pan-KRAS (KD = 6.9, 4.5, 32, 26, 4.3 nM for WT, G12C, G12D, G12V and G13D mutant KRAS, respectively)

BI-2865 is a port aminoalcohol substituent and terminal pin connection equivalent that is labeled. E62 and the R68 side chain of BI-2865 (5 days) have a direct ionic link with BI-2865, as demonstrated by the cocrystal structure of BI-2865 bound to KRAS. G12C, G12D, or G12V expression is inhibited when IL-13 is present. Water-mediated hydrogen bonding network formed by average IC50 and Q61 backbone locations in BaF3 cells with mutant KRAS during their growth [1]. In the neighborhood of 140 nM.

BI-2865 improved the vivo efficacy of paclitaxel in P-gp overexpressing xenografts[2]
To further test the capability of BI-2865 on impairing MDR in vivo, the established P-gp overexpressing KBv200 cells were subcutaneously planted into the 3-week female BALB/C nude mice. When tumors grew near to 100 mm3 in volume, we grouped mice into four with randomization and gave indicated administration once every two days. As the result shown, there were no statistically markable differences in tumor growth analyzed in mice alone treated with saline, 15 mg/kg paclitaxel or 30 mg/kg BI-2865, while the co-administration of paclitaxel and BI-2865 significantly suppressed growth of tumor volume and weight without damaging mice body weight gain (Fig. 2A-D). Collectively, these results verified that BI-2865 could notably augment the therapeutic efficacy of paclitaxel in MDR xenografts mediated by P-gp overexpression in vivo.

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Researchers found BI-2865 notably fortified response of P-gp-driven MDR cancer cells to the administration of chemo-drugs including paclitaxel, vincristine and doxorubicin, while such an effect was not observed in their parental sensitive cells and BCRP or MRP1-driven MDR cells. Importantly, the mice vivo combination study has verified that BI-2865 effectively improved the anti-tumor action of paclitaxel without toxic injury. In mechanism, BI-2865 prompted doxorubicin accumulating in carcinoma cells by directly blocking the efflux function of P-gp, which more specifically, was achieved by BI-2865 competitively binding to the drug-binding sites of P-gp. What's more, at the effective MDR reversal concentrations, BI-2865 neither varied the expression and location of P-gp nor reduced its downstream AKT or ERK1/2 signaling activity.
Conclusions: This study uncovered a new application of BI-2865 as a MDR modulator, which might be used to effectively, safely and specifically improve chemotherapeutic efficacy in the clinical P-gp mediated MDR refractory cancers.[2]

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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ITC[1]
Calorimetric experiments of the pan-KRASi (BI-2865) were performed on a MicroCal PEAQ-ITC instrument. Protein solutions were buffer exchanged by dialysis into buffer containing 20 mM HEPES pH 7.6, 130 mM sodium chloride, 2 mM magnesium chloride and 0.5 mM TCEP. All measurements were carried out at 23 °C. Titrand and titrator concentrations were adjusted to 3% DMSO. The cell was loaded with protein solutions in the range of 0 to 40 μM. All injections were performed using an initial injection of 0.5 μl followed by 19 injections of 2 μl of compound in the range of 100–500 µM. The data were analysed with the MicroCal PEAQ-ITC analysis software package (v.1.1.0.1262). The first data point was excluded from the analysis. Thermodynamic parameters were calculated by the following formula: ΔG = ΔH − TΔS = −RTlnKd, where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding, respectively, T is the temperature, and R is the universal gas constant (Supplementary Fig. 2).

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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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P-gp ATPase assay[2]
The activity of P-gp ATPase was evaluated via performing a colorimetric assay as described previously. Crude membranes were separated from P-gp overexpressing High Five insect cells. The crude membrane protein (100 µg protein/mL) was incubated with varying doses of BI-2865 (0–5 µM) with or without 0.3 mM of sodium orthovanadate (Na3VO4) in the pH 6.8 buffer (composed of 50 mM KCl, 2 mM EDTA, 10 mM MgCl2, 1 mM DTT and 5 mM sodium azide) at 37 °C for 5 min. Then added Mg-ATP solution (5 mM) to initiate the ATP hydrolysis, and the reaction was kept for 20 min at 37 °C. Afterwards, 30 µL of 10% SDS solution was added to end the reaction. Followed by an addition of detection reagent (containing 10% ascorbic acid, 15 mM zinc acetate and 35 mM ammonium molybdate), incubating another 20 min at 37 °C, then measuring the absorbance of the mixture at 750 nm via the 96-well Fisher Scientific Multiskan FC Microplate Reader. The release of inorganic phosphate was quantified at the standard curve. The final BI-2865-stimulated P-gp ATPase activation was defined as the variation between the released amounts of inorganic phosphate from ATP in the presence and absence of Na3VO4.

Cell viability assay and cell proliferation assays[1]
Individual cancer cell lines[1]
The cells were seeded in 96-well plates at 2,000 cells per well in triplicates (at the minimum) and treated with the indicated concentrations of BI-2865. After 72 h, cell viability was assayed by CellTiter-Glo Luminescent Cell Viability Assay. The background value (media without cells) was subtracted from the raw data and fold change was calculated relative to time zero.

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Isogenic BaF3 cells[1]
In brief, BaF3 cells were transduced with a virus derived from plasmids expressing KRAS G12C, G12D or G12V mutants (pMSCV-KRAS-PGK-Puro-IRES-GFP). The transduction efficacy was monitored by fluorescence-activated cell sorting. Cells were selected in puromycin (1 µg ml−1) and Il-3 (10 ng ml−1) for 1–2 weeks or until control cells were dead. This was followed by withdrawal of Il-3 and several passages in the absence of Il-3. Integration of the exogenous KRAS was confirmed by sequencing. To determine the effect of drug treatment on proliferation, 1,500 cells were plated in 384-well plates in 60 µl of Roswell Park Memorial Institute medium (10% FCS) and kept overnight at 37 °C. Cell viability was determined as above.

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High-throughput screen of the 274 cell line panel[1]
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 ofBI-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.

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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The cytotoxicity of BI-2865 and its MDR removal effect in vitro were tested by MTT assays, and the corresponding reversal function in vivo was assessed through the P-gp mediated KBv200 xenografts in mice. BI-2865 induced alterations of drug discharge and reservation in cells were estimated by experiments of Flow cytometry with fluorescent doxorubicin, and the chemo-drug accumulation in xenografts' tumor were analyzed through LC-MS. Mechanisms of BI-2865 inhibiting P-gp substrate's efflux were analyzed through the vanadate-sensitive ATPase assay, [125I]-IAAP-photolabeling assay and computer molecular docking. The effects of BI-2865 on P-gp expression and KRAS-downstream signaling were detected via Western blotting, Flow cytometry and/or qRT-PCR. Subcellular localization of P-gp was visualized by Immunofluorescence.[2]

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

[2]. BI-2865, a pan-KRAS inhibitor, reverses the P-glycoprotein induced multidrug resistance in vitro and in vivo. Cell Commun Signal. 2024 Jun 13;22(1):325

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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Background: Multidrug resistance (MDR) limits successful cancer chemotherapy. P-glycoprotein (P-gp), BCRP and MRP1 are the key triggers of MDR. Unfortunately, no MDR modulator was approved by FDA to date. Here, we will investigate the effect of BI-2865, a pan-KRAS inhibitor, on reversing MDR induced by P-gp, BCRP and MRP1 in vitro and in vivo, and its reversal mechanisms will be explored.
Methods: The cytotoxicity of BI-2865 and its MDR removal effect in vitro were tested by MTT assays, and the corresponding reversal function in vivo was assessed through the P-gp mediated KBv200 xenografts in mice. BI-2865 induced alterations of drug discharge and reservation in cells were estimated by experiments of Flow cytometry with fluorescent doxorubicin, and the chemo-drug accumulation in xenografts' tumor were analyzed through LC-MS. Mechanisms of BI-2865 inhibiting P-gp substrate's efflux were analyzed through the vanadate-sensitive ATPase assay, [125I]-IAAP-photolabeling assay and computer molecular docking. The effects of BI-2865 on P-gp expression and KRAS-downstream signaling were detected via Western blotting, Flow cytometry and/or qRT-PCR. Subcellular localization of P-gp was visualized by Immunofluorescence.
Results: We found BI-2865 notably fortified response of P-gp-driven MDR cancer cells to the administration of chemo-drugs including paclitaxel, vincristine and doxorubicin, while such an effect was not observed in their parental sensitive cells and BCRP or MRP1-driven MDR cells. Importantly, the mice vivo combination study has verified that BI-2865 effectively improved the anti-tumor action of paclitaxel without toxic injury. In mechanism, BI-2865 prompted doxorubicin accumulating in carcinoma cells by directly blocking the efflux function of P-gp, which more specifically, was achieved by BI-2865 competitively binding to the drug-binding sites of P-gp. What's more, at the effective MDR reversal concentrations, BI-2865 neither varied the expression and location of P-gp nor reduced its downstream AKT or ERK1/2 signaling activity.
Conclusions: This study uncovered a new application of BI-2865 as a MDR modulator, which might be used to effectively, safely and specifically improve chemotherapeutic efficacy in the clinical P-gp mediated MDR refractory cancers.[2]

C23H27N7O2S

465.571182489395

465.19

C, 59.34; H, 5.85; N, 21.06; O, 6.87; S, 6.89

2937327-93-8

2937327-93-8

168268166

Light yellow to brown solid

1.38±0.1 g/cm3(Temp: 20 °C; Press: 760 Torr)(Predicted)

708.7±70.0 °C(Predicted)

4.1

1

10

5

33

752

3

C12CCC[C@](C)(C3ON=C(C4=NC=CC(O[C@H]([C@@H]5CCCN5C)C)=N4)N=3)C=1C(C#N)=C(N)S2

MIUFORKWYHBPRW-HMFCALDFSA-N

InChI=1S/C23H27N7O2S/c1-13(15-6-5-11-30(15)3)31-17-8-10-26-20(27-17)21-28-22(32-29-21)23(2)9-4-7-16-18(23)14(12-24)19(25)33-16/h8,10,13,15H,4-7,9,11,25H2,1-3H3/t13-,15-,23-/m0/s1

(4S)-2-amino-4-methyl-4-[3-[4-[(1S)-1-[(2S)-1-methylpyrrolidin-2-yl]ethoxy]pyrimidin-2-yl]-1,2,4-oxadiazol-5-yl]-6,7-dihydro-5H-1-benzothiophene-3-carbonitrile

BI-2865; BI2865; BI-2865; 2937327-93-8; SCHEMBL26167321; BI 2865

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)

DMSO: ~250 mg/mL (536.98 mM)

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.)