Chemical optimization of an orally bioavailable macrocyclic peptide KRAS inhibitors that achieve KRAS selectivity by recognizing a single amino acid difference

Macrocyclic peptides represent a promising modality for targeting intracellular proteins that are traditionally considered "undruggable" by small molecules or antibodies. Their tunable physicochemical properties allow for engineering of membrane permeability and metabolic stability, enabling oral bioavailability and intracellular engagement. A classic example is cyclosporine A, which exhibits both oral absorption and intracellular activity. To expand the drug-like potential of this class, we developed an mRNA display platform optimized for the selection of bioactive cyclic peptides with favorable pharmacokinetic profiles. This platform enables the identification of peptides capable of passive cell membrane permeation and oral absorption without the need for permeation enhancers. Using this technology, we previously reported LUNA18 (Paluratide), a mid-sized cyclic peptide that binds reversibly to GDP-bound RAS isoforms (KRAS, NRAS, HRAS), inhibiting their signaling (J. Am. Chem. Soc. 2023, 145, 16610). In this presentation, we report the structural optimization research that led to the development of AUBE00, a next-generation pan-KRAS inhibitor derived from the LUNA18 scaffold. AUBE00 was engineered to achieve KRAS isoform selectivity by targeting His95, a residue uniquely present in KRAS but absent in NRAS and HRAS. KRAS selectivity was achieved through a targeted interaction with His95, a KRAS-specific amino acid residue. A key challenge in this process was the inherent flexibility of the cyclic peptide scaffold. To address this, we introduced a partial fixation by bridging the peptide backbone near His95, enabling a precise interaction with the residue. As a result, we achieved approximately 14-fold selectivity over NRAS and 10-fold selectivity over HRAS. Importantly, this structural optimization was accomplished while maintaining the favorable pharmacokinetic profile of the parent compound, LUNA18. AUBE00 is an 11-residue hydrophobic cyclic peptide lacking dissociable side chains. It exhibits high membrane permeability (Caco-2 Papp = 2.2 × 10⁻⁶ cm/s) and strong binding affinity to KRAS (K D = 92 pM). Notably, AUBE00 demonstrates moderate oral bioavailability (F = 24-65%) across three animal species without specialized formulation. In mouse xenograft models, oral administration of AUBE00 resulted in robust antitumor efficacy. A Phase 1 clinical trial in patients with advanced solid tumors is currently ongoing.