Discovery of K-Ras inhibitors for the treatment of cancer

Abstract

Ras is a small GTPase that functions as a molecular switch, cycling between inactive (GDP-bound) and active (GTP-bound) states. Mutations in Ras fix the protein in the active state and endow tumor cells with the ability to proliferate, evade apoptosis and metastasis. Indeed, aberrant K-Ras signaling plays a role in 30% of all human cancers, with the highest incidence of activating mutations found in pancreatic (70-90%), colon (30-50%), and lung (20-30%) carcinomas. Thus, Ras inhibition represents an attractive therapeutic strategy for many cancers. However, Ras activation and signaling is accomplished primarily through protein-protein interactions. Such protein interfaces typically lack well-defined binding pockets and have been difficult to target with small molecules. My goal during my graduate studies was to discover potent Ras inhibitors using state-of-the-art methodologies such as fragment-based methods and structure-based design. To achieve this goal, I started by screening a fragment library of 11,000 compounds using NMR in which molecules were identified that bind to K-Ras. Based on the crystal structures of protein-ligand complexes, these compounds were found to bind to a hydrophobic pocket that is absent in ligand-free K-Ras. Using structure-based design, we synthesized analogs of the fragment hits with improved binding affinity as well as functional activity in a SOS-catalyzed nucleotide exchange assay. To further optimize the inhibitors, a second-site screen was conducted using a cysteine tethering strategy to block the first pocket. Using this approach, fragments that bind in a distinct pocket were identified. In addition, small molecules were also found that bind to the Ras/SOS complex and inhibit Ras signaling downstream, which can be used as an alternative approach to target K-Ras. This work provides an important starting point and a template for the rational design of more potent chemical probes for elucidating new insights into Ras signaling and for discovering Ras inhibitors for the treatment of Ras-driven cancers.