孤儿细胞毒素是一种机制未知或模糊的小分子。揭示这些化合物的机制可能导致有用的生物研究工具和新的治疗方法。在特定情况下，DNA错配修复缺陷结肠癌细胞系HCT116已被用作前向基因筛选中的工具，用于鉴定耐药突变，最终导致靶标的确定。为了扩大这种方法的应用性，我们构建了具有可诱导错配修复缺陷的癌细胞系，从而提供了对突变的时间控制。通过在具有低或高突变率的细胞中筛选抗药表型，我们增加了鉴定抵抗突变的特异性和敏感性。利用这种可诱导突变的系统，我们确定了多个孤儿细胞毒素的靶标，包括天然产物和高通量筛选中出现的化合物，从而为未来的机制研究提供了强大的工具。版权所有©2023爱思唯尔有限公司。保留所有权利。

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.Copyright © 2023 Elsevier Ltd. All rights reserved.