Over the past several decades, direct inhibition of the RAS family of oncogenic proteins has been a significant challenge in the field of drug development. Despite accounting for approximately 30% of all human cancers through its three mutated forms (KRAS, HRAS, and NRAS), no approved pan-RAS therapies currently exist. Among these, KRAS mutations constitute about 85% of all mutations and drive some of the deadliest cancers, including pancreatic, colorectal, and lung cancers. Due to the lack of well-defined binding pockets, intrinsic flexibility, involvement in multiple protein-protein interactions, and picomolar affinity for the endogenous ligand GTP, KRAS has been regarded as undruggable.
The successful approval of KRAS G12C inhibitors has sparked enthusiasm for the development of inhibitors targeting other KRAS subtypes. To address a broader patient population, investigations into other more common KRAS mutations, such as G12D and G12V, are also underway. Nevertheless, identifying effective small molecule inhibitors remains a formidable endeavor. As the KRAS signaling pathway predominantly relies on intracellular protein-protein interactions (PPIs) often involving large binding surfaces, medicinal chemists are exploring larger molecular modalities including nucleic acids, peptides, antibodies, or non-immunoglobulin proteins.
Why the Interest in Cyclic Peptide Inhibitors?
Molecules that inhibit challenging targets like intracellular protein-protein interactions have the potential to become a potent class of novel drugs, broadening the range of therapeutic targets. One approach to targeting intracellular proteins that are difficult to reach is the development of medium-sized cyclic peptides. It has been reported that drugs with molecular weights as high as 1200 g/mol can inhibit intracellular targets and/or have oral bioavailability. A classic example of an orally available cyclic peptide drug is cyclosporin A (with approximately 30% oral bioavailability), discovered in 1969 and widely used as an immunosuppressant. Cyclosporin A binds to cyclophilins and then forms intracellular PPIs with calcineurin.
Merck’s PCSK9 inhibitor, currently in Phase IIb clinical trials, is another example of the successful development of a medium-sized cyclic peptide inhibitor. PCSK9 is an extracellular protein targeted by antibody drugs. A PCSK9 cyclic peptide inhibitor was obtained through mRNA display technology screening. After obtaining hits through screening, a metabolically stable PCSK9 inhibitor was optimized. Further modifications led to MK-0616, a cyclic compound with improved hydrophilicity and approximately 100,000-fold increased inhibitory activity compared to the original hit. MK-0616 exhibited low clearance, relying more on renal clearance than hepatic clearance.
As a result, cyclic peptides have attracted the attention of numerous companies. They are typically larger than traditional small molecules (500-3000 Da), better suited for exploiting the flat and hydrophobic interface features of PPIs, and can exhibit antibody-like binding affinity and specificity within a fraction of their molecular weight. This is the theoretical basis for the high selectivity and fewer off-target effects observed in large cyclic peptide inhibitors, which also retain many desirable small molecule characteristics, such as low immunogenicity and resistance to protein degradation.
Development of Cyclic Peptide KRAS Inhibitors
In 2017, as depicted in Figure 1, Sakamoto et al. reported a 19-residue cyclic peptide, KRpep-2d, discovered and synthetically assembled through phage display technology. KRpep-2d selectively bound to GDP-bound K-Ras (G12D) and GTP-bound K-Ras (G12D) with subnanomolar KD values, inhibiting the exchange of GDP and GTP in K-Ras (G12D) with an IC50 value of 1.6 nM. This was the first K-Ras (G12D)-targeting peptide exhibiting in vivo anti-cancer activity. KRpep-2d contained a disulfide bond between two Cys residues, crucial for its binding and inhibitory activities within its structure. However, this bond would break under intracellular reducing conditions. In the presence of the reducing agent dithiothreitol, KRpep-2d’s inhibitory activity significantly decreased. KRpep-2d’s in vitro cell growth inhibitory activity showed selectivity toward cells expressing K-Ras (G12D).
The X-ray crystal structure information of the K-Ras (G12D)/KRpep-2d complex was used to predict alternative amino acid residues in KRpep-2d. A second-generation cyclic peptide, KS-36, was synthesized with significant changes, such as replacing the disulfide bond with an amide bond and cyclizing the side chains of Dap5 and Asp15 to form a cyclic structure. In competitive binding experiments, KS-36 exhibited approximately 30-fold stronger binding activity to K-Ras (G12D) compared to KRpep-2d. At a concentration of 30 µM, the growth inhibition rates of A427 cells by KS-36 and KRpep-2d were 33.1% and 56.4%, respectively.
Considering the binding activity, lipophilicity, water solubility, and synthesis cost of the amino acid substitutions in KS-36, a combination of these substitutions was selected for further study. This led to the development of a third-generation cyclic peptide, KS-58, with half the molecular weight of the previous version. At a peptide concentration of 30 µM, KS-58 exhibited growth inhibition rates of 21.1% in A427 cells and 50.1% in PANC-1 cells (human pancreatic cancer with G12D mutation).
Marina Buyanova et al. reported a class of bicyclic pan-RAS inhibitors. Prior to this, they developed a bicyclic peptide inhibitor, cyclorasin B3, which selectively bound to Ras-GTP with moderate affinity and blocked its interaction with downstream effector proteins. However, cyclorasin B3 lacked cell permeability or biological activity. Cyclorasin B3 was optimized to develop an effective pan-Ras inhibitor, cyclorasin B4-27, which formed a cyclic peptide through a truxillic acid linker connecting three amino acid side chains.
Recently, a JACS article reported the development process from optimized hits to the clinical pan-KRAS inhibitor LUNA18. LUNA18, a cyclic peptide targeting intracellular proteins, achieved various levels of oral bioavailability (21-47%) without the need for special formulations. It is currently in Phase I clinical trials, expected to conclude by March 31, 2025. The indication is for locally advanced or metastatic solid tumors. mRNA display libraries have proven to be useful tools for obtaining cyclic peptide hits. As shown in Figure 4, the discovery of AP8747 from an mRNA display library led to a substantial enhancement of cellular activity from >20000 nM to 1.4 nM through modifications to side chains. Particularly noteworthy is LUNA18’s significant cellular activity against various KRAS mutant types, including LS180 (colorectal cancer, KRASG12D), GSU (colorectal cancer, KRASG12D), and NCI-H441 (colorectal cancer, KRASG12D), indicating its effectiveness against different KRAS mutations.
In a comprehensive review, Paul M. Levine et al. summarized methods for preclinical drug discovery using cyclic peptide screening. The article also listed cyclic peptide drugs approved by the FDA in the last 20 years. Due to challenges in optimizing membrane permeability, most FDA-approved cyclic peptides target extracellular proteins. This includes significant drugs such as daptomycin and linaclotide. Utilizing various complex screening technologies to generate cyclic peptide libraries, such as phage display, mRNA display, split protein circular ligation of peptides and proteins, and computational screening, the development of cyclic peptide drugs is at the forefront of modern drug discovery. A common method to improve the proteolytic stability of cyclic peptides is to introduce additional ring constraints, leading to bicyclic peptides. Another frequently used approach involves incorporating non-natural amino acids, such as D-amino acids.
Challenges in Developing Cyclic Peptide KRAS Inhibitors
Through the examples of several cyclic KRAS inhibitors mentioned above, the primary challenges faced by these peptide inhibitors include: the susceptibility of amide bonds to poor stability in plasma, limited cell permeability leading to low cellular activity, low oral bioavailability necessitating injection, and low activity preventing in vivo studies. Currently, the clinical pan-KRAS inhibitor LUNA18 remains the forefront example in this field of cyclic peptide development. It demonstrates the best inhibitory effect on the KRAS G12D subtype mutation and can be administered orally.
References:
- Sakamoto, K., et al., Generation of KS-58 as the first K-Ras(G12D)-inhibitory peptide presenting anti-cancer activity in vivo, Scientific Reports, 2020, 10, 21671.
- Tanada, M., et al., Development of Orally Bioavailable Peptides Targeting an Intracellular Protein: From a Hit to a Clinical KRAS Inhibitor, J. Am. Chem. Soc., 2023, 145, 30, 16610-16620.
- Buyanova, M., et al., Discovery of a Bicyclic Peptidyl Pan-Ras Inhibitor, J. Med. Chem., 2021, 64, 13038−13053.
- Li, X., et al., Cyclic Peptide Screening Methods for Preclinical Drug Discovery, J. Med. Chem., 2022, 65, 11913−11926.