Targeting K-Ras in CRC

对结直肠癌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，在40%的人类CRC中发生突变。事实上，Ras蛋白作为控制细胞增殖、生长、分化和凋亡的分子开关发挥着重要作用。通过激活突变、过表达或上游激活来解除Ras信号通路的管制在许多人类肿瘤中很常见。因此，K-Ras代表了CRC和各种其他癌症的高度验证和重要靶点。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，但以K-Ras本身为靶点是理想的。然而，KRas被认为是一个较差的药物靶点。为了验证K-Ras能否被小分子药物化，我们克隆、表达、同位素标记和纯化了K-Ras (G12D)，并利用NMR对GDP和gtp结合的K-Ras进行了片段筛选。在这些筛选中，我们发现了100多个与K-Ras结合的小分子，这表明K-Ras可能是一个可药物治疗的靶标。此外，我们获得了几个K-Ras结合到屏幕中识别的hit的x射线晶体结构，我们目前正在使用它来指导K-Ras抑制剂的合成。我们建议发现能与K-Ras有效结合、抑制其功能并对体内CRC肿瘤模型高效的小分子，目的是发现一种适合进入CRC临床试验的化合物。我们假设一种K-Ras抑制剂将对治疗结直肠癌患者非常有效。

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.