The Stat3 inhibitor F0648-0027 is a potential therapeutic against rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease characterized by chronic joint inflammation, pain and joint destruction, leading to alteration in activities of daily living, yet pathological mechanisms underlying the condition are not fully clarified. To date, various therapeutic agents have been developed as RA therapy including DMARDs and/or biological agents that target inflammatory cytokines or inhibit JAK. Here we asked whether inhibiting signal transducer and activator of transcription 3 (Stat3) activity would antagonize RA. Stat3 forms dimers when activated and undergoes nuclear translocalization; thus we screened approximately 4.9 million small compounds as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening. We identified 15 as strong candidates as potential blockers of protein-protein interactions required for Stat3 dimerization using in silico screening from those compounds. Four of the 15 significantly inhibited expression of IL-6 and RANKL, both of which are direct targets of Stat3, induced by IL-6. Among four, one compound, F0648-0027, significantly inhibited arthritis development without apparent adverse effects in vivo in collagen-induced arthritis model mice. F0648-0027 also significantly blocked Stat3 phosphorylation and nuclear localization following IL-6 stimulation of fibroblasts. These data suggest that Stat3 is a target for collagen-induced arthritis in mice, and that F0648-0027 could serve as a therapeutic reagent against comparable conditions in humans.

Efficacy and safety of glecaprevir and pibrentasvir in Japanese patients with hepatitis C virus infection aged 75 years or older.

BACKGROUND: It is estimated that approximately 50% of patients with hepatitis C virus (HCV) infection in Japan are currently over 75 years old. However, patients aged ≥ 75 years are typically underrepresented in clinical trials of direct-acting antivirals. This study aimed to evaluate the efficacy and safety of glecaprevir and pibrentasvir (G/P) treatment in Japanese patients with HCV infection aged ≥ 75 years. METHODS: This multicenter, retrospective study included 271 Japanese patients with HCV infection from 12 centers in Miyazaki Prefecture, Japan. Demographic, clinical, virological, and adverse events (AEs) data obtained during and after G/P treatment were collected from medical records. The patients were divided into two groups: younger (n = 199, aged < 75 years) and older (n = 72, aged ≥ 75 years). Virological data and AEs were analyzed according to the age group. RESULTS: In intention-to-treat (ITT) and per-protocol analyses, the overall sustained virological response 12 (SVR12) rates were 93% and 98.8%, respectively. Two patients in the older group and 14 patients in the younger group dropped out before SVR12 assessment. Although patients in the older group tended to have liver cirrhosis, 95.8% in the older group and 92% in the younger group achieved SVR12 in the ITT analysis (P = 0.404). In total, 48 (17.7%) patients experienced treatment-related AEs. Common AEs during treatment included pruritus, headache, and fatigue. The AEs were not significantly different between the two groups. CONCLUSIONS: Compared with younger patients, older patients showed similar virological response and tolerance to G/P treatment.

Synthesis and Evaluation of a Novel Series of 2,7-Substituted-6-tetrazolyl-1,2,3,4-tetrahydroisoquinoline Derivatives as Selective Peroxisome Proliferator-Activated Receptor γ Partial Agonists.

A novel series of 7-substituted-2-[3-(2-furyl)acryloyl]-6-tetrazolyl-1,2,3,4-tetrahydroisoquinoline derivatives were synthesized to clarify structure-activity relationships for peroxisome proliferator-activated receptor γ (PPARγ) partial agonist activity and identify more efficacious PPARγ partial agonists with minor adverse effects. Among the derivatives synthesized, compound 26v with a 2-(2,5-dihydropyrrol-1-yl)-5-methyloxazol-4-ylmethoxy group at the 7-position of the tetrahydroisoquinoline structure exhibited stronger PPARγ agonist and antagonist activities (EC50 = 6 nM and IC50 = 101 nM) than previously reported values for compound 1 (EC50 = 13 nM and IC50 = 512 nM). Compound 26v had very weak protein tyrosine phosphatase 1B (PTP1B) inhibitory activity and showed higher oral absorption (Cmax = 11.4 µg/mL and area under the curve (AUC) = 134.7 µg·h/mL) than compound 1 (Cmax = 7.0 µg/mL and AUC = 63.9 µg·h/mL) in male Sprague-Dawley (SD) rats. A computational docking calculation revealed that 26v bound to PPARγ in a similar manner to that of compound 1. In male Zucker fatty rats, 26v and pioglitazone at 10 and 30 mg/kg for 4 weeks similarly reduced plasma triglyceride levels, increased plasma adiponectin levels, and attenuated increases in plasma glucose levels in the oral glucose tolerance test, while only pioglitazone decreased hematocrit values. In conclusion, 6-tetrazolyl-1,2,3,4-tetrahydroisoquinoline derivatives provide a novel scaffold for selective PPARγ partial agonists and 26v attenuates insulin resistance possibly by adiponectin enhancements with minor adverse effects.

Identification of novel EED-EZH2 PPI inhibitors using an in silico fragment mapping method.

Enhancer of zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is overexpressed in many cancers. Numerous EZH2 inhibitors have been developed as anticancer agents, but recent studies have also focused on protein-protein interaction (PPI) between embryonic ectoderm development (EED) and EZH2 as a novel drug discovery target. Because EED indirectly enhances EZH2 enzymatic activity, EED-EZH2 PPI inhibitors suppress the methyltransferase activity and inhibit cancer growth. By contrast to the numerous promising EZH2 inhibitors, there are a paucity of EED-EZH2 PPI inhibitors reported in the literature. Here, we aimed to discover novel EED-EZH2 PPI inhibitors by first identifying possible binders of EED using an in-house knowledge-based in silico fragment mapping method. Next, 3D pharmacophore models were constructed from the arrangement pattern of the potential binders mapped onto the EED surface. In all, 16 compounds were selected by 3D pharmacophore-based virtual screening followed by docking-based virtual screening. In vitro evaluation revealed that five of these compounds exhibited inhibitory activities. This study has provided structural insights into the discovery and the molecular design of novel EED-EZH2 PPI inhibitors using an in silico fragment mapping method.

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

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.

(S)-1,2,3,4-Tetrahydroisoquinoline Derivatives Substituted with an Acidic Group at the 6-Position as a Selective Peroxisome Proliferator-Activated Receptor γ Partial Agonist.

A novel series of 2,6,7-substituted 3-unsubstituted 1,2,3,4-tetrahydroisoquinoline derivatives were synthesized to find a peroxisome proliferator-activated receptor γ (PPARγ) partial agonist. Among the derivatives, (E)-7-[2-(cyclopent-3-eny)-5-methyloxazol-4-ylmethoxy]-2-[3-(2-furyl)acryloyl]-6-(1H-tetrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline (20g) exhibited potent partial agonist activity (EC50 = 13 nM, maximal response 30%) and very weak protein tyrosine phosphatase 1B (PTP1B) inhibition (IC50 = 1100 nM), indicating a selective PPARγ partial agonist. A computational docking calculation revealed that 20g bound to PPARγ in a similar manner to that of known partial agonists. In male and female KK-Ay mice with insulin resistance and hyperglycemia, 20g at 30 mg/kg for 7 d significantly reduced plasma glucose levels, but not triglyceride levels. The effects of 20g were similar to those of pioglitazone at 10 mg/kg. In conclusion, the 2,6,7-substituted 1,2,3,4-tetrahydroisoquinoline with an acidic group at the 6-position provides a novel scaffold for selective PPARγ partial agonists and 20g exerted anti-diabetic effects via the partial activation of PPARγ.

A 3D-QSAR Analysis of CDK2 Inhibitors Using FMO Calculations and PLS Regression.

We report a three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis of CDK2 inhibitors using fragment molecular orbital (FMO) calculations and partial least squares (PLS) regression. In our analysis, fragment binding energies of individual amino acids and fragment binding energy of a single ligand in a protein-ligand complex are evaluated by FMO calculations and used as descriptors in PLS regression to estimate biological activities of the ligands. The analysis was applied to the system of CDK2 protein and its inhibitors and the effectiveness of the method was tested. Application of the 3D-QSAR model demonstrated that it offered good predictive ability and was able to predict not only biological activity of ligands but also identify important amino acid residues which could be targeted in order to improve ligand activity.

Characterizing the selectivity of ER α-glucosidase inhibitors.

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.

[Identifying Inhibitors of USP7-HDM2 Protein-Protein Interaction (PPI) by the in Silico Fragment-mapping Method].

Proteolysis mediated by the ubiquitin-proteome system plays an important role in cancer. Recently, a deubiquitinating enzyme, ubiquitin-specific protease 7 (USP7) has attracted attention as a key regulator of the p53-human double minute 2 (HDM2) pathway in cancer cells. Although some USP7 enzyme inhibitors have been identified, issues related to activity and selectivity prevent their therapeutic application. In this study, we aimed to search for novel USP7-HDM2 protein-protein interaction (PPI) inhibitors that do not affect the USP7 enzyme activity. Using the fragment-mapping program Fsubsite and the canonical subsite-fragment database (CSFDB) developed in our laboratory, we mapped a variety of fragments onto USP7 protein and constructed 3D-pharmacophore models based on the arrangement patterns of the mapped fragments. Finally, we performed 3D pharmacophore-based virtual screening of a commercial compound database and successfully selected promising USP7-HDM2 PPI inhibitor candidates.

Structure-based virtual screening for novel chymase inhibitors by in silico fragment mapping.

The term chymase refers to a family of chymotrypsin-like serine proteases stored within the secretory granules of mast cells. Recently, a variety of small molecule inhibitors for chymase have been developed with a primary focus on the treatment of cardiovascular diseases. Despite the expected therapeutic benefit of these chymase inhibitors, they have not been used clinically. Here, we attempted to identify new chymase inhibitors using a multistep structure-based virtual screening protocol combined with our knowledge-based in silico fragment mapping technique. The mapping procedure identified fragments with novel modes of interaction at the oxyanion hole of chymase. Next, we constructed a three-dimensional (3D) pharmacophore model and retrieved eight candidate chymase inhibitors from a commercial database that included approximately five million compounds. This selection was achieved using a multistep virtual screening protocol, which combined a 3D pharmacophore-based search, docking calculations, and analyses of binding free energy. One of the eight compounds exhibited concentration-dependent chymase inhibitory activity, which could be further optimized to develop more potent chymase inhibitors.

DFT study of the influence of acetyl groups of cellulose acetate on its intrinsic birefringence and wavelength dependence.

The effect of the acetyl groups of cellulose acetate (CA) on its intrinsic birefringence and its wavelength dependence was investigated using density functional theory (DFT). Seven types of CA repeating-unit models that differ in their degree of substitution (DS) and substitution sites were used in the calculations. The results suggested that the intrinsic birefringence (Δn°) and its wavelength dependence significantly depended on the conformations of the acetyl group at C6. Additionally, the intrinsic birefringence of CA films was estimated as the ensemble average of the calculated Δn° values of the conformers. The increase in the DS of CA led to a more negative intrinsic birefringence and a larger wavelength dependence. The computational results were in good qualitative agreement with the experimental results and suggested that conformational variety and/or its control would be important factors for the design of optical films containing CA.

2-Acyl-3-carboxyl-tetrahydroisoquinoline Derivatives: Mixed-Type PTP1B Inhibitors without PPARγ Activation.

A novel series of 2-acyl-3-carboxyl-tetrahydroisoquinoline derivatives were synthesized and biologically evaluated. Among them, (S)-2-{(E)-3-furan-2-ylacryloyl}-7-[(2E,4E)-5-(2,4,6-trifluorophenyl)penta-2,4-dienyloxy]-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (compound 17u) was identified as a potent protein tyrosine phosphatase 1B (PTP1B) inhibitor without peroxisome proliferator-activated receptor (PPAR) γ activation: PTP1B inhibition IC50=0.19 µM and PPARγ ΕC50>10 µM. Compound 17u exhibited mixed-type inhibition for PTP1B, and this mode of inhibition was rationalized by computational ligand docking into the catalytic and allosteric sites of PTP1B. Compound 17u also showed high oral absorption at 10 mg/kg (per os (p.o.), Cmax=4.67 µM) in rats, significantly reduced non-fasting plasma glucose and triglyceride levels with no side effects at 30 mg/kg/d (p.o.) for 4 weeks, and attenuated elevations in plasma glucose levels in the oral glucose tolerance test performed 24 h after its final administration in db/db mice. In conclusion, the substituted 2-acyl-3-carboxyl-tetrahydroisoquinoline is a novel scaffold of mixed-type PTP1B inhibitors without PPARγ activation, and compound 17u has potential as an efficacious and safe anti-diabetic drug as well as a useful tool for investigations on the physiological and pathophysiological effects of mixed-type PTP1B inhibition.

Design of a New α-1-C-Alkyl-DAB Derivative Acting as a Pharmacological Chaperone for ß-Glucocerebrosidase Using Ligand Docking and Molecular Dynamics Simulation.

Some point mutations in ß-glucocerebrosidase cause either improper folding or instability of this protein, resulting in Gaucher disease. Pharmacological chaperones bind to the mutant enzyme and stabilize this enzyme; thus, pharmacological chaperone therapy was proposed as a potential treatment for Gaucher disease. The binding affinities of α-1-C-alkyl 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives, which act as pharmacological chaperones for ß-glucocerebrosidase, abruptly increased upon elongation of their alkyl chain. In this study, the primary causes of such an increase in binding affinity were analyzed using protein⁻ligand docking and molecular dynamics simulations. We found that the activity cliff between α-1-C-heptyl-DAB and α-1-C-octyl-DAB was due to the shape and size of the hydrophobic binding site accommodating the alkyl chains, and that the interaction with this hydrophobic site controlled the binding affinity of the ligands well. Furthermore, based on the aromatic/hydrophobic properties of the binding site, a 7-(tetralin-2-yl)-heptyl-DAB compound was designed and synthesized. This compound had significantly enhanced activity. The design strategy in consideration of aromatic interactions in the hydrophobic pocket was useful for generating effective pharmacological chaperones for the treatment of Gaucher disease.

In silico fragment-mapping method: a new tool for fragment-based/structure-based drug discovery.

Here, we propose an in silico fragment-mapping method as a potential tool for fragment-based/structure-based drug discovery (FBDD/SBDD). For this method, we created a database named Canonical Subsite-Fragment DataBase (CSFDB) and developed a knowledge-based fragment-mapping program, Fsubsite. CSFDB consists of various pairs of subsite-fragments derived from X-ray crystal structures of known protein-ligand complexes. Using three-dimensional similarity-matching between subsites on one protein and another, Fsubsite compares the surface of a target protein with all subsites in CSFDB. When a local topography similar to the subsite is found on the surface, Fsubsite places a fragment combined with the subsite in CSFDB on the target protein. For validation purposes, we applied the method to the apo-structure of cyclin-dependent kinase 2 (CDK2) and identified four compounds containing three mapped fragments that existed in the list of known inhibitors of CDK2. Next, the utility of our fragment-mapping method for fragment-growing was examined on the complex structure of tRNA-guanine transglycosylase with a small ligand. Fsubsite mapped appropriate fragments on the same position as the binding ligand or in the vicinity of the ligand. Finally, a 3D-pharmacophore model was constructed from the fragments mapped on the apo-structure of heat shock protein 90-α (HSP90α). Then, 3D pharmacophore-based virtual screening was carried out using a commercially available compound database. The resultant hit compounds were very similar to a known ligand of HSP90α. As a result of these findings, this in silico fragment-mapping method seems to be a useful tool for computational FBDD and SBDD.

Linear Discriminant Analysis for the in Silico Discovery of Mechanism-Based Reversible Covalent Inhibitors of a Serine Protease: Application of Hydration Thermodynamics Analysis and Semi-empirical Molecular Orbital Calculation.

We recently reported that the Gibbs free energy of hydrolytic water molecules (ΔGwat) in acyl-trypsin intermediates calculated by hydration thermodynamics analysis could be a useful metric for estimating the catalytic rate constants (kcat) of mechanism-based reversible covalent inhibitors. For thorough evaluation, the proposed method was tested with an increased number of covalent ligands that have no corresponding crystal structures. After modeling acyl-trypsin intermediate structures using flexible molecular superposition, ΔGwat values were calculated according to the proposed method. The orbital energies of antibonding π* molecular orbitals (MOs) of carbonyl C=O in covalently modified catalytic serine (Eorb) were also calculated by semi-empirical MO calculations. Then, linear discriminant analysis (LDA) was performed to build a model that can discriminate covalent inhibitor candidates from substrate-like ligands using ΔGwat and Eorb. The model was built using a training set (10 compounds) and then validated by a test set (4 compounds). As a result, the training set and test set ligands were perfectly discriminated by the model. Hydrolysis was slower when (1) the hydrolytic water molecule has lower ΔGwat; (2) the covalent ligand presents higher Eorb (higher reaction barrier). Results also showed that the entropic term of hydrolytic water molecule (-TΔSwat) could be used for estimating kcat and for covalent inhibitor optimization; when the rotational freedom of the hydrolytic water molecule is limited, the chance for favorable interaction with the electrophilic acyl group would also be limited. The method proposed in this study would be useful for screening and optimizing the mechanism-based reversible covalent inhibitors.

Validation of molecular force field parameters for peptides including isomerized amino acids.

Recently, stereoinversions and isomerizations of amino acid residues in the proteins of living beings have been observed. Because isomerized amino acids cause structural changes and denaturation of proteins, isomerizations of amino acid residues are suspected to cause age-related diseases. In this study, AMBER molecular force field parameters were tested by using computationally generated nonapeptides and tripeptides including stereoinverted and/or isomerized amino acid residues. Energy calculations by using density functional theory were also performed for comparison. Although the force field parameters were developed by parameter fitting for l-α-amino acids, the accuracy of the computational results for d-amino acids and ß-amino acids was comparable to those for l-α-amino acids. The conformational energies for tripeptides calculated by using density functional theory were reproduced more accurately than those for nonapeptides calculated by using the molecular mechanical force field. The evaluations were performed for the ff99SB, ff03, ff12SB, and the latest ff14SB force field parameters.

Exploration of DPP-IV inhibitors with a novel scaffold by multistep in silico screening.

Dipeptidyl peptidase-IV (DPP-IV), an enzyme that degrades incretins-hormones that promote insulin secretion-is a therapeutic target for type 2 diabetes, with a number of its inhibitors having been launched as therapies for diabetes. Since adverse effects of these inhibitors have recently been reported, the development of novel DPP-IV inhibitors with higher efficacy and safety is required. We, therefore, screened for novel DPP-IV inhibitors using the combination of an in silico drug discovery technique and a DPP-IV assay system. We initially selected seven candidate compounds as DPP-IV inhibitors from a database consisting of four million compounds by a multistep in silico screening procedure combining pharmacophore-based screening, docking calculation and the analysis of three-dimensional quantitative structure-activity relationship. We then measured the inhibitory activity of the selected compounds and identified a hit compound. In addition, we discuss the structure-activity relationship between the binding mode model and inhibitory activity of the hit compound.

Novel Non-carboxylate Benzoylsulfonamide-Based Protein Tyrosine Phosphatase 1B Inhibitors with Non-competitive Actions.

A novel series of benzoylsulfonamide derivatives were synthesized and biologically evaluated. Among them, 4-(biphenyl-4-ylmethylsulfanylmethyl)-N-(hexane-1-sulfonyl)benzamide (compound 18K) was identified as a protein tyrosine phosphatase 1B (PTP1B) inhibitor with potent and selective inhibitory activity against PTP1B (IC50=0.25 µM). Compound 18K functioned as a non-competitive inhibitor and bound to the allosteric site of PTP1B. It also showed high oral absorption in mice (the maximum drug concentration (Cmax)=45.5 µM at 30 mg/kg), rats (Cmax=53.6 µM at 30 mg/kg), and beagles (Cmax=37.8 µM at 10 mg/kg), and significantly reduced plasma glucose levels at 30 mg/kg/d (per os (p.o.)) for one week with no side effects in db/db mice. In conclusion, the substituted benzoylsulfonamide was shown to be a novel scaffold of a non-competitive and allosteric PTP1B inhibitor, and compound 18K has potential as an efficacious and safe anti-diabetic drug as well as a useful tool for investigations of the physiological and pathophysiological effects of allosteric PTP1B inhibition.

Gibbs Free Energy of Hydrolytic Water Molecule in Acyl-Enzyme Intermediates of a Serine Protease: A Potential Application for Computer-Aided Discovery of Mechanism-Based Reversible Covalent Inhibitors.

In order to predict the potencies of mechanism-based reversible covalent inhibitors, the relationships between calculated Gibbs free energy of hydrolytic water molecule in acyl-trypsin intermediates and experimentally measured catalytic rate constants (kcat) were investigated. After obtaining representative solution structures by molecular dynamics (MD) simulations, hydration thermodynamics analyses using WaterMap™ were conducted. Consequently, we found for the first time that when Gibbs free energy of the hydrolytic water molecule was lower, logarithms of kcat were also lower. The hydrolytic water molecule with favorable Gibbs free energy may hydrolyze acylated serine slowly. Gibbs free energy of hydrolytic water molecule might be a useful descriptor for computer-aided discovery of mechanism-based reversible covalent inhibitors of hydrolytic enzymes.

In silico analyses of essential interactions of iminosugars with the Hex A active site and evaluation of their pharmacological chaperone effects for Tay-Sachs disease.

The affinity of a series of iminosugar-based inhibitors exhibiting various ring sizes toward Hex A and their essential interactions with the enzyme active site were investigated. All the Hex A-inhibiting iminosugars tested formed hydrogen bonds with Arg178, Asp322, Tyr421 and Glu462 and had the favorable cation-π interaction with Trp460. Among them, DMDP amide (6) proved to be the most potent competitive inhibitor with a Ki value of 0.041 µM. We analyzed the dynamic properties of both DMDP amide (6) and DNJNAc (1) in aqueous solution using molecular dynamics (MD) calculations; the distance of the interaction between Asp322 and 3-OH and Glu323 and 6-OH was important for stable interactions with Hex A, reducing fluctuations in the plasticity of the active site. DMDP amide (6) dose-dependently increased intracellular Hex A activity in the G269S mutant cells and restored Hex A activity up to approximately 43% of the wild type level; this effect clearly exceeded the border line treatment for Tay-Sachs disease, which is regarded as 10-15% of the wild type level. This is a significantly greater effect than that of pyrimethamine, which is currently in Phase 2 clinical trials. DMDP amide (6), therefore, represents a new promising pharmacological chaperone candidate for the treatment of Tay-Sachs disease.