Early identification of promiscuous attributes of aldose reductase inhibitors using a DMSO-perturbation assay.

Aldose reductase (AR) inhibitors are used clinically to treat long-term diabetic complications. Previous studies reported a series of AR inhibitory candidates, but unfortunately the mode of inhibition was poorly described due mainly to the lack of readily available methods for evaluating the specificity. The present study examined the AR inhibitory effects of novel synthetic hydantoins and their structural relatives, some of which were obtained from chemically engineered extracts of natural plants, and discovered several novel AR inhibitors with moderate inhibitory activity. The identified inhibitors were then subjected to a two-step mechanistic characterization using a detergent-addition assay and our novel dimethyl sulfoxide (DMSO)-perturbation assay. The detergent-addition assay revealed aggregation-based inhibitors, and the subsequent DMSO-perturbation assay identified nonspecific binding inhibitors. Thus, the present study demonstrates the usefulness of the DMSO-perturbation screen for identifying nonspecific binding characteristics of AR inhibitors.

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.

Ginnalin B induces differentiation markers and modulates the proliferation/differentiation balance via the upregulation of NOTCH1 in human epidermal keratinocytes.

The red maple and sugar maple (Acer rubrum and A. saccharum, respectively) contain acertannins (ginnalins and maplexins), galloylated derivatives of 1,5-anhydro-d-glucitol (1,5-AG, 1). These compounds have a variety of potential medicinal properties and we have shown that some of them promote the expression of ceramide synthase 3. We now report on the beneficial effects of ginnalin B, (6-O-galloyl-1,5-AG, 5), leading to acceleration of skin metabolism and reduction of the turnover time. Ginnalin B dose-dependently increased the relative amount of keratin 10, keratin 1, and filaggrin gene, with maximal increase of 1.7-, 2.9, and 5.2-fold at 100 µM, respectively. The validation study showed that it had superior capacity to induce multiple stages of keratinocyte differentiation and significantly elevated the immunostaining site of keratin 10 and filaggrin in a 3-dimensional cultured human skin model, by 1.2 and 2.8-fold, respectively. Furthermore, ginnalin B caused the arrest of proliferation at the G0/G1 phase but it did not induce apoptotic cell death in normal human keratinocytes. Molecular studies revealed that ginnalin B up-regulated the levels of NOTCH1 and a concomitant increase p21 expression. Ginnalin B, therefore, represents a new class of promising functional and medical cosmetic compound and it could contribute to the maintenance of homeostasis of the epidermis.

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.

Multivalency To Inhibit and Discriminate Hexosaminidases.

A set of multivalent polyhydroxylated acetamidoazepanes based on ethylene glycol, glucoside, or cyclodextrin scaffolds was prepared. The compounds were assessed against plant, mammalian, and therapeutically relevant hexosaminidases. Multimerization was shown to improve the inhibitory potency with synergy, and to fine tune the selectivity profile between related hexosaminidases.

Discovery of hyaluronidase inhibitors from natural products and their mechanistic characterization under DMSO-perturbed assay conditions.

The present study discovered four novel hyaluronan-degrading enzyme (hyaluronidase) inhibitors including chikusetsusaponins and catechins through the activity-guided separation of Panax japonicus and Prunus salicina, respectively. Although the discovery resulted in identification of usual frequent hitters, subsequent mechanistic characterizations under our DMSO-perturbed assay conditions and related protocols revealed that chikusetusaponin IV would serve as an aggregating and non-specific binding inhibitor, while (-)-epicatechin would interact specifically with enzyme at the catalytic site or more likely at a kind of catechin-binding site with a relatively week inhibitory activity. The latter description might provide a possible explanation for the well-known fact that a series of catechin have been described as frequent hitters in biological assays with a moderate activity. Thus, the present study demonstrated a practical and robust methodology to characterize initial screening hits mechanistically molecule-by-molecule in the early stage of natural product-based drug discovery.

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.

Strategy for designing selective α-l-rhamnosidase inhibitors: Synthesis and biological evaluation of l-DMDP cyclic isothioureas.

This study shows that the cyclization of l-DMDP thioureas to bicyclic l-DMDP isothioureas improved α-l-rhamnosidase inhibition which was further enhanced by increasing the length of the alkyl chain. The addition of a long alkyl chain, such as decyl or dodecyl, to the nitrogen led to the production of highly potent inhibitors of α-l-rhamnosidase; it also caused broad inhibition spectrum against ß-glucosidase and ß-galactosidase. In contrast, the corresponding N-benzyl-l-DMDP cyclic isothioureas display selective inhibition of α-l-rhamnosidase; 3',4'-dichlorobenzyl-l-DMDP cyclic isothiourea (3r) was found to display the most potent and selective inhibition of α-l-rhamnosidase, with IC50 value of 0.22µM, about 46-fold better than the positive control 5-epi-deoxyrhamnojirimycin (5-epi-DRJ; IC50=10µM) and occupied the active-site of this enzyme (Ki=0.11µM). Bicyclic isothioureas of ido-l-DMDP did not inhibit α-l-rhamnosidase. These new mimics of l-rhamnose may affect other enzymes associated with the biochemistry of rhamnose including enzymes involved in progression of tuberculosis.

Interpreting the behavior of concentration-response curves of hyaluronidase inhibitors under DMSO-perturbed assay conditions.

Hyaluronan-degrading enzyme (hyaluronidase) is involved in tumor growth and inflammation, and as such, hyaluronidase inhibitors have received recent attention as potential therapeutics. The previous studies have successfully discovered a wide range of inhibitors, but unfortunately most of them are dissimilar to original ligand hyaluronan and the mode of action is poorly understood. The present study mechanistically characterized these structurally unrelated inhibitors by interpreting the behavior of concentration-response curves under several in vitro assay conditions. Detergent-addition conditions definitely identified aggregation-based inhibitors. Subsequently, DMSO-perturbed conditions, though preliminary, highlighted the inhibitors that might bind to enzyme non-specifically. Here, an intriguing implication of the latter description is that DMSO-perturbed conditions would generate non-productive but not-denatured enzyme that is an assembly of effective species to capture non-specific binding molecules, and thereby would attenuate their inhibitory activities.

Synthesis of new tricyclic thiolactams as potent antitumor agent for pancreatic cancer.

We synthesized the novel tricyclic thiolactams 2a-d, 3d-k, having a benzyl or substituted benzyl substituent on the nitrogen of indole subunit, and their preferential cytotoxicity under both nutrient-deprived medium (NDM) and Dulbecco's modified Eagle's medium (DMEM) was evaluated against a human pancreatic cancer cell line PANC-1. Among the tested compounds, the 4'-hydroxy derivative 3d showed the most potent cytotoxicity in NDM (PC50 1.68µM) although the moderate preferential cytotoxicity (PC50 1.68µM in NDM vs PC50 20µM in DMEM). The 3'-hydroxy derivative 3e exhibited the most preferential cytotoxicity (PC50 1.96µM in NDM vs less than 50% inhibition at 30µM in DMEM). The benzyl 2a and halogenated benzyl derivatives 2b,c showed no cytotoxicity in NDM. In addition, the indole (10, PC50 173.7µM), lactone (11, PC50 131.7µM), and lactam (12, PC50 44.8µM) derivatives showed week or moderate cytotoxicity in NDM. These results indicated that the hydroxy group on the benzyl substituent and tricyclic thiolactam ring were essential for the cytotoxicity in NDM against PANC-1 cell line. Moreover, 3'-hydroxy derivative 3e compound exhibited antitumor activity against the pancreatic ductal adenocarcinoma (PDAC) xenograft model in vivo.

Docking study and biological evaluation of pyrrolidine-based iminosugars as pharmacological chaperones for Gaucher disease.

We report on the synthesis and biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) derivatives as pharmacological chaperones for Gaucher disease. The parent compound, DAB, did not show inhibition of human ß-glucocerebrosidase but showed moderate intestinal α-glucosidase inhibition; in contrast, extension of α-1-C-alkyl chain length gave a series of highly potent and selective inhibitors of the ß-glucocerebrosidase. Our design of α-1-C-tridecyl-DAB (5j) produced a potent inhibitor of the ß-glucocerebrosidase, with IC50 value of 0.77 µM. A molecular docking study revealed that the α-1-C-tridecyl group has a favorable interaction with the hydrophobic pocket and the sugar analogue part (DAB) interacted with essential hydrogen bonds formed to Asp127, Glu235 and Glu340. Furthermore, α-1-C-tridecyl-DAB (5j) displayed enhancement of activity at an effective concentration 10-times lower than isofagomine. α-1-C-Tridecyl-DAB therefore provides the first example of a pyrrolidine iminosugar as a new class of promising pharmacological chaperones with the potential for treatment of Gaucher disease.

Fluorinated and Conformationally Fixed Derivatives of L-HomoDMDP: Synthesis and Glycosidase Inhibition.

Fluorinated and conformationally fixed derivatives of L-homoDMDP, i.e., 2,5-dideoxy-2,5-imino-DL-glycero-L-manno-heptitol, have been synthesized from d-xylose-derived cyclic nitrone 10 with oxazolidinone 19 or 28 and oxazinanone 22 or 32 as key intermediates. An evaluation of glycosidase inhibition showed replacement of the C-6 hydroxyl groups with fluoride in L-homoDMDP and its C-6 epimer did not have a significant influence on α-glucosidase inhibition by these iminosugars, while replacement of an amino group with a cyclic carbamate group in most conformationally fixed derivatives led to a sharp decrease in the level of glycosidase inhibition, revealing the importance of the free amino group in interaction of enzymes with these molecules.

Design and Synthesis of Labystegines, Hybrid Iminosugars from LAB and Calystegine, as Inhibitors of Intestinal α-Glucosidases: Binding Conformation and Interaction for ntSI.

This paper identifies the required configuration and orientation of α-glucosidase inhibitors, miglitol, α-1-C-butyl-DNJ, and α-1-C-butyl-LAB for binding to ntSI (isomaltase). Molecular dynamics (MD) calculations suggested that the flexibility around the keyhole of ntSI is lower than that of ctSI (sucrase). Furthermore, a molecular-docking study revealed that a specific binding orientation with a CH-π interaction (Trp370 and Phe648) is a requirement for achieving a strong affinity with ntSI. On the basis of these results, a new class of nortropane-type iminosugars, labystegines, hybrid iminosugars of LAB and calystegine, have been designed and synthesized efficiently from sugar-derived cyclic nitrones with intramolecular 1,3-dipolar cycloaddition or samarium iodide catalyzed reductive coupling reaction as the key step. Biological evaluation showed that our newly designed 3(S)-hydroxy labystegine (6a) inherited the selectivity against intestinal α-glucosidases from LAB, and its inhibition potency was 10 times better than that of miglitol. Labystegine, therefore, represents a promising new class of nortropane-type iminosugar for improving postprandial hyperglycemia.

3-Fluoroazetidinecarboxylic Acids and trans,trans-3,4-Difluoroproline as Peptide Scaffolds: Inhibition of Pancreatic Cancer Cell Growth by a Fluoroazetidine Iminosugar.

Reverse aldol opening renders amides of 3-hydroxyazetidinecarboxylic acids (3-OH-Aze) unstable above pH 8. Aze, found in sugar beet, is mis-incorporated for proline in peptides in humans and is associated with multiple sclerosis and teratogenesis. Aze-containing peptides may be oxygenated by prolyl hydroxylases resulting in potential damage of the protein by a reverse aldol of the hydroxyazetidine; this, rather than changes in conformation, may account for the deleterious effects of Aze. This paper describes the synthesis of 3-fluoro-Aze amino acids as hydroxy-Aze analogues which are not susceptible to aldol cleavage. 4-(Azidomethyl)-3-fluoro-Aze and 3,4-difluoroproline are new peptide building blocks. trans,trans-2,4-Dihydroxy-3-fluoroazetidine, an iminosugar, inhibits the growth of pancreatic cancer cells to a similar degree as gemcitabine.

Nine of 16 stereoisomeric polyhydroxylated proline amides are potent ß-N-acetylhexosaminidase inhibitors.

All 16 stereoisomeric N-methyl 5-(hydroxymethyl)-3,4-dihydroxyproline amides have been synthesized from lactones accessible from the enantiomers of glucuronolactone. Nine stereoisomers, including all eight with a (3R)-hydroxyl configuration, are low to submicromolar inhibitors of ß-N-acetylhexosaminidases. A structural correlation between the proline amides is found with the ADMDP-acetamide analogues bearing an acetamidomethylpyrrolidine motif. The proline amides are generally more potent than their ADMDP-acetamide equivalents. ß-N-Acetylhexosaminidase inhibition by an azetidine ADMDP-acetamide analogue is compared to an azetidine carboxylic acid amide. None of the amides are good α-N-acetylgalactosaminidase inhibitors.

Synthesis and biological evaluation of α-1-C-4'-arylbutyl-L-arabinoiminofuranoses, a new class of α-glucosidase inhibitors.

A series of α-1-C-4'-arylbutyl-L-arabinoiminofuranoses 3 with functional groups attached to the phenyl ring, which are potential α-glycosidase inhibitors, was designed and synthesized by using a Negishi cross-coupling reaction as the key reaction. Arylbutyl derivatives 3a-e showed potent inhibitory activities against intestinal maltase. Among them, difluorophenylbutyl derivative 3e showed good inhibition activities against intestinal isomaltase and sucrase as compared to those of 1 and commercial drugs.

Synthesis of the enantiomers of XYLNAc and LYXNAc: comparison of ß-N-acetylhexosaminidase inhibition by the 8 stereoisomers of 2-N-acetylamino-1,2,4-trideoxy-1,4-iminopentitols.

The enantiomers of XYLNAc (2-N-acetylamino-1,2,4-trideoxy-1,4-iminoxylitol) are prepared from the enantiomers of glucuronolactone; the synthesis of the enantiomers of LYXNAc (2-N-acetylamino-1,2,4-trideoxy-1,4-iminolyxitol) from an L-arabinono-δ-lactone and a D-ribono-δ-lactone is reported. A comparison is made of the inhibition of ß-N-acetylhexosaminidases (HexNAcases) and α-N-acetylgalactosaminidase (α-GalNAcase) by 8 stereoisomeric 2-N-acetylamino-1,2,4-trideoxy-1,4-iminopentitols; their N-benzyl derivatives are better inhibitors than the parent compounds. Both XYLNAc and LABNAc are potent inhibitors against HexNAcases. None of the compounds show any inhibition of α-GalNAcase.

Docking and SAR studies of calystegines: binding orientation and influence on pharmacological chaperone effects for Gaucher's disease.

We report on the identification of the required configuration and binding orientation of nor-tropane alkaloid calystegines against ß-glucocerebrosidase. Calystegine B2 is a potent competitive inhibitor of human lysosomal ß-glucocerebrosidase with Ki value of 3.3 µM. A molecular docking study revealed that calystegine B2 had a favorable van der Waals interactions (Phe128, Trp179, and Phe246) and the hydrogen bonding (Glu235, Glu340, Asp127, Trp179, Asn234, Trp381 and Asn396) was similar to that of isofagomine. All calystegine isomers bound into the same active site as calystegine B2 and the essential hydrogen bonds formed to Asp127, Glu235 and Glu340 were maintained. However, their binding orientations were obviously different. Calystegine A3 bound to ß-glucocerebrosidase with the same orientations as calystegine B2 (Type 1), while calystegine B3 and B4 had different binding orientations (Type 2). It is noteworthy that Type 1 orientated calystegines B2 and A3 effectively stabilized ß-glucocerebrosidase, and consequently increased intracellular ß-glucocerebrosidase activities in N370S fibroblasts, while Type 2 orientated calystegines B3 and B4 could not keep the enzyme activity. These results clearly indicate that the binding orientations of calystegines are changed by the configuration of the hydroxyl groups on the nor-tropane ring and the suitable binding orientation is a requirement for achieving a strong affinity to ß-glucocerebrosidase.

General synthesis of sugar-derived azepane nitrones: precursors of azepane iminosugars.

A general and efficient method has been developed for the synthesis of sugar-derived azepane nitrones starting from aldohexoses, with an intramolecular condensation of aldehyde and hydroxylamine as the key step. Through this strategy, each aldohexose produced a pair of azepane nitrones, which are precursors of various azepane iminosugars.

Synthesis of eight stereoisomers of pochonicine: nanomolar inhibition of ß-N-acetylhexosaminidases.

Pochonicine, the first naturally occurring polyhydroxylated pyrrolizidine containing an acetamidomethyl group, which was isolated from Pochonia suchlasporia var. suchlasporia TAMA 87, together with its enantiomer and their C-1 and/or C-3 epimers, have been synthesized from the sugar-derived cyclic nitrones 9D and 9L, respectively. An in-depth NMR study showed that both the (1)H and (13)C NMR spectra of the synthetic pochonicines (1D and 1L) matched very well with those of natural pochonicine in D2O, which unequivocally determined the relative configuration of the natural product as 1D or 1L. In addition, comparison of the optical rotations of the synthetic pochonicines and that of the natural product, but more convincingly their glycosidase inhibition profiles, confirmed the absolute configuration of natural pochonicine as 1R,3S,5R,6R,7S,7aR. Thereby, the structure of natural pochonicine was unequivocally determined as (+)-(1R,3S,5R,6R,7S,7aR)-pochonicine (1D). Glycosidase inhibition experiments showed that natural pochonicine 1D and its epimers 2D, 3D, and 4D all are powerful inhibitors of hexosaminidases (five ß-N-acetylglucosaminidases and two ß-N-acetylgalactosaminidases) while their enantiomers 1L, 2L, 3L, and 4L are much weaker inhibitors of the same enzymes. (-)-3-epi-Pochonicine (2L) was found to be a potent and selective inhibitor of α-l-rhamnosidase. None of the compounds showed any inhibition of α-GalNAcase.