评估酶活性的分析测定和鸟氨酸脱羧酶抑制剂的筛选

L. Tinoco, Bruno da Silva Santos, Jhones Matheus da Silva Soares, Fernanda G. Finelli
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引用次数: 1

摘要

鸟氨酸脱羧酶(ODC)催化鸟氨酸脱羧产生腐胺,这是多胺(腐胺、亚精胺和精胺)代谢的第一步,多胺是真核细胞中必不可少的生长因子。ODC作为同源二聚体具有活性,并依赖于5′-磷酸吡哆醛(PLP)作为辅因子。多胺浓度的增加与致癌作用有关。因此,人们对鉴定该途径的抑制剂作为潜在的化疗和化学预防剂非常感兴趣。哺乳动物ODC最著名的抑制剂是α-二氟甲基鸟氨酸(DFMO),这是一种高度选择性的化合物,可使Cys-360(ODC活性位点的残基)烷基化。尽管DFMO最初是为治疗癌症而开发的,但世界卫生组织建议将其与硝呋替莫联合用于治疗人类非洲锥虫病。考虑到ODC作为治疗各种类型癌症和其他传染病的有前景的靶点的重要性,选择正确的方法来筛选潜在的抑制剂有助于加速新药的发现。文献中有几种测定ODC活性的方法。其中,我们可以提到放射性标记物分析、使用辅助酶检测CO2或H2O2释放的比色分析、腐胺衍生的色谱分离、质谱和光谱技术。在这篇综述中,将描述所使用的主要分析方法,强调其优缺点,并确定最有前途的高通量筛选方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Analytical assays to evaluate enzymatic activity and screening of inhibitors for ornithine decarboxylase
Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to produce putrescine, the first step in the metabolism of polyamines (putrescine, spermidine, and spermine), which are essential growth factors in eukaryotic cells. ODC is active as a homodimer and depends on pyridoxal 5′-phosphate (PLP) as a cofactor. An increase in the concentration of polyamines has been associated with carcinogenesis. Therefore, there is much interest in identifying inhibitors of this pathway as potential chemotherapeutic and chemopreventive agents. The best-known inhibitor of mammalian ODC is α-difluoromethylornithine (DFMO), a highly selective compound that alkylates Cys-360 (a residue of the ODC active site). Although DFMO was initially developed for the treatment of cancer, the World Health Organization recommends its use in combination with nifurtimox for the treatment of human African trypanosomiasis. Considering the importance of ODC as a promising target for the treatment of various types of cancer and other infectious diseases, choosing the right method for screening potential inhibitors can help to accelerate the discovery of new drugs. Several methods for the determination of ODC activity are found in the literature. Among these, we can mention analysis with radioactive markers, colorimetric assays using auxiliary enzymes to detect CO2 or H2O2 release, chromatographic separations with putrescine derivatization, mass spectrometry, and spectroscopic techniques. In this review, the main analysis methods used will be described, highlighting their advantages and disadvantages, as well as identifying the most promising methods for high-throughput screening.
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