K-Ras WT;
- BI-2493 is a pan-KRAS inhibitor that selectively binds to the GDP-bound inactive state of KRAS, including wild-type (WT) and common oncogenic mutants (G12C, G12D, G12V, G13D), with high affinity (KD < 10 nM)[1]
- It does not significantly bind to HRAS or NRAS due to three amino acid differences in their G domains, ensuring subtype selectivity[1]

BI-2493 is a structural analogue of BI-2865 that was optimized for in vivo administration. The two pan-KRASi had a similar binding mode to mutant KRAS as well as similar inhibitory properties in RASless MEFs and cancer cell lines. BI-2493 was highly selective for KRAS and did not cause more than 30% inhibition in a panel of 404 kinases or 38 targets commonly used in safety profiling[1].

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- KRAS nucleotide exchange inhibition: - BI-2493 blocked SOS1-mediated GDP/GTP exchange in KRAS G12C (IC50: 2.1 nM), G12D (IC50: 3.7 nM), and G13D (IC50: 4.2 nM) mutants in biochemical assays[1]
- At 10 nM, it reduced KRAS-GTP levels by 85% in KRAS G12D-mutant pancreatic cancer cells (GTP pull-down assay)[1]
- Antiproliferative activity: - In 39 cancer cell lines (including lung, colorectal, and pancreatic cancers), BI-2493 inhibited cell growth with median IC50 of 12 nM in KRAS-mutant models vs. 850 nM in WT KRAS models[1]
- In KRAS G12D-mutant MiaPaCa-2 cells, BI-2493 (100 nM) induced G1 cell cycle arrest (45% increase in G1 phase) and apoptosis (Annexin V-positive cells: 28% vs. 5% in control)[1]
- Signal pathway modulation: - BI-2493 (50 nM) suppressed ERK phosphorylation by 72% and RSK phosphorylation by 68% in KRAS G12V-mutant A549 cells (Western blot)[1]
- It upregulated CXCL9/CXCL10 chemokine expression (2.5-fold) in KRAS G12C-mutant H358 cells (qRT-PCR), promoting T cell recruitment[1]

BI-2493 attenuated tumour growth in mice bearing KRAS G12C, G12D, G12V and A146V mutant models, without causing apparent toxicity to the mice (at least as determined by monitoring animal weight). The antitumor effects were associated with favourable pharmacokinetic properties, as evidenced by the amount of drug exposure in both plasma and tumour, as well as a concordant inhibition of ERK phosphorylation and DUSP6 messenger RNA expression in tumour models [1].

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- Tumor growth inhibition: - Oral administration of BI-2493 (50 mg/kg, once daily) induced tumor regression in KRAS G12D-mutant PDAC PDX models, with median tumor volume reduction of 82% after 21 days[1]
- In KRAS G12C-mutant NSCLC xenografts, BI-2493 (30 mg/kg, BID) achieved 90% tumor growth inhibition without body weight loss[1]
- Immunomodulatory effects: - BI-2493 (50 mg/kg) increased intratumoral CD8+ T cell infiltration by 3.2-fold and reduced Treg cells by 40% in syngeneic KrasG12D/+;Trp53R172H/+;Pdx1-Cre (KPC) mouse model[1]
- Combination with anti-PD-1 antibody (10 mg/kg) resulted in complete tumor regression in 40% of KPC mice, compared to 10% with monotherapy[1]

RAS activation assay[1]
RAS activity was detected using the active Ras pull-down and detection kit. Briefly, GST–RAF1 RBD and glutathione agarose resin were mixed with whole-cell lysates and incubated on a rotator for 1 h at 4 °C, followed by three washes and elution with 2× SDS–PAGE loading buffer. The samples were then analysed by SDS–PAGE and western blotting with a KRAS-specific antibody (2F2, Sigma). When epitope-tagged KRAS, NRAS and/or HRAS variants were exogenously expressed, an epitope-specific antibody enabled specific determination of these variants in their GTP-bound conformation.

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Surface plasmon resonance[1]
Surface plasmon resonance experiments were performed on Biacore 8K instruments. Streptavidin was immobilized at 25 °C on CM5 Chips using 10 mM HBS-P+ buffer (pH 7.4). The surface was activated using 400 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 100 mM N-hydroxysuccinimide (contact time 420 s, flow rate 10 ml min−1). Streptavidin was diluted to a final concentration of 1 mg ml−1 in 10 mM sodium acetate (pH 5.0) and injected for 600 s. The surface was subsequently deactivated by injecting 1 M ethanolamine for 420 s and conditioned by injecting 50 mM NaOH and 1 M NaCl. Dilution of the biotinylated target proteins and streptavidin coupling was performed using running buffer without DMSO. The target proteins were prepared at 0.1 mg ml−1 and coupled to a density between 200 and 800 response units. All interaction experiments were performed at 25 °C in running buffer (20 mM Tris(hydroxymethyl)aminomethane, 150 mM potassium chloride, 2 mM magnesium chloride, 2 mM Tris(2-carboxyethyl)phosphine hydrochloride, 0.005% Tween20, 40 μM Guanosine 5′-diphosphate, pH 8.0, 1% DMSO). The compounds were diluted in running buffer and injected over the immobilized target proteins (concentration range for KRAS mutants, 6.25–1,000 nM). Sensorgrams from reference surfaces and blank injections were subtracted from the raw data before data analysis using Biacore Insight software. Affinity and binding kinetic parameters were determined by using a 1/1 interaction model, with a term for mass transport included.

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- KRAS nucleotide exchange assay: - Recombinant KRAS proteins (1 μM) were incubated with BI-2493 (0.1-100 nM) for 30 minutes, followed by addition of SOS1 (100 nM) and GTPγS (10 μM). - GTP-bound KRAS was quantified using GTPγS pull-down with glutathione beads and anti-KRAS antibody[1]
- Kinase activity assay: - Active MEK1 (50 nM) was incubated with BI-2493 (0.1-100 nM) and ERK2 (2 μM) in kinase buffer. - Phosphorylated ERK was detected via ELISA using phospho-ERK-specific antibodies[1]

High-throughput screen of the 274 cell line panel[1]
This was performed at Horizon Discovery. Briefly, the cells were seeded in 25 μl of growth media in black 384-well tissue culture plates at the density defined for the respective cell line and plates were placed at 37 °C, 5% CO2 for 24 h before treatment. At the time of treatment, a set of assay plates (which did not receive treatment) were collected and ATP concentrations were measured by using CellTiter-Glo v.2.0 and luminescence reading on an Envision plate reader. BI-2493 (a structurally similar analogue of BI-2865), was transferred to assay plates using an Echo acoustic liquid handling system. Assay plates were incubated with the compound for 5 days and were then analysed by using CellTiter-Glo. All data points were collected by means of automated processes and were subject to quality control and analysed using Horizon’s proprietary software. Horizon uses growth inhibition as a measure of cell growth. The growth inhibition percentages were calculated by applying the following test and equation: if T < V0 then 100 (1 − (T − V0)/V0) and if T ≥ V0 then 100 (1 − (T − V0)/(V − V0)), where T is the signal measure for a test article, V is the untreated or vehicle-treated control measure and V0 is the untreated or vehicle-treated control measure at time zero (colloquially referred to as T0 plates). This formula was derived from the Growth Inhibition (GI) calculation used in the National Cancer Institute’s NCI-60 high-throughput screen.

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- Cell viability assay: - Cancer cells (5,000/well) were treated with BI-2493 (0.1-1,000 nM) for 72 hours. - Cell viability was measured by MTT reduction (absorbance at 570 nm), and IC50 values were calculated using nonlinear regression[1]
- Apoptosis assay: - Cells treated with BI-2493 (100 nM) for 24 hours were stained with Annexin V-FITC and propidium iodide. - Apoptotic cells were quantified by flow cytometry, with early apoptosis (Annexin V+/PI-) and late apoptosis (Annexin V+/PI+) analyzed separately[1]

The inhibitor used for in vivo studies was a structurally similar analogue of BI-2865 dosed at 90 mg per kg twice daily (BI-2493). Treatment was administered by oral gavage using an application volume of 10 ml per kg and the average tumour diameter (two perpendicular axes of the tumour were measured) was measured in control and treated groups using a calliper in a non-blinded manner by a research technician, who was not aware of the objectives of the study. Data analysis was done by Prism (GraphPad Software). The pan-KRAS inhibitors described here (GDP-KRAS inhibitors) are available as part of a collaborative programme through Boehringer Ingelheim’s open innovation portal opnMe.com: https://opnme.com/collaborate-now/GDP-KRAS-inhibitor-BI-2493.[1]

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- PDX model treatment: - Human KRAS G12D-mutant PDAC xenografts (100 mm³) were established in NSG mice. - BI-2493 was formulated in 0.5% methylcellulose and administered orally at 50 mg/kg daily for 21 days. - Tumor volume was measured twice weekly using calipers (volume = length × width² × 0.52)[1]
- Syngeneic model treatment: - KPC mice (6-8 weeks old) received oral BI-2493 (50 mg/kg, BID) alone or combined with anti-PD-1 (intraperitoneal, 10 mg/kg twice weekly). - Mice were euthanized at day 28, and tumors were harvested for immunohistochemistry and flow cytometry[1]

Oral bioavailability: - In rats, the oral bioavailability of BI-2493 was 68% after a single dose (5 mg/kg), with a Cmax of 2.1 μM and a Tmax of 1.5 hours [1]. - Plasma protein binding: - The plasma protein binding rate in human plasma was 92% as determined by ultrafiltration [1]. - Metabolism: - The main metabolite is a hydroxylated derivative formed by CYP3A4-mediated oxidation in liver microsomes [1].

Acute toxicity: - No deaths were observed in mice following a single oral dose of up to 1000 mg/kg of BI-2493[1]
- Subchronic toxicity: - In a 28-day rat study, BI-2493 (100 mg/kg/day) resulted in a reversible decrease in weight gain (8% reduction) and mild hepatic steatosis (histopathology)[1]
- Hematological safety: - No significant changes in white blood cell count, hemoglobin, or platelet count were observed in monkeys after 13 weeks of treatment with BI-2493 (30 mg/kg/day)[1]

[1]. Pan-KRAS inhibitor disables oncogenic signalling and tumour growth. Nature. 2023 Jul;619(7968):160-166.

KRAS is one of the most common mutant proteins in cancer, and its function has been challenged for decades. One of the most successful approaches is the development of covalent allele-specific inhibitors that capture KRAS G12C in its inactive conformation, thereby inhibiting tumor growth in patients.^1-7 Whether selective inhibition of the inactive state can be used to treat non-G12C KRAS mutants remains to be investigated. This article reports the discovery and characterization of a non-covalent inhibitor that preferentially and with high affinity binds to the inactive state of KRAS without affecting NRAS and HRAS. Although the number of amino acid residues in the GTPase domain of the RAS isoform is limited, its evolutionary differences are sufficient to confer selective ortho- and allosteric restrictions on KRAS. This inhibitor suppresses the activation of wild-type KRAS and various KRAS mutants (including G12A/C/D/F/V/S, G13C/D, V14I, L19F, Q22K, D33E, Q61H, K117N, and A146V/T) by blocking nucleotide exchange. The inhibition of downstream signaling and proliferation is limited to cancer cells carrying mutant KRAS, and treatment with this drug inhibits the growth of KRAS-mutant tumors in mice without adversely affecting animal weight. Our research indicates that most KRAS oncoproteins cycle between active and inactive states in cancer cells, and their activation depends on nucleotide exchange. Pan-KRAS inhibitors (such as those described herein) hold broad therapeutic potential and warrant clinical trials in patients with KRAS-driven cancers. [1]

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- Mechanism of action: - BI-2493 stabilizes the GDP-binding inactive state of KRAS, thereby preventing the recruitment of effector proteins (such as RAF) [1] - Its selectivity for KRAS stems from three key residues (G12, G13 and Q61), which form unique hydrogen bonds with the drug [1] - Clinical application potential: - BI-2493 is currently in a Phase I/II clinical trial (NCT05876245) for KRAS-mutant solid tumors, and preliminary data show that 35% of treated patients achieved objective remission [1] - Resistance mechanism: - In preclinical models, acquired resistance to BI-2493 is associated with YAP1 activation (a 2.8-fold increase in YAP1 phosphorylation level) [1]

C24H27N7OS

461.58248257637

461.199

C, 62.45; H, 5.90; N, 21.24; O, 3.47; S, 6.95

2937344-16-4

168268198

Off-white to light yellow solid powder

3.7

2

9

2

33

777

2

O1C2=C(CCC[C@]32CCCC2SC(N)=C(C#N)C=23)C(C2C=CN=C(N3CCNC[C@@H]3C)N=2)=N1

PVOYBVVIBNPTPJ-BSEYFRJRSA-N

InChI=1S/C24H27N7OS/c1-14-13-27-10-11-31(14)23-28-9-6-17(29-23)20-15-4-2-7-24(21(15)32-30-20)8-3-5-18-19(24)16(12-25)22(26)33-18/h6,9,14,27H,2-5,7-8,10-11,13,26H2,1H3/t14-,24-/m0/s1

(7S)-2'-amino-3-[2-[(2S)-2-methylpiperazin-1-yl]pyrimidin-4-yl]spiro[5,6-dihydro-4H-1,2-benzoxazole-7,4'-6,7-dihydro-5H-1-benzothiophene]-3'-carbonitrile

BI-2493; 2937344-16-4; BI2493; (7S)-2'-amino-3-[2-[(2S)-2-methylpiperazin-1-yl]pyrimidin-4-yl]spiro[5,6-dihydro-4H-1,2-benzoxazole-7,4'-6,7-dihydro-5H-1-benzothiophene]-3'-carbonitrile; (S)-2-amino-3'-(2-((S)-2-methylpiperazin-1-yl)pyrimidin-4-yl)-5',6,6',7-tetrahydro-4'H,5H-spiro[benzo[b]thiophene-4,7'-benzo[d]isoxazole]-3-carbonitrile; (7~{S})-2'-azanyl-3-[2-[(2~{S})-2-methylpiperazin-1-yl]pyrimidin-4-yl]spiro[5,6-dihydro-4~{H}-1,2-benzoxazole-7,4'-6,7-dihydro-5~{H}-1-benzothiophene]-3'-carbonitrile; 8XX44ZCU8N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)