| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| Other Sizes |
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| Targets |
K-Ras WT
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| ln Vitro |
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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| ln Vivo |
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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| Enzyme Assay |
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. 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. |
| Cell Assay |
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. |
| Animal Protocol |
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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| References | |
| Additional Infomation |
KRAS is one of the most commonly mutated proteins in cancer, and efforts to directly inhibit its function have been continuing for decades. The most successful of these has been the development of covalent allele-specific inhibitors that trap KRAS G12C in its inactive conformation and suppress tumour growth in patients1-7. Whether inactive-state selective inhibition can be used to therapeutically target non-G12C KRAS mutants remains under investigation. Here we report the discovery and characterization of a non-covalent inhibitor that binds preferentially and with high affinity to the inactive state of KRAS while sparing NRAS and HRAS. Although limited to only a few amino acids, the evolutionary divergence in the GTPase domain of RAS isoforms was sufficient to impart orthosteric and allosteric constraints for KRAS selectivity. The inhibitor blocked nucleotide exchange to prevent the activation of wild-type KRAS and a broad range of KRAS mutants, including G12A/C/D/F/V/S, G13C/D, V14I, L19F, Q22K, D33E, Q61H, K117N and A146V/T. Inhibition of downstream signalling and proliferation was restricted to cancer cells harbouring mutant KRAS, and drug treatment suppressed KRAS mutant tumour growth in mice, without having a detrimental effect on animal weight. Our study suggests that most KRAS oncoproteins cycle between an active state and an inactive state in cancer cells and are dependent on nucleotide exchange for activation. Pan-KRAS inhibitors, such as the one described here, have broad therapeutic implications and merit clinical investigation in patients with KRAS-driven cancers.[1]
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| Molecular Formula |
C24H27N7OS
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|---|---|
| Molecular Weight |
461.58248257637
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| Exact Mass |
461.199
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| Elemental Analysis |
C, 62.45; H, 5.90; N, 21.24; O, 3.47; S, 6.95
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| CAS # |
2937344-16-4
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| PubChem CID |
168268198
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
3.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
33
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| Complexity |
777
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| Defined Atom Stereocenter Count |
2
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| SMILES |
O1C2=C(CCC[C@]32CCCC2SC(N)=C(C#N)C=23)C(C2C=CN=C(N3CCNC[C@@H]3C)N=2)=N1
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| InChi Key |
PVOYBVVIBNPTPJ-BSEYFRJRSA-N
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| InChi Code |
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
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| Chemical Name |
(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
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| Synonyms |
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;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1665 mL | 10.8324 mL | 21.6647 mL | |
| 5 mM | 0.4333 mL | 2.1665 mL | 4.3329 mL | |
| 10 mM | 0.2166 mL | 1.0832 mL | 2.1665 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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