K-RAS is known as the most frequently mutated oncogene. However, the development of conventional K-RAS inhibitors has been extremely challenging, with a mutation-specific inhibitor reaching clinical trials only recently. Targeted proteolysis has emerged as a new modality in drug discovery to tackle undruggable targets. Our laboratory has developed a system for targeted proteolysis using peptidic high-affinity binders, called “AdPROM.” Here, we used CRISPR/Cas9 technology to knock in a GFP tag on the native K-RAS gene in A549 adenocarcinoma (A549^GFPKRAS) cells and constructed AdPROMs containing high-affinity GFP or H/K-RAS binders. Expression of GFP-targeting AdPROM in A549^GFPKRAS led to robust proteasomal degradation of endogenous GFP-K-RAS, while expression of anti-HRAS-targeting AdPROM in different cell lines resulted in the degradation of both GFP-tagged and untagged K-RAS, and untagged H-RAS. Our findings imply that endogenous RAS proteins can be targeted for proteolysis, supporting the idea of an alternative therapeutic approach to these undruggable targets.

K-RAS 被称为最常发生突变的癌基因。然而，传统 K-RAS 抑制剂的开发一直极具挑战性，一种突变特异性抑制剂直到最近才进入临床试验。靶向蛋白水解已成为药物发现的一种新模式，以解决无法成药的目标。我们的实验室开发了一种使用肽类高亲和力结合剂进行靶向蛋白水解的系统，称为“AdPROM”。在这里，我们使用 CRISPR/Cas9 技术在 A549 腺癌细胞 (A549 ^{GFPKRAS ) 的天然}K-RAS基因上敲入 GFP 标签，并构建了含有高亲和力 GFP 或 H/K-RAS 结合剂的 AdPROM。^{A549 GFPKRAS}中 GFP 靶向 AdPROM 的表达导致内源性 GFP-K-RAS 的强烈蛋白酶体降解，而抗 HRAS 靶向 AdPROM 在不同细胞系中的表达导致 GFP 标记和未标记的 K-RAS 以及未标记的 H-RAS 的降解。我们的研究结果表明，内源性 RAS 蛋白可以作为蛋白水解的靶点，这支持了对这些不可成药靶点的替代治疗方法的想法。