Potential Pharmacological Chaperones Research Articles

Synthesis of Uronic Acid 1-Azasugars as Putative Inhibitors of α-Iduronidase, β-Glucuronidase and Heparanase.

1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited β-glucuronidase (β-GLU). The d-GlcA-configured 9 was a potent inhibitor of β-GLU and a moderate inhibitor of the endo-β-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.

In silico identification and characterization of potential orthosteric and allosteric pharmacological chaperones of the NAGLU enzyme and evaluation of their chaperone effect in vitro

In silico identification and characterization of potential orthosteric and allosteric pharmacological chaperones of the NAGLU enzyme and evaluation of their chaperone effect in vitro

P.0868 New glucocerebrosidase potential pharmacological chaperones tested on primary macrophages derived from patients with Gaucher Disease and Parkinson's disease

P.0868 New glucocerebrosidase potential pharmacological chaperones tested on primary macrophages derived from patients with Gaucher Disease and Parkinson's disease

Pharmacological Chaperones for β-Galactosidase Related to GM1 -Gangliosidosis and Morquio B: Recent Advances.

A short survey on selected β-galactosidase inhibitors as potential pharmacological chaperones for GM1 -gangliosidosis and Morquio B associated mutants of human lysosomal β-galactosidase is provided highlighting recent developments in this particular area of lysosomal storage disorders and orphan diseases.

Identification of Natural Products as Potential Pharmacological Chaperones for Protein Misfolding Diseases.

Defective protein folding and accumulation of misfolded proteins is associated with neurodegenerative, cardiovascular, secretory, and metabolic disorders. Efforts are being made to identify small-molecule modulators or structural-correctors for conformationally destabilized proteins implicated in various protein aggregation diseases. Using a metastable-reporter-based primary screen, we evaluated pharmacological chaperone activity of a diverse class of natural products. We found that a flavonoid glycoside (C-10, chrysoeriol-7-O-β-D-glucopyranoside) stabilizes metastable proteins, prevents its aggregation, and remodels the oligomers into protease-sensitive species. Data was corroborated with additional secondary screen with disease-specific pathogenic protein. In vitro and cell-based experiments showed that C-10 inhibits α-synuclein aggregation which is implicated in synucleinopathies-related neurodegeneration. C-10 interferes in its structural transition into β-sheeted fibrils and mitigates α-synuclein aggregation-associated cytotoxic effects. Computational modeling suggests that C-10 binds to unique sites in α-synuclein which may interfere in its aggregation amplification. These findings open an avenue for comprehensive SAR development for flavonoid glycosides as pharmacological chaperones for metastable and aggregation-prone proteins implicated in protein conformational diseases.

EU-OPENSCREEN: A Novel Collaborative Approach to Facilitate Chemical Biology

Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN’s compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN.

Characterization of two potential pharmacological chaperones for N-acetylgalactosamine-6-sulfate sulfates (GALNS) enzyme

Characterization of two potential pharmacological chaperones for N-acetylgalactosamine-6-sulfate sulfates (GALNS) enzyme

New perspectives for pharmacological chaperoning treatment in methylmalonic aciduria cblB type

Methylmalonic aciduria cblB type (MMA cblB) is caused by the impairment of ATP:cob(I)alamin adenosyltransferase (ATR), the enzyme responsible for the synthesis of adenosylcobalamin (AdoCbl) from cob(I)alamin. No definitive treatment is available for patients with this condition and novel therapeutic strategies are therefore much needed. Recently, we described a proof-of-concept regarding the use of pharmacological chaperones as a treatment. This work describes the effect of two potential pharmacological chaperones - compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2-phenylacetamide) and compound VI (4-(4-(4-fluorophenyl)-5-methyl-1H-pyrazol-3-yl)benzene-1,3-diol) - on six ATR mutants, including the most common, p.Arg186Trp. Comprehensive functional analysis identified destabilizing (p.Arg186Gln, p.Arg190Cys, p.Arg190His, p.Arg191Gln and p.Glu193Lys) and oligomerization (p.Arg186Trp and p.Arg191Gln) mutations. In a cellular model overexpressing the destabilizing/oligomerization mutations, compounds V and VI had a positive effect on the stability and activity of all ATR variants. When provided in combination with hydroxocobalamin a more positive effect was obtained than with the compounds alone, even in mutations previously described as B12 non-responsive. In addition, a normal oligomerization profile was recovered after treatment of the p.Arg186Trp mutant with both compounds. These promising results confirm MMA cblB type as a conformational disorder and hence, pharmacological chaperones as a new therapeutic option alone or in combination with hydroxocobalamin for many patients with MMA cblB.

Synthesis and evaluation of N-alkylated analogues of aza-galacto-fagomine – potential pharmacological chaperones for Krabbe disease

Seven novel alkylated or acylated analogues of hexahydropyridazine aza-galacto-fagomine (AGF) was prepared and studied as glycosidase inhibitors with the aim of increasing inhibitory potency and selectivity. The enzyme galactocerebrosidase, implicated in Krabbe disease, was found to be potently inhibited by n-butyl N2-alkylated AGF.

5-Fluoro derivatives of 4-epi-isofagomine as d-galactosidase inhibitors and potential pharmacological chaperones for GM1-gangliosidosis as well as Fabry's disease

Electrophilic fluorination of an exocyclic methoxymethylene enol ether derived from N-tert-butyloxycarbonyl-1,5-dideoxy-1,5-imino-3,4-O-isopropylidene-d-erythro-pent-2-ulose (11) provided the 5-fluoro derivative of the powerful β-galactosidase inhibitor 4-epi-isofagomine (8). This structural alteration, in combination with N-alkylation, led to considerably improved α-galactosidase selectivity. New compounds may serve as leads en route to new pharmacological chaperones for Fabry's disease.

Potential pharmacological chaperones targeting cancer-associated MCL-1 and Parkinson disease-associated α-synuclein.

Pharmacological chaperones are small molecules that bind to proteins and stabilize them against thermal denaturation or proteolytic degradation, as well as assist or prevent certain protein-protein assemblies. These activities are being exploited for the development of treatments for diseases caused by protein instability and/or aberrant protein-protein interactions, such as those found in certain forms of cancers and neurodegenerative diseases. However, designing or discovering pharmacological chaperones for specific targets is challenging because of the relatively featureless protein target surfaces, the lack of suitable chemical libraries, and the shortage of efficient high-throughput screening methods. In this study, we attempted to address all these challenges by synthesizing a diverse library of small molecules that mimic protein α-helical secondary structures commonly found in protein-protein interaction surfaces. This was accompanied by establishing a facile "on-bead" high-throughput screening method that allows for rapid and efficient discovery of potential pharmacological chaperones and for identifying novel chaperones/inhibitors against a cancer-associated protein, myeloid cell leukemia 1 (MCL-1), and a Parkinson disease-associated protein, α-synuclein. Our data suggest that the compounds and methods described here will be useful tools for the development of pharmaceuticals for complex-disease targets that are traditionally deemed "undruggable."

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.

Pharmacological chaperones increase residual β-galactocerebrosidase activity in fibroblasts from Krabbe patients

Krabbe disease or globoid cell leukodystrophy is a degenerative, lysosomal storage disease resulting from the deficiency of β-galactocerebrosidase activity. This enzyme catalyzes the lysosomal hydrolysis of galactocerebroside and psychosine. Krabbe disease is inherited as an autosomal recessive trait, and many of the 70 disease-causing mutations identified in the GALC gene are associated with protein misfolding. Recent studies have shown that enzyme inhibitors can sometimes translocate misfolded polypeptides to their appropriate target organelle bypassing the normal cellular quality control machinery and resulting in enhanced activity. In search for pharmacological chaperones that could rescue the β-galactocerebrosidase activity, we investigated the effect of α-Lobeline or 3′,4′,7-trihydroxyisoflavone on several patient-derived fibroblast cell lines carrying missense mutations, rather than on transduced cell lines. Incubation of these cell lines with α-lobeline or 3′,4′,7-trihydroxyisoflavone leads to an increase of β-galacocerebrosidase activity in p.G553R+p.G553R, in p.E130K+p.N295T and in p.G57S+p.G57S mutant forms over the critical threshold. The low but sustained expression of β-galactocerebrosidase induced by these compounds is a promising result; in fact, it is known that residual enzyme activity of only 15–20% is sufficient for clinical efficacy. The molecular interaction of the two chaperones with β-galactocerebrosidase is also supported by in silico analysis. Collectively, our combined in silico–in vitro approach indicate α-lobeline and 3′,4′,7-trihydroxyisoflavone as two potential pharmacological chaperones for the treatment or improvement of quality of life in selected Krabbe disease patients.

Stereoselective synthesis of 1-C-alkyl iminogalactitol derivatives, potential chaperones for galactosidase-linked LSDs: a real challenge

1-C-Alkyl iminogalactitol derivatives are highly desirable target compounds as potential pharmacological chaperones for the treatment of galactosidase-linked lysosomal storage disorders (LSDs). The synthesis of such compounds from d-galactose by a C-1 chain extension process by way of an open-chain 6-amino d-gulo nonulose intermediate was complicated by unexpected deoxygenation reactions leading to 3-deoxy iminogalactitol derivatives and epimers; an alternative, stereocontrolled synthesis from an l-tagatose derivative by C-6 chain-extension was therefore developed, which involved addition of organometallic reagents onto t-butanesulfinylimine intermediates in the key step.

On the design of broad based screening assays to identify potential pharmacological chaperones of protein misfolding diseases.

Correcting aberrant folds that develop during protein folding disease states is now an active research endeavor that is attracting increasing attention from both academic and industrial circles. One particular approach focuses on developing or identifying small molecule correctors or pharmacological chaperones that specifically stabilize the native fold. Unfortunately, the limited screening platforms available to rapidly identify or validate potential drug candidates are usually inadequate or slow because the folding disease proteins in question are often transiently folded and/or aggregation-prone, complicating and/or interfering with the assay outcomes. In this review, we outline and discuss the numerous platform options currently being employed to identify small molecule therapeutics for folding diseases. Finally, we describe a new stability screening approach that is broad based and is easily applicable toward a very large number of both common and rare protein folding diseases. The label free screening method described herein couples the promiscuity of the GroEL binding to transient aggregation-prone hydrophobic folds with surface plasmon resonance enabling one to rapidly identify potential small molecule pharmacological chaperones.

Development of a pharmacorphore model for pharmacological chaperones targeting mutant trafficking-deficient CNG channels

Complete colorblindness (achromatopsia) is caused by autosomal recessively inherited mutations in the retinal phototransduction pathway, predominantly in the CNGA3- and CNGB3-subunit of the cyclic nucleotide-gated (CNG) channels in cone photoreceptors. CNGA3, which is mutated in about 25% of the achromatopsia patients, mainly harbors missense mutations which frequently impair the folding and/or trafficking of the mutant CNGA3-channels [1]. Pharmacological chaperones stabilizing the folding of the mutant protein may be used to overcome this folding-/trafficking-deficiency. More than 50 compounds were evaluated in their ability to restore signal transduction using a calcium imaging-based bioassay utilizing the CNGA3-mutant E228K [2]. With this data we created several pharmacophore models using Schrodinger Phase [3], which describe the chemical features of potential pharmacological chaperones targeting achromatopsia. We used several approaches leading to different pharmacophore hypotheses: a) Training with the complete set of experimental data (see Figure ​Figure11) Figure 1 Pharmacophore depicting potential features of CNG channel-chaperones. b) Training with only dihydropyridines since this group showed the highest experimental activity, and c) Training with a data set excluding dihydropyridines. Our in-house database TueScreen, which includes ZINC12 [4], was screened to identify potentially active compounds. As a result, several potential molecule classes could be found that may be useful as pharmacological chaperones to improve folding/trafficking of mutant CNG-channels. We will experimentally validate these predictions in a calcium imaging-based bioassay.

On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases

On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases

The Two Faces of Iminoalditols: Powerful Inhibitors Trigger Glycosidase Activation

Understanding and controlling carbohydrate processing enzymes (CPE) have been major issues and challenges for chemists, biochemists and clinical practitioners alike. One of the most powerful families of substances for probing active sites as well as allosteric interactions with CPEs are basic sugar analogues, in particular iminoalditols. This compound class presents a basic trivalent nitrogen instead of oxygen in the sugar ring as the common feature. Depending on the task, such molecules may show two faces, acting as powerful competitive inhibitors or as folding templates for the same CPE protein. When applied at sub-inhibitory concentration iminoalditols and derivatives thereof have become attractive as pharmacological chaperones for the treatment of lysosomal storage diseases. As such these structures can restore protein activity by assisting correct folding of mutant enzymes thus facilitating transportation to the lysosome and consequently substrate hydrolysis. This review surveys iminoalditol structures which have recently been investigated as potential pharmacological chaperones for the treatment of lysosomal storage diseases.

Syntheses of p-nitrophenyl 3- and 4-thio-β- d-glycopyranosides

Thioglycosides have proved to be useful, enzymatically stable analogs of glycosides for structural and mechanistic studies and their synthesis is considerably simplified through the use of thioglycoligases. As part of an investigation into the use of thioglycosides as potential pharmacological chaperones, and as components of glycoproteins and glycolipids, the syntheses of p-nitrophenyl 3-thio-β- d-galactopyranoside, phenyl 1,4-dithio-β- d-glucopyranoside, p-nitrophenyl 4-thio-β- d-mannopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-4-thio-β- d-mannopyranoside are described.

Click Chemistry Approach to New N-Substituted Aminocyclitols as Potential Pharmacological Chaperones for Gaucher Disease

New N-alkylaminocyclitols bearing a 1,2,3-triazole system at different positions of the alkyl chain have been prepared as potential GCase pharmacological chaperones using click chemistry approaches. Among them, compounds 1d and 1e, with the shorter spacer (n = 1) between the alkyltriazolyl system and the aminocyclitol core, were the most active ones as GCase inhibitors, revealing a determinant effect of the location of the triazole ring on the activity. Furthermore, SAR data and computational docking models indicate a correlation between lipophilicity and enzyme inhibition and suggest "extended" and "bent" potential binding modes for the compounds. In the "bent" mode, the most active compounds could establish a hydrogen-bond interaction between the triazole moiety and enzyme residue Q284. Such an interaction would be precluded in compounds with a longer spacer between the triazole and the aminocyclitol core.