RESUMO
Autosomal recessive loss-of-function mutations in N-glycanase 1 (NGLY1) cause NGLY1 deficiency, the only known human disease of deglycosylation. Patients present with developmental delay, movement disorder, seizures, liver dysfunction and alacrima. NGLY1 is a conserved cytoplasmic component of the Endoplasmic Reticulum Associated Degradation (ERAD) pathway. ERAD clears misfolded proteins from the ER lumen. However, it is unclear how loss of NGLY1 function impacts ERAD and other cellular processes and results in the constellation of problems associated with NGLY1 deficiency. To understand how loss of NGLY1 contributes to disease, we developed a Drosophila model of NGLY1 deficiency. Loss of NGLY1 function resulted in developmental delay and lethality. We used RNAseq to determine which processes are misregulated in the absence of NGLY1. Transcriptome analysis showed no evidence of ER stress upon NGLY1 knockdown. However, loss of NGLY1 resulted in a strong signature of NRF1 dysfunction among downregulated genes, as evidenced by an enrichment of genes encoding proteasome components and proteins involved in oxidation-reduction. A number of transcriptome changes also suggested potential therapeutic interventions, including dysregulation of GlcNAc synthesis and upregulation of the heat shock response. We show that increasing the function of both pathways rescues lethality. Together, transcriptome analysis in a Drosophila model of NGLY1 deficiency provides insight into potential therapeutic approaches.
Assuntos
Defeitos Congênitos da Glicosilação/genética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Acetilglucosamina/biossíntese , Animais , Deficiências do Desenvolvimento/metabolismo , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Glicosilação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Convulsões/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/genéticaRESUMO
N-Glycanase deficiency, or NGLY1 deficiency, is an extremely rare human genetic disease. N-Glycanase, encoded by the gene NGLY1, is an important enzyme involved in protein deglycosylation of misfolded proteins. Deglycosylation of misfolded proteins precedes the endoplasmic reticulum (ER)-associated degradation (ERAD) process. NGLY1 patients produce little or no N-glycanase (Ngly1), and the symptoms include global developmental delay, frequent seizures, complex hyperkinetic movement disorder, difficulty in swallowing/aspiration, liver dysfunction, and a lack of tears. Unfortunately, there has not been any therapeutic option available for this rare disease so far. Recently, a proposed molecular mechanism for NGLY1 deficiency suggested that endo-ß-N-acetylglucosaminidase (ENGase) inhibitors may be promising therapeutics for NGLY1 patients. Herein, we performed structure-based virtual screening utilizing FDA-approved drug database on this ENGase target to enable repurposing of existing drugs. Several Proton Pump Inhibitors (PPIs), a series of substituted 1H-benzo [d] imidazole, and 1H-imidazo [4,5-b] pyridines, among other scaffolds, have been identified as potent ENGase inhibitors. An electrophoretic mobility shift assay was employed to assess the inhibition of ENGase activity by these PPIs. Our efforts led to the discovery of Rabeprazole Sodium as the most promising hit with an IC50 of 4.47±0.44µM. This is the first report that describes the discovery of small molecule ENGase inhibitors, which can potentially be used for the treatment of human NGLY1 deficiency.
Assuntos
Inibidores Enzimáticos/farmacologia , Doenças Genéticas Inatas/tratamento farmacológico , Inibidores da Bomba de Prótons/farmacologia , Bombas de Próton/metabolismo , Rabeprazol/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Doenças Genéticas Inatas/genética , Humanos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/antagonistas & inibidores , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Estrutura Molecular , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/deficiência , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Inibidores da Bomba de Prótons/síntese química , Inibidores da Bomba de Prótons/química , Rabeprazol/síntese química , Rabeprazol/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis. Xyloside mediated modulation of biological functions depends on the extent of priming activity and fine structures of primed GAG chains. In earlier studies, copper (Cu) catalyzed synthesis of click-xylosides and their priming activity were extensively documented. In the current study, ruthenium (Ru) mediated catalysis was employed to synthesize xylosides with a 1,5-linkage between the xylose and the triazole ring instead of a 1,4-linkage as found in Cu-catalyzed click-xyloside synthesis. Mono- and bis-click-xylosides were synthesized using each catalytic method and their glycosaminoglycan priming activity was assessed in vitro using a cellular system. Ru-catalyzed click-xylosides showed a higher priming activity as measured by incorporation of radioactive sulfate into primed glycosaminoglycan chains. This study demonstrates that altering the linkage of the aglycone to the triazole ring changes the priming activity. Computational modeling provides a molecular rationale for higher priming ability of Ru-mediated click-xylosides. Higher GAG priming activity is attributed to the formation of more stable interactions between the 1,5-linked xylosides and ß-1,4-galactosyltransferase 7 (ß4GalT7).
Assuntos
Cobre/química , Glicosaminoglicanos/química , Glicosídeos/química , Rutênio/química , Sítios de Ligação , Catálise , Química Click , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Glicosaminoglicanos/síntese química , Glicosídeos/síntese química , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de ProteínaRESUMO
Glycosaminoglycans (GAGs) play crucial roles in several biological processes including cell division, angiogenesis, anticoagulation, neurogenesis, axon guidance and growth, and viral and bacterial infections among others. The GAG cleaving hydrolases/lyases play a major role in the control of GAG structures, functions, and turn over. Dysregulation of GAG cleaving enzymes in vivo are linked to a number of human diseases including cancer, diabetes, atherosclerosis, arthritis, inflammation, and cardiovascular diseases. Several GAG cleaving enzymes are widely used for studying GAG glycobiology: heparitinases, chondroitinases, heparanases, hyaluronidases, and keratanases. Herein, we describe a method to synthesize four distinct nanometal surface energy transfer (NSET)-based gold-GAG-dye conjugates (nanosensors). Heparin, chondroitin sulfate, heparan sulfate, and hyaluronic acid are covalently linked with distinct fluorescent dyes and then immobilized on gold nanoparticles (AuNPs) to build nanosensors that serve as excellent substrates for GAG cleaving enzymes. Upon treatment of nanosensors with their respective GAG cleaving enzymes, dye-labeled oligosaccharides/disaccharides are released from AuNPs resulting in enhanced fluorescence recovery. These nanosensors have a great promise as diagnostic tools in various human pathophysiological conditions for detecting dysregulated expression of GAG cleaving enzymes and also as a sensitive analytical tool for assessing the quality control of pharmaceutical grade heparin polysaccharides that are produced in millions of small- and medium-sized animal slaughter houses worldwide.
Assuntos
Nanopartículas Metálicas , Animais , Sulfatos de Condroitina , Glicosaminoglicanos , Ouro , Heparina , Heparitina Sulfato , HumanosRESUMO
Pharmaceutical heparin's activity arises from a key high affinity and high selectivity antithrombin binding motif, which forms the basis for its use as an anticoagulant. The current problems with the supply of pig heparin raises the emphasis of understanding heparin biosynthesis so as to control and advance recombinantly expressed agent that could bypass the need for animals. Unfortunately, much remains to be understood about the generation of the antithrombin-binding motif by the key enzyme involved in its biosynthesis, 3-O-sulfotransferase-1 (3OST-1). In this work, we present a novel computational approach to understand recognition of oligosaccharide sequences by 3OST-1. Application of combinatorial virtual library screening (CVLS) algorithm on hundreds of tetrasaccharide and hexasaccharide sequences shows that 3OST-1 belongs to the growing number of proteins that recognize glycosaminoglycans with very high selectivity. It prefers very well defined pentasaccharide sequences carrying distinct groups in each of the five residues to generate the antithrombin binding motif. CVLS also identifies key residues including His271, Arg72, Arg197 and Lys173, which interact with 6-sulfate, 5-COO¯, 2-/6-sulfates and 2-sulfate at the -2, -1, +2, and +1 positions of the precursor pentasaccharide, respectively. Additionally, uncharged residues, especially Gln163 and Asn167, were also identified as playing important roles in recognition. Overall, the success of CVLS in predicting 3OST-1 recognition characteristics that help engineer selectivity lead to the expectation that recombinant enzymes could be designed to help resolve the current problems in the supply of anticoagulant heparin.
RESUMO
Heparin has been in clinical use as an anticoagulant for the last eight decades and used worldwide in more than 100 million medical procedures every year. This lifesaving drug is predominantly obtained from ~700 million pig intestines or bovine organs through millions of small and medium-sized slaughterhouses. However, the preparations from animal sources have raised many safety concerns, including the contamination of heparin with potential pathogens, proteins, and other impurities. In fact, contaminated heparin preparations caused 149 deaths in several countries, including the United States, Germany, and Japan in 2008, highlighting the need for implementing sensitive and simple analytical techniques to monitor and safeguard the heparin supply chain. The contaminant responsible for the adverse effects in 2008 was identified as oversulfated chondroitin sulfate (OSCS). We have developed a very sensitive, facile method of detecting OSCS in heparin lots using a nanosensor, a gold nanoparticle-heparin dye conjugate. The sensor is an excellent substrate for heparitinase enzyme, which cleaves the heparin polymer into smaller disaccharide fragments, and therefore facilitates recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, because OSCS inhibits the enzyme. The newly designed nanosensor can detect as low as 1 × 10-9% (w/w) OSCS, making it the most sensitive tool available to date for the detection of trace amounts of OSCS in pharmaceutical heparins. In this report, we describe a simple methodology for the preparation of nanosensor and its application in the detection of OSCS contaminants.
Assuntos
Técnicas Biossensoriais/instrumentação , Heparina/análise , Nanotecnologia/instrumentação , Fluorescência , Ouro/química , Nanopartículas Metálicas/químicaRESUMO
SecA ATPase plays a crucial role in translocation of membrane and secreted polypeptides and proteins in bacteria and therefore a perfect target for novel antimicrobial drug design. Herein, we generated QSAR models with an alignment-independent method. The optimum model obtained for the training set was statistically significant with cross-validation regression coefficient (q2) value of 0.40 and correlation coefficient (r2) value of 0.89. These results suggest that this 3D-QSAR model can be used to guide the development of new SecA inhibitors.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Modelos Estatísticos , Conformação Proteica , Relação Quantitativa Estrutura-Atividade , Canais de Translocação SEC , Proteínas SecARESUMO
The aim of the present study is to synthesize Pluronic F127-polyethylenimine-folate (PF127-PEI-FA) copolymer, construct a mixed micelle system with PF127-PEI-FA copolymer and Pluronic P123 (PP123) and to evaluate the potential of these mixed micelles as an oral drug delivery system for paclitaxel (PTX). The results of intestinal absorption revealed that the PTX-loaded micelles displayed superior permeability across intestinal barrier than free drug and PF127-PEI-FA/PP123 mixed micelles exhibited the strongest permeability across intestinal barrier. These results were also proved by the studies on cytotoxicity and cell uptake tests. The mechanism was demonstrated in connection with inhibition of the efflux mediated by intestinal P-glycoprotein (P-gp) and enhancement of the electrostatic interaction of positive micelles with the negative intestinal epithelial cells, thereby promoting the permeation across the intestinal wall. The presence of verapamil and Pluronic both improved the intestinal absorption of PTX, which further certified the effect of Pluronic on P-gp inhibition. Pharmacokinetic study demonstrated that the area under the plasma concentration-time curve (AUC(0â36 h)) of PTX-loaded micelles was three times greater than the PTX solution (dissolved in a 50/50 (vol/vol) mixture of Cremophore EL/dehydrated ethanol) (p < 0.05). In general PF127-PEI-FA/PP123 mixed micelles were proved to be potential oral drug delivery system for PTX.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Portadores de Fármacos/síntese química , Ácido Fólico/análogos & derivados , Paclitaxel/administração & dosagem , Poloxâmero/síntese química , Polietilenoimina/análogos & derivados , Animais , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/farmacologia , Varredura Diferencial de Calorimetria , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Composição de Medicamentos , Ácido Fólico/síntese química , Ácido Fólico/química , Humanos , Absorção Intestinal , Masculino , Micelas , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Paclitaxel/farmacocinética , Paclitaxel/farmacologia , Tamanho da Partícula , Poloxâmero/química , Polietilenoimina/síntese química , Polietilenoimina/química , Ratos , Ratos Wistar , Solubilidade , Propriedades de Superfície , Difração de Raios XRESUMO
Retigeric acid B (RB) has been reported to exhibit its anti-tumor activity in vitro and in vivo. Here, we found that RB significantly inhibited activity of topoisomerase IIα (Topo IIα), leading to remarkable DNA damage in prostate cancer (PCa) cells as evidenced by a strong induction of γH2AX and DNA fragmentation. Activation of ATM and ATR sequentially led to induction of phospho-Chk1/2 and phospho-Cdc25 in response to RB. Blockade of ATM/ATR signaling resulted in the attenuation of RB-induced γH2AX, and partially rescued RB-mediated cell death. RB treatment also resulted in inactivation of DNA repair proteins such as phospho-BRCA1, impairment of HR, and NHEJ repair as indicated by DNA end-joining assays. Meanwhile, a stress-responsive gene activating transcription factor 3 (ATF3) was noted for its predominant expression in response to RB-induced DNA damage. Knockdown of ATF3 inhibited the RB-induced expression changes of cell cycle- and apoptosis-related genes such as DR5, DDIT4, CDC25A, GADD45A, and partially blocked RB-mediated inhibition on cell proliferation and induction of apoptosis, suggesting the crucial involvement of ATF3 in this event. Microarray data displayed that RB caused changes of genes required for damaged-DNA binding and repair, as well as ATF3 and its target genes. Our data firstly demonstrated that RB was a novel DNA Topo II inhibitor and triggered cell death by inducing DNA damage and stress-response, suggesting a promising anticancer agent.