Sarver Ronald W, Rogers Joseph M, Epps Dennis E
Structural and Medicinal Chemistry, Pharmacia Corporation, Kalamazoo, MI 49001, USA.
J Biomol Screen. 2002 Feb;7(1):21-8. doi: 10.1177/108705710200700104.
We used a temperature-jump isothermal denaturation procedure with various methods of detection to evaluate the quality of putative inhibitors of MurB discovered by high-throughput screening. Three optical methods of detection-ultraviolet hyperchromicity of absorbance, fluorescence of bound dyes, and circular dichroism-as well as differential scanning calorimetry were used to dissect the effects of two chemical compounds and a natural substrate on the enzyme. The kinetics of the denaturation process and binding of the compounds detected by quenching of flavin fluorescence were used to quantitate the dose dependencies of the ligand effects. We found that the first step in the denaturation of MurB is the rapid loss of flavin from the active site and that the two chemical inhibitors appeared to destabilize the interaction of the cofactor with the enzyme but stabilize the global unfolding. The kinetics of the denaturation process as well as the loss of flavin fluorescence on binding established that both compounds had nanomolar affinities for the enzyme. We showed that coupling of the various detection methods with isothermal denaturation yields a powerful regimen to provide analytical data for assessing inhibitor specificity for a protein target.
我们采用温度跃升等温变性程序，并结合多种检测方法，来评估通过高通量筛选发现的MurB假定抑制剂的质量。使用了三种光学检测方法——吸光度的紫外增色、结合染料的荧光以及圆二色性——以及差示扫描量热法，来剖析两种化合物和一种天然底物对该酶的影响。通过黄素荧光猝灭检测到的变性过程动力学和化合物结合情况，用于定量配体效应的剂量依赖性。我们发现，MurB变性的第一步是黄素从活性位点快速丢失，并且这两种化学抑制剂似乎会破坏辅因子与酶的相互作用，但会稳定整体解折叠。变性过程的动力学以及结合时黄素荧光的丧失表明，这两种化合物对该酶都具有纳摩尔亲和力。我们表明，将各种检测方法与等温变性相结合，可产生一种强大的方案，为评估蛋白质靶点的抑制剂特异性提供分析数据。
