Andreotti Giuseppina, Monticelli Maria, Cubellis Maria Vittoria
Istituto di Chimica Biomolecolare -CNR, Pozzuoli, Italy.
Dipartimento di Biologia, Università Federico II, Napoli, Italy.
Drug Test Anal. 2015 Sep;7(9):831-4. doi: 10.1002/dta.1798. Epub 2015 Apr 5.
Thermal shift assay can be used for the high-throughput screening of pharmacological chaperones. These drugs are small molecules that bind a mutant protein and stabilize it. We demonstrated the robustness, reproducibility and versatility of the method using two molecules that are in clinical trial for Fabry or Pompe disease, Deoxygalactonojirimycin and N-Butyldeoxynojirimycin, and their target enzymes, lysosomal alpha-galactosidaseA and alpha-glucosidase, as test cases. We assessed the influence of solvents and of scanning rate on the measures. We showed that a value that is equivalent to the melting temperature can be obtained by the first derivatives of raw data. We discuss the advantages of the method and the precaution to be taken in running the experiments.
热位移分析可用于药物伴侣分子的高通量筛选。这些药物是与突变蛋白结合并使其稳定的小分子。我们以两种正在进行法布里病或庞贝病临床试验的分子——脱氧半乳糖野尻霉素和N - 丁基脱氧野尻霉素，以及它们的靶酶溶酶体α - 半乳糖苷酶A和α - 葡萄糖苷酶作为测试案例，证明了该方法的稳健性、可重复性和通用性。我们评估了溶剂和扫描速率对测量结果的影响。我们表明，通过原始数据的一阶导数可以获得与熔解温度等效的值。我们讨论了该方法的优点以及进行实验时应采取的预防措施。
