was restored of GAA towas the small; same even level the as LAB most inreducedα-1-C-heptyl-LAB. α-1-C-ethyl-LAB In contrast, was only the 21-fold influence lower on ER than glucosidase LAB (Table inhibition 1). Furthermore, is large. α the-1-C inhibition-Ethyl-LAB of showedGAA was 193-fold restored weaker to the inhibition same level than as LAB,LAB andin α the-1-C inhibitory-heptyl-LAB. activity In contrast, was not the fully influence restored on even ER glucosidase inhibition is large. α-1-C-Ethyl-LAB showed 193-fold weaker inhibition than LAB, and the inhibitory activity was not fully restored even when the alkyl chain was extended to heptyl.

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Molecules 2020, 25, x FOR PEER REVIEW 5 of 13 when the alkyl chain was extended to heptyl. Consequently, α-1-C-heptyl-LAB displayed a remarkable Consequently, α-1-C-heptyl-LAB displayed a remarkable selectivity index (SI) of 168.2, which is selectivityMolecules index 2020 (SI), 25 of, x FOR 168.2, PEER which REVIEW is 31-fold better relative to the parent LAB (Table15 ).of 13 31-fold better relative to the parent LAB (Table 1). Consequently, α-1-C-heptyl-LAB displayed a remarkable selectivity index (SI) of 168.2, which is Table 1. IC50 values (µM) for α-1-C-alkyl-LAB and related compounds against processing ER Table 1.31-fold IC50 bettervalues relative (µM) forto the α-1- parentC-alkyl-LAB LAB (Table and 1). related compounds against processing ER αα-glucosidase-glucosidase II (ER II (ER α-Glaaseα-Glaase II) and II) lysosomal and lysosomal acid α-glucosidase acid α-glucosidase (GAA). (GAA). Table 1. IC50 values (µM) for α-1-C-alkyl-LAB and related compounds against processing ER α-glucosidase II (ER α-Glaase II) and lysosomal acid α-glucosidaseIC50 IC(μM)50 ( (GAA).µM) Selectivity SelectivityIndex Index n = Compounds ER α-Glcaseα II GAA ER α-Glcase II/GAAα n = Compounds ER -GlcaseIC II50 (μM) GAASelectivity ER Index-Glcase II/GAA 0 LAB 2.8 ± 0.22 0.52 ± 0.032 5.4 0n = LAB Compounds 2.8 ER α0.22-Glcase II 0.52 GAA 0.032 ER α-Glcase II/GAA 5.4 ± ± 11 α-1-αC0-1- -Methyl-LABC-Methyl-LAB LAB 2626 ± 2.0 2.8 2.0 ± 0.22 2.0 ± 0.52 0.12 2.0 ± 0.0320.12 13.0 5.4 13.0 ± ± 22 α-1-α1-1-C -Ethyl-LABC-Ethyl-LABα-1-C-Methyl-LAB 541 541 ± 26 26 26 ± 2.0 11 ± 2.01.1 11 ± 0.121.1 49.2 13.0 49.2 ± ± 33 α-1-α2C-1- -Propyl-LABC-Propyl-LABα-1-C-Ethyl-LAB 413413 ± 50 541 50 ± 26 7.4 ± 0.31 117.4 ± 1.10.31 55.8 49.2 55.8 ± ± 44 α-1-α3-1-C -Butyl-LABC-Butyl-LABα-1-C-Propyl-LAB 228228 ±13 413 13 ± 50 1.6 ± 0.040 7.41.6 ± 0.31 0.040 142.5 55.8 142.5 ± ± 5 α4-1- C-Pentyl-LABα-1-C-Butyl-LAB 180 22819 ±13 1.6 1.1 ± 0.0400.088 142.5 163.6 5 α-1-C-Pentyl-LAB 180 ± 19± 1.1 ± 0.088± 163.6 6 α5-1- C-Hexyl-LABα-1-C-Pentyl-LAB 100 1806.2 ± 19 1.10.71 ± 0.0880.029 163.6 140.8 6 α-1-C-Hexyl-LAB 100 ± 6.2± 0.71 ± 0.029± 140.8 7 α6-1- C-Heptyl-LABα-1-C-Hexyl-LAB 74 1003.7 ± 6.2 0.71 0.44 ± 0.0290.044 140.8 168.2 7 α-1-C-Heptyl-LAB 74 ± 3.7± 0.44 ± 0.044± 168.2 7 α-1-C-Heptyl-LAB 74 ± 3.7 0.44 ± 0.044 168.2 DNJDNJ 8.0 8.0 ± 0.550.55 0.15 ± 0.15 0.0032 0.0032 53.3 53.3 DNJ ± 8.0 ± 0.55 0.15 ± ±0.0032 53.3

Figure 3. The eﬀect of alkyl chain length on inhibitory activities against GAA (blue bar) and ER Figure 3. The effect of alkyl chain length on inhibitory activities against GAA (blue bar) and ER α-glucosidaseα-glucosidase II (red bar).II (red Inhibition bar). Inhibition strength strength of GAAof GAA in eachin each alkyl alkyl chain chain waswas indicated by by log log (1/IC50). Figuren: α-1- 3.C The-alkyl-chain(1/IC effect50). n: ofα-1- alkyl length.C-alkyl-chain chain length length. on inhibitory activities against GAA (blue bar) and ER α-glucosidase II (red bar). Inhibition strength of GAA in each alkyl chain was indicated by log 2.3. Analysis of GAA- Ligand Stable Binding Pose by Using MD Calculation 2.3.(1/IC Analysis50). n: α of-1- GAA-C-alkyl-chain Ligand length. Stable Binding Pose by Using MD Calculation MD simulation can elucidate the dynamic changes in the interaction between target enzyme MD simulation can elucidate the dynamic changes in the interaction between target enzyme and 2.3. Analysisand of GAA- the ligands. Ligand Maintaining Stable Binding a stable Pose structure by Using in MD the Calculationactive site is an important factor for strong the ligands.recognition Maintaining by the aenzyme. stable To structure understand in thethe structural active site basis is anof the important interaction factor of α-1- forC-alkyl-LAB strong recognition by theMD enzyme. simulationwith GAA, To can we understand firstelucidate studied the the dynamic structuralprotein-ligand change basis dockings ofin the analyses interaction of α-1- Cbetween-alkyl-LAB of α-1- Ctarget-alkyl-LAB by using enzyme three with GAA, andwe ﬁrstthe ligands. studiedGAA crystalMaintaining the protein-ligandstructures a stable(PDB ID:structure docking 5NN5, 5NN6,in analyses the andactive of5NN8), αsite-1- Ciswhich-alkyl-LAB an importanthave reported by factor using as thefor three complex strong GAA crystal recognitionstructures by the enzyme. of GAA To with understand DNJ, miglitol, the andstructural acarbose, basis respectively. of the interaction They commonly of α-1- containC-alkyl-LAB several structures (PDB ID: 5NN5, 5NN6, and 5NN8), which have reported as the complex structures of GAA with GAA, waterwe first molecules studied in the the protein-ligandligand-binding site. docking Therefore, analyses we considered of α-1-C-alkyl-LAB these water bymolecules using threein the GAAwith crystal DNJ,docking miglitol,structures analyses. and (PDB acarbose,The ID: docking 5NN5, respectively.analyses 5NN6, were and performed They5NN8), commonly whichusing the have grids contain reported generated several as from the water thecomplex crystal molecules in structuresthe ligand-binding ofstructures GAA with with site. DNJ, two Therefore, miglitol,to four water and we acarbose,molecule considered corespectively.mbinations these water in They 5NN5 molecules commonly and 5NN6 incontain and the the docking several crystal analyses. structure with two water molecules in 5NN8 (see Supporting Information S2). By including water waterThe docking molecules analyses in the ligand-binding were performed site. using Therefore, the grids we considered generated these from water the crystal molecules structures in the with two molecules in the docking analyses, the docking poses of the co-crystalized ligands could be correctly dockingto four analyses. waterreproduced. molecule The Thedocking best combinations docking analyses score were inposes 5NN5performed were andselected using 5NN6 from the andmany grids the poses generated crystal generated structurefrom using the each crystal with grid two water structuresmolecules withand in 5NN8energy-minimized two to (see four Supporting water to select molecule one Information complex combinations structure S2). By inwith including5NN5 the bestand binding water5NN6 interactionmoleculesand the crystalenergy. in the docking structureanalyses, with the two docking water posesmolecules of the in 5NN8 co-crystalized (see Supporting ligands Information could be S2). correctly By including reproduced. water The best moleculesdocking scorein the posesdocking were analyses, selected the fromdocking many poses poses of the generated co-crystalized using ligands each gridcould and be correctly energy-minimized reproduced.to select one The complex best docking structure score poses with thewere best selected binding from interactionmany poses energy.generated Molecular using each dynamicgrid (MD) and energy-minimized to select one complex structure with the best binding interaction energy. simulations were carried out using the docking structure as initial structures. In the result of the MD simulation, the root mean square deviation (RMSD) of the protein heavy-atoms, compared with the initial structure, initially increased, and then became almost stable. The 50 ligand binding MoleculesMolecules2020, 25 2020, 2843, 25, x FOR PEER REVIEW 6 of 13 6 of 13

Molecular dynamic (MD) simulations were carried out using the docking structure as initial conformations,structures. sampledIn the result equally of the MD in time simulation, intervals the fromroot mean the trajectories square deviation between (RMSD) 24 nsof the and protein 48 ns, were presentedheavy-atoms, in Figure compared4. Furthermore, with the initial Figure structure,5 shows initially the relationship increased, and between then became the almost extension stable. of the α-1-C-alkylThe 50 chain ligand length binding and conformations, the variation sampled of the ligandequally bindingin time intervals pose by from using the the trajectories RMSD as the between 24 ns and 48 ns, were presented in Figure 4. Furthermore, Figure 5 shows the relationship evaluation standard. We evaluated the variation of ligand binding pose from the viewpoint of the whole between the extension of the α-1-C-alkyl chain length and the variation of the ligand binding pose by ligandusing (light the orange) RMSD andas the the evaluation pyrrolidine standard. ring partWe eval (orange).uated the Among variation these of ligand compounds, binding thepose parent from LAB and α-1-theC viewpoint-methyl-LAB of the remainedwhole ligand the (light stable orange) ligand-binding and the pyrrolidine conformations, ring part (orange). and their Among average these RMSD valuescompounds, were both lessthe thanparent 1.5Å LAB in theand ligand-bindingα-1-C-methyl-LAB site remained (Figure4 A,Bthe andstable Figure ligand-binding5). In contrast, the introductionconformations, of short and their (ethyl average and propyl) RMSD values groups were reduced both less the than ligand-binding 1.5Å in the ligand-binding conformation site stability and the(Figure positions 4A,B ofand the Figure pyrrolidine 5). In contrast, ring and the introd the overalluction orientationsof short (ethyl moved and propyl) drastically groups (Figurereduced4 C,D). They resultedthe ligand-binding in the high conformation variation ofstability the position and the andpositions theorientation of the pyrrolidine of both ring the and whole the overall ligand and orientations moved drastically (Figure 4C,D). They resulted in the high variation of the position and the pyrrolidine ring moiety (Figure5). Furthermore, we found that the ligand-binding conformation the orientation of both the whole ligand and the pyrrolidine ring moiety (Figure 5). Furthermore, we stability gradually improved with increasing length of the alkyl chain; a much longer alkyl chain than found that the ligand-binding conformation stability gradually improved with increasing length of butyl isthe required alkyl chain; for a achieving much longer them alkyl (Figure chain than4E–H). butyl Therefore, is required the for introduction achieving them of (Figure longer 4E–H). (hexyl and heptyl)Therefore, groups reducedthe introduction the ﬂuctuations of longer of(hexyl the ligand-binding and heptyl) groups conformation, reduced the and fluctuations it was observed of the that the positionsligand-binding of the pyrrolidineconformation, ring and were it was very observed stable that (Figure the positions4G,H). of the pyrrolidine ring were very stable (Figure 4G,H).

FigureFigure 4. The 4. binding The binding poses ofposes the of LAB the and LABα -1-andC-alkyl-LAB α-1-C-alkyl-LAB derivatives derivatives extracted extracted from from the molecularthe dynamicsmolecular (MD) trajectory:dynamics (MD) (A) LAB, trajectory: (B) α-1- (aC)-methyl-LAB, LAB, (b) α-1- (CC-methyl-LAB,) α-1-C-ethyl-LAB, (c) α-1- (DC-ethyl-LAB,) α-1-C-propyl-LAB, (d) Molecules 2020, 25, x FOR PEER REVIEW 7 of 13 (E) α-1-α-1-C-butyl-LAB,C-propyl-LAB, (F ) α(e-1-) Cα-pentyl-LAB,-1-C-butyl-LAB, (G )(fα) -1-αC-1--hexyl-LAB,C-pentyl-LAB, (H ) (αg-1-) Cα-heptyl-LAB.-1-C-hexyl-LAB, (h) α-1-C-heptyl-LAB.

Figure 5. Eﬀect of alkyl chain length on variation of ligand binding pose on GAA. Light orange: whole Figure 5. Effect of alkyl chain length on variation of ligand binding pose on GAA. Light orange: ligand (excludingwhole ligand hydrogen). (excluding Orange: hydrogen). pyrrolidine Orange: pyrrolidin ring parte ring (excludingpart (excluding hydrogen). hydrogen). Root Root mean mean square deviation (RMSD)square deviation value (RMSD) calculated value for calculated 50 extracted for 50 extracted from 24 from ns 24 to ns 48 to ns. 48 ns.

2.4. Hydrogen Bonding Interactions and Cation-π Interactions between GAA and DNJ, LAB, and α-1-C-heptyl-LAB In order to investigate the interaction between GAA and the ligands, we modelled the three-dimensional structures of the complex structures by GAA with LAB and α-1-C-heptyl-LAB, using the docking protocol. As shown in Figure 6B, the positively charged nitrogen and the hydroxyl groups of LAB formed hydrogen bonds with Asp404, Asp518, Arg600, and Asp616. Furthermore, the pyrrolidine ring part of LAB had the favorable cation-π interaction with Trp376 and Trp516 and ionic interaction with Asp404, Asp443, Asp518, and Asp616. The schematic diagram of hydrogen bonding interactions and cation-π interactions between GAA and LAB was similar to that of DNJ (Figure 6A), except for the cation-π interaction with Phe649 and ionic interaction with His674 of DNJ. In contrast, α-1-C-heptyl-LAB formed clearly different interactions with GAA (Figure 6C). It formed the additional cation-π interaction and hydrogen bonds with Trp481, whereas it lacked hydrogen bonds with Asp616 and Arg600 and ionic interaction with Asp616. Furthermore, it is notable that the heptyl group of α-1-C-heptyl-LAB formed hydrophobic interaction with Trp481, Phe525, and Met519 of the alkyl chain storage pocket of GAA. Figure 6D showed the 25 binding poses of LAB (blue) and α-1-C-heptyl-LAB (green) obtained from the MD simulations. Both compounds reduced the fluctuations of the ligand-binding conformation, and the positions of the pyrrolidine ring were very stable. Therefore, the number of hydrogen bonds formed by α-1-C-heptyl-LAB is smaller than that of DNJ and LAB, but it was thought to be strongly affected by hydrophobic interactions.

Molecules 2020, 25, 2843 7 of 13

2.4. Hydrogen Bonding Interactions and Cation-π Interactions between GAA and DNJ, LAB, and α-1-C-heptyl-LAB In order to investigate the interaction between GAA and the ligands, we modelled the three-dimensional structures of the complex structures by GAA with LAB and α-1-C-heptyl-LAB, using the docking protocol. As shown in Figure6B, the positively charged nitrogen and the hydroxyl groups of LAB formed hydrogen bonds with Asp404, Asp518, Arg600, and Asp616. Furthermore, the pyrrolidine ring part of LAB had the favorable cation-π interaction with Trp376 and Trp516 and ionic interaction with Asp404, Asp443, Asp518, and Asp616. The schematic diagram of hydrogen bonding interactions and cation-π interactions between GAA and LAB was similar to that of DNJ (Figure6A), except for the cation- π interaction with Phe649 and ionic interaction with His674 of DNJ. In contrast, α-1-C-heptyl-LAB formed clearly different interactions with GAA (Figure6C). It formed the additional cation-π interaction and hydrogen bonds with Trp481, whereas it lacked hydrogen bonds with Asp616 and Arg600 and ionic interaction with Asp616. Furthermore, it is notable that the heptyl group of α-1-C-heptyl-LAB formed hydrophobic interaction with Trp481, Phe525, and Met519 of the alkyl chain storage pocket of GAA. Figure6D showed the 25 binding poses of LAB (blue) and α-1-C-heptyl-LAB (green) obtained from the MD simulations. Both compounds reduced the fluctuations of the ligand-binding conformation, and the positions of the pyrrolidine ring were very stable. Therefore, the number of hydrogen bonds formed by α-1-C-heptyl-LAB is smaller than that of DNJ and LAB, but it was thought to be strongly affected by hydrophobic interactions.

2.5. Comparison of GAA and ER α-Glucosidase II Focusing on LAB-alkyl Chain Storage Pocket On the basis of these findings, we hypothesized that the difference in the storage sites of the alkyl chains would also affect the selectivity of GAA and ER α-glucosidase II. Based on this hypothesis, we first identified each storage site (Figure7). At first, the crystal structures of GAA (PDB ID: 5NN5) and ER α-glucosidase II (PDB ID: 5IEE) were superposed using the Protein Structure Alignment tool in Maestro. Subsequently, the complex structure of each enzyme with α-1-C-hexyl-LAB was extracted from the MD simulation and superimposed on them (Figure7). Figure7A showed the amino acids of GAA and ER α-glucosidase II within 5Å surrounding the α-1-C-hexyl-LAB. The amino acids that differ between GAA (magenta) and ER α-glucosidase II (blue) were displayed in the square box. As shown in Figure2, the amino acid residues surrounding the sugar recognition site of both enzymes were almost common. In sharp contrast, it was revealed that the amino acid residues surrounding the α-1-C-hexyl-LAB binding part in each active site was clearly different. We focused especially on three amino acids: Ala284, Leu650, and Ser676 (red solid square box), which composed the LAB-alkyl chain storage pocket. In case of ER α-glucosidase II, these amino acids were replaced by Phe307, Phe674, and His700. As shown in Figure7B,C, the substitution of these three amino acids causes a large difference in the opening part (red dotted circle) of each active site. Similarly, from the view of the outside of the opening, it can be seen that GAA has a wider opening than ER α-glucosidase II (Figure7E,F). Figure7D showed the superimposition of both protein surface. Consequently, the space of the alkyl chain storage pocket in ER α-glucosidase II was narrower than GAA, and it cannot accommodate the extension of alkyl chains. Molecules 2020, 25, 2843 8 of 13 Molecules 2020, 25, x FOR PEER REVIEW 8 of 13

Figure 6. SchematicSchematic diagram diagram of of the the interactions interactions between between GAA GAA and and ( (AA)) DNJ, DNJ, ( (BB)) LAB LAB and and ( (CC)) α-1-C-heptyl-LAB.-heptyl-LAB. DNJ: DNJ: the the complex crysta crystall structure (PDB ID: 5NN5). LAB LAB and and α-1--1-C-heptyl-LAB:-heptyl-LAB: the complex complex structure structure showing showing the the best best binding binding energy energy among among 50 complex 50 complex structures structures obtained obtained from MDfrom simulations. MD simulations. The red Thedashed red lines dashed represent lines representhydrogen-bond hydrogen-bond interactions. interactions. The green dashed The green lines representdashed lines cation- representπ interactions. cation-π Theinteractions. blue dashed The lines blue repr dashedesent ionic lines interactions. represent ionic The interactions.orange arcs (dashedThe orange line) arcs represent (dashed line)hydrophobic represent hydrophobicinteractions. interactions.The labels Theof amino labels ofacids amino involved acids involved in the hydrogen-bondingin the hydrogen-bonding and the and cation- theπ cation- interactionsπ interactions are colored are coloredin red and in redgreen, and respectively. green, respectively. (D) The 25(D )binding The 25 binding poses of poses LAB of (blue) LAB (blue) and α and-1-Cα-heptyl-LAB-1-C-heptyl-LAB (green) (green) obtained obtained from from the the MD MD simulations. simulations. The molecularmolecular surfacesurface of of GAA GAA is displayedis displayed using using Sybyl-X Sybyl-X 2.2.1 2.2.1 (Certara (Certara USA, USA, Inc., Princeton,Inc., Princeton, NJ, USA). NJ, USA).

2.5. Comparison of GAA and ER α-Glucosidase II Focusing on LAB-alkyl Chain Storage Pocket. On the basis of these findings, we hypothesized that the difference in the storage sites of the alkyl chains would also affect the selectivity of GAA and ER α-glucosidase II. Based on this hypothesis, we first identified each storage site (Figure 7). At first, the crystal structures of GAA (PDB ID: 5NN5) and ER α-glucosidase II (PDB ID: 5IEE) were superposed using the Protein Structure Alignment tool in Maestro. Subsequently, the complex structure of each enzyme with α-1-C-hexyl-LAB was extracted from the MD simulation and superimposed on them (Figure 7). Figure 7A showed the amino acids of GAA and ER α-glucosidase II within 5Å surrounding the α-1-C-hexyl-LAB. The amino acids that differ between GAA (magenta) and ER α-glucosidase II (blue) were displayed in the square box. As shown in Figure 2, the amino acid residues surrounding

Molecules 2020, 25, x FOR PEER REVIEW 9 of 13 the sugar recognition site of both enzymes were almost common. In sharp contrast, it was revealed that the amino acid residues surrounding the α-1-C-hexyl-LAB binding part in each active site was clearly different. We focused especially on three amino acids: Ala284, Leu650, and Ser676 (red solid square box), which composed the LAB-alkyl chain storage pocket. In case of ER α-glucosidase II, these amino acids were replaced by Phe307, Phe674, and His700. As shown in Figure 7B,C, the substitution of these three amino acids causes a large difference in the opening part (red dotted circle) of each active site. Similarly, from the view of the outside of the opening, it can be seen that GAA has a wider opening than ER α-glucosidase II (Figure 7E,F). Figure 7D showed the superimposition of both protein surface. Consequently, the space of the alkyl chain storage pocket in ER α-glucosidase II was narrower than GAA, and it cannot accommodate the extension of alkyl Molecules 2020, 25, 2843 9 of 13 chains.

FigureFigure 7. 7. DifferenceDifference of of LAB LAB-alkyl-alkyl chainchain storagestorage pocket pocket in in GAA GAA and and ER ERα-glucosidase α-glucosidase II. ( AII.) The(A) aminoThe aminoacids acids of GAA of andGAA ER andα-glucosidase ER α-glucosidase II within II 5Å wi surroundingthin 5Å surrounding the α-1-C -hexyl-LAB. the α-1-C-hexyl The- aminoLAB. The acids aminothat diaciffdser that between differ GAA between (magenta) GAA (magenta) and ER αand-glucosidase ER α-glucosidase II (blue) II were(blue) displayed were displayed in the in square the squarebox. (Bbox) The. (B protein) The protein surface ofsurface GAA andof GAA the characteristic and the characteristic three amino three acids, amino which acids, composed which the composedopening partthe (redopening dotted part circle) (red of dotted the active circle) site. of (C )the The active protein site surface. (C) ofThe ER pαrotein-glucosidase surface II of and ER the α-characteristicglucosidase II three and aminothe characteristic acids which composedthree amino the acids opening which part composed (red dotted the circle) opening of the part active (red site. dotted(D) The circle) superimposition of the active ofsite. each (D protein) The superimposition surface of GAA of and each ER proteinα-glucosidase surface II.of DockingGAA and pose ER of α-αg-1-lucosidaseC-hexyl-LAB II. Docking against pose GAA of was α-1 superimposed-C-hexyl-LAB withagainst them. GAA (E )was GAA superimposed protein surface with from them. the view(E) GAAof the protein outside surface of the openingfrom the partview of of the the active outside site. of (F )the ER openingα-glucosidase part of II proteinthe active surface site. from(F) ER the α-viewglucosidase of the outside II protein of thesurface opening from part the ofview the of active the outside site. of the opening part of the active site. 2.6. GAA Stabilization Effect of LAB, α-1-C-ethyl-LAB and α-1-C-heptyl-LAB 2.6. GAA Stabilization Effect of LAB, α-1-C-ethyl-LAB and α-1-C-heptyl-LAB With respect to LAB and α-1-C-heptyl-LAB having a different binding pocket in the With respect to LAB and α-1-C-heptyl-LAB having a different binding pocket in the sugar-recognition site toward GAA, we next investigated the effect of these compounds on the sugar-recognition site toward GAA, we next investigated the effect of these compounds on the stabilization of this enzyme at 47 C (Figure8). GAA was incubated in 100 mM McIlvaine bu ffer stabilization of this enzyme at 47 °C (◦Figure 8). GAA was incubated in 100 mM McIlvaine buffer (pH (pH 6.8) containing 0.25% sodium taurocholate and 0.1% Triton X-100 for 0, 20, 40, and 60 min, 6.8) containing 0.25% sodium taurocholate and 0.1% Triton X-100 for 0, 20, 40, and 60 min, and the and the remaining enzyme activity was determined with 4-methylumbelliferl-α-d-glucopyranoside as remaining enzyme activity was determined with 4-methylumbelliferl-α-D-glucopyranoside as substrate. The enzyme activity was 83% lost within 60 min under incubation without test samples, substrate. The enzyme activity was 83% lost within 60 min under incubation without test samples, while the incubation with α-1-C-heptyl-LAB effectively stabilized GAA. The enzyme activity remained while the incubation with α-1-C-heptyl-LAB effectively stabilized GAA. The enzyme activity over 94% even under the incubation for 60 min in the presence of 10 µM α-1-C-heptyl-LAB (Figure8). remained over 94% even under the incubation for 60 min in the presence of 10 µM α-1-C-heptyl-LAB This behavior is similar to that of 10 µM LAB, but the stabilization effect was slightly weaker than LAB (89% activity remained at 60 min). Therefore, it is considered that α-1-C-heptyl-LAB forms an

appropriate hydrogen ion network in the sugar-recognition site, which is diﬀerent from LAB and DNJ, and stabilizes GAA. In contrast, 10 µM α-1-C-ethyl-LAB could not suﬃciently protect the enzyme activity (50% activity remained at 60 min). These results correlate with stability in the MD analysis. Molecules 2020, 25, x FOR PEER REVIEW 10 of 13

(Figure 8). This behavior is similar to that of 10 µM LAB, but the stabilization effect was slightly weaker than LAB (89% activity remained at 60 min). Therefore, it is considered that α-1-C-heptyl-LAB forms an appropriate hydrogen ion network in the sugar-recognition site, which is different from LAB and DNJ, and stabilizes GAA. In contrast, 10 µM α-1-C-ethyl-LAB could not sufficiently protect the enzyme activity (50% activity remained at 60 min). These results correlate Molecules 2020, 25, 2843 10 of 13 with stability in the MD analysis.

100 non treat 90 LAB 10 μM

α 80 -1-C-ethyl-LAB 10 μM α-1-C-heptyl-LAB 10 μM 70 60 50 40 30 20 R esidual activity (% ) activity R esidual 10 0 0 204060

incubation time (min)

Figure 8. Eﬀect of LAB, α-1-C-ethyl-LAB, and α-1-C-heptyl-LAB on the in vitro thermostability.

GAAFigure was 8. Effect incubated of LAB, at 47α-1-◦CC in-ethyl-LAB, 100 mM sodium and α-1- citrateC-heptyl-LAB buffer (pH on 6.8) the containing in vitro thermostability. 1 mg/mL of bovine GAA serumwas incubated albumin (BSA)at 47 °C at variousin 100 mM incubation sodium times citrat withe buffer or without (pH 6.8) iminosugars. containing Each 1 mg/mL value representsof bovine theserum mean albuminSEM ((BSA)n = 3). at various incubation times with or without iminosugars. Each value ± represents the mean ± SEM (n = 3). 3. Experimental Section 3. Experimental Section 3.1. Chemistry 3.1. Chemistry1,4-Dideoxy-1,4-imino- l-arabinitol (LAB) was prepared from l-arabinose, by an identical method to that used for the synthesis of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) [18–21]. α-1-C-Alkyl-LAB with 1,4-Dideoxy-1,4-imino-L-arabinitol (LAB) was prepared from L-arabinose, by an identical various lengths of side chains from C1 to C7 at the 1-position were synthesized, as previously described [8]. method to that used for the synthesis of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) [18–21]. 3.2.α-1- EnzymeC-Alkyl-LAB Inhibition with Assays various lengths of side chains from C1 to C7 at the 1-position were synthesized, as previously described [8]. The inhibitory activity toward lysosomal acid α-glucosidase (GAA) was measured with Myozyme (Genzyme;3.2. Enzyme Boston,Inhibition MA, Assays USA) as the enzyme source, and 4-methylumbelliferyl-α-d-glucopyranoside (Sigma-Aldrich Co.; St. Louis, Mo, USA) as substrate. The reaction mixture consisted 100 mM McIlvaine The inhibitory activity toward lysosomal acid α-glucosidase (GAA) was measured with buffer (pH 6.8), 0.25% sodium taurocholate and 0.1% Triton X-100 (Nacalai Tesque Inc.; Kyoto, Japan), Myozyme (Genzyme; Boston, MA, USA) as the enzyme source, and and the appropriate amount of enzyme. The reaction mixture was pre-incubated at 0 ◦C for 45 min, 4-methylumbelliferyl-α-D-glucopyranoside (Sigma-Aldrich Co.; St. Louis, Mo, USA) as substrate. and the reaction was started by the using 4 mM substrate solution, followed by incubation at 37 ◦C for The reaction mixture consisted 100 mM McIlvaine buffer (pH 6.8), 0.25% sodium taurocholate and 30 min. The reaction was stopped by the addition of 1.6 mL of the solution of 400 mM Glycine-NaOH 0.1% Triton X-100 (Nacalai Tesque Inc.; Kyoto, Japan), and the appropriate amount of enzyme. The solution (pH 10.6). The released 4-methylumbelliferone was measured (excitation 362 nm, emission reaction mixture was pre-incubated at 0 °C for 45 min, and the reaction was started by the using 4 450 nm) with a F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). ER α-glucosidase II mM substrate solution, followed by incubation at 37 °C for 30 min. The reaction was stopped by the was prepared from the rat liver according to the method of Hino et al. [22]. For ER α-glucosidase II addition of 1.6 mL of the solution of 400 mM Glycine-NaOH solution (pH 10.6). The released activities, 4-nitrophenyl-α-d-glucopyranoside (Sigma-Aldrich Co.; St. Louis, MO, USA) was used 4-methylumbelliferone was measured (excitation 362 nm, emission 450 nm) with a F-4500 as substrate. The reaction mixture contained phosphate buffer (pH 6.8), 0.25% sodium taurocholate, fluorescence spectrophotometer (Hitachi, Tokyo, Japan). ER α-glucosidase II was prepared from the and the appropriate amount of enzyme. The reaction mixture was pre-incubated at 0 ◦C for 30 min, rat liver according to the method of Hino et al. [22]. For ER α-glucosidase II activities, and the reaction was started by the using 4 mM substrate solution, followed by incubation at 37 ◦C for 4-nitrophenyl-α-D-glucopyranoside (Sigma-Aldrich Co.; St. Louis, MO, USA) was used as substrate. 30 min. The reaction was stopped by adding 0.4 mL of 400 mM Na2CO3. The released p-nitrophenol The reaction mixture contained phosphate buffer (pH 6.8), 0.25% sodium taurocholate, and the was measured spectrometrically at 400 nm. appropriate amount of enzyme. The reaction mixture was pre-incubated at 0 °C for 30 min, and the 3.3.reaction MD Calculationswas started by the using 4 mM substrate solution, followed by incubation at 37 °C for 30 The crystal structures of GAA with deoxynojirimycin, miglitol, and acarbose (PDB ID: 5NN5, 5NN6 and 5NN8) were downloaded from the Protein Data Bank. The preparations of the protein structures were performed using Maestro Protein Preparation Wizard (Schrödinger, LLC, New York, NY, USA). After removing water molecules other than those used in docking analysis, hydrogen atoms were added at pH 6.8. The docking analyses were performed with the standard precision (SP) mode of Glide (version 7.9, Molecules 2020, 25, 2843 11 of 13

Schrödinger, LLC, New York, NY, USA) using the grids generated from the crystal structures with two to four water molecule combinations in 5NN5 and 5NN6 and the crystal structure with two water molecules in 5NN8. The best docking score poses were selected for each grid and energy-minimized to select one complex structure with the best binding interaction energy. These docking structures were solvated in a cubic simulation box using a simple point charge (SPC) water model with periodic boundary conditions. The net charge of the system was neutralized by addition of sodium and chloride ions. MD simulations were performed in 48 ns at 310 K and 1 bar and NPT ensemble using Desmond (version 5.4, Schrödinger, LLC, New York, NY, USA) [23,24]. Electrostatic interactions were calculated using the Particle Mesh Ewald with a cut-off distance of 9.0Å. MD simulations were performed using the OPLS3 force field. All calculations were performed using the Schrödinger Suite 2018-2 (Schrödinger, LLC, New York, NY, USA).

3.4. Thermostability of Lysosomal Acid α-Glucosidase (GAA)

The enzyme GAA was incubated at 47 ◦C in 100 mM McIlvaine buﬀer (pH 6.8) containing 0.25% sodium taurocholate and 0.1% Triton X-100 for 0, 20, 40, and 60 min. After incubation, the remaining GAA activity was assayed immediately using 4 mM 4-methylumbelliferyl-α-d-glucopyranoside as substrate.

4. Conclusions Iminosugars competitively inhibit glycosidases due to homology with the corresponding substrate. Therefore, piperidine iminosugars that faithfully mimic the stereochemistry of the terminal hexopyranose cleaved by exo-glucosidase are expected to show strong inhibition. However, it is difficult to define subtle differences in the isoenzyme that are localized in multiple organs and tissues. The present work elucidated the following features: (a) GAA and ER α-glucosidase II have a high degree of structural and sequence similarity at the glucose-recognition site; (b) LAB had the same level of inhibitory activity (IC50 = 0.52 µM) as DNJ (IC50 = 0.15 µM), and does not change recognition by GAA; (c) in contrast, the selectivity index (ER α-Glc II/GAA) of LAB was lower than DNJ, thus, the selectivity toward GAA cannot be obtained simply by changing the ring structure; (d) α-1-C-heptyl-LAB showed the most potent inhibition of GAA, with an IC50 value of 0.44 µM, furthermore, it displayed a remarkable selectivity (SI value of 168.2), which is 31-fold better than the parent LAB; (e) the MD simulation suggested that the ligand-binding conformation stability gradually improved with the increasing length of the alkyl chain, and a much longer alkyl chain than butyl is required for achieving this; (f) docking calculations suggested that the binding conformations and orientations of α-1-C-heptyl-LAB and monosaccharide mimics (DNJ and LAB) in GAA are clearly different—It had the lower number of hydrogen bonds, but it formed the characteristic hydrophobic interactions; (g) the amino acid residues surrounding the α-1-C-hexyl-chain storage part in each active site was clearly different—the space of ER α-glucosidase II was narrower than GAA, and it cannot accommodate the extension of alkyl chains; (h) α-1-C-heptyl-LAB improved the thermostability of GAA in vitro at the same level as LAB. Thus, our findings suggested that focusing on the alkyl chain storage site could improve the selectivity toward GAA and the ligand-binding conformation stability.

Supplementary Materials: The following are available online, Figure S1: Interactions of deoxynojirimycin with GAA and ER α-glucosidase. Figure S2: Water molecules found in the ligand-binding site. The water molecules surrounded by dashed circles were used for the docking analyses. Author Contributions: Y.N. and Y.Y. performed the chemical syntheses, analyzed the data. M.H. and I.A. performed the glycosidase inhibition assay; A.K. wrote the original draft; I.N. and S.H. performed MD calculations and analyzed the validated the experimental data. A.K., R.J.N., and G.W.J.F. designed the experiments and reviewed the manuscript. All authors approved the ﬁnal manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by a Grant-in-Aid for Scientiﬁc Research (C) from the Japanese Society for the Promotion of Science (JSPS KAKENHI Grant Number JP17K08362) (A.K.). Molecules 2020, 25, 2843 12 of 13

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds 1,4-dideoxy-1,4-imino-L-arabinitol (LAB) and deoxynojirimycin (DNJ) are available from the authors.

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