PYK2 (IC50 = 637 nM)
PYK2 (proline-rich tyrosine kinase 2) – IC50 = 637 nM (enzyme assay). [1]
FAK (focal adhesion kinase) – no IC50 reported. [1]
p38 MAP kinase – reduced activity compared to BIRB796 (selectivity profiling showed improved selectivity over p38). [1]

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].

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PF-4618433 (637 nM IC50) exhibited improved PYK2 potency, reduced p38 activity, and superior overall kinase selectivity relative to BIRB796, as shown in a diverse kinase selectivity panel (percent inhibition at 10 μM). [1]
PF-4618433 treatment of hMSC cultures (days 1-7) produced a dose-dependent increase in both alkaline phosphatase activity and mineralization (calcein-labeled nodules), demonstrating osteogenic activity. [1]
PF-4618433 released from P1000:G10 hydrogel significantly inhibited Pyk2 tyrosine kinase activity at 0.1, 0.3 and 0.5 μM compared to no-drug control (p<0.05), with comparable efficacy to freshly prepared inhibitor. [2]
PF-4618433 (0.1 and 0.3 μM) significantly increased osteoblast precursor proliferation compared to untreated control (p<0.05), with 0.1 μM showing the highest activity. [2]
PF-4618433 (0.1 and 0.3 μM) significantly increased ALP activity in osteoblasts at day 7. [2]
PF-4618433 (0.1 and 0.3 μM) enhanced calcium deposition (mineralization) at day 21, whereas PF-431396 showed no effect on mineralization at these concentrations. [2]
Hydrogel-released PF-4618433 (0.1 and 0.5 μM) markedly enhanced ALP activity compared to control (p<0.05), with activity comparable to freshly prepared inhibitor. [2]
Hydrogel-released PF-4618433 (0.5 μM) enhanced Ca²⁺ nodule formation (Alizarin Red S staining), indicating increased osteoblastic differentiation and mineralization. [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.

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PYK2 kinase inhibition assay: Kinase assays for PYK2 were performed using unactivated full-length, C-terminal his-tagged human PYK2 protein. Test compounds were preincubated with enzyme for 1 h prior to the addition of 500 μM ATP. IC50 determinations were generated from 8-point dose response curves. Selectivity profiling against a diverse panel of kinases was performed and reported as average percent inhibition of duplicate tests at 10 μM. [1]
Surface plasmon resonance (SPR) kinetics: PYK2 catalytic domain was biotinylated and immobilized on a neutravidin surface. Kinetic analyses were performed at 25°C in 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 1 mM MnCl₂, 0.05% Tween 20, 3% DMSO. Compounds were injected at 50-100 μL/min. PF-4618433 bound with on-rate constants of 10³/Ms (characteristic of DFG-out inhibitors). Data were processed using Scrubber 2 software and kinetic analyses performed using BiaEval software. [1]
NMR spectroscopy: ¹⁵N-Phe-labeled PYK2 kinase domain (100 μM) was analyzed by TROSY spectra in the absence and presence of PF-4618433 (150 μM). The compound stabilized the DFG-out conformation, resulting in sharpening of TROSY spectra and appearance of the missing Phe peak in the DFG loop. [1]
Kinase inhibition assay (PYK2 expressed in cells): 293VnR cells were transfected with Pyk2 cDNA, treated with released PF-4618433 (0.1, 0.3, 0.5 μM) for 2 hours, then Pyk2 was immunoprecipitated and a tyrosine kinase activity assay was performed using a commercial Universal Tyrosine Kinase Assay Kit following the manufacturer's protocol. [2]

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 .

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Proliferation assay (MTS): BMSCs were plated at 2×10³ cells/well in 96-well plates with PF-4618433 (0-0.3 μM) for 24 hours. MTS reagent was added and absorbance measured at 490 nm. Experiments performed in triplicate and repeated three times. [2]
ALP activity assay: BMSCs (4×10⁴ cells) were differentiated with ascorbic acid (50 μM) and β-glycerophosphate (5 mM) in presence or absence of PF-4618433 (0.1, 0.3 μM) for 7 days. Cell lysates were assayed with p-nitrophenyl phosphate substrate (2 mg/mL in 1.5 M alkaline buffer); reaction stopped with 20 mM NaOH; absorbance at 405 nm. ALP activity normalized to total protein by BCA assay. [2]
Mineralization assay (Alizarin Red S): BMSCs were differentiated for up to 21 days with PF-4618433 (0.1, 0.3 μM), fixed and stained with 40 mM Alizarin Red S (pH 4.2). Bound stain was extracted with 1% cetyl pyridinium chloride in 10 mM sodium phosphate (pH 7.0) and absorbance measured at 562 nm. [2]
Cytotoxicity assay (ISO 10993-5): MC3T3-E1 cells (1.5×10³/well) were cultured with eluates from hydrogels for 24 hours, then MTS assay performed. Cell viability ≥70% considered non-cytotoxic. [2]
Hydrogel drug release: AMC or PF-4618433-loaded hydrogels were soaked in PBS at 37°C, aliquots collected at various time points, and fluorochrome concentration measured. Release profile followed Fickian diffusion (Higuchi equation, R²>0.96). PF-4618433 release from P1000:G10 showed initial burst (1-4 h) followed by slower release, with 100% release achieved by days 3-4. [2]

PF-4618433 is a diarylurea DFG-out inhibitor of PYK2, derived from optimization of BIRB796. The compound features a pyridinyloxymethylpyrazole moiety replacing the N-morpholinylethoxynaphthalene of BIRB796, which improved PYK2 potency (IC50 = 637 nM) and reduced p38 activity. In the co-crystal structure with PYK2 (1.95 Å resolution, PDB code 3FZT), the pyrazole ring rotates ~40° relative to the naphthyl ring in BIRB796 to form a hydrogen bond with catalytic Lys-457. Unlike BIRB796, Leu-504 remains in a similar conformation to the apo structure, allowing the pyridyl nitrogen to form a hydrogen bond with the backbone amide of Tyr-505 in the hinge loop. Three additional residues in the activation loop (Leu-570-Arg-572 following the DFG motif) are visible in the co-crystal structure, and the guanidium group of Arg-572 is stabilized by a stacking interaction with the tolyl group of PF-4618433. The compound binds with slow on-rate kinetics (10³/Ms) characteristic of DFG-out inhibitors. Selectivity profiling showed improved overall selectivity compared to BIRB796 and classical inhibitors such as PF-431396. PF-4618433 promotes osteogenesis in hMSC cultures and has been incorporated into a PEGDA-gelatin hydrogel (P1000:G10) for controlled release, with the released compound retaining its bioactivity. [1][2]

[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]

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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.)