Pharmacological Chaperone Activity Of Glycosidase Inhibitors&Antitumor Effects Of Histone Deacetylase Inhibitors

Enzymes involve in almost all of our life process, so their activities play a vital role in maintaining the normal operation of the body. Many diseases are caused by disorder of the activity of some enzymes, so it has been widely recognized in recently years that enzymes are promising targets for drug development. So far, enzyme inhibitor drugs supply a third of drugs clinical applied, and as a mainly source of new drugs.Glycosidases catalyze the hydrolysis of the glycosidic linkage to release smaller sugars. They widely involve in digesting of food, polysaccharides and glycoconjugates metabolism, glycosidase inhibitors can inhibit the activity of glycosidase, and block the decomposition of carbohydrates. they have important therapeutic value against some glycometabolism disorder diseases, such as lysosomal storage diseases, diabetes, etc. Meanwhile, The study of the glycosidase inhibitors contribute to better understanding of the the hydrolytic mechanism and biological functions of glycosylation, revealing the pathogenesises of Alzheimer’s disease, cancer, and other major diseases. Surrounding the development of enzyme inhibitor drugs, we carried out the topic:design, screening and mechanism analysis of glycosidase inhibitors and HDAC inhibitors. These results provide a basis for the development of the targeted drugs.Gaucher disease, the highest incidence of lysosomal storage diseases, is caused by the mutations in acid β-glucocerebrosidase (GCase). The mutation of GCase leads to the earlier degradation of GCase in endoplasmic reticulum, so GCase cannot be located in Iysosome, eventually, cause the accumulation of glucosylceramide (GlcCer). Based on the mechanism of competitive enzymes inhibitors, people put forward the hypothesis of pharmacological chaperone therapy (PCT), which refers to the use of competitive inhibitors to specifically bind and potentially stabilize catalytically competent GCase variants at neutral pH in the ER, assisting with the folding of these mutant proteins and helping these proteins pass the quality control checks for trafficking to the lysosome. Due to the high substrate concentrations and low pH in the lysosome, the pharmacological chaperones (PCs) would be displaced from the active site of the enzyme, and the stored GlcCer would be degraded. According to the catalytic mechanism of GCase, we synthesized a series of glucoimidazoles. By biological activity screening, we found all of the compounds had strong inhibitory activity, high selectivity, good cell membrane permeability, and low cytotoxicity. A cell-based assay using patient-derived lymphoblasts (N370S or L444P mutation) demonstrated that the administration of these compounds can significantly increase GCase activity. Interestingly, the moderate inhibitor, compound11, which bears a3,3-dimethyl-N-phenyl-4-amide-l-butyl substituent, had the greatest effect on activity, yielding a2.1-fold increase in N370S lymphoblasts at2.5μM and a1.2-fold increase in L444P at0.5μM following a three-day incubation. Next, computer docking studies and a protease protection assay were used to elucidate the ligand-enzyme interactions responsible for the chaperone activity of11. Western blot and immuno-fluorescence assays verified the trafficking of the restored GCase to the lysosome.O-linked N-acetylglucosamine (O-GlcNAc) modification, which involes in a variety of cellular processes, is an essential posttranslational modification in metazoans. O-GlcNAcase (OGA) is the only glycosidase which is responsible for the removal of O-GlcNAc. The study found that O-GlcNAcylation of tau in the brains of alzheimer’s patients is severe insufficient. So, We wanted to improve the level of O-GlcNAcby OGA inhibitors. We designed and synthesized series of compounds. By enzyme activity screening, we got a powerful selective OGA inhibitor (Kj=5.9μM) which effectively induced more cellular hyper-O-GlcNAcylation than PUGNAc. Using piecewise expression of OGA in vitro, we obtained a specific nOGA (N r terminal fragment of OGA during apoptosis) inhibitor (Kj=48μM). These OGA inhibitors contributed to revealing the mechanism of O-GlcNAc modifications, and pathogenesises of diseases for the abnormalities of O-GlcNAc modification, in order to provide a basis for the development of the relevant drugs.HDACs (Histone deacetylases) are a kind of key enzymes which play fundamental roles in the regulation of gene expression and maintenance of chromatin structures. When the histones are in low acetylation state, which can condense chromatin, promote cell growth, differentiation and inhibit the expression of apoptosis related genes, finally cause the occurrence of tumor. Histone deacetylase inhibitors (HDACi) can reverse the hypoacetylation status of histones, thereby inducing the tumor cell differentiation and apoptosis. It has been widely recognized in recently years that HDACs are promising targets for cancer therapy. We synthesized a novel calss of HDAC inhibitors. After in vitro enzyme activity and cell toxicity screening, we discovered a compound, Z-13, which showed about one order of magnitude more potent than SAHA (suberoylanilide hydroxamic acid) in both enzymatic and cellular assays. Based on the research of antitumor mechanism of Z-13, we found that it inhibited the proliferation of tumor cell in vitro and in vivo mainly by inducing cell apoptosis and cell cycle arrest. We also want to know whether SAHA can enhance the growth inhibitory effect induced by taxol against breast cancer cell. By experiment, we found that SAHA can enhance taxol-induced cell death against human breast cancer cells, especially in taxol-resistant and multi-resistant breast cancer cells. Based on BALB/c nude mice breast cancer xenograft model, the synergetic effect was also observed in the in vivo xenograft tumor model. The combination of SAHA and taxol may have therapeutic potential in the treatment of breast cancer.