1 Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund University, Lund, Sweden.
2 Chemical Biology and Therapeutics group, Department of Experimental Medical Science, Lund University, Lund, Sweden.
SLAS Discov. 2019 Mar;24(3):304-313. doi: 10.1177/2472555218823531.
Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.
Diamond-Blackfan 贫血（DBA）是一种由核糖体蛋白基因突变引起的骨髓衰竭综合征。发病机制尚不清楚，但涉及红系前体细胞的严重增殖减少。由于目前的 DBA 治疗方法无效且伴有严重的副作用，因此迫切需要针对特定疾病的治疗方法。我们假设可以通过表型小分子筛选揭示潜在缺陷的可用药分子途径。因此，使用来自强力霉素诱导的 DBA 小鼠模型的 c-kit+ 胎肝红系祖细胞开发了筛选测定法。向培养基中添加强力霉素可诱导表型并将增殖减少至正常水平的<10%，因此增殖的恢复可用作筛选的简单读出。在这里，我们描述了测定原理和验证微滴定板兼容测定法的努力及其在 3871 种已注释化合物的初步筛选中的应用。十种化合物显示出浓度依赖性活性，我们报告了其中一种化合物的简要后续研究。总之，我们建立了一种用于筛选可挽救 DBA 中红细胞生成的分子的强大、可扩展的测定法。
