FAK/focal adhesion kinase (IC50 = 2 nM); PYK2/proline-rich tyrosine kinase 2 (IC50 = 11 nM); BRD4 (Kd = 445 nM)

A PYK2 Inhibitor Increases Bone Formation and Prevents Bone Loss in OVX Rats. [3]
We next tested whether the pharmacological modulation of PYK2 activity may impact bone mass in the OVX rats, an established preclinical disease model of postmenopausal osteoporosis, using PF-431396 (PF-46), a potent pyrimidine-based PYK2 inhibitor having an IC50 of 31 nM against the recombinant PYK2 enzyme (Fig. 5A and SI Methods). Four-month-old OVX rats were treated daily for 28 days with vehicle, PF-431396 (PF-46) (10 and 30 mg/kg), or EE, an antiresorptive agent. Pharmacokinetic studies indicated that the free plasma concentration of PF-431396 covers the PYK2 IC50 for at least 8 h at the high dose (data not shown). As shown in μCT images of the distal femur metaphases (Fig. 5B), vehicle-treated OVX rats had less trabecular bone mass than the sham controls. Both EE and PF-431396 counteracted OVX-induced bone loss, completely preserving total bone content and total bone density (Fig. 5 C and D). In agreement with previous studies, the vehicle-treated OVX rats exhibited high bone turnover characterized by increased bone formation (mineralizing surface per bone surface and bone formation rate per bone surface) and bone resorption (osteoclast surface and serum CTX) compared with sham controls (Table 1 and Fig. 5 E and F). Treatment of OVX rats with EE suppressed the high bone turnover as evidenced by decreased bone resorption and formation relative to vehicle treatment. In contrast to EE, both doses of PF-431396 significantly increased bone formation rate, which was accompanied by an elevation in mineralizing surface and mineral apposition rate (Table 1 and Fig. 5E), suggesting that PF-431396 promotes osteoblast recruitment and activity. Consistent with this, PF-431396 increased alkaline phosphatase activity in 7-day hMSC cultures (P.C.B. and L.B., data not shown). Although high-dose PF-431396 decreased osteoclast surface (a referent parameter of bone resorption) at the proximal tibia, it did not alter serum CTX (a systemic biomarker of bone resorption) at either dose after both 2 weeks (data not shown) and 4 weeks (Fig. 5F) of treatment. These results showed that PF-431396, a potent PYK2 inhibitor, prevents bone loss induced by estrogen deficiency in rats primarily by stimulating bone formation, providing independent pharmacological confirmation for the function of PYK2 in regulating bone formation.

IC50 Determination for the PYK2 Inhibitor.[3]
A reaction mixture containing 150 pM PYK2 domain enzyme, 30 mM peptide substrate, and 10 mM DTT in assay buffer [50 mM Hepes (pH 7.0), 1 mM MgCl2, and 0.1% BSA] was dispensed to all wells of a 384-well assay plate. Compounds diluted in dimethyl-sulfoxide (DMSO) were dispensed in triplicate into the assay plate. Included in each assay was a DMSO-only (positive) control and a potent PYK2 inhibitor (negative) control used to set maximum and minimum responses, respectively. ATP diluted in assay buffer was dispensed to the entire plate to a final concentration of 50 mM (»kM for ATP). The assay plate was then incubated at 30°C for 2 h. A stop/detection mixture containing 45 mM EDTA, 10´ PTK green tracer, and 10´ antiphosphotyrosine antibody was added to each well of the plate. The detection reaction was incubated at room temperature in the dark for 1 h, and then fluorescence polarization was read on a Analyst GT by using a fluorescence polarization protocol (485-nm excitation and 530-nm emission with a 505-nm dichroic filter). IC50 determination was calculated from the dose-response curve by using GraphPad Prism 4 or similar proprietary software and fitting to a sigmoidal dose-response curve.

Cell Spreading [2]
Tissue culture plates were coated overnight at 4 °C with a rat anti-mouse LFA-1 monoclonal Ab or with fibronectin and then blocked with phosphate-buffered saline containing 2% bovine serum albumin for 1 h. A20 cells (105 cells in 0.5 ml of RPMI 1640 medium with 2% fetal calf serum and 50 μm 2-mercaptoethanol) were pretreated with DMSO or PF-431396 for 45 min, added to the coated wells, and incubated at 37 °C. Cells scored as spread were phase dark and had an elongated or irregular shape with obvious membrane processes.

Sprague–Dawley female rats were used for the PYK2 pharmacology studies. Rats were sham-operated or ovariectomized at 4.5–5 months of age. Beginning the day after surgery, animals were dosed every day by oral gavage with either vehicle (20% β-cyclodextrin in water) or the PYK2 inhibitor (PF-431396 (PF-46)) at 10 or 30 mg/kg in vehicle for 28 consecutive days.[3]

[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]. B cell receptor-induced phosphorylation of Pyk2 and focal adhesion kinase involves integrins and the Rap GTPases and is required for B cell spreading. J Biol Chem. 2009 Aug 21;284(34):22865-77.
[3]. Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis. Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10619-24.

N-methyl-N-[2-[[[2-[(2-oxo-1,3-dihydroindol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]methanesulfonamide is a sulfonamide.

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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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Signaling by the B cell receptor (BCR) promotes integrin-mediated adhesion and cytoskeletal reorganization. This results in B cell spreading, which enhances the ability of B cells to bind antigens and become activated. Proline-rich tyrosine kinase (Pyk2) and focal adhesion kinase (FAK) are related cytoplasmic tyrosine kinases that regulate cell adhesion, cell morphology, and cell migration. In this report we show that BCR signaling and integrin signaling collaborate to induce the phosphorylation of Pyk2 and FAK on key tyrosine residues, a modification that increases the kinase activity of Pyk2 and FAK. Activation of the Rap GTPases is critical for BCR-induced integrin activation as well as for BCR- and integrin-induced reorganization of the actin cytoskeleton. We now show that Rap activation is essential for BCR-induced phosphorylation of Pyk2 and for integrin-induced phosphorylation of Pyk2 and FAK. Moreover Rap-dependent phosphorylation of Pyk2 and FAK required an intact actin cytoskeleton as well as actin dynamics, suggesting that Rap regulates Pyk2 and FAK via its effects on the actin cytoskeleton. Importantly B cell spreading induced by BCR/integrin co-stimulation or by integrin engagement was inhibited by short hairpin RNA-mediated knockdown of either Pyk2 or FAK expression and by treatment with PF-431396, a chemical inhibitor that blocks the kinase activities of both Pyk2 and FAK. Thus Pyk2 and FAK are downstream targets of the Rap GTPases that play a key role in regulating B cell morphology.[2]

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Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.[3]

C22H21F3N6O3S

506.5

506.134

C, 52.17; H, 4.18; F, 11.25; N, 16.59; O, 9.48; S, 6.33

717906-29-1

11598628

Beige solid powder

1.5±0.1 g/cm3

1.643

1.36

3

11

7

35

854

0

S(C([H])([H])[H])(N(C([H])([H])[H])C1=C([H])C([H])=C([H])C([H])=C1C([H])([H])N([H])C1C(C(F)(F)F)=C([H])N=C(N=1)N([H])C1C([H])=C([H])C2=C(C=1[H])C([H])([H])C(N2[H])=O)(=O)=O

POJZIZBONPAWIV-UHFFFAOYSA-N

InChI=1S/C22H21F3N6O3S/c1-31(35(2,33)34)18-6-4-3-5-13(18)11-26-20-16(22(23,24)25)12-27-21(30-20)28-15-7-8-17-14(9-15)10-19(32)29-17/h3-9,12H,10-11H2,1-2H3,(H,29,32)(H2,26,27,28,30)

N-Methyl-N-[2-[[[2-[(2,3-dihydro-2-oxo-1H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]methanesulfonamide

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)

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.)