RESUMEN
Current therapies used in minimal change disease (MCD) were originally designed to cure other diseases. They are only partially efficient, and present inconvenient side effects. Therefore, understanding the molecular mechanisms implicated in the pathogenesis of proteinuria in MCD could lead to new therapeutic strategies. A new experimental transgenic rat model of human MCD was generated. These NPHS2-Angptl4 transgenic rats over-express two different forms of the glycoprotein Angptl4 from the podocyte. The majority of the protein shows a lack of sialylation that is implicated in the pathogenesis of proteinuria. Supplementation of ManNAc, a precursor of sialic acid, significantly reduces albuminuria in those rats by increasing sialylation of the hyposialylated form of Angptl4. After treatment of the first episode of MCD with glucocorticoids in patients, ManNAc could be used as a maintenance drug, especially to reduce the frequency and intensity of relapse. ManNAc is a promising therapeutic agent for patients with MCD.
Asunto(s)
Angiopoyetinas/genética , Hexosaminas/uso terapéutico , Nefrosis Lipoidea/terapia , Proteinuria/genética , Proteína 4 Similar a la Angiopoyetina , Animales , Humanos , Nefrosis Lipoidea/genética , Síndrome Nefrótico/genética , Síndrome Nefrótico/terapia , Ratas , Ratas Transgénicas , Terapias en InvestigaciónRESUMEN
Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant GM1 ß-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations.
Asunto(s)
Galactosilceramidasa/genética , Hexosaminas/uso terapéutico , Leucodistrofia de Células Globoides/tratamiento farmacológico , Leucodistrofia de Células Globoides/genética , Adulto , Edad de Inicio , Animales , Células COS , Células Cultivadas , Niño , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Galactosilceramidasa/antagonistas & inhibidores , Galactosilceramidasa/química , Humanos , Lactante , Leucodistrofia de Células Globoides/patología , Chaperonas Moleculares/uso terapéuticoRESUMEN
Patients with GNE myopathy, a progressive and debilitating disease caused by a genetic defect in sialic acid biosynthesis, rely on supportive care and eventually become wheelchair-bound. To elucidate whether GNE myopathy is treatable at a progressive stage of the disease, we examined the efficacy of sialic acid supplementation on symptomatic old GNE myopathy mice that have ongoing, active muscle degeneration. We examined the therapeutic effect of a less metabolized sialic acid compound (6'-sialyllactose) or free sialic acid (N-acetylneuraminic acid) by oral, continuous administration to 50-week-old GNE myopathy mice for 30 weeks. To evaluate effects on their motor performance in living mice, spontaneous locomotion activity on a running wheel was measured chronologically at 50, 65, 72 and 80 weeks of age. The size, force production, and pathology of isolated gastrocnemius muscle were analysed at the end point. Sialic acid level in skeletal muscle was also measured. Spontaneous locomotion activity was recovered in 6'-sialyllactose-treated mice, while NeuAc-treated mice slowed the disease progression. Treatment with 6'-sialyllactose led to marked restoration of hyposialylation in muscle and consequently to robust improvement in the muscle size, contractile parameters, and pathology as compared to NeuAc. This is due to the fact that 6'-sialyllactose is longer working as it is further metabolized to free sialic acid after initial absorption. 6'-sialyllactose ameliorated muscle atrophy and degeneration in symptomatic GNE myopathy mice. Our results provide evidence that GNE myopathy can be treated even at a progressive stage and 6'-sialyllactose has more remarkable advantage than free sialic acid, providing a conceptual proof for clinical use in patients.
Asunto(s)
Miopatías Distales/tratamiento farmacológico , Lactosa/análogos & derivados , Envejecimiento/patología , Péptidos beta-Amiloides/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Células Cultivadas , Creatina Quinasa/metabolismo , Modelos Animales de Enfermedad , Miopatías Distales/patología , Ensayo de Inmunoadsorción Enzimática , Hexosaminas/uso terapéutico , Lactosa/efectos adversos , Lactosa/farmacocinética , Lactosa/uso terapéutico , Ratones , Contracción Muscular/fisiología , Músculo Esquelético/patología , Mutación/genética , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/uso terapéutico , Fragmentos de Péptidos/metabolismo , FenotipoRESUMEN
Inflammation and tissue degeneration play key roles in numerous rheumatic diseases, including osteoarthritis (OA). Efforts to reduce and effectively repair articular cartilage damage in an osteoarthritic environment are limited in their success due to the diseased environment. Treatment strategies focused on both reducing inflammation and increasing tissue production are necessary to effectively treat OA from a tissue-engineering perspective. In this work, we investigated the anti-inflammatory and tissue production capacity of a small molecule 3,4,6-O-tributanoylated-N-acetylglucosamine (3,4,6-O-Bu3GlcNAc) previously shown to inhibit the nuclear factor κB (NFκB) activity, a key transcription factor regulating inflammation. To mimic an inflammatory environment, chondrocytes were stimulated with interleukin-1ß (IL-1ß), a potent inflammatory cytokine. 3,4,6-O-Bu3GlcNAc exposure decreased the expression of NFκB target genes relevant to OA by IL-1ß-stimulated chondrocytes after 24 h of exposure. The capacity of 3,4,6-O-Bu3GlcNAc to stimulate extracellular matrix (ECM) accumulation by IL-1ß-stimulated chondrocytes was evaluated in vitro utilizing a three-dimensional hydrogel culturing system. After 21 days, 3,4,6-O-Bu3GlcNAc exposure induced quantifiable increases in both sulfated glycosaminoglycan and total collagen. Histological staining for proteoglycans and type II collagen confirmed these findings. The increased ECM accumulation was not due to the hydrolysis products of the small molecule, n-butyrate and N-acetylglucosamine (GlcNAc), as the isomeric 1,3,4-O-tributanoylated N-acetylglucosamine (1,3,4-O-Bu3GlcNAc) did not elicit a similar response. These findings demonstrate that a novel butanoylated GlcNAc derivative, 3,4,6-O-Bu3GlcNAc, has the potential to stimulate new tissue production and reduce inflammation in IL-1ß-induced chondrocytes with utility for OA and other forms of inflammatory arthritis.
Asunto(s)
Cartílago/citología , Ácidos Grasos Volátiles/química , Ácidos Grasos Volátiles/uso terapéutico , Hexosaminas/química , Osteoartritis/terapia , Ingeniería de Tejidos/métodos , Acetilglucosamina/farmacología , Animales , Butiratos/farmacología , Bovinos , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Hexosaminas/uso terapéutico , Inmunohistoquímica , Interleucina-1beta/farmacologíaRESUMEN
The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. Non-allosteric GNE gene mutations cause the muscular disorder GNE myopathy (also known as hereditary inclusion body myopathy), whose exact pathology remains unknown. Increased knowledge of GNE regulation, including isoform regulation, may help elucidate the pathology of GNE myopathy. While eight mRNA transcripts encoding human GNE isoforms are described, we only identified two mouse Gne mRNA transcripts, encoding mGne1 and mGne2, homologous to human hGNE1 and hGNE2. Orthologs of the other human isoforms were not identified in mice. mGne1 appeared as the ubiquitously expressed, major mouse isoform. The mGne2 encoding transcript is differentially expressed and may act as a tissue-specific regulator of sialylation. mGne2 expression appeared significantly increased the first 2 days of life, possibly reflecting the high sialic acid demand during this period. Tissues of the knock-in Gne p.M712T mouse model had similar mGne transcript expression levels among genotypes, indicating no effect of the mutation on mRNA expression. However, upon treatment of these mice with N-acetylmannosamine (ManNAc, a Gne substrate, sialic acid precursor, and proposed therapy for GNE myopathy), Gne transcript expression, in particular mGne2, increased significantly, likely resulting in increased Gne enzymatic activities. This dual effect of ManNAc supplementation (increased flux through the sialic acid pathway and increased Gne activity) needs to be considered when treating GNE myopathy patients with ManNAc. In addition, the existence and expression of GNE isoforms needs consideration when designing other therapeutic strategies for GNE myopathy.
Asunto(s)
Hexosaminas/uso terapéutico , Complejos Multienzimáticos/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Miopatías Distales/tratamiento farmacológico , Miopatías Distales/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutación Missense , Especificidad de Órganos , Estructura Secundaria de ProteínaRESUMEN
AIM: To study the antitumor effect of anti-NprPSA monoclonal antibody (mAb) in combination with ManNPr, a precursor of N-propionyl PSA, in multiple myeloma (MM), and to explore the mechanisms of the action. METHODS: Human multiple myeloma cell line RPMI-8226 was tested. The cells were pre-treated with ManNPr (1, 2, and 4 mg/mL), and then incubated with anti-NprPSA mAb (1 mg/mL). Cell apoptosis in vitro was detected using MTT assay and flow cytometry. BALB/c nude mice were inoculated sc with RPC5.4 cells. On 5 d after the injection, the mice were administered sc with anti-NprPSA mAb (200 µg/d) and ManNPr (5 mg/d) for 8 d. The tumor size and body weight were monitored twice per week. TUNEL assay was used for detecting apoptosis in vivo. The apoptotic pathway involved was examined using Western blot analysis and caspase inhibitor. RESULTS: Treatment of RPMI-8226 cells with anti-NprPSA mAb alone failed to inhibit cell growth in vitro. In RPMI-8226 cells pretreated with ManNPr, however, the mAb significantly inhibited the cell proliferation, decreased the viability, and induced apoptosis, which was associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. In the mouse xenograft model, treatment with the mAb in combination with ManNPr significantly inhibited the tumor growth, and induced significant apoptosis as compared to treatment with the mAb alone. Moreover, apoptosis induced by the mAb in vivo resulted from the activation of the caspases and poly(ADP-ribose) polymerase. CONCLUSION: The anti-NprPSA mAb in combination with ManNPr is an effective treatment for in vitro and in vivo induction of apoptosis in multiple myeloma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Hexosaminas/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Profármacos/uso terapéutico , Ácidos Siálicos/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Hemocianinas/inmunología , Hexosaminas/administración & dosificación , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Profármacos/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor-associated carbohydrate antigens (TACAs) are useful targets for the development of cancer vaccines or immunotherapies. However, a major obstacle in this application of TACAs is their poor immunogenicity. To overcome the problem, a new immunotherapeutic strategy combining synthetic vaccines made of artificial TACA derivatives and metabolic glycoengineering of cancer cells to express the artificial TACA derivatives was explored. Using a murine leukemia model FBL3 with GM3 antigen as the target, it was shown that artificial GM3 N-phenylacetyl derivative (GM3NPhAc) elicited robust antigen-specific T cell-dependent immunity and that N-phenylacetyl-D-mannosamine (ManNPhAc) as the biosynthetic precursor of GM3NPhAc selectively glycoengineered cancer cells to express GM3NPhAc both in vitro and in vivo. It was also demonstrated that GM3NPhAc-specific antisera and antibodies mediated strong cytotoxicity to ManNPhAc-treated FBL3 cell. Furthermore, vaccination with a conjugate vaccine made of GM3NPhAc followed by ManNPhAc treatment could significantly suppress tumor growth and prolong the survival of tumor-bearing mouse. These results have proved the feasibility of the new cancer immunotherapeutic strategy, as well as its efficacy to cure cancer, which is of general significance.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Gangliósido G(M3)/análogos & derivados , Leucemia/terapia , Animales , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Antineoplásicos/uso terapéutico , Vacunas contra el Cáncer/síntesis química , Vacunas contra el Cáncer/inmunología , Ingeniería Celular , Línea Celular Tumoral , Femenino , Gangliósido G(M3)/síntesis química , Gangliósido G(M3)/inmunología , Hexosaminas/uso terapéutico , Inmunoterapia , Leucemia/inmunología , Leucemia/mortalidad , Ratones , Ratones Endogámicos C57BL , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Resultado del Tratamiento , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/uso terapéuticoRESUMEN
Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades Renales/genética , Animales , Biomarcadores/metabolismo , Carbohidrato Epimerasas/genética , Proteínas Portadoras/genética , Suplementos Dietéticos , Evaluación Preclínica de Medicamentos/métodos , Hexosaminas/uso terapéutico , Humanos , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/embriología , Glomérulos Renales/metabolismo , Glomérulos Renales/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Microscopía Electrónica , Mutación , Ácido N-Acetilneuramínico/fisiología , Podocitos/metabolismo , Podocitos/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sialoglicoproteínas/metabolismoRESUMEN
ß-Galactosidase deficiency is a group of lysosomal lipid storage disorders with an autosomal recessive trait. It causes two clinically different diseases, G(M1) -gangliosidosis and Morquio B disease. It is caused by heterogeneous mutations in the GLB1 gene coding for the lysosomal acid ß-galactosidase. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant ß-galactosidase proteins. In this study, we performed genotype analyses of patients with ß-galactosidase deficiency and identified 46 mutation alleles including 9 novel mutations. We then examined the NOEV effect on mutant ß-galactosidase proteins by using six strains of patient-derived skin fibroblast. We also performed mutagenesis to identify ß-galactosidase mutants that were responsive to NOEV and found that 22 out of 94 mutants were responsive. Computational structural analysis revealed the mode of interaction between human ß-galactosidase and NOEV. Moreover, we confirmed that NOEV reduced G(M1) accumulation and ameliorated the impairments of lipid trafficking and protein degradation in ß-galactosidase deficient cells. These results provided further evidence to NOEV as a promising chaperone compound for ß-galactosidase deficiency.
Asunto(s)
Fibroblastos/efectos de los fármacos , Gangliosidosis GM1/tratamiento farmacológico , Hexosaminas/farmacología , beta-Galactosidasa/química , beta-Galactosidasa/metabolismo , Animales , Células Cultivadas , Estabilidad de Enzimas , Fibroblastos/enzimología , Gangliosidosis GM1/enzimología , Expresión Génica , Vectores Genéticos , Hexosaminas/química , Hexosaminas/uso terapéutico , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucopolisacaridosis IV/genética , Mutación Missense/genética , Estructura Terciaria de Proteína , Relación Estructura-Actividad , beta-Galactosidasa/genéticaRESUMEN
We have proposed a chemical chaperone therapy for lysosomal diseases, based on a paradoxical phenomenon that an exogenous competitive inhibitor of low molecular weight stabilizes the target mutant molecule and restores its catalytic activity as a molecular chaperone intracellularly. After Fabry disease experiments, we investigated a new synthetic chaperone compound N-octyl-4-epi-beta-valienamine (NOEV) in a GM1-gangliosidosis model mice. Orally administered NOEV entered the brain through the blood-brain barrier, enhanced beta-galactosidase activity, reduced the substrate storage, and clinically improved neurological deterioration. We hope that chemical chaperone therapy will prove useful for some patients with GM1-gangliosidosis and potentially other lysosomal storage diseases with central nervous system involvement.
Asunto(s)
Gangliosidosis GM1/tratamiento farmacológico , Hexosaminas/uso terapéutico , Chaperonas Moleculares/uso terapéutico , Animales , Fibroblastos/enzimología , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/genética , Hexosaminas/química , Humanos , Ratones , Chaperonas Moleculares/químicaRESUMEN
Certain low-molecular-weight substrate analogs act both as in vitro competitive inhibitors of lysosomal hydrolases and as intracellular enhancers (chemical chaperones) by stabilization of mutant proteins. In this study, we performed oral administration of a chaperone compound N-octyl-4-epi-beta-valienamine to G(M1)-gangliosidosis model mice expressing R201C mutant human beta-galactosidase. A newly developed neurological scoring system was used for clinical assessment. N-Octyl-4-epi-beta-valienamine was delivered rapidly to the brain, increased beta-galactosidase activity, decreased ganglioside G(M1), and prevented neurological deterioration within a few months. No adverse effect was observed during this experiment. N-Octyl-4-epi-beta-valienamine will be useful for chemical chaperone therapy of human G(M1)-gangliosidosis.
Asunto(s)
Gangliosidosis GM1/tratamiento farmacológico , Gangliosidosis GM1/fisiopatología , Hexosaminas/uso terapéutico , Chaperonas Moleculares/uso terapéutico , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/fisiopatología , Animales , Encéfalo/metabolismo , Gangliosidosis GM1/metabolismo , Hexosaminas/farmacocinética , Humanos , Inmunohistoquímica , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Chaperonas Moleculares/farmacocinética , Mutación , Sistema Nervioso/metabolismo , Concentración Osmolar , Distribución Tisular , beta-Galactosidasa/deficiencia , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
This article reviews synthesis and structures of carbaglycosylamines, a group of carbocyclic sugar analogues. Some unsaturated derivatives are known to be potent glycosidase inhibitors. Among them, N-octyl-4-epi-beta-valienamine as a lysosomal beta-galactosidase inhibitor is currently undergoing a new molecular therapeutic trial (chemical chaperone therapy) for control of the human beta-galactosidase deficiency disorder, G(M1)-gangliosidosis.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Animales , Ciclohexenos/química , Ciclohexenos/farmacología , Ciclohexenos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Gangliosidosis GM1/tratamiento farmacológico , Hexosaminas/química , Hexosaminas/farmacología , Hexosaminas/uso terapéutico , Humanos , Ratones , Chaperonas Moleculares/química , Chaperonas Moleculares/farmacología , Chaperonas Moleculares/uso terapéutico , Relación Estructura-ActividadRESUMEN
A new study by Galeano and colleagues in this issue of the JCI reports the first glomerular disease caused by a genetic defect in sialic acid biosynthesis (see the related article beginning on page 1585). Mice that harbor mutations in the Gne/Mnk gene produce lower amounts of sialic acid, suffer from hematuria, proteinuria, and structural defects in the glomerulus and die within days after birth. Remarkably, the lesion can be reversed through dietary addition of N-acetylmannosamine, a sialic acid precursor, raising the intriguing possibility that this approach might have therapeutic benefit in patients with glomerular disease.
Asunto(s)
Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Ácido N-Acetilneuramínico/biosíntesis , Animales , Modelos Animales de Enfermedad , Femenino , Hexosaminas/uso terapéutico , Humanos , Enfermedades Renales/tratamiento farmacológico , Glomérulos Renales/metabolismo , Ratones , Ratones Mutantes , Modelos Biológicos , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , EmbarazoRESUMEN
Mutations in the key enzyme of sialic acid biosynthesis, uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (Gne(M712T/M712T)) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in Gne(M712T/M712T) muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the Gne(M712T/M712T) pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities. These findings establish this Gne(M712T/M712T) knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.
Asunto(s)
Hexosaminas/uso terapéutico , Enfermedades Renales/genética , Enfermedades Renales/metabolismo , Ácido N-Acetilneuramínico/biosíntesis , Proteinuria/genética , Proteinuria/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Enfermedades Renales/tratamiento farmacológico , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Microscopía Electrónica , Modelos Biológicos , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Embarazo , Proteinuria/tratamiento farmacológicoRESUMEN
We propose a new molecular therapeutic approach to lysosomal diseases with severe neurological manifestations. Some low-molecular-weight compounds, acting as competitive inhibitors of a lysosomal enzyme in vitro, were found to stabilize and restore catalytic activities of the enzyme molecule as a molecular chaperone. We started this trial first in Fabry disease (generalized vasculopathy) using galactose and 1-deoxygalactonojirimycin, and then in beta-galactosidase deficiency disorders (beta-galactosidosis) with generalized neurosomatic and/or systemic skeletal manifestations (GM(1)-gangliosidosis and Morquio B disease), using a newly developed chemical compound N-octyl-4-epi-beta-valienamine (NOEV). Administration of this chaperone compound resulted in elevation of intracellular enzyme activity in cultured fibroblasts from patients and genetically engineered model mice. In addition, substrate storage was improved after NOEV had been transported into the brain tissue via the blood-brain barrier. We hope this new approach (chemical chaperone therapy) will be useful for certain patients with beta-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Gangliosidosis GM1/tratamiento farmacológico , Hexosaminas/farmacología , Chaperonas Moleculares/metabolismo , beta-Galactosidasa/deficiencia , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Gangliósido G(M1)/metabolismo , Gangliosidosis GM1/metabolismo , Hexosaminas/uso terapéutico , Humanos , Ratones , Ratones Transgénicos , Mutación , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
We performed screening of beta-galactosidase-deficient fibroblasts for possible chemical chaperone therapy using N-octyl-4-epi-beta-valienamine (NOEV) in patients with GM1-gangliosidosis and Morquio B disease (beta-galactosidosis). Fibroblasts were cultured with NOEV for 4 days and beta-galactosidase activity was measured. Mutation analysis was performed simultaneously. Two separate criteria were set for evaluation of the chaperone effect: a relative increase of enzyme activity (more than 3-fold), and an increase up to more than 10% normal enzyme activity. Among the 50 fibroblast strains tested, more than 3-fold increase was achieved in 17 cell strains (34%), and more than 10% normal activity in 10 (20%). Both criteria were satisfied in 6 (12%), and either of them in 21 (42%). Juvenile GM1-gangliosidosis was most responsive, and then infantile GM1-gangliosidosis. This enhancement was mutation-specific. We estimate that the NOEV chaperone therapy will be effective in 20-40% of the patients, mainly in juvenile and infantile GM1-gangliosidosis patients. A molecular design may produce mutation-specific chaperone compounds for the other disease phenotypes. This cellular screening will be useful for identification of human patients with beta-galactosidase deficiency for chaperone therapy to be started in the near future.
Asunto(s)
Fibroblastos/efectos de los fármacos , Gangliosidosis GM1/patología , Hexosaminas/farmacología , Chaperonas Moleculares/farmacología , Mucopolisacaridosis IV/patología , Arginina/genética , Células Cultivadas , Ciclohexenos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Gangliosidosis GM1/genética , Genotipo , Glutamina/genética , Hexosaminas/uso terapéutico , Humanos , Chaperonas Moleculares/uso terapéutico , Mucopolisacaridosis IV/genética , Mutación , Fenotipo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The effects of glucosamine hydrochloride, glucosamine sulfate, N-acetylglucosamine, glucosamine pentaacetate, and combinations of glucosamine hydrochloride and N-acetylglucosamine with diclofenac sodium on the endogenous glucosamine (aminosugar) content in the blood and kidney of rats with experimental nephropathy have been studied. The development of inflammatory and destructive processes in kidneys is accompanied by an increase in the aminosugar content in the blood serum and the corresponding decrease in renal tissues. This fact can be used as a nonspecific diagnostic criterion for the level of kidney damage and for the efficacy of pharmacotherapy. Normalization of these indices in the case of successful therapy was observed, which was most pronounced upon the administration of glucosamine hydrochloride, N-acetylglucosamine, and their combinations with diclofenac sodium.
Asunto(s)
Acetilglucosamina/sangre , Glomerulonefritis Membranosa/tratamiento farmacológico , Glucosamina/análogos & derivados , Glucosamina/uso terapéutico , Hexosaminas/uso terapéutico , Animales , Biomarcadores/sangre , Diclofenaco/uso terapéutico , Modelos Animales de Enfermedad , Quimioterapia Combinada , RatasRESUMEN
OBJECTIVE: To compare the inhibitory effects of glucosamine and mannosamine on articular cartilage degradation and the effects on chondrocyte viability in vitro. SAMPLE POPULATION: Bovine articular cartilage explants. PROCEDURES: Explants were cultured in commercial medium for 48 hours. Cartilage was exposed to medium containing 10% fetal bovine serum, 10 microg of lipopolysaccharide/mL, and 0.5, 1.0, 2.5, 5.0, and 10.0 mg of glucosamine or mannosamine/mL for 24 hours. Nitric oxide (NO) production (nitrite concentration) and proteoglycan (PG) release (PG concentration) in media were measured. Cartilage extracts were analyzed via zymography to detect gelatinolytic activity. At the end of the experiment, explants were assessed for chondrocyte viability. RESULTS: Addition of lipopolysaccharide resulted in increased NO production and PG release, but no increase in gelatinolytic activity, compared with controls. Glucosamine and mannosamine at concentrations as low as 0.5 mg/mL inhibited NO production. Glucosamine inhibited PG release at a minimum concentration of 1.0 mg/mL, whereas mannosamine inhibited PG release at a concentration of 0.5 mg/mL. Concentrations of glucosamine < or = 5.0 mg/mL did not adversely affect chondrocyte viability; however, at a concentration of 10.0 mg/mL, cell death was evident. Mannosamine had a toxic effect at a concentration of 5.0 mg/mL and was associated with pronounced chondrocyte death at a concentration of 10.0 mg/mL. CONCLUSIONS AND CLINICAL RELEVANCE: Glucosamine and mannosamine inhibit selected indices of bovine articular cartilage degradation at concentrations that do not affect chondrocyte viability. The potential for cytotoxic effects at higher concentrations underscores the importance of establishing appropriate dosage regimens for these aminomonosaccharides.
Asunto(s)
Cartílago/efectos de los fármacos , Enfermedades de los Bovinos/fisiopatología , Glucosamina/farmacología , Hexosaminas/farmacología , Osteoartritis/veterinaria , Animales , Bovinos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucosamina/uso terapéutico , Hexosaminas/uso terapéutico , Técnicas In Vitro , Lipopolisacáridos , Metaloproteinasas de la Matriz/metabolismo , Óxido Nítrico/metabolismo , Osteoartritis/tratamiento farmacológico , Osteoartritis/fisiopatología , Proteoglicanos/metabolismoRESUMEN
We synthesized a galactose derivative, N-octyl-4-epi-beta-valienamine (NOEV), for a molecular therapy (chemical chaperone therapy) of a human neurogenetic disease, beta-galactosidosis (GM1-gangliosidosis and Morquio B disease). It is a potent inhibitor of lysosomal beta-galactosidase in vitro. Addition of NOEV in the culture medium restored mutant enzyme activity in cultured human or murine fibroblasts at low intracellular concentrations, resulting in a marked decrease of intracellular substrate storage. Short-term oral administration of NOEV to a model mouse of juvenile GM1-gangliosidosis, expressing a mutant enzyme protein R201C, resulted in significant enhancement of the enzyme activity in the brain and other tissues. Immunohistochemical stain revealed a decrease in the amount of GM1 and GA1 in neuronal cells in the fronto-temporal cerebral cortex and brainstem. However, mass biochemical analysis did not show the substrate reduction observed histochemically in these limited areas in the brain probably because of the brief duration of this investigation. Chemical chaperone therapy may be useful for certain patients with beta-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.