KRAS G12C inhibitor | BI-0474

The KRAS oncoprotein is a GTPase acting as a key node in intracellular signaling pathways that are involved in cell growth and survival^3,4. In normal cells, KRAS functions as a molecular switch, alternating between an inactive GDP-bound and an active GTP-bound state⁵. Transition between these states is modulated by guanine-nucleotide exchange factors (GEFs) such as SOS, and GTP hydrolysis, which is catalyzed by GTPase-activating proteins (GAPs), to inactivate KRAS. GTP-bound KRAS allows binding of effector proteins to trigger downstream signaling pathways, including the RAF-MEK-ERK (MAPK) pathway^6-9. Activating mutations in KRAS are the most frequent oncogenic driver events in cancer¹⁰. Oncogenic KRAS mutations have been shown to shift the equilibrium between the two KRAS states towards the active, GTP-bound form. However, the major oncogenic versions of KRAS still undergo GAP-mediated GTP/GDP exchange and reactivation¹¹. The oncogenic hotspot position 12, located at the lip of the switch II pocket, offers a covalent attachment point for KRAS^G12C inhibitors.

X-ray structure of the complex of BI-0474 with KRAS^G12C (PDB code 8AFB)¹

BI-0474 shows single digit nM biochemical activity in a KRAS^G12C::SOS1 PPI assay and an antiproliferative effect at low nM concentrations in KRAS^G12C mutated cell lines like NCI-H358. As BI-0474 is a KRAS^G12C selective covalent inhibitor, it only has µM activity in the corresponding KRAS^G12D biochemical assay as well as µM antiproliferative activity against a KRAS^G12D mutated cell line. The corresponding negative control shows almost 200-fold lower biochemical activity in the KRAS^G12C::SOS1 assay.

^a BI-0474 is highly selective for KRAS^G12C compared to the KRAS^G12D mutant, as measured in vitro using an AlphaScreen assay. Details of the experimental procedure can be found in reference 1.

^{b, c} BI-0474 shows potent antiproliferative activity on NCI-H358 cells carrying a G12C KRAS mutation. However, an antiproliferative effect on GP2D cells carrying a KRAS^G12D mutation was only observed in concentrations above 4 μM. Details of the experimental procedure can be found in reference 1.

BI-0474 shows medium predicted clearance and a high Caco-2 efflux ratio.

BI-0474 shows decent exposure upon 25mg/kg po dosing as well as 50mg/kg ip dosing (higher ip doses not tested). Oral dosing of 100mg/kg did not lead to significantly increased exposure compared to 25mg/kg.

^bmouse iv 1mg/kg (solution in 25% HP-b-CD, acidified with 0.1M HCl to pH6)
^cmouse oral 25mg/kg (suspension in Natrosol 0.5% or methyl cellulose)
^dmouse ip 50mg/kg (solution in 25% HP-b-CD, acidified with 0.1M HCl to pH6)

Target occupancy (TO) as well as pharmacodynamic (PD) biomarker modulation were assessed in the KRAS^G12C mutant NCI-H358 cell line-derived xenograft model after a daily treatment with 40 mg/kg ip on 3 consecutive days. As shown in Figure 4, MS-based TO analysis showed a strong and treatment-induced reduction of unmodified KRAS^G12C protein on the third day of consecutive treatment. This was confirmed by the analysis of RAS-GTP and p-ERK levels. Apoptosis induction was detectable in tumors in all 5 treated animals 6h post-dose on day 3, indicating that KRAS^G12C inhibition leads to induction of programmed cell death in this xenograft model. Anti-tumor efficacy was also assessed in this model, but as BI-0474 has not been optimized towards high oral bioavailability, it was administered ip once weekly or twice weekly on two consecutive days to explore whether infrequent systemic administration of covalent KRAS^G12C inhibitors could be efficacious. At 40 mg/kg BI-0474 showed anti-tumor efficacy in both dose groups with 68 % and 98 % tumor growth inhibition (TGI), respectively. Both dose groups showed some body weight loss, which is less pronounced for the low dose group compared to the high dose group.

BI-0474 shows efficacy and PD biomarker modulation in an NCI-H358 cell line-derived non-small cell lung cancer xenograft model; left: treatment with BI-0474 leads to TO (decrease of unmodified KRAS^G12C protein levels measured by MS), reduction of RAS-GTP and pERK levels, as well as apoptosis induction in vivo; right: BI-0474 shows efficacy in NCI-H358 xenograft (mean tumor volumes and mean body weight change shown vs. control (green circles), 40 mg/kg BI-0474 ip once/week (q7d, grey squares), 40 mg/kg BI-0474 ip twice/week on 2 consecutive days (qdx2, white triangles)); Details of the experimental procedures can be found in reference 1.

The negative control BI-0473 is a diastereomer of BI-0474 and also carries an electrophilic acrylamide warhead. However due to the inverted stereo center close to the amino-cyano-thiophene, which serves as one of the key pharmacophores, the non-covalent binding contribution is significantly reduced, resulting in a 200-fold lower biochemical activity compared to BI-0474.

BI-0473 which serves as a negative control

As expected for a covalent KRAS^G12C inhibitor the compound shows a strong selectivity for KRAS^G12C vs other mutations (see KRAS^G12C vs. KRAS^G12D AlphaScreen data in the provided in vitro activity table). In the SafetyScreen44^TM at a high concentration of 10µM the compound hit 8 of 41 targets.

Selectivity data available	BI-0474	BI-0473
SafetyScreen44™ with kind support of	Yes 8/41 hits @10µM	in preparation
Invitrogen®	No	No
DiscoverX®	No	No
Dundee	No	No

Download selectivity data: 
BI-0474_selectivityData.xlsx

BI-0474 is a covalent KRAS^G12C inhibitor showing high cellular activity and in vivo efficacy after intraperitoneal administration. It was discovered using structure-based design approaches, by optimizing non-covalent binding of a fragment hit¹.

2 D structure formats available

KRAS G12C Inhibitor | BI-0474.png

KRAS G12C Inhibitor | BI-0474.smiles

KRAS G12C Inhibitor | BI-0474.sdf

Negative control | BI-0473.png

Negative control | BI-0473.smiles

Negative control | BI-0473.sdf

When you plan a publication, please use the following acknowledgement: 
BI-0474 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://opnme.com.

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