Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

Abstract

Targeting undruggable intracellular proteins with peptides: novel on-target macrocyclic peptide inhibitors of KRAS with broad inhibition of proliferation of multiple KRAS-dependent cancer cell lines.

Accelerated Identification of Cell Active KRAS Inhibitory Macrocyclic Peptides using Mixture Libraries and Automated Ligand Identification System (ALIS) Technology

“…Building from this work and recognizing the potential to address liabilities identified with this peptide, we recently disclosed our discovery of optimized macrocyclic peptide inhibitors based on this scaffold offering multiple improved properties: prolonged metabolic stability, enhanced cell permeability, and validated on-target cellular activity. 24 Our studies substantiated the value of this novel peptide series as an alternate approach toward seeking KRAS-inhibitory chemical matter that could progress the field beyond the recent successes with the KRAS G12C mutant protein. We reported 24 that the KRpep-2d peptide series might inhibit KRAS signaling in at least two distinct ways, by directly blocking the interaction with KRAS effectors (e.g., RAF) as well as by indirectly preventing these interactions by blocking the conversion of the GDP (off) state to the GTP (on) state.…”

“…Additionally, as we previously reported, KRpep-2d ( 1 ) exhibited inadequate proteolytic stability and its polycationic character was responsible for off-target effects, namely mast cell degranulation (MCD). 24 Importantly, appropriate retention of arginine-rich analogues on the SEC column of the ALIS system could be compromised and the rate of false positives could be elevated. 32 Such polycationic peptides also tend to behave poorly on LC-MS systems and carry a higher risk of detection issues.…”

“…Metabolite identification studies in cell homogenate suggested that the backbone amide bond was a soft spot for proteolysis in this series. 24 Therefore, we hypothesized that the introduction of unnatural substitutions at these positions could improve the protease stability of the macrocycle. This initial set of combinations was small enough to allow for synthesis optimization but large enough to cover a diverse side chain chemical space as well as explore some potential limitations of the ALIS experiments on macrocyclic peptides.…”

“…We had also demonstrated that the redox-sensitive disulfide cyclization motif of these macrocycles was a barrier to achieving cellular activity but that this issue could be addressed by disulfide replacement with a linkage that preserved binding affinity. 24 We applied changes to our new analogues to improve overall proteolytic/redox stability after completion of multiple rounds of ALIS-based potency optimization (vide infra).…”

“…We also demonstrated that these cell permeable macrocycles were able to inhibit cell growth and block pERK signaling in the low micromolar range in a variety of KRAS G12D , KRAS G12C , and KRAS G12V cancer lines, but not in KRAS WT cell lines, without inducing cytotoxicity (LDH leakage). 24 We also identified off-target challenges inherent in using polyarginine-based cell entry strategies employed by the KRpep-2d chemotype, which contains eight flanking arginine residues. This necessitates continued investment in identifying cell active peptides with reduced arginine burden, which we speculated would require specific modifications to the macrocyclic (non-arginine) core of this peptide.…”