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Pralsetinib (GAVRETO; BLU667; BLU-667) is a novel, highly potent, selective, FDA approved RET inhibitor with anticancer activity. Its approval for the treatment of metastatic non-small cell lung cancer (NSCLC) exhibiting RET fusion was granted in 2020. At an IC50 of 0.3–0.4 nM, pralisetinib inhibits RET in WT RET, RET mutants V804L, V804M, M918T, and CCDC6-RET fusion. RET fusions, predicted resistant mutants, and mutations are all effectively and selectively inhibited by BLU-667. Lung and thyroid cancer are among the many malignancies that are primarily caused by RET fusions; our findings also indicate that certain colon and breast cancers are largely motivated by RET. Through concurrently focusing on the main driver and anticipated resistant mutations that make cancer cells immune to current, approved medication treatments. Pralsetinib (GAVRETO) was given accelerated approval by the Food and Drug Administration on September 4, 2020, for use in adult patients with metastatic RET fusion-positive non-small cell lung cancer (NSCLC) that was identified by an FDA-approved test.
Physicochemical Properties
| Molecular Formula | C27H32FN9O2 |
| Molecular Weight | 533.6005 |
| Exact Mass | 533.27 |
| Elemental Analysis | C, 60.77; H, 6.04; F, 3.56; N, 23.62; O, 6.00 |
| CAS # | 2097132-94-8 |
| Related CAS # | trans-Pralsetinib;2097132-93-7 |
| PubChem CID | 129073603 |
| Appearance | White to off-white solid powder |
| LogP | 3.1 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 8 |
| Heavy Atom Count | 39 |
| Complexity | 816 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | CC1=CC(=NN1)NC2=NC(=NC(=C2)C)C3CCC(CC3)(C(=O)N[C@@H](C)C4=CN=C(C=C4)N5C=C(C=N5)F)OC |
| InChi Key | GBLBJPZSROAGMF-SIYOEGHHSA-N |
| InChi Code | InChI=1S/C27H32FN9O2/c1-16-11-22(33-23-12-17(2)35-36-23)34-25(31-16)19-7-9-27(39-4,10-8-19)26(38)32-18(3)20-5-6-24(29-13-20)37-15-21(28)14-30-37/h5-6,11-15,18-19H,7-10H2,1-4H3,(H,32,38)(H2,31,33,34,35,36)/t18-,19?,27?/m0/s1 |
| Chemical Name | N-[(1S)-1-[6-(4-fluoropyrazol-1-yl)pyridin-3-yl]ethyl]-1-methoxy-4-[4-methyl-6-[(5-methyl-1H-pyrazol-3-yl)amino]pyrimidin-2-yl]cyclohexane-1-carboxamide |
| Synonyms | BLU-667; BLU 667; BLU667; Gavreto; Pralsetinib |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets |
RET (IC50 = 0.4 nM); RET V804L (IC50 = 0.3 nM); RET V804M (IC50 = 0.4 nM); RET M918T (IC50 = 0.4 nM); CCDC6-RET (IC50 = 0.4 nM) RET (Rearranged during transfection) (IC₅₀ = 0.4 nM for WT RET, 0.3–0.4 nM for RET V804L/V804M/M918T, 0.4 nM for CCDC6-RET fusion)[1] |
| ln Vitro |
BLU-667 is at least a hundred times more selective for RET than 96% of the kinases tested (371 kinases in total). BLU-667 selectively inhibits RET signaling in cancers with altered RET across various lineages. In comparison to multikinase inhibitors, BLU-667-mediated RET pathway inhibition also more effectively suppresses the proliferation of RET-modified cell lines[1]. BLU-667 inhibited RET autophosphorylation in KIF5B-RET Ba/F3 cells with a cellular IC₅₀ of 5 nM, which was at least 10-fold more potent than cabozantinib, vandetanib, and RXDX-105.[1] In RET-driven cell lines (LC2/ad, MZ-CRC-1, TT), BLU-667 inhibited phosphorylation of RET, SHC, and ERK1/2 at concentrations ≤10 nM.[1] BLU-667 decreased transcript levels of ERK-regulated genes DUSP6 and SPRY4 in a dose-dependent manner, without affecting GSK3B expression.[1] BLU-667 suppressed proliferation of RET-altered cell lines more potently than multikinase inhibitors (MKIs).[1] BLU-667 retained potent activity against RET gatekeeper mutants V804L, V804M, and V804E in cellular proliferation assays.[1] |
| ln Vivo |
BLU-667 effectively suppresses NSCLC and thyroid cancer xenograft growth in vivo when driven by different RET mutations and fusions, but does not inhibit VEGFR2. Throughout the in vivo trials, BLU-667 is well tolerated[1]. BLU-667 showed dose-dependent antitumor activity in KIF5B-RET Ba/F3 allografts and KIF5B-RET V804L allografts at doses as low as 10 mg/kg twice daily.[1] BLU-667 demonstrated potent activity in RET C634W MTC xenografts, KIF5B-RET NSCLC PDX, and CCDC6-RET colorectal cancer PDX models.[1] In KIF5B-RET NSCLC PDX, BLU-667 induced sustained inhibition of RET phosphorylation and downstream MAPK target genes DUSP6/SPRY4, unlike cabozantinib which showed transient suppression.[1] BLU-667 did not alter circulating VEGFA or soluble VEGFR2 levels in vivo, indicating no significant VEGFR2 inhibition.[1] |
| Enzyme Assay |
BLU-667 is tested for inhibitory activity against a panel of 371 kinases at 300 nmol/L. The 23 kinases that exhibit >50% inhibition at 300 nmol/L are chosen for complete 10-point concentration-response curves, utilizing BLU-667 (1 μmol/L maximum concentration) at 200 μmol/L ATP to produce biochemical IC50 (Reaction Biology Corp). The reaction is initiated with 33P-ATP (10 mCi/mL), and kinase activity is then detected through filter-binding methodology. A radiometric kinase activity assay was used to measure inhibition of 371 kinases by BLU-667 at 300 nM. IC₅₀ values were determined for kinases inhibited >50%.[1] BLU-667 was screened against a panel of kinases to assess selectivity; it showed >100-fold selectivity for RET over 96% of tested kinases.[1] |
| Cell Assay |
Following a 48-hour exposure to compound concentrations ranging from 25 µM to 95.4 pM, the proliferation of KIF5B-RET Ba/F3 cells is measured using Cell Titer Glo. BrdU incorporation is used to measure the proliferation of TT, MZ-CRC-1, TPC-1, or LC2/ad cells after four days of compound exposure. Ba/F3 cells expressing KIF5B-RET were treated with BLU-667 or comparators for 90 minutes, and RET autophosphorylation was measured using an AlphaLISA assay.[1] RET-driven cell lines were treated with BLU-667, and phosphorylation of RET, SHC, and ERK1/2 was assessed by immunoblotting.[1] For gene expression analysis, cells were treated for 7 hours, RNA was extracted, and qRT-PCR was performed for DUSP6, SPRY4, and GSK3B.[1] |
| Animal Protocol |
Mice: BALB/c nude mice are injected subcutaneously with TT cells, KIF5B-RET V804L Ba/F3 cells, or KIF5B-RET Ba/F3 cells into the right flank. Mice are given vehicle, 3 mg/kg, 10 mg/kg, or 30 mg/kg of Pralsetinib (Blu667) twice a day, or 60 mg/kg of XL184 or 60 mg/kg of Pralsetinib (Blu667; given orally) once a day for all experiments. Allograft and xenograft models were established in immunodeficient mice using RET-altered cell lines or patient-derived xenografts.[1] Mice were dosed twice daily with vehicle, BLU-667 (3, 10, or 30 mg/kg), or once daily with BLU-667 (60 mg/kg) or cabozantinib (60 mg/kg).[1] Tumor volumes were measured regularly, and tumor lysates were collected at specified time points for immunoblotting and qPCR analysis.[1] |
| Toxicity/Toxicokinetics |
In clinical cases, BLU-667 was well tolerated with only grade 1 adverse events reported, including transient leukopenia, nausea, hyperphosphatemia, constipation, dry skin, and rash.[1] |
| References |
[1]. Cancer Discov . 2018 Jul;8(7):836-849. |
| Additional Infomation |
Pralsetinib is a Kinase Inhibitor. The mechanism of action of pralsetinib is as a Rearranged during Transfection (RET) Inhibitor. See also: Pralsetinib (annotation moved to). BLU-667 is a highly potent and selective RET inhibitor designed to target oncogenic RET alterations, including fusions and mutations, with minimal off-target activity against VEGFR2.[1] In a phase I clinical trial, BLU-667 induced rapid and durable responses in patients with RET-altered NSCLC and MTC, including those previously treated with MKIs.[1] BLU-667 demonstrated activity against RET gatekeeper mutations that confer resistance to cabozantinib and vandetanib.[1] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~100 mg/mL (~187.4 mM) Water: <1 mg/mL Ethanol: ~13 mg/mL (~24.4 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.69 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: ≥ 2.5 mg/mL (4.69 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8741 mL | 9.3703 mL | 18.7406 mL | |
| 5 mM | 0.3748 mL | 1.8741 mL | 3.7481 mL | |
| 10 mM | 0.1874 mL | 0.9370 mL | 1.8741 mL |