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| Targets |
PRKACA (protein kinase cAMP-activated catalytic subunit alpha) – IC50 = 0.3 nM (biochemical assay); Kd = 3.3 nM; cellular IC50 = 36.6 nM (inhibition of VASP Ser157 phosphorylation in forskolin-stimulated Huh7 cells) [1][2]
ROCK2 – IC50 = 12.7 nM (42-fold selective over PRKACA) [1][2] AKT1 – IC50 = 2120 nM; AKT2 – IC50 = 4910 nM; AKT3 – IC50 = 475 nM [1][2] |
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| ln Vitro |
BLU2864 inhibited PRKACA catalytic activity with an IC50 of 0.3 nM in biochemical assays using Kemptide peptide substrate and Km concentrations of ATP (5 μM). [1][2]
BLU2864 inhibited PRKACA cellular activity with an IC50 of 36.6 nM, determined from inhibition of VASP Ser157 phosphorylation in forskolin-stimulated Huh7 cells. [1][2] BLU2864 demonstrated good to moderate selectivity against closely related AGC kinase family members and excellent overall kinome selectivity, with an S(10) selectivity score of 0.057. Kinome-wide selectivity was assessed at 3 μM across a panel of 400 human kinases. Kd measurement showed most potent binding affinity to PRKACA at 3.3 nM, with Kd < 100 nM for only 10 non-mutant kinases. [1][2] BLU2864 (40 nM and 200 nM) inhibited forskolin-induced in vitro cystogenesis of mIMCD3 cells cultured in Matrigel by 72% and 100%, respectively, relative to control. [1] BLU2864 (200 nM) inhibited forskolin-induced ex vivo cystogenesis by 50-53% and inhibited CREB phosphorylation in Pkd1RC/RC metanephric organ cultures without evidence of toxicity. [1] In Pkd1RC/RC mice treated daily for 5 days with BLU2864 (45 mg/kg by oral gavage), kidney basal and total PKA activities were suppressed by 74% and 87% at 3 hours, and by 46% and 56% at 15 hours, respectively, compared to controls. [1] In Pkd1RC/RC mice treated with BLU2864 (30 mg/kg daily by oral gavage from 4-16 weeks of age), renal basal and total PKA activities at sacrifice (1-2 hours after dosing) were 69% and 84% lower than controls. Kidney AQP-2 and p-AQP-2, PCNA, pro-proliferative signaling pathways (Src, ERK1/2, mTOR, GSK3β, AKT), and transcription factors (CREB, STAT3, Pax2) downstream from PKA activation were downregulated. Gli1 and Gli2 were increased, while Gli3A and Gli3R were unchanged. [1] In vitro, BLU2864 (40 nM and 200 nM; 5 d) suppresses cystogenesis caused by forskolin [1]. |
| ln Vivo |
In mice harboring FLC PDX tumors, oral administration of BLU2864 at 30 mg/kg once daily (QD) for 34 days inhibited tumor growth by 45.3% (P = 0.0005) compared to vehicle control. [2]
In pharmacokinetic/pharmacodynamic studies, BLU2864 at 30 mg/kg QD reduced phosphorylated VASP to 27% of baseline 2 hours after dosing; phosphorylated VASP levels fully recovered by 24 hours post-administration. [2] In Pkd1RC/RC mice (F1 129S6/Sv x C57BL/6 background) treated with BLU2864 (30 mg/kg daily by oral gavage from 4-16 weeks of age), kidney volumes measured by MRI were significantly lower in treated mice at 15-16 weeks compared to controls. Kidney weights, kidney weights as percent of body weights, and cyst indices were also lower in treated mice. Urine outputs were higher in treated mice. Plasma creatinine and urea concentrations were not significantly different. Liver volumes were not different between groups. [1] In Pkd1RC/RC mice, BLU2864 (oral gavage; 45 mg/kg; once daily; 5 days) inhibits renal PKA activity [1]. In Pkd1RC/RC mice, BLU2864 (oral gavage; 30 mg/kg; once daily; 5 days) reduces PKA activity and enhances PKD [1]. FLC tumor growth is inhibited in vivo by BLU2864 (oral gavage; 30 mg/kg and 75 mg/kg; once daily; 34 days) [2]. |
| Enzyme Assay |
PRKACA biochemical inhibition assay: PRKACA enzyme (0.007 ng/mL) was added to each well of a 384-well plate containing 1 μM Kemptide peptide substrate (5-FAM-LRRASLG), Km concentrations of ATP (5 μM), and a concentration series of test compounds (1% final DMSO) in assay buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM MgCl2, 1 mM DTT) and incubated for 90 minutes at 25°C. The reaction was stopped with Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 35 mM EDTA, 0.2% Coating Reagent 3). Results were read with an electrophoretic mobility shift platform and IC50 was calculated using a 4-parameter fit. [1][2]
ROCK2 assay: ROCK2 enzyme (0.006 ng/mL) was used under similar conditions. [1][2] AKT1-3 assays: Test compounds were diluted in 100% DMSO using 3-fold dilution steps. Final compound concentration ranged from 10 μM to 0.056 nM, with 1% final DMSO. Staurosporine was used as reference compound. [1][2] Kinome-wide selectivity profiling: BLU2864 was tested at 3 μM concentration across a panel of 400 human kinases using KINOMEscan methodology. For follow-up, Kd was determined for all non-mutant kinases bound with >90% occupancy at the 3 μM screening concentration. [1][2] PKA activity assay in kidney lysates: PKA activity was assayed using a 32P base and biotinylated Kemptide peptide substrate. 2 μg of protein lysate was incubated with γ32P-labeled phosphate and biotinylated Kemptide in the absence (basal activity) or presence of 0.01 mM cAMP (total activity). The 32P-labeled biotinylated substrate was recovered using perforated SAM2 Biotin Capture Membranes. PKA activity was measured by scintillation to determine radioactive counts/minute. [1] |
| Cell Assay |
PRKACA cellular activity assay (HTRF): Phosphorylation of VASP on Serine 157 was used as readout. Huh7 cells were plated at 2 × 10⁴ cells per well in 384-well plates in serum-free and phenol-free DMEM and incubated overnight. Test compounds (0.24% DMSO final) were added and cells incubated for 4 hours. Forskolin (5 μM final) was added and plates incubated for 30 minutes. Cells were lysed with lysis buffer containing Halt protease cocktail inhibitors. Lysate was transferred to a 384-well proxi plate and premixed antibody solution (phospho-VASP cryptate antibody and phospho-VASP d2 antibody) was added. Fluorescence emission at 665 nm and 620 nm was read on an EnVision instrument. [1][2]
In vitro three-dimensional cystogenesis assay: mIMCD3 cells were cultured in Matrigel in the presence of 100 μM N6,2'-O-dibutyryladenosine-3',5'-cAMP or 10 μM forskolin to induce cyst formation, in the presence or absence of BLU2864 (40 nM, 200 nM). Compounds or vehicle were added daily. Images were collected every 3 hours using an automated system that measures average cyst areas. [1] Ex vivo metanephric organ culture: Pkd1RC/RC metanephric organ cultures were treated with BLU2864 (200 nM). Cyst formation and CREB phosphorylation were assessed. [1] Western blot analysis: Cells or tissues were lysed in PhosphoSafe Extraction Reagent with Halt protease inhibitor cocktail. Proteins were resolved by SDS-PAGE and electrotransferred to nitrocellulose membranes. Immunodetection was performed using antibodies against PRKACA, pVASP, pCREB, PCNA, Src, p-Src, ERK1/2, p-ERK1/2, GSK3β, p-GSK3β, AKT, p-AKT, STAT3, p-STAT3, CREB, p-CREB, Pax2, AQP-2, p-AQP-2, Gli1, Gli2, Gli3, and loading controls. [1] Cell Viability Assay [1] Cell Types: mIMCD3 Cell Tested Concentrations: 40 nM and 200 nM Incubation Duration: 5 days Experimental Results: Forskolin-induced in vitro cyst formation of mIMCD3 cells cultured in Matrigel was inhibited at 40 and 200 nM concentrations, respectively. 72% and 100%, respectively, relative to the control. |
| Animal Protocol |
Animal/Disease Models: Pkd1RC/RC mice [1]
Doses: 45 mg/kg Route of Administration: po (oral gavage); 45 mg/kg; one time/day; 5 days Experimental Results: Kidney basis of BLU2864-treated mice compared with control group and total PKA activity were inhibited by 74% and 87%, respectively, at 3 h, and by 46% and 56%, respectively, at 15 h. Animal/Disease Models: Pkd1RC/RC mice [1] Doses: 30 mg/kg Doses: po (oral gavage); 30 mg/kg; one time/day; 5 days Experimental Results: At 15 weeks, BLU2864-treated mice had higher urine output than Control group. demonstrated lower kidney weight, kidney volume as a percentage of body weight, and cyst index. Compared with the control group, renal basal and total PKA activities were diminished by 69% and 84%, respectively, in BLU2864-treated mice. Animal/Disease Models: FLC PDX tumor-bearing mice [2] Doses: 30 mg/kg and 75 mg/kg Route of Administration: po (oral gavage); 30 mg/kg and 75 mg/kg; one time/day; 34-day Experimental Results: Day 3 At 34 days, tumor growth was inhibited by 48.5% (P=0.003) and 45.3% (P=0.0005), respectively. |
| ADME/Pharmacokinetics |
BLU2864 has a cLogP value of 3.4, topological polar surface area of 90.9 angstroms, plasma protein binding fraction of 98%, and is compliant with Lipinski's Rule of 5. [1]
Following oral administration of BLU2864 at 30 mg/kg and 45 mg/kg once daily in NOD-SCID mice, plasma concentrations peaked within 2-4 hours of dosing. [2] Following oral administration of BLU2864 at 30 mg/kg QD, phosphorylated VASP was reduced to 27% of baseline 2 hours after dosing and fully recovered by 24 hours. [2] In Pkd1RC/RC mice treated with BLU2864 (45 mg/kg daily for 5 days by oral gavage), plasma concentrations at 3 and 15 hours after the last dose were close to the IC90 and IC50 values, respectively. [1] |
| Toxicity/Toxicokinetics |
In a 28-day rat tolerability study, BLU2864 dose levels up to 15 mg/kg/day were well tolerated with no clinical signs noted. Animals in the top dose group (30 mg/kg) showed reduced activity after a few days, followed by death after 6-7 days. The cause of death was not identified. The highest tolerated dose of BLU2864 for more than 3 weeks of continuous dosing was established as 30 mg/kg QD in mice. [2]
In Pkd1RC/RC mice treated with BLU2864 (30 mg/kg daily by oral gavage from 4-16 weeks of age), weekly body weights were similar in treated and control groups, and no evidence of toxicity was associated with pharmacologic PKA downregulation. [1] |
| References | |
| Additional Infomation |
BLU2864 is a highly selective, ATP-competitive inhibitor of PRKACA, identified and optimized by Blueprint Medicines from a library of >10,000 agnostically designed kinase inhibitors. It is one of the first selective small molecule PRKACA enzyme inhibitors reported. BLU2864 has been used to validate PRKACA as a therapeutic target in fibrolamellar carcinoma (FLC) and polycystic kidney disease (PKD). In FLC PDX models, BLU2864 treatment reduced tumor growth by approximately 45% and reversed an FLC-specific gene signature. In Pkd1RC/RC mice, BLU2864 inhibited renal PKA activity and ameliorated polycystic kidney disease. [1][2]
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| Molecular Formula |
C24H19F3N4O2
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|---|---|
| Molecular Weight |
452.428475618362
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| Exact Mass |
452.146
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| Elemental Analysis |
C, 63.71; H, 4.23; F, 12.60; N, 12.38; O, 7.07
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| CAS # |
2810747-89-6
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| PubChem CID |
164603354
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
3.2
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
33
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| Complexity |
715
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CC1=CNC2=NC=CC(=C12)C3=NC=C(C=C3)C(=O)N[C@H]4[C@H](CC5=C4C(=CC(=C5)C(F)F)F)O
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| InChi Key |
VRPOVDLLZCQZEG-RXVVDRJESA-N
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| InChi Code |
InChI=1S/C24H19F3N4O2/c1-11-9-30-23-19(11)15(4-5-28-23)17-3-2-12(10-29-17)24(33)31-21-18(32)8-13-6-14(22(26)27)7-16(25)20(13)21/h2-7,9-10,18,21-22,32H,8H2,1H3,(H,28,30)(H,31,33)/t18-,21-/m0/s1
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| Chemical Name |
N-[(1R,2S)-5-(difluoromethyl)-7-fluoro-2-hydroxy-2,3-dihydro-1H-inden-1-yl]-6-(3-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)pyridine-3-carboxamide
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| Synonyms |
BLU2864; BLU-2864; BLU 2864; orb1819252; SCHEMBL27203788; SCHEMBL29854562;
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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) |
DMSO: ~100 mg/mL (221.0 mM)
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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 | 2.2103 mL | 11.0514 mL | 22.1029 mL | |
| 5 mM | 0.4421 mL | 2.2103 mL | 4.4206 mL | |
| 10 mM | 0.2210 mL | 1.1051 mL | 2.2103 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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