D3S-001, a KRAS G12C Inhibitor with Rapid Target Engagement Kinetics, Inhibits Nucleotide Cycling and Demonstrates Robust Preclinical and Clinical Activity

Background Introduction

The KRAS (Kirsten rat sarcoma viral oncogene homolog) gene mutation is one of the oncogenic drivers in various solid tumors (such as non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), etc.). KRAS mutations are mainly single-base missense mutations, with common locations including the mutation from glycine (G) to cysteine © in the 12th codon, namely KRAS G12C, which is the predominant mutation form in about 14% of adenocarcinomas in NSCLC, as well as 0.5% to 4% of squamous cell carcinomas.

Over the past few decades, the discovery and development of targeted therapies against KRAS have been a challenging task. KRAS is a GTPase that cycles between a GTP-bound active conformation and a GDP-bound inactive conformation by hydrolyzing guanosine triphosphate (GTP) into guanosine diphosphate (GDP). This enzyme is different from protein kinases, which use ATP (adenosine triphosphate) as a substrate. Due to the picomolar affinity of KRAS for GTP/GDP and the lack of obvious small molecule drug binding sites, it is difficult to target with small molecule inhibitors. In recent years, medicinal chemistry research has cleverly identified compounds that can form covalent adducts with the mutant cysteine residue at G12c of KRAS, and induced a concealed allosteric pocket under the GDP-bound conformation of the KRAS protein, which marked a major discovery in considering KRAS as a “drug-resistant” protein.

However, first-generation KRAS G12C inhibitors such as Sotorasib and Adagrasib have shown somewhat insufficient effects in clinical application. Although they can trap the KRAS protein in its GDP-bound inactive state, thereby inhibiting its oncogenic signaling and tumor growth, their clinical response depth and duration are limited. This has sparked controversy over whether targeting only the GDP-bound form of KRAS can completely block this oncogenic driver.

Research Overview

The paper “D3S-001, a KRAS G12C Inhibitor with Rapid Target Engagement Kinetics, Overcomes Nucleotide Cycling, and Demonstrates Robust Preclinical and Clinical Activities” authored by Jing Zhang, Sun Min Lim, Mi Ra Yu, Cheng Chen, Jia Wang, Wenqian Wang, Haopeng Rui, Jingtao Lu, Shun Lu, Tony Mok, Zhi Jian Chen, and Byoung Chul Cho, etc., was published in the journal “Cancer Discovery”. The paper researched the characteristics of a new generation KRAS G12C inhibitor—D3S-001 and its improvements over previous inhibitors.

Research Process

a) Detailed Research Process

The main work of the study includes several steps: 1. Analysis of early KRAS G12C inhibitors: Molecular activity, covalent potency, and cell target binding kinetics data analysis of early KRAS G12C inhibitors ARS-853, ARS-1620, Sotorasib, and Adagrasib was conducted. This part of the study employed techniques such as Ras-binding domain pull-down assay, immunoblot analysis, surface plasmon resonance (SPR), and liquid chromatography-mass spectrometry/tandem mass spectrometry (LC-MS/MS). 2. Discovery and Characterization Evaluation of D3S-001: Through a series of biochemical and cell biology experiments, the characteristics of the novel KRAS G12C inhibitor D3S-001 were discovered and evaluated. The evaluation includes the compound’s covalent potency, target binding kinetics in cells, and toxicity selectivity features. 3. In Vivo and In Vitro Antitumor Activity Assays: The antitumor activity of D3S-001 was evaluated in various cancer cell lines and animal models, including NSCLC, CRC, and pancreatic cancer. Tumor volume measurement and Homogeneous Time-Resolved Fluorescence (HTRF), etc., were used to analyze its inhibition of KRAS downstream signaling and cell proliferation.

b) Detailed Main Results

1. D3S-001 shows significantly improved covalent potency and target binding kinetics:
   • D3S-001 demonstrated significantly better covalent potency in several tests compared to Sotorasib and Adagrasib (kinact/ki value reached 1.43 × 10^6 M^-1s^-1).
   • Through SPR methods, it was found that D3S-001 had significantly better reversible affinity for GDP-bound KRAS G12C than previous inhibitors, achieved through high kon and low koff. In terms of cell target engagement efficiency, D3S-001 also showed a faster execution rate.
2. D3S-001 maintains high efficiency inhibition and overcomes nucleotide cycling in the presence of growth factors:
   • Under EGF (epidermal growth factor) stimulation, D3S-001 still achieved highly efficient inhibition of KRAS G12C, almost unaffected, while the inhibitory effects of Sotorasib and Adagrasib significantly decreased. Additionally, D3S-001 could also overcome the effect of other growth factors such as HGF (hepatocyte growth factor) mediated KRAS GDP to GTP transition.
3. Significant antitumor activity in vitro and in vivo in various tumor models:
   • In cell lines such as NCI-H358 and MIA PaCa2, D3S-001 showed higher inhibitory activity on ERK phosphorylation levels and cell proliferation.
   • In animal models including NCI-H358 and MIA PaCa2, D3S-001 demonstrated dose-dependent antitumor effects, achieving nearly complete tumor regression at a dose of 30 mg/kg. In addition, in multiple patient-derived tumor samples (PDOs), D3S-001 demonstrated significant tumor inhibition effects.

c) Research Conclusions and Significance

1. Scientific Value: The research indicates that D3S-001, through improved covalent potency and enhanced target engagement efficiency, can overcome the previous inadequacies of KRAS G12C inhibitors in clinical practice. The drug is capable of effectively inhibiting KRAS G12C even in the presence of growth factors, avoiding the impact of nucleotide cycling.

2. Application Value: D3S-001 has demonstrated potential application value in the treatment of tumors with the KRAS G12C mutation. Its highly efficient cell target engagement and antitumor activity give it a significant advantage in the treatment of various cancers.

d) Research Highlights

1. Improved Covalent Potency: By enhancing covalent potency and selectivity, D3S-001 significantly outperforms previous KRAS G12C inhibitors in antitumor activity, which has been validated in clinical and in vitro experiments.
2. Efficient Inhibition in the Presence of Growth Factors: Unlike Sotorasib and Adagrasib, D3S-001 is able to efficiently inhibit KRAS G12C even in the presence of growth factors, suggesting advantages under both physiological and pathological conditions.
3. Broad Applicability and Efficiency: In a variety of cancer animal models (including patient-derived tumor samples with different genetic backgrounds), D3S-001 has demonstrated robust antitumor effects and sustained tumor control.

Conclusion

Through a series of comprehensive biochemical and cell experiments, D3S-001 demonstrated significant improvements in covalent potency, rapid cellular target engagement kinetics, superior antitumor activity, and specificity. Additionally, its highly effective action in multiple tumor models further proves the potential application prospect of D3S-001 in the treatment of KRAS G12C mutant cancers. This discovery brings new hope to future cancer treatment, especially in the KRAS G12C mutation domain.