PRKACA (protein kinase cAMP-activated catalytic subunit alpha) – IC50 = 1 nM (biochemical assay); Kd = 4 nM; cellular IC50 = 25.0 nM (inhibition of VASP Ser157 phosphorylation in forskolin-stimulated Huh7 cells) [1]
ROCK2 – IC50 = 83.1 nM (83-fold selective over PRKACA) [1]
AKT1 – IC50 = 1540 nM; AKT2 – IC50 = 3780 nM; AKT3 – IC50 = 397 nM [1]

BLU0588 inhibited PRKACA catalytic activity with an IC50 of 1 nM in biochemical assays using Kemptide peptide substrate and Km concentrations of ATP (5 μM). [1]
BLU0588 inhibited PRKACA cellular activity with an IC50 of 25.0 nM, determined from inhibition of VASP Ser157 phosphorylation in forskolin-stimulated Huh7 cells. [1]
In FLC PDX spheroid cultures, BLU0588 treatment led to a dose-dependent reduction in phosphorylated VASP, indicative of inhibited PRKACA signaling. Proliferation was not substantially inhibited by BLU0588 treatment in the FLC PDX spheroid model. [1]
BLU0588 treatment (1.5 μM for 1 day or 14 days) reversed an FLC-specific gene signature, leading to downregulation of genes overexpressed in FLC (CPS1 and G6PC) and upregulation of genes underexpressed in FLC (PYGL and SHH). The IC50 values for these gene expression effects were between 55 nM and 180 nM. [1]
BLU0588 modulated gene expression in FLC PDX cells, with 817 protein-coding genes differentially expressed vs DMSO control (323 negative fold changes, 494 positive fold changes). The list of BLU0588-modulated genes showed significant overlap with PRKACA shRNA-modulated genes (N = 206, Fisher’s exact test P < 2.2 × 10⁻¹⁶), defining a signature of PRKACA inhibition. [1]
BLU0588 demonstrated good to moderate selectivity against closely related AGC kinase family members and excellent overall kinome selectivity, with an S(10) selectivity score of 0.047. Kinome-wide selectivity was assessed at 3 μM across a panel of 400 human kinases. At the 3 μM screening concentration, BLU0588 bound with >90% occupancy to a limited set of kinases. Kd measurement showed most potent binding affinity to PRKACA at 4 nM, with Kd < 100 nM for only 9 non-mutant kinases. [1]

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BLU0588 (1.5 μM, 1 day or 14 days) reverses gene signatures specific to FLC, leading to the downregulation of FLC-overexpressed genes (like CPS1 and G6PC, top) and FLC-underexpressed genes [1]. BLU0588 (0-312.5 nM) dose-dependently lowers pVASP in FLC PDX cells[1].

In mice harboring FLC PDX tumors, oral administration of BLU0588 at 30 mg/kg once daily (QD) for 34 days inhibited tumor growth by 48.5% (P = .003) compared to vehicle control. [1]
In pharmacokinetic/pharmacodynamic studies, BLU0588 at 30 mg/kg QD reduced phosphorylated VASP to 19% of baseline phosphorylation levels 4 hours after dosing; at 75 mg/kg QD, phosphorylated VASP was reduced to 4% of baseline. Phosphorylated VASP levels fully recovered by 24 hours post-administration of 30 mg/kg QD. [1]

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The oral BLU0588 (30-75 mg/kg) efficiently suppresses PRKACA downstream signaling and, within 24 hours, phosphorylated VASP levels recover to baseline levels[1]. Over the course of three weeks, mice were given a maximum tolerated dose of 30 mg/kg QD of BLU0588[1]. In mice, BLU0588 (30 mg/kg, once day, oral) suppresses the formation of tumors[1].

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]
ROCK2 assay: ROCK2 enzyme (0.006 ng/mL) was used under similar conditions. [1]
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]
Kinome-wide selectivity profiling: BLU0588 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]

PRKACA cellular activity assay (HTRF): Phosphorylation of VASP on Serine 157 was used as readout of PRKACA cellular activity. Huh7 cells were plated at 2 × 10⁴ cells per well in 384-well optiwell 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 cryptic antibody and phospho-VASP d2 antibody) was added. Fluorescence emission at 665 nm and 620 nm was read on an EnVision instrument. [1]
FLC PDX spheroid culture: FLC PDX tumors were dissociated with collagenase B, mouse cells depleted using a Mouse Cell Depletion Kit. Dissociated cells were cultured as spheroids in DMEM/F-12 containing 20 ng/mL EGF, 10 ng/mL FGF, 2% B27, and 1% N2 on low-attachment plates. For BLU0588 treatment, spheroids were treated with BLU0588 for 4 hours (for Western blot) or 14 days (for proliferation and gene expression). [1]
Cell proliferation assay: FLC PDX spheroids were treated with BLU0588 at a range of concentrations, staurosporine, or DMSO vehicle. Proliferation was measured after 14 days using CellTiter-Glo Luminescent Cell Viability Assay. [1]
Quantitative PCR: Total RNA was extracted using RNeasy Plus Mini Kit and reverse transcribed to cDNA. Expression of PYGL, SHH, CPS1, and G6PC was detected using TaqMan Gene Expression Assays. Relative quantification was calculated using the 2⁻ΔΔCt method and normalized to housekeeping control. [1]
Transcriptome sequencing: RNA sequencing was performed on FLC PDX cells treated with BLU0588 or DMSO. Differential gene expression was assessed using DESeq2 with false discovery rate threshold of 0.01 and absolute log2 fold change of 1. [1]
Western blot analysis: Cells were lysed in PhosphoSafe Extraction Reagent with Halt protease inhibitor cocktail. Proteins were resolved by SDS-PAGE on 20% gradient gels and electrotransferred to nitrocellulose membranes. Immunodetection was performed using primary antibodies against PRKACA, pVASP, pCREB, ROCK, and β-actin loading control. [1]

Animal/Disease Models: female NOD-SCID (severe combined immunodeficient) mouse harboring FLC PDX tumors (6-8weeks old, FLC PDX shRNA cell lines or Hep3B cells were implanted)[1]
Doses: 30 mg/kg
Route of Administration: Orally, one time/day, 34 days
Experimental Results: Inhibited tumor growth in mice, by day 34 tumor growth was inhibited by 48.5%.

Following oral administration of BLU0588 at 30 mg/kg and 75 mg/kg once daily in mice, plasma concentrations peaked within 2-4 hours of dosing. For BLU0588-treated mice, phosphorylated VASP was reduced to 19% and 4% of baseline phosphorylation levels with 30 mg/kg QD and 75 mg/kg QD, respectively, 4 hours after dosing; phosphorylated VASP levels fully recovered by 24 hours post-administration of 30 mg/kg QD. [1]

In a 28-day rat tolerability study, 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 in these animals was not identified. The highest tolerated dose of BLU0588 for more than 3 weeks of continuous dosing was established as 30 mg/kg QD in mice. [1]

[1]. Evaluation of Protein Kinase cAMP-Activated Catalytic Subunit Alpha as a Therapeutic Target for Fibrolamellar Carcinoma. Gastro Hep Advances. 2022 Nov 8;2(3):307-321.

BLU0588 is a novel selective PRKACA small molecule kinase inhibitor developed through iterative medicinal chemistry optimization from screening hits identified from a proprietary library of more than 10,000 chemically diverse kinase inhibitors. It is an ATP-competitive inhibitor. BLU0588 treatment led to a dose-dependent reduction in phosphorylated VASP in FLC PDX spheroid cultures, and in vivo treatment reduced FLC PDX tumor growth by approximately 50%. BLU0588 and BLU2864 are the first selective small molecule PRKACA enzyme inhibitors reported. [1]

C26H25N5O

423.509605169296

423.205

2810747-78-3

164603520

White to yellow solid powder

3.3

2

4

4

32

667

2

C1CCN(C1)[C@@H]2CC3=CC=CC=C3[C@H]2NC(=O)C4=CN=C(C=C4)C5=C6C=CNC6=NC=C5

YXSWSKFLTNJZEO-DNQXCXABSA-N

InChI=1S/C26H25N5O/c32-26(18-7-8-22(29-16-18)20-9-11-27-25-21(20)10-12-28-25)30-24-19-6-2-1-5-17(19)15-23(24)31-13-3-4-14-31/h1-2,5-12,16,23-24H,3-4,13-15H2,(H,27,28)(H,30,32)/t23-,24-/m1/s1

N-[(1R,2R)-2-pyrrolidin-1-yl-2,3-dihydro-1H-inden-1-yl]-6-(1H-pyrrolo[2,3-b]pyridin-4-yl)pyridine-3-carboxamide

BLU0588; BLU 0588; BLU-0588; orb1819253; SCHEMBL27203841;

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.)