Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
Cell Death Dis. 2013 May 2;4(5):e619. doi: 10.1038/cddis.2013.143.
Development of drugs targeting Bcl-2 relatives and caspases, for treating diseases including cancer and inflammatory disorders, often involves measuring interactions with recombinant target molecules, and/or monitoring cancer cell killing in vitro. Here, we present yeast-based methods for evaluating drug-mediated inhibition of Bcl-2 relatives or caspases. Active Bax and caspases kill Saccharomyces cerevisiae, and pro-survival Bcl-2 proteins can inhibit Bax-induced yeast death. By measuring the growth or adenosine triphosphate content of transformants co-expressing Bax with pro-survival Bcl-2 relatives, we found that the Bcl-2 antagonist drugs ABT-737 or ABT-263 abolished Bcl-2 or Bcl-xL function and reduced Bcl-w activity, but failed to inhibit Mcl-1, A1 or the poxvirus orthologs DPV022 and SPPV14. Using this technique, we also demonstrated that adenoviral E1B19K was resistant to these agents. The caspase inhibitor Q-VD-OPh suppressed yeast death induced by caspases 1 and 3. Yeast engineered to express human apoptotic regulators enable simple, automatable assessment of the activity and specificity of candidate drugs targeting Bcl-2 relatives or caspases.
开发针对 Bcl-2 相关蛋白和半胱天冬酶的药物，用于治疗癌症和炎症性疾病等疾病，通常涉及测量与重组靶分子的相互作用，以及/或监测体外癌细胞杀伤。在这里，我们提出了基于酵母的方法来评估药物介导的 Bcl-2 相关蛋白或半胱天冬酶的抑制作用。活性 Bax 和半胱天冬酶可杀死酿酒酵母，而存活的 Bcl-2 蛋白可以抑制 Bax 诱导的酵母死亡。通过测量与存活的 Bcl-2 相关蛋白共表达 Bax 的转化体的生长或三磷酸腺苷含量，我们发现 Bcl-2 拮抗剂药物 ABT-737 或 ABT-263 消除了 Bcl-2 或 Bcl-xL 的功能，并降低了 Bcl-w 的活性，但未能抑制 Mcl-1、A1 或痘病毒直系同源物 DPV022 和 SPPV14。使用这项技术，我们还证明了腺病毒 E1B19K 对这些药物具有抗性。半胱天冬酶抑制剂 Q-VD-OPh 抑制了半胱天冬酶 1 和 3 诱导的酵母死亡。表达人类凋亡调节剂的酵母可用于简单、自动化评估针对 Bcl-2 相关蛋白或半胱天冬酶的候选药物的活性和特异性。
