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 implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.[1]

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Pyk2 is a non-receptor tyrosine kinase that belongs to the family of focal adhesion kinases. Studies from our laboratory and others demonstrated that mice lacking the Pyk2 gene (Ptk2B) have high bone mass, which was due to increased osteoblast activity, as well as decreased osteoclast activity. It was previously reported that a chemical inhibitor that targets both Pyk2 and its homolog FAK, led to increased bone formation in ovariectomized rats. In the current study, we developed a hydrogel containing poly(ethylene glycol) diacrylate (PEGDA) and gelatin which was curable by visible-light and was suitable for the delivery of small molecules, including a Pyk2-targeted chemical inhibitor. We characterized several critical properties of the hydrogel, including viscosity, gelation time, swelling, degradation, and drug release behavior. We found that a hydrogel composed of PEGDA1000 plus 10% gelatin (P1000:G10) exhibited Bingham fluid behavior that can resist free flowing before in situ polymerization, making it suitable for use as an injectable carrier in open wound applications. The P1000:G10 hydrogel was cytocompatible and displayed a more delayed drug release behavior than other hydrogels we tested. Importantly, the Pyk2-inhibitor-hydrogel retained its inhibitory activity against the Pyk2 tyrosine kinase, and promoted osteoblast activity and mineral deposition in vitro. Overall, our findings suggest that a Pyk2-inhibitor based hydrogel may be suitable for the treatment of craniofacial and appendicular skeletal defects and 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.)