A Systematic Investigation of Iminosugar Click Clusters as Pharmacological Chaperones for the Treatment of Gaucher Disease

A series of 18 mono‐ to 14‐valent iminosugars with different ligands, scaffolds, and alkyl spacer lengths have been synthesized and evaluated as inhibitors and pharmacological chaperones of β‐glucocerebrosidase (GCase). Small but significant multivalent effects in GCase inhibition have been observed for two iminosugar clusters. Our study provides strong confirmation that compounds that display the best affinity for GCase are not necessarily the best chaperones. The best chaperoning effect observed for a deprotected iminosugar cluster has been obtained with a tetravalent 1‐deoxynojirimycin (DNJ) analogue (3.3‐fold increase at 10 μM ). In addition, our study provides the first evidence of the high potential of prodrugs for the development of potent pharmacological chaperones. Acetylation of a trivalent DNJ derivative, to give the corresponding acetate prodrug, leads to a pharmacological chaperone that produces higher enzyme activity increases (3.0‐fold instead of 2.4‐fold) at a cellular concentration (1 μM ) reduced by one order of magnitude.     

Iminosugar‐Based Galactoside Mimics as Inhibitors of Galactocerebrosidase: SAR Studies and Comparison with Other Lysosomal Galactosidases

Several families of iminosugar‐based galactoside mimics were designed, synthesized, and evaluated as galactocerebrosidase (GALC) inhibitors. They were also tested as inhibitors of lysosomal β‐ and α‐galactosidases in order to find new potent and selective pharmacological chaperones for treatment of the lysosomal storage disorder, Krabbe disease. Whereas 1‐C‐alkyl imino‐L ‐arabinitols are totally inactive toward the three enzymes, 1‐C‐alkyl imino‐D ‐galactitols were found to be active only toward α‐galactosidase A. Finally, 1‐N‐iminosugars provided the best results, as 4‐epi‐isofagomine was found to be a good inhibitor of both lysosomal β‐galactosidase and GALC. Further elaboration of this structure is required to achieve selectivity between these two galactosidases.     

Human Acid β‐Glucosidase Inhibition by Carbohydrate Derived Iminosugars: Towards New Pharmacological Chaperones for Gaucher Disease

A collection of carbohydrate‐derived iminosugars belonging to three structurally diversified sub‐classes (polyhydroxylated pyrrolidines, piperidines, and pyrrolizidines) was evaluated for inhibition of human acid β‐glucosidase (glucocerebrosidase, GCase), the deficient enzyme in Gaucher disease. The synthesis of several new pyrrolidine analogues substituted at the nitrogen or α‐carbon atom with alkyl chains of different lengths suggested an interpretation of the inhibition data and led to the discovery of two new GCase inhibitors at sub‐micromolar concentration. In the piperidine iminosugar series, two N‐alkylated derivatives were found to rescue the residual GCase activity in N370S/RecNcil mutated human fibroblasts (among which one up to 1.5‐fold). This study provides the starting point for the identification of new compounds in the treatment of Gaucher disease.     

A Fluorescent sp2-iminosugar with pharmacological chaperone activity for gaucher disease: synthesis and intracellular distribution studies

Gaucher disease (GD) is the most prevalent lysosomal-storage disorder, it is caused by mutations of acid β-glucosidase (β-glucocerebrosidase; β-Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp(2)-iminosugar type, including the 6-thio-N'-octyl-(5N,6S)-octyliminomethylidene derivative (6S-NOI-NJ), behaved as very selective competitive inhibitors of the lysosomal β-Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine-thiazolidine bicyclic skeleton and incorporates a dansyl group in the N'-substituent, namely 6-thio-(5N,6S)-[4-(N'-dansylamino)butyliminomethylidene]nojirimycin (6S-NDI-NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S-NDI-NJ is mainly located in lysosome-related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S-NDI-NJ in the lysosome is related to the β-Glu concentration level. 6S-NDI-NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S-NDI-NJ uptake in fibroblasts showed that high concentrations of D-glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose-transporter-independent mechanisms.     

N‐Alkyl‐, 1‐C‐Alkyl‐, and 5‐C‐Alkyl‐1,5‐dideoxy‐1,5‐imino‐(l)‐ribitols as Galactosidase Inhibitors

A series of 1,5‐dideoxy‐1,5‐imino‐(l )‐ribitol (DIR) derivatives carrying alkyl or functionalized alkyl groups were prepared and investigated as glycosidase inhibitors. These compounds were designed as simplified 4‐epi‐isofagomine (4‐epi‐IFG) mimics and were expected to behave as selective inhibitors of β‐galactosidases. All compounds were indeed found to be highly selective for β‐galactosidases versus α‐glycosidases, as they generally did not inhibit coffee bean α‐galactosidase or other α‐glycosidases. Some compounds were also found to be inhibitors of almond β‐glucosidase. The N‐alkyl DIR derivatives were only modest inhibitors of bovine β‐galactosidase, with IC₅₀ values in the 30–700 μm range. Likewise, imino‐l ‐ribitol substituted at the C1 position was found to be a weak inhibitor of this enzyme. In contrast, alkyl substitution at C5 resulted in enhanced β‐galactosidase inhibitory activity by a factor of up to 1000, with at least six carbon atoms in the alkyl substituent. Remarkably, the ‘pseudo‐anomeric’ configuration in this series does not appear to play a role. Human lysosomal β‐galactosidase from leukocyte lysate was, however, poorly inhibited by all iminoribitol derivatives tested (IC₅₀ values in the 100 μm range), while 4‐epi‐IFG was a good inhibitor of this enzyme. Two compounds were evaluated as pharmacological chaperones for a GM1‐gangliosidosis cell line (R301Q mutation) and were found to enhance the mutant enzyme activity by factors up to 2.7‐fold.     

Glucocerebrosidase Enhancers for Selected Gaucher Disease Genotypes by Modification of α‐1‐C‐Substituted Imino‐D‐xylitols (DIXs) by Click Chemistry

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9‐DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α‐1‐C‐propargyl‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as β‐glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX‐28 (α‐1‐C‐[(1‐(3‐trimethylsilyl)propyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl]‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol), bearing the 3‐trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM . Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non‐competitive inhibitors, with the exception of the mixed‐type inhibitor DIX‐28.     

Chaperone Activity of Bicyclic Nojirimycin Analogues for Gaucher Mutations in Comparison with N‐(n‐nonyl)Deoxynojirimycin

Gaucher disease (GD), the most prevalent lysosomal storage disorder, is caused by mutations of lysosomal β‐glucosidase (acid β‐Glu, β‐glucocerebrosidase); these mutations result in protein misfolding. Some inhibitors of this enzyme, such as the iminosugar glucomimetic N‐(n‐nonyl)‐1‐deoxynojirimycin (NN‐DNJ), are known to bind to the active site and stabilize the proper folding for the catalytic form, acting as “chemical chaperones” that facilitate transport and maturation of acid β‐Glu. Recently, bicyclic nojirimycin (NJ) analogues with structure of sp² iminosugars were found to behave as very selective, competitive inhibitors of the lysosomal β‐Glu. We have now evaluated the glycosidase inhibitory profile of a series of six compounds within this family, namely 5‐N,6‐O‐(N′‐octyliminomethylidene‐NJ (NOI‐NJ), the 6‐thio and 6‐amino‐6‐deoxy derivatives (6S‐NOI‐NJ and 6N‐NOI‐NJ) and the corresponding galactonojirimycin (GNJ) counterparts (NOI‐GNJ, 6S‐NOI‐GNJ and 6N‐NOI‐GNJ), against commercial as well as lysosomal glycosidases. The chaperone effects of four selected candidates (NOI‐NJ, 6S‐NOI‐NJ, 6N‐NOI‐NJ, and 6S‐NOI‐GNJ) were further evaluated in GD fibroblasts with various acid β‐Glu mutations. The compounds showed enzyme enhancement on human fibroblasts with N188S, G202R, F213I or N370S mutations. The chaperone effects of the sp² iminosugar were generally stronger than those observed for NN‐DNJ; this suggests that these compounds are promising candidates for clinical treatment of GD patients with a broad range of β‐Glu mutations, especially for neuronopathic forms of Gaucher disease.     

6‐Amino‐6‐deoxy‐5,6‐di‐N‐(N′‐octyliminomethylidene)nojirimycin: Synthesis,Biological Evaluation,and Crystal Structure in Complex with Acid β‐Glucosidase

6‐Amino‐6‐deoxy‐5,6‐di‐ N ‐( N ′‐octyliminomethylidene)nojirimycin , a reducing analogue of N‐nonyl‐1‐deoxynojirimycin, proved to be a potent and very selective inhibitor of β‐glucosidases, including human acid β‐glucosidase. Structural studies of the enzyme–inhibitor complex showed a binding mode in which the anomeric hydroxy group is accommodated in the “wrong” α configuration.

     

New Insights into the Pharmacological Chaperone Activity of C2‐Substituted Glucoimidazoles for the Treatment of Gaucher Disease

Mutations in acid β‐glucocerebrosidase (GCase) lead to the accumulation of the sphingolipid glucosylceramide, thereby resulting in Gaucher disease (GD). Active‐site‐specific competitive GCase inhibitors are effective pharmacological chaperones (PCs) that act as folding agents for mutant GCase folding in the endoplasmic reticulum. In this study, we prepared a series of glucoimidazole C2‐substituent derivatives, and evaluated their inhibition and PC properties with GCase. A cell‐based assay with patient‐derived lymphoblasts (N370S or L444P mutations) demonstrated that administration of these compounds can significantly increase GCase activity. Interestingly, the 3,3‐dimethyl‐N‐phenyl‐4‐amide‐1‐butyl‐substituted moderate inhibitor 11 had the greatest effect on activity: 2.1‐fold increase in N370S lymphoblasts at 2.5 μM and 1.2‐fold increase in L444P at 0.5 μM following a three‐day incubation. Computer docking studies and a protease protection assay were used to elucidate the ligand–enzyme interactions responsible for the chaperone activity of 11 . Western blot and immuno‐fluorescence assays verified restoration of GCase trafficking to the lysosome. Together, these results indicate that 11 is a promising PC for GD treatment and provide direct evidence of the mechanism of GCase chaperoning.     

A Highly Potent and Selective Caspase 1 Inhibitor that Utilizes a Key 3‐Cyanopropanoic Acid Moiety

Herein, we examine the potential of a nitrile‐containing propionic acid moiety as an electrophile for covalent attack by the active‐site cysteine residue of caspase 1. The syntheses of several cyanopropanate‐containing small molecules based on the optimized peptidic scaffold of prodrug VX‐765 were accomplished. These compounds were found to be potent inhibitors of caspase 1 (IC₅₀ values ≤1 nM ). Examination of these novel small molecules against a caspase panel demonstrated an impressive degree of selectivity for caspase 1 inhibition over other caspase isozymes. Assessment of hydrolytic stability and selected ADME properties highlighted these agents as potentially useful tools for studying caspase 1 down‐regulation in various settings, including in vivo analyses.     

Design and Synthesis of A‐Ring Simplified Pyripyropene A Analogues as Potent and Selective Synthetic SOAT2 Inhibitors

Currently, pyripyropene A, which is isolated from the culture broth of Aspergillus fumigatus FO‐1289, is the only compound known to strongly and selectively inhibit the isozyme sterol O‐acyltransferase 2 (SOAT2). To aid in the development of new cholesterol‐lowering or anti‐atherosclerotic agents, new A‐ring simplified pyripyropene A analogues have been designed and synthesized based on total synthesis, and the results of structure–activity relationship studies of pyripyropene A. Among the analogues, two A‐ring simplified pyripyropene A analogues exhibited equally efficient SOAT2 inhibitory activity to that of natural pyripyropene A. These new analogues are the most potent and selective SOAT2 inhibitors to be used as synthetic compounds and attractive seed compounds for the development of drug for dyslipidemia, including atherosclerotic disease and steatosis.     

Brain-Derived Neurotrophic Factor (BDNF) Is Associated with Platelet Activity and Bleeding Tendency in Patients with Gaucher Disease

Bleeding tendency, a prominent feature of patients with Gaucher disease (GD), is associated with abnormal platelet function. Brain-derived neurotrophic factor (BDNF) is a protein with neuroprotective potential stored in alpha granules of circulating platelets. Here we studied BDNF levels in 50 patients with type I GD (GD1) and their correlation with platelet activity and bleeding tendency. Flow cytometry was used to test unstimulated and stimulated measurement of platelet surface-activated expression of αIIbβ3 integrin, P-selectin and lysosomal-associated membrane protein (LAMP3/CD63). Serum and plasma BDNF levels were quantified using ELISA. The bleeding history was recorded by a bleeding questionnaire. Serum BDNF levels were positively correlated with platelet count and moderately correlated with unstimulated and stimulated platelet P-selectin expression. Patients with more than one bleeding manifestation were shown to have lower serum BDNF levels, albeit similar platelet count. Plasma BDNF levels were significantly elevated in splenectomized patients and showed a moderate positive correlation with stimulated platelet CD63 expression. These observations demonstrate the first association between BDNF levels in the peripheral blood with platelet dysfunction and increased bleeding manifestation. The role of measuring serum BDNF for assessing platelet alpha degranulation defects and bleeding risk in patients with GD and the general population needs further study.     

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.     

Design,Synthesis, and Biological Evaluation of Coumarin Derivatives Tethered to an Edrophonium‐like Fragment as Highly Potent and Selective Dual Binding Site Acetylcholinesterase Inhibitors

A large series of substituted coumarins linked through an appropriate spacer to 3‐hydroxy‐N,N‐dimethylanilino or 3‐hydroxy‐N,N,N‐trialkylbenzaminium moieties were synthesized and evaluated as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The highest AChE inhibitory potency in the 3‐hydroxy‐N,N‐dimethylanilino series was observed with a 6,7‐dimethoxy‐3‐substituted coumarin derivative, which, along with an outstanding affinity (IC₅₀=0.236 nM ) exhibits excellent AChE/BChE selectivity (SI>300 000). Most of the synthesized 3‐hydroxy‐N,N,N‐trialkylbenzaminium salts display an AChE affinity in the sub‐nanomolar to picomolar range along with excellent AChE/BChE selectivities (SI values up to 138 333). The combined use of docking and molecular dynamics simulations permitted us to shed light on the observed structure–affinity and structure–selectivity relationships, to detect two possible alternative binding modes, and to assess the critical role of π–π stacking interactions in the AChE peripheral binding site.     

Design of a Highly Selective and Potent Class of Non‐planar Estrogen Receptor β Agonists

Selective activation of the estrogen receptor β (ERβ) could be a safe approach to hormone replacement therapy for both women and men, in contrast to the estrogens currently used for women which activate both ERβ and ERα, occasionally causing severe side effects. The selective ERβ agonist AC‐131 has shown efficacy in animal models of Parkinson’s disease and neuropathic pain. With the use of AC‐131 as template, herein we report the discovery, synthesis, and structure–activity relationship (SAR) study of a new class of dihydrobenzofurans as potent and selective ERβ agonists. The SAR was established by enantioselective synthesis, molecular modeling, and whole‐cell‐based functional assays. The most potent diastereomer, cis‐ 10 ‐SR, was shown to have an EC₅₀ value of <1 nM , potency 100‐fold higher than that of AC‐131. Even more interestingly, compound trans‐ 10 ‐SS exhibited 1000‐fold ERβ/ERα selectivity while still maintaining good potency (～10 nM ). In addition, trans‐ 10 ‐SS showed only partial agonist activity (30–60 % Eff.) toward ERα at 10 μM . This unprecedented selectivity could be rationalized by molecular modeling. Compound trans‐ 10 ‐SS appears to be the first molecule to take advantage of both conservative amino acid differences found in the α‐ and β‐faces of the binding cavities of ERα and ERβ.