PYK2 (IC50 = 637 nM)

In hMSC cultures, PF-4618433 (0.1-1.0 μM; 7 days) stimulates osteogenesis. Alkaline phosphatase (ALP) activity and mineralization are increased by PF-4618433 in a drug-dependent way [1]. At dosages of 0.1 and 0.3 μM, PF-4618433 (0.0125-0.3 μM; 14 or 21 days) improves calcium deposition [2]. μM; 24 hours) increases the proliferation of osteoblasts [2].

Crystallization—Crystallization of apo-PYK2, with the BIRB796, with PF-431396, and with the PF-4618433 (PF-46) inhibitor was achieved using a 5 mg/ml protein stock. For co-crystallization experiments, inhibitor (from a DMSO stock of 30 mm) was used to give a final concentration of between 0.5-1 mm. Hanging drops of 2 μl + 2 μl were setup over 750 μl of well solution in standard Linbro plates and incubated at 22 °C. Initial screening using Hampton screens produced a condition, 0.1 m bis-Tris: pH 6.0-7.0, 0.2 m MgCl2, 20-27% PEG 3350, 1 mm TCEP, which gave needle crystals that were optimized by refinement of the conditions as well as multiple rounds of streak and micro-seeding. For the inhibitor co-complex crystallization, the optimized crystals were further soaked overnight in 3 mm inhibitor to increase the occupancy of the inhibitor[1].

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Alkaline phosphatase (ALP) activity assay [2]
BMSC were plated at 4 × 10~4 cells and differentiated into mature osteoblasts with 50 μM ascorbic acid (AA) and 5 mM β-glycerol phosphate (β-GP) in the presence or absence of various concentration of PF-43 or PF-4618433 (PF-46) for 7 days. ALP activity assay was assayed by adding cell lysate to the ALP substrate containing 2 mg/mL p-nitrophenyl phosphate in 1.5 M alkaline buffer as previously reported. The enzymatic reaction was stopped by adding 20 mM NaOH, and optical absorbance at 405 nm was recorded using a spectrophotometer. ALP activity was normalized by total protein concentration using a Pierce™ BCA protein assay kit. Experiments was performed in triplicate and repeated three times.

Cell Proliferation Assay[2] Cell
Cell Types: mouse bone marrow-derived mesenchymal stem cells (BMSC)
Tested Concentrations: 0.1, 0.3 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Compared with the untreated group or control group, cell proliferation activity increased Dramatically .

[1]. Structural Characterization of Proline-Rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design. J Biol Chem. 2009 May 8; 284(19): 13193-201.

[2]. A Pyk2 Inhibitor Incorporated Into a PEGDA-gelatin Hydrogel Promotes Osteoblast Activity and Mineral Deposition. Biomed Mater. 2019 Feb 27; 14(2): 025015.

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic non-receptor tyrosine kinase involved in multiple signaling pathways. It is a negative regulator of bone formation and is considered a potential drug target for treating osteoporosis. We resolved the high-resolution structures of human PYK2 kinase domains in complexes with different inhibitors, confirming its classic bifoliate kinase structure and revealing conformational variability of the DFG ring. Our structural analysis explains the lack of selectivity of the classic kinase inhibitor PF-431396 within the FAK family. Importantly, novel DFG-out conformations induced by two diarylurea inhibitors (BIRB796 and PF-4618433) reveal a unique subclass of non-receptor tyrosine kinases characterized by the gating amino acid Met-502 and a unique hinge ring conformation Leu-504. This is the first instance in the DFG-out conformation where leucine residues in the hinge ring block the ATP binding site. Our structural, biophysical, and pharmacological studies have shown that the unique features of the DFG motif, including the variability of the Leu-504 hinge loop, can be used to develop selective protein kinase inhibitors. [1] Pyk2 is a non-receptor tyrosine kinase belonging to the focal adhesion kinase family. Studies in our lab and other labs have shown that mice lacking the Pyk2 gene (Ptk2B) have higher bone mass due to increased osteoblast activity and decreased osteoclast activity. It has been previously reported that a chemical inhibitor that targets both Pyk2 and its homologue FAK can increase bone formation in ovariectomized rats. In this study, we developed a hydrogel containing polyethylene glycol diacrylate (PEGDA) and gelatin that can be cured by visible light and is suitable for delivering small molecule drugs, including Pyk2-targeting chemical inhibitors. We characterized several key properties of the hydrogel, including viscosity, gel time, swelling, degradation, and drug release behavior. We found that the hydrogel composed of PEGDA1000 and 10% gelatin (P1000:G10) exhibited Bingham fluid properties, resisting free flow before in-situ polymerization, making it suitable as an injection carrier for the treatment of open wounds. The P1000:G10 hydrogel had good cell compatibility and slower drug release compared to other hydrogels we tested. Importantly, the Pyk2 inhibitor hydrogel maintained its inhibitory activity against Pyk2 tyrosine kinase and promoted osteoblast activity and mineral deposition in vitro. Overall, our results suggest that Pyk2 inhibitor-based hydrogels may be suitable for the treatment of craniofacial and limb bone defects and for targeted bone regeneration. [2]

C24H27N7O2

445.52

445.222

C, 64.70; H, 6.11; N, 22.01; O, 7.18

1166393-85-6

1166393-85-6

25203958

White to off-white solid powder

1.3±0.1 g/cm3

587.6±50.0 °C at 760 mmHg

309.2±30.1 °C

0.0±1.6 mmHg at 25°C

1.649

4.86

3

5

7

33

635

0

O=C(NC1C=C(COC2C=NC=CC=2)NN=1)NC1=CC(C(C)(C)C)=NN1C1C=CC(C)=CC=1

NJARPUHZDSAXPL-UHFFFAOYSA-N

InChI=1S/C24H27N7O2/c1-16-7-9-18(10-8-16)31-22(13-20(30-31)24(2,3)4)27-23(32)26-21-12-17(28-29-21)15-33-19-6-5-11-25-14-19/h5-14H,15H2,1-4H3,(H3,26,27,28,29,32)

1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[5-(pyridin-3-yloxymethyl)-1H-pyrazol-3-yl]urea

PF-4618433; PF4618433; 1-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(3-((pyridin-3-yloxy)methyl)-1H-pyrazol-5-yl)urea; 1-[5-tert-butyl-2-(4-methylphenyl)-1,2-dihydro-3H-pyrazol-3-ylidene]-3-{3-[(pyridin-3-yloxy)methyl]-1H-pyrazol-5-yl}urea; CHEMBL1084269; 1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[5-(pyridin-3-yloxymethyl)-1H-pyrazol-3-yl]urea; PF 4618433

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: ~100 mg/mL (~224.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.61 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.61 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.61 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)