The discovery of an ideal groove on the smooth surface of the KRAS protein seemed to be an impossible mission until 2013, when it was shown that the switch-II pocket (S-IIP) is potentially targetable when KRAS proteins have the G12C substitution [24].

In an attempt to overcome the challenges of inhibiting RAS directly, Shokat and co-workers explored a novel approach to covalently target the reactive cysteine-12 of KRAS^G12C, which is located in close proximity to both the nucleotide pocket and a previously unrecognized surface groove in the switch-II region (S-IIP). Interestingly, the covalent modification of the mutant C12 only occurs in GDP-bound KRAS, as S-IIP is absent in GTP-bound KRAS [10,24]. The formation of an irreversible and covalent bond between the inhibitor and the cysteine residue of KRAS^G12C irreversibly disrupts both switch-I and switch-II, reduces the affinity of KRAS for guanosine nucleotides despite the presence of GTP, and allows for the persistent disruption of the adducted protein, which locks KRAS^G12C in the GDP-bound conformation. Notably, these inhibitors rely on the intrinsic GTPase of mutant KRAS^G12C, i.e., the KRAS^G12C protein is still able to alternate between their active and inactive state, as GTP-bound KRAS is the predominant form in KRAS^G12C-mutant cancer cells. Such a mutant-specific strategy enables the selective inhibition of KRAS^G12C while sparing the other KRAS mutants (e.g., G12D, G12S, G12S, G13D, Q61H, etc.) or KRAS wild-type cells (e.g., normal tissue), potentially overcoming the toxicological challenges elicited by the nonselective inhibition of KRAS-driven cell growth [24].

ARS853. The first G12C-selective inhibitor was developed based on the so-called compound 12, which is described as the most potent candidate in the primary report. ARS853 covalently binds to cysteine residue 12 with high affinity and can signficantly affect the active KRAS protein as well as its downstream effectors, such as the RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways, by inducing apoptosis and decreasing cell proliferation in vitro [25,26]. The high selectivity of ARS853 has also been demonstrated by the fact that its inhibitory effects can be abrogated by the ectopic expression of KRAS^G12C substitution [26].

ARS1620. Compared to ARS853, which was successful in in vitro studies, the second-generation inhibitor ARS1620 has improved pharmacological properties (e.g., metabolic stability), making it the first inhibitor with in vivo efficacy [27]. ARS1620 was not only optimized for in vivo stability, but also exhibited an intensive potency in G12C occupation and the effective blockade of RAS signaling [28]. In a Mia.paca-2 cell line xenograft, tumor growth was completely inhibited at a drug dose of 200 mg/kg, and the protein levels of RAS-GTP, phospho-ERK, phospho-AKT, and phospho-S6 were all significantly downregulated. In comparison, ARS1620 shows no effects in KRAS^G12V mutant xenografts.

AMG510 (sotorasib). Derived from ARS1620, this third-generation sotorasib is the first G12C inhibitor to enter and complete clinical trials. Based on the promising results of the NCT03600883 trial and an ongoing phase III clinical trial (CodeBreaK 200; https://doi.org/10.1016/j.jtho.2020.10.137 accessed on 21 November 2021), AMG510 was recently approved by the FDA (the United States Food and Drug Administration) for the treatment of locally advanced or metastatic NSCLC [29,30]. Compared to ARS853 and ARS1620, a breakthrough of AMG510 is that its binding induces an alternative orientation of histidine 95 (His-95), which provides a novel surface groove on KRAS^G12C that can be occupied by the aromatic rings of AMG510, leading to the enhanced interaction of AMG510 with KRAS^G12C via van der Waals contacts and the improved potency of AMG510. Indeed, AMG510 exhibits a 10-fold increase in efficacy and improved kinetics compared to ARS1620. The onset of the maximal inhibition of the MAPK pathway occurs 2–4 h after exposure to AMG510, which maintains the inhibition of phospho-ERK in vivo for up to 48 h [29].

The safety and tolerability profile of AMG510 was demonstrated in a phase I study [31] that included 59 cases of NSCLC, 42 cases of colorectal cancer, and 28 cases of other tumors. The total 129 patients were divided into four cohorts with planned doses of 180, 360, 720, and 960 mg per day, respectively. The mean elimination half-life of the drug was 5.5 h. Treatment-related adverse effects occurred in 73 patients (56.58%), of which 15 patients had grade 3 or 4 effects, e.g., an increase in alanine aminotransferase (ALT), diarrhea, anemia, an increase in aspartate aminotransferase (AST), an increase in blood alkaline phosphatase, hepatitis, a decrease in lymphocyte count, an increase in γ-glutamyltransferase, and hyponatremia. The treatment showed high potential anticancer activity, especially in the subgroup of NSCLC: 19 patients had a confirmed partial response and 33 had stable disease. That is, the confirmed response rate was 32.2% and disease control (including objective response and stable disease) was 88.1%. In this subgroup, the median progression-free survival was 6.3 months. In addition, 34 patients with NSCLC in the 960 mg cohort had a more satisfactory disease control rate of 91.2%.

In a phase II study of 126 NSCLC patients, the efficacy of sotorasib was further evaluated [32]. While complete responses were observed in four cases only, a partial response occurred in 42 cases and stable disease in 54 cases. Although the reported overall objective response rate was 37.1% and the disease control rate was 80.6%, the median progression-free survival (PFS) was 6.8 months. Treatment-related adverse events (TRAEs) were observed in 69.8% of patients, with 20.6% of patients experiencing severe TRAEs (twenty-five grade 3 events and one grade 4 event).

MRTX849 (adagrasib). MRTX849 is another third-generation drug for targeting KRAS^G12C that may well receive final approval. MRTX849 has a relatively long half-life of 17 to 48 h [33,34], and its efficacy in discriminating KRAS^G12C activity was widely confirmed in KRAS^G12C-mutant cell lines under both 2D and 3D conditions. On the contrary, non-KRAS^G12C-mutant cell lines showed IC₅₀ values greater than 1 μM in 2D and greater than 3 μM under 3D conditions [33]. An RNA sequencing analysis of a xenograft mouse model confirmed that ERK-dependent transcription was blocked, accompanied by the reactivation of receptor tyrosine kinase (RTK)- and ERK-dependent signaling.

Based on these in vitro and in vivo data, a phase I/II clinical trial (NCT03785249) was then conducted. Adagrasib is also well tolerated and provides great benefits to patients with KRAS^G12C-mutant cancers [35,36]. In 110 patients with colorectal cancer or NSCLC, treatment-relative adverse events occurred in 85% of cases, including 33 patients with grade 3 or 4 adverse events. Clinical activity was evaluable in 51 patients, of whom 23 patients had a partial response and 26 patients had stable disease. Overall, the disease control rate was up to 96%. In addition, an objective response rate of 43% was confirmed in 14 phaseI/Ib patients with a longer follow-up.

Other G12C inhibitors. GDC-6036, JNJ-74699157 (ARS-3248), and LY3499446 are other inhibitors in the field. The latter two candidates have been enrolled in clinical trials without much reporting of preclinical evidence. The clinical trial of JNJ-74699157 (NCT04006301) was reported to be completed after less than a year, with only 10 participants recruited and no result published. The study of LY3499446 (NCT04165031) was terminated at the end of 2020 due to an unexpected toxicity finding.