Small Molecules as Molecular Probes

小分子作为分子探针

• 批准号: 7593531
• 负责人: Kenneth Kirk
• 金额: $ 31.39万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593531
• 关键词: Acids; Active Sites; Acylation; Amides; Amino Acids; Antibiotic Resistance; Antibiotics; Area; Arsenic; Azides; Bacteria; Benzene; Binding; Binding Proteins; Biological; Biological Assay; Biological Process; Biology; Biotin; Breast Cancer Cell; Cancer cell line; Carbon; Cell model; Chemicals; Chemistry; Chloride Ion; Chlorides; Cleaved cell; Codon Nucleotides; Communities; Coupling; Cysteine; Development; Enzymatic Biochemistry; Enzymes; Escherichia coli; Esters; Future; Glucosamine; Glycosides; Guanosine Triphosphate; Hydrolysis; In Vitro; Link; Lithium; MCF7 cell; Modification; Molecular Probes; Object Attachment; Peptides; Phenotype; Plant Resins; Polyacrylamide Gel Electrophoresis; Positioning Attribute; Post-Translational Protein Processing; Property; Proteins; Pyruvate Kinase; Reaction; Reagent; Research; Research Design; Research Project Grants; Ribosomal RNA; Serine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptavidin; Succinic Acid; Succinic Acids; Sulfhydryl Compounds; Threonine; Tin; Transferase; Tubulin; UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; Western Blotting; Work; X-Ray Crystallography; analog; aspartic semialdehyde; base; carbene; design; enolate; epimerization; inhibitor/antagonist; molecular modeling; oxophenylarsine; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; phosphonate; polymerization; ribosomal protein S12; small molecule; tool

Our group continues to develop new mechanistic probes using a rational approach to compound development. Beta-Methylthioaspartic Acid: Beta-methylthioaspartic acid occurs at position 88 in E. coli ribosomal protein S12, a position that is a mutational hotspot resulting in both antibiotic-resistant and antibiotic-sensitive phenotypes. In bacteria, S12 binds to 16S rRNA in regions associated with the fidelity of codon recognition. This posttranslational modification is highly conserved phylogenetically and thus should be both structurally and functionally important. Research designed to determine the biological function of beta-methylthioaspartic will involve elucidation of the enzymology of this modification. Critical to this work is having available synthetic beta-methylaspartic acid as well as derivatives designed for peptide incorporation. Our contribution to this project involves carrying out the synthesis of these amino acid derivatives. The key installation of the beta-methylthio functionality was accomplished by reaction of the lithium enolate of 2-benzyloxycarbonylamino-succinic acid 1-methyl ester 4-tert-butyl ester with 1-methyldisulfanyl-2,4-dinitro-benzene. Acid hydrolysis cleaved the three protecting groups to give the target compound as a mixture of diastereomers. In order to prepare orthogonally protected material, the same sequence was carried out using the 1-benzyl ester. Careful chromatographic separation provides the orthogonally protected compound. Attempts to selectively remove the benzyl ester to provide the free acid suitable for peptide coupling have as yet been unsuccessful but are continuing. Alternate orthogonal protecting groups will be explored if necessary. Biotin conjugate of 4-aminophenylarsenoxide. An arsenic-biotin conjugate was synthesized by coupling the pentafluorophenol ester of biotin with 4-aminophenylarsenoxide. Previous studies suggest that phenylarsenoxide reacts with closely spaced cysteine (Cys) residues of proteins with high Cys content and accessible sulfhydryl (SH) groups. The biotin conjugate prepared by our group was used to examine these interactions. In a collaborative project, a human breast cancer cell line MCF-7 was examined as a cellular model to explore arsenic-binding proteins and the mechanism of binding. Arsenic-binding proteins were eluted with streptavidin resin from arsenic-biotin treated MCF-7 cells, separated by polyacrylamide gel electrophoresis, and identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Arsenic-binding properties of two of these proteins, -tubulin and pyruvate kinase M2 (PKM2), were studied further in vitro and the biological consequences of this binding was evaluated. Binding assay with Western blotting confirmed binding of -tubulin and PKM2 by arsenic in a concentration-dependent manner. Arsenic binding inhibited tubulin polymerization, but surprisingly had no effect on PKM2 activity. Molecular modeling showed that binding of Cys12 alone or vicinal Cys residues (Cys12 and Cys213) of -tubulin by arsenic blocked the active site for access of GTP, which is necessary for tubulin polymerization. In contrast, all Cys residues of PKM2 were far away from the active site of the enzyme. In summary, this study confirmed -tubulin and PKM2 as arsenic-binding proteins in MCF-7 cells. Functional consequence of such binding may depend on whether arsenic binding causes conformational changes or blocks active sites of target proteins. Future synthetic work will include acylation of 4-aminophenylarsenoxide with propyonyl chloride to afford the propyonyl amide. This will be used for click chemistry with biotin azide, either before or after interaction with cellular protein. Analogues of UDP-Glc-NAc: potential inhibitors of O-linked GlcNAc transferase (OTG). We have prepared the phosphonate analogue of GlcNAc from a key C-allyl glycoside of GlcNAc. The phosphonate proved to be devoid of activity as an inhibitor of OTG transferase. This was surprising, so crystals of key intermediates were prepared. X-ray crystallography ruled out epimerization during the synthesis, confirming the alpha-configuration of the final product. The lack of activity provides further evidence that OTG has strict structural requirements in substrate recognition. Inhibitors of O-GlcNAcAse. The products of OGT-catalyzed transfer of GlcNAc are O-linked conjugates with serine and threonine residues in proteins. We are continuing the syntheses of amino acid and peptide beta-C-linked conjugates of UDP-Glc-NAc. Coupling of a tributyl tin derivative of N,N-diactyl-tribenzyl glucosamine with N-t-Boc methyl aspartate semialdehyde constitutes the key carbon-carbon bond forming step. Subsequent functionalization will include deoxygenation or fluorodeoxygenation to produce methylene, fluoromethylene and difluoromethylene isosteres of O-linked GlcNAc conjugates.

我们的团队继续使用合理的化合物开发方法来开发新的机械探针。 β-甲硫基天冬氨酸： β-甲硫基天冬氨酸出现在大肠杆菌核糖体蛋白 S12 的第 88 位，该位置是导致抗生素耐药和抗生素敏感表型的突变热点。在细菌中，S12 在与密码子识别保真度相关的区域与 16S rRNA 结合。这种翻译后修饰在系统发育上高度保守，因此在结构和功能上都应该很重要。旨在确定 β-甲硫基天冬氨酸生物学功能的研究将涉及阐明这种修饰的酶学。这项工作的关键是拥有可用的合成 β-甲基天冬氨酸以及设计用于肽掺入的衍生物。我们对这个项目的贡献包括进行这些氨基酸衍生物的合成。 β-甲硫基官能团的关键安装是通过2-苄氧基羰基氨基-琥珀酸1-甲酯4-叔丁酯的烯醇锂与1-甲基二硫基-2,4-二硝基-苯的反应完成的。酸水解裂解三个保护基团，得到目标化合物，为非对映异构体的混合物。为了制备正交保护的材料，使用1-苄酯进行相同的顺序。仔细的色谱分离提供了正交保护的化合物。选择性除去苄酯以提供适合肽偶联的游离酸的尝试尚未成功，但仍在继续。如有必要，将探索替代的正交保护基团。 4-氨基苯氧化砷的生物素缀合物。 通过生物素的五氟苯酚酯与4-氨基苯氧化砷的偶联合成了砷-生物素缀合物。先前的研究表明，氧化苯砷与具有高 Cys 含量和可接近的巯基 (SH) 基团的蛋白质中间隔紧密的半胱氨酸 (Cys) 残基发生反应。我们小组制备的生物素缀合物用于检查这些相互作用。在一个合作项目中，将人类乳腺癌细胞系 MCF-7 作为细胞模型进行检查，以探索砷结合蛋白及其结合机制。用链霉亲和素树脂从砷生物素处理的 MCF-7 细胞中洗脱砷结合蛋白，通过聚丙烯酰胺凝胶电泳分离，并通过基质辅助激光解吸电离质谱 (MALDI-MS) 进行鉴定。在体外进一步研究了其中两种蛋白质 - 微管蛋白和丙酮酸激酶 M2 (PKM2) 的砷结合特性，并评估了这种结合的生物学后果。 Western blotting 结合测定证实砷以浓度依赖性方式结合β-微管蛋白和PKM2。砷结合抑制微管蛋白聚合，但令人惊讶的是对 PKM2 活性没有影响。 分子模型表明，砷单独结合 Cys12 或 β-微管蛋白的邻近 Cys 残基（Cys12 和 Cys213）会阻断 GTP 进入的活性位点，而 GTP 是微管蛋白聚合所必需的。相反，PKM2 的所有 Cys 残基都远离酶的活性位点。总之，本研究证实β微管蛋白和PKM2是MCF-7细胞中的砷结合蛋白。这种结合的功能结果可能取决于砷结合是否引起构象变化或阻断靶蛋白的活性位点。 未来的合成工作将包括用丙酰氯酰化 4-氨基苯氧化砷以得到丙酰酰胺。这将用于在与细胞蛋白质相互作用之前或之后与生物素叠氮化物进行点击化学。 UDP-Glc-NAc 类似物：O-连接 GlcNAc 转移酶 (OTG) 的潜在抑制剂。 我们从 GlcNAc 的关键 C-烯丙基糖苷制备了 GlcNAc 的膦酸酯类似物。事实证明，该膦酸酯缺乏作为 OTG 转移酶抑制剂的活性。这是令人惊讶的，因此制备了关键中间体的晶体。 X 射线晶体学排除了合成过程中的差向异构化，证实了最终产品的 α 构型。活性的缺乏进一步证明OTG在底物识别中具有严格的结构要求。 O-GlcNAcAse 抑制剂。 OGT 催化 GlcNAc 转移的产物是与蛋白质中丝氨酸和苏氨酸残基的 O 连接缀合物。我们正在继续合成 UDP-Glc-NAc 的氨基酸和肽 β-C 连接缀合物。 N,N-二酰基-三苄基葡糖胺的三丁基锡衍生物与N-t-Boc甲基天冬氨酸半醛的偶联构成了关键的碳-碳键形成步骤。随后的官能化将包括脱氧或氟脱氧以产生O-连接的GlcNAc缀合物的亚甲基、氟亚甲基和二氟亚甲基电子等排体。