Targeting K-Ras in CRC

CRC的有效治疗仍然是一个关键的未满足的医疗需求。CRC最重要的驱动因素之一是K-Ras，它在40%的人类CRC中发生突变。事实上，Ras蛋白作为分子开关发挥重要作用，控制细胞增殖、生长、分化和凋亡。Ras信号通路通过激活突变、过表达或上游激活的失调在许多人类肿瘤中是常见的。因此，K-Ras代表了CRC和各种其他癌症的高度有效和重要的靶标。尽管已经尝试了许多不同的方法来靶向K-Ras信号通路中的上游或下游蛋白，但靶向K-Ras本身将是理想的。为了测试K-Ras是否可以用小分子药物化，我们克隆、表达、同位素标记和纯化K-Ras（G12 D），并使用NMR对GDP和GTP结合的K-Ras进行基于片段的筛选。在这些筛选中，我们鉴定了超过100种与K-Ras结合的小分子，这表明K-Ras可能是一种可药物化的靶标。此外，我们还获得了与筛选中识别的命中物结合的K-Ras的几种X射线晶体结构，我们目前正在使用这些晶体结构来指导K-Ras抑制剂的合成。我们建议发现与K-Ras有效结合，抑制其功能，并且对体内CRC肿瘤模型高度有效的小分子，目的是发现适合进入CRC临床试验的化合物。我们假设K-Ras抑制剂将非常有效地治疗CRC患者。 目标1。从我们基于片段的筛选和最近使用基于迭代结构的设计确定的K-RAS/抑制剂复合物的三维结构中产生紧密结合K-RAS（nM）的先导化合物。 目标二。优化先导K-RAS结合剂在生化测定中的K-RAS抑制作用及其对结肠癌细胞的细胞活性。 目标3。优先考虑具有优良药物特性的K-RAS抑制剂，这些抑制剂在体内有效，使用新的干细胞衍生的他莫昔芬诱导型Cre驱动（Lrig 1-CreERT 2）小鼠激活小鼠结肠中的突变KRAS。在资助期结束前选择一种适合结肠癌临床试验的化合物。

Effective therapies for CRC still remain a critical unmet medical need. One of the most important drivers of CRC is K-Ras, which is mutated in 40% of human CRCs. Indeed, Ras proteins play essential roles as molecular switches, controlling cell proliferation, growth, differentiation, and apoptosis. Deregulation of the Ras signaling pathway by activating mutations, overexpression, or upstream activation is common in many human tumors. Thus, K-Ras represents a highly validated and important target for CRC and a wide variety of other cancers. Although a number of different approaches have been attempted to target upstream or downstream proteins in the K-Ras signaling pathway, it would be ideal to target K-Ras itself However, KRas is considered to be a poor drug target. In order to test whether K-Ras could be druggable with a small molecule, we cloned, expressed, isotopically labeled, and purified K-Ras (G12D) and conducted a fragment-based screen on GDP- and GTP-bound K-Ras using NMR. In these screens we identified over 100 small molecules that bind to K-Ras, suggesting that K-Ras may be a druggable target. In addition, we obtained several X-ray crystal structures of K-Ras bound to the hits identified in the screen, which we are currently using to guide the synthesis of K-Ras inhibitors. We propose to discover small molecules that potently bind to K-Ras, inhibit its functions, and are highly efficacious against in vivo CRC tumor models with the goal of discovering a compound that is suitable for entry into a CRC clinical trial. We hypothesize that a K-Ras inhibitor will be highly effective for treating CRC patients. Aim 1. Generate lead compounds that bind K-RAS tightly (nM) from our fragment-based screens and recently determined three-dimensional structures of K-RAS/inhibitor complexes using iterative structure-based design. Aim 2. Optimize lead K-RAS binders for their K-RAS inhibitory effects in biochemical assays and their cell-based activities against colon cancer cells. Aim 3. Prioritize K-RAS inhibitors with excellent pharmaceutical properties that are efficacious in vivo using a new stem cell-derived, tamoxifen-inducible Cre driver (Lrig1-CreERT2) mouse to activate mutant KRAS in the mouse colon. Select a compound that is suitable for a clinical trial in colon cancer by the end of the granting period.