RESUMO
Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid ß-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent ß-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of ß-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.
Assuntos
Ciclopentanos/farmacologia , Galactosidases/metabolismo , Imino Piranoses/farmacologia , Lisossomos/enzimologia , Chaperonas Moleculares/metabolismo , Cristalização , Ciclopentanos/síntese química , Ciclopentanos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Galactosidases/antagonistas & inibidores , Humanos , Imino Piranoses/síntese química , Imino Piranoses/química , Ligantes , Lisossomos/efeitos dos fármacos , Conformação Molecular , Proteínas Mutantes/metabolismoRESUMO
From 1,2;3,4-di-O-isopropylidene-d-galactopyranose, a preliminary series of highly functionalized amino(hydroxymethyl)cyclopentanes was easily available. These amine-containing basic carbasugars featuring the d-galacto configuration are potent inhibitors of the GH20 ß-d-hexosaminidases probed and may bear potential as regulators of N-acetyl-d-hexosaminidase activities in vivo.
Assuntos
Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Cristalografia por Raios X , Ciclopentanos/síntese química , Ciclopentanos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Conformação Molecular , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of ß-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.
Assuntos
Ciclopentanos/química , Ciclopentanos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Gangliosidose GM1/tratamento farmacológico , beta-Galactosidase/antagonistas & inibidores , Aminação , Animais , Bovinos , Gangliosidose GM1/enzimologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Metilação , beta-Galactosidase/metabolismoRESUMO
By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,ß-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of ß-galactosidases, is outlined. In line with reported data, the (C-5aR) epimer was found a highly potent experimental pharmacological chaperone for GM1-associated human lysosomal ß-galactosidase mutant R201C.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Piranoses/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/metabolismo , Humanos , Imino Piranoses/síntese química , Imino Piranoses/química , Lisossomos/enzimologia , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger on similar structures, these amine containing basic carbasugars are potent inhibitors of ß-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.
Assuntos
Ciclopentanos/química , Ciclopentanos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Gangliosidose GM1/genética , Mutação , beta-Galactosidase/antagonistas & inibidores , Ciclopentanos/síntese química , Inibidores Enzimáticos/síntese química , Gangliosidose GM1/enzimologia , Humanos , Modelos Moleculares , Conformação Molecular , beta-Galactosidase/genéticaRESUMO
From an easily available partially protected analog of 1-deoxy-L-gulo-nojirimycin, by chain-branching at C-4 and suitable modification, lipophilic analogs of the powerful ß-D-galactosidase inhibitor 4-epi-isofagomine have been prepared. New compounds exhibit considerably improved inhibitory activities when compared with the unsubstituted parent compound and may serve as leads toward new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Imino Piranoses/síntese química , beta-Galactosidase/antagonistas & inibidores , 1-Desoxinojirimicina/química , Gangliosidose GM1/tratamento farmacológico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imino Piranoses/química , Mucopolissacaridose IV/tratamento farmacológico , beta-Galactosidase/químicaRESUMO
From an easily available partially protected formal derivative of 1-deoxymannojirimycin, by hydroxymethyl chain-branching and further elaboration, lipophilic analogs of the powerful ß-d-galactosidase inhibitor 4-epi-isofagomine have become available. New compounds exhibit improved inhibitory activities comparable to benchmark compound NOEV (N-octyl-epi-valienamine) and may serve as leads towards improved and more selective pharmacological chaperones for GM1-gangliosidosis.
Assuntos
Inibidores Enzimáticos/farmacologia , Gangliosidose GM1/enzimologia , Imino Piranoses/farmacologia , Lisossomos/enzimologia , beta-Galactosidase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Gangliosidose GM1/patologia , Humanos , Imino Piranoses/síntese química , Imino Piranoses/química , Lisossomos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , beta-Galactosidase/metabolismoRESUMO
Unlike replacement therapy by infusion of exogenous recombinant lysosomal enzymes, pharmacological chaperones aim at a gain of function of endogenous gene products. Deficits resulting from missense mutations may become treatable by small, competitive inhibitors binding to the catalytical site and thus correcting the erroneous conformation of mutant enzymes. This may prevent their premature degradation and normalize intracellular trafficking as well as biological half-life. A major limitation currently arises from the huge number of individual missense mutations and the lack of knowledge on the structural requirements for specific interaction with mutant protein domains. Our previous work on mutations of the ß-galactosidase (ß-gal) gene, causing GM1 gangliosidosis (GM1) and Morquio B disease (MBD), respectively, characterized clinical phenotypes as well as biosynthesis, intracellular transport and subcellular localization of mutants. We recently identified an effective chaperone, DL-HexDGJ (Methyl 6-{[N(2)-(dansyl)-N(6)-(1,5-dideoxy-D-galactitol-1,5-diyl)- L-lysyl]amino} hexanoate), among a series of N-modified 1-deoxygalactonojirimycin derivatives carrying a dansyl group in its N-acyl moiety. Using novel and flexible synthetic routes, we now report on the effects of two oligofluoroalkyl-derivatives of 1-deoxygalactonojirimycin, Ph(TFM)(2)OHex-DGJ (N-(α,α-di-trifluoromethyl) benzyloxyhexyl-1,5-dideoxy-1,5-imino-D: -galactitol) and (TFM)(3)OHex-DGJ (N-(Nonafluoro-tert-butyloxy)hexyl-1,5-dideoxy-1,5-imino-D: -galactitol) on the ß-gal activity of GM1 and MBD fibroblasts. Both compounds are competitive inhibitors and increase the residual enzyme activities up to tenfold over base line activity in GM1 fibroblasts with chaperone-sensitive mutations. Western blots showed that this was due to a normalization of protein transport and intralysosomal maturation. The fact that the novel compounds were effective at very low concentrations (0.5-10 µM) in the cell culture medium as well as their novel chemical character suggest future testing in animal models. This may contribute to new aspects for efficient and personalized small molecule treatment of lysosomal storage diseases.
Assuntos
Gangliosidose GM1/genética , Mucopolissacaridose IV/genética , Álcoois Açúcares/química , beta-Galactosidase/genética , Alelos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Éxons , Gangliosidose GM1/tratamento farmacológico , Genótipo , Humanos , Doenças por Armazenamento dos Lisossomos/terapia , Lisossomos/metabolismo , Modelos Químicos , Modelos Genéticos , Chaperonas Moleculares/metabolismo , Mucopolissacaridose IV/tratamento farmacológico , Mutação , FenótipoRESUMO
G(M1)-gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid beta-galactosidase (beta-Gal) degrades two functionally important molecules, G(M1)-ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable beta-Gal precursors, normally transported and processed to mature, intralysosomal beta-Gal. In accordance with the MBD phenotype, elevated residual activity against G(M1)-ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor proteins rapidly degraded by endoplasmic reticulum-associated protein degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid beta-galactosidase in vitro, and describes its effects on activity, protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of beta-Gal precursors.