A Benzothiophene Inhibitor of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Inhibits Tumor Necrosis Factor Production and Has Oral Anti-Inflammatory Efficacy in Acute and Chronic Models of Inflammation

Activation of the p38 kinase pathway in immune cells leads to the transcriptional and translational regulation of proinflammatory cytokines. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), a direct downstream substrate of p38 kinase, regulates lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF ) and interleukin-6 (IL-6) production through modulating the stability and translation of these mRNAs. Developing small-molecule inhibitors of MK2 may yield anti-inflammatory efficacy with a different safety profile relative to p38 kinase inhibitors. This article describes the pharmacologic properties of a benzothiophene MK2 inhibitor, PF3644022 [(10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12tetrahydro-8H-[1,4]diazepino[5 ,6 :4,5]thieno[3,2-f]quinolin-8one]. PF-3644022 is a potent freely reversible ATP-competitive compound that inhibits MK2 activity (Ki 3 nM) with good selectivity when profiled against 200 human kinases. In the human U937 monocytic cell line or peripheral blood mononuclear cells, PF-3644022 potently inhibits TNF production with similar activity (IC50 160 nM). PF-3644022 blocks TNF and IL-6 production in LPS-stimulated human whole blood with IC50 values of 1.6 and 10.3 M, respectively. Inhibition of TNF in U937 cells and blood correlates closely with inhibition of phospho-heat shock protein 27, a target biomarker of MK2 activity. PF-3644022 displays good pharmacokinetic parameters in rats and is orally efficacious in both the rat acute LPS-induced TNF model and the chronic streptococcal cell wall-induced arthritis model. Dose-dependent inhibition of TNF production in the acute model and inhibition of paw swelling in the chronic model is observed with ED50 values of 6.9 and 20 mg/kg, respectively. PF-3644022 efficacy in the chronic inflammation model is strongly correlated with maintaining a Cmin higher than the EC50 measured in the rat LPS-induced TNF model. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by an imbalance of proinflammatory and anti-inflammatory cytokines, autoimmunity, joint inflammation, and eventual joint destruction (McInnes and Schett, 2007). Evidence supporting the role of the proinflammatory cytokines TNF , IL-1 , and IL-6 has been demonstrated in both animal models and human clinical trials (Dayer et al., 2001; Scott and Kingsley, 2006; Hennigan and Kavanaugh, 2008). Use of biologic therapeutics that neutralize these cyThis study was sponsored by Pfizer Inc. Portions of this work were presented as: Daniels JS, Lai Y, Davis JW, South SA, Stevens JC, Mourey RJ, and Anderson DR (2008) Inhibition of hepatobiliary transporters by a novel kinase inhibitor contributes to liver toxicity in nonclinical species at Great Lakes Drug Metabolism Discussion Group Meeting; 2008 May 1–2; Indianapolis, IN. Great Lakes Drug Metabolism Discussion Group, University of Michigan, Ann Arbor, MI. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.110.166173. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: MAPK, mitogen-activated protein kinase; MAPKAP, MAPK-activated protein; MK2, MAPKAP kinase 2; MNK, MAPK-interacting kinase; PF-3644022, (10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8H-[1,4]diazepino[5 ,6 :4,5]thieno[3,2-f]quinolin-8-one; RA, rheumatoid arthritis; TNF, tumor necrosis factor; IL-1, interleukin-1; PRAK, p38-regulated and activated kinase; LPS, lipopolysaccharide; SCW, streptococcal cell wall; rSCW, rat SCW; MSK, mitogenand stress-activated protein kinase; HSP27, heat shock protein 27; hPBMC, human peripheral blood mononuclear cell; HWB, human whole blood; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PK-PD, pharmacokinetic-pharmacodynamic; BE, biochemical efficiency; DMSO, dimethyl sulfoxide; JNK, c-Jun N-terminal kinase; ERK, extracellular signalregulated kinase; AUC, area under the curve; CL, clearance; ff, free fraction. 0022-3565/10/3333-797–807$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 333, No. 3 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 166173/3590513 JPET 333:797–807, 2010 Printed in U.S.A. 797 http://jpet.aspetjournals.org/content/suppl/2010/03/17/jpet.110.166173.DC1 Supplemental material to this article can be found at: at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from tokines has shown some clinical success in reducing joint pain and inflammation while retarding joint destruction (Smolen and Steiner, 2003). Limits to the use of biologics in RA include the high cost of protein pharmaceuticals, parenteral administration, loss of efficacy over time, risk of infection, and a significant portion of patients who show partial or no response to these agents. Therefore, development of orally active small-molecule inhibitors that target signaling pathways regulating inflammatory cytokine production could add significant value to unmet medical need. In 1994, p38 kinase was identified as a target that regulates inflammatory cytokine biosynthesis (Lee et al., 1994). Since then, kinases have been hotly pursued as drugable targets that regulate inflammation signaling pathways (Gaestel et al., 2007, 2009). Activation of the p38 kinase pathway in immune cells leads to the transcriptional and translational regulation of proinflammatory cytokine synthesis (Winzen et al., 1999). Many p38 kinase inhibitors have subsequently been developed that demonstrate inhibition of TNF , IL-1 , and IL-6 production and display anti-inflammatory efficacy in animal models (Pettus and Wurz, 2008). In clinical trials, many p38 kinase inhibitors were discontinued because of unacceptable safety profiles, namely elevated liver enzymes with significant incidence of skin rash (Dominguez et al., 2005). Of the compounds that advanced to testing in RA patients, initial short-term efficacy was observed, but was subsequently lost with further treatment presumably because of feedback control of the p38 kinase network (Hammaker and Firestein, 2010). It is possible, therefore, that other targets upstream or downstream in the p38 kinase pathway may avoid this feedback loop while exhibiting enhanced safety. Activated p38 kinase directly phosphorylates and activates the mitogen-activated protein kinase-activated protein (MAPKAP) kinases MK2, MK3, and MK5 (also known as PRAK) (Gaestel, 2006). Before 1999, it was unclear which downstream p38 kinase pathway components regulated TNF production. In MK2( / ) knockout mice, TNF levels were reduced approximately 90% through a post-transcriptional mechanism, demonstrating that MK2 is essential for lipopolysachharide (LPS)-induced TNF biosynthesis (Kotlyarov et al., 1999). IL-6 and interferonwere also significantly reduced, whereas IL-1 showed only modest reduction (Kotlyarov et al., 1999). MK2 regulates LPS-stimulated TNF and IL-6 production through modulating the stability and translation of TNF and IL-6 mRNAs via AU-rich elements in the 3 untranslated region (Neininger et al., 2002). Interestingly, MK3( / ) mice showed little reduction in LPS-stimulated TNF levels, whereas the MK2( / )MK3( / ) double knockout mouse exhibited complete inhibition, indicating that MK2 is the major MAPKAP kinase regulating TNF production (Ronkina et al., 2007). No effect on TNF synthesis or other cytokines was observed in PRAK( / ) mice (Shi et al., 2003). MK2( / ) mice were also resistant to collageninduced arthritis in a murine model of RA (Hegen et al., 2006). MK2, therefore, is an attractive target for development of anti-inflammatory kinase inhibitors. The MK2 knockout mouse has been useful in defining MK2’s role in inflammation. Having a potent and selective MK2 kinase inhibitor as an investigative tool, however, would be advantageous for further exploring the biology of MK2 and the p38 kinase pathway. Potent MK2 inhibitors have been recently described (Anderson et al., 2005, 2007, 2009a,b; Trujillo et al., 2007; Wu et al., 2007; Goldberg et al., 2008; Schlapbach et al., 2008; Xiong et al., 2008; Keminer et al., 2009), but few show nanomolar potency in cells (Schlapbach et al., 2008; Anderson et al., 2009b). Developing potent, selective MK2 inhibitors that have optimized pharmacologic properties for activity in blood or in vivo has been extremely difficult, with just one compound from the pyrrolopyridine series reported to have oral efficacy in blocking TNF production in LPS-challenged rats (Anderson et al., 2007). PF-3644022 [(10R)-10-methyl-3-(6-methylpyridin-3-yl)-9, 10,11,12-tetrahydro-8H-[1,4]diazepino[5 ,6 :4,5]thieno[3,2-f] quinolin-8-one] represents a potent and selective benzothiophene MK2 inhibitor, the first MK2 inhibitor described with oral efficacy in both acute and chronic models of inflammation. This ATP-competitive compound potently inhibits MK2 enzyme activity with good selectivity across 200 human kinases. PF-3644022 potently inhibits LPS-stimulated TNF production in cells and blood and when dosed orally in LPSchallenged rats. PF-3644022 exhibits good pharmacokinetic properties, demonstrating efficacy in the streptococcal cell wall (SCW)-induced arthritis model. Materials and Methods Preparation of PF-3644022. PF-3644022 was prepared by the Pfizer Discovery Medicinal Chemistry Department (Chesterfield, MO) as described (Anderson et al., 2009b). The molecular weight and formula of the parent compound are 374.47 g/mol and C21H18N4OS. The free base form was used for all dosing studies and calculation of solution concentration. Fresh 10 mM PF-3644022 stock concentrations were made in 100% dimethyl sulfoxide (DMSO) to support enzyme and cell studies and kept at room temperature for no more than 2 weeks. Generation of Recombinant Protein Kinases. MAPKAP kinase family recombinant proteins were generated in-house. N-terminally truncated MK2 (amino acids 45–400) was expressed and purified as described (Schindler et al., 2002). To support compound binding studies to MK2, N-terminally biotinylated MK2 (amino acids 45–371) was expressed in Escherichia coli by using the BirA expression system (Smith et al., 1998). MK3 (GenBank accession no. U43784), PRAK (GenBank accession no. AF032437), and MNK1 (GenBank accession no. AB000409) were expressed in E. coli as glutathione S-transferase fusion proteins and affinity-purified over glutathione-Sepharose (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The glutathione S-transferase purification tag was removed by thrombin cleavage, and the proteins were further purified to homogeneity over Mono Q-sepharaose (GE Healthcare). The C-terminal kinase domains of MSK1 (amino acids 367–802) (GenBank accession no. AF074393) and MSK2 (amino acids 351–772) (GenBank accession no. AJ010119) were expressed as N-terminal 6 His-tagged fusion proteins in baculovirus-infected Sf9 insect cells. MSK1 and MSK2 were purified to near homogeneity over Ni-NTAagarose (QIAGEN, Valencia, CA). In Vitro Kinase Assays and Inhibition Kinetics. Activated p38 prepared according to Hope et al. (2009) was used to activate recombinant MAPKAP kinases by incubation at a 1:50 M ratio with 250 M ATP for 1 h at 30°C. The kinase activity of MK2 was followed by using fluorescently labeled heat shock protein 27 (HSP27) peptide (fluorescein isothiocyanate-KKKALSRQLSVAA) and a Caliper LabChip 3000 (Caliper Life Sciences, Hopkinton, MA). Phosphorylated peptide was separated from substrate peptide electrophoretically and quantified. All kinase reactions were performed at room temperature in 20 mM HEPES, containing 10 mM MgCl2, 1 mM dithiothreitol, 0.01% bovine serum albumin, and 0.0005% Tween 20, pH 7.5. Unless specified otherwise, the reactions were initiated by the 798 Mourey et al. at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from addition of enzyme. For endpoint experiments, reactions were terminated during the linear phase by the addition of 30 mM EDTA. The kinase selectivity experiments were performed with the MgATP concentration fixed at the Km(app) determined for each enzyme. To determine the mechanism of action of PF-3644022 binding, the initial velocities in the presence and absence of PF-3644022 with ATP as the varied substrate while the HSP27 peptide concentration was held constant. The data were fit to the competitive inhibition model (eq. 1), noncompetitive inhibition model (eq. 2), or an uncompetitive inhibition model (eq. 3). In these equations, Vmax is the maximum velocity, Km is the Michaelis–Menton constant for the varied substrate, S is the concentration of the varied substrate, I is the concentration of the inhibitor, and Kis and Kii are the slope and intercept inhibition constants, respectively. The best fit was based on an F test and resulted in the lowest standard errors for the inhibition constants. The apparent inhibition constants (Ki) were determined with GraFit 5.0 (Erithacus Software, Horley, Surrey, UK). v Vmax S/ Km 1 I/Kis S (1) v Vmax S/ Km 1 I/Kis S 1 I/Kii (2) v Vmax S/ Km S 1 I/Kii (3) Other MAPKAP enzymes were assayed for activity by using an ion exchange separation method for the detection of P-labeled product peptide as described (Anderson et al., 2007). PF-3644022 was evaluated for inhibition of 200 human kinases by using an in-house 30 kinase selectivity panel (Card et al., 2009) and 170 kinases from the Upstate Kinase Profiler service (Millipore Corporation, Billerica, MA). The kinase selectivity experiments were performed with the MgATP concentration fixed at the Km(app) determined for each enzyme. MK2 Inhibitor Binding Studies. Surface plasmon resonance spectroscopy using standard Biacore methodology on a Biacore 3000 instrument (GE Healthcare) was used to follow real-time binding kinetics of PF-3644022 to immobilized biotinylated MK2. The binding studies were performed at 25°C by using a running buffer of 10 mM HEPES, 150 mM NaCl, 0.005% P20 detergent with a flow rate of 60 l/min. PF-3644022 (1–100 nM) was injected over immobilized MK2 for 4 min and then dissociation followed for 15 min. For biotinMK2 capture, a CM5 chip (GE Healthcare) was first preconditioned and then streptavidin was immobilized by using amine coupling with N-hydroxysuccinimide and N-ethyl-N -(3-dimethylaminopropyl) carbodiimide according to Biacore methods. For kinetic analyses, sensorgrams were double reference-subtracted, and BiaEvaluation (Biacore) was used to determine the association and dissociation constants by using a Langmuir 1:1 model. Cell-Based Assays. The U937 human premonocytic cell line was obtained from the American Type Culture Collection (Manassas, VA) and differentiated to a monocyte/macrophage phenotype with phorbol myristate acetate (Sigma-Aldrich, St. Louis, MO) as described (Burnette et al., 2009). Human peripheral blood mononuclear cells (hPBMCs) were prepared from venous blood of donors collected anonymously with informed consent at an on-site clinic. Venous blood was collected into sodium heparin tubes, and hPBMCs were isolated by density gradient centrifugation using Histopaque 1077 (Sigma-Aldrich) per the manufacturer’s directions. U937 cells and hPBMCs were cultured as described (Burnette et al., 2009; Hope et al., 2009). The ability of PF-3644022 to inhibit LPS-stimulated cytokine production in U937 cells and hPBMCs was evaluated after a 1-h pretreatment of compound in cell culture media containing less than 1% DMSO final concentration. All cell incubations were done at 37°C. Culture media TNF levels were measured 4 h after LPS stimulation at 100 ng/ml by using an electrochemoluminescence MesoScale Discovery TNF kit (MesoScale Discovery, Gaithersburg, MD). In hPBMCs, TNF , IL-1, IL-6, and IL-8 were measured at 16 h after LPS stimulation by using a four-plex human cytokine MSD plate (MesoScale Discovery). For mitogen-activated protein kinase (MAPK) signaling studies in U937 cells, cells were pretreated with PF-3644022 at varying concentrations for 1 h before LPS stimulation at 100 ng/ml for 30 min. Cell lysates were prepared and analyzed for phosphoSer78-HSP27, phospho-p38, and phospho-c-Jun N-terminal kinase (JNK) levels by Western blot analysis as described (Anderson et al., 2007). Quantitation of Western blots was performed with Alexaconjugated secondary antibodies (Invitrogen, Carlsbad, CA) and fluorescent LI-COR (LI-COR Biosciences, Lincoln, NE) scanning. MK2 activity in U937 cells, measured by monitoring the phosphorylation of the MK2 substrate HSP27, was quantitated in cell lysates by using a phospho-Ser82 HSP27 and total HSP27 MSD kit. Phospho-HSP27 levels were normalized in cell lysates to total HSP27 protein levels. LPS-Stimulated Human Whole Blood. Venous blood from human donors was collected in sodium heparin tubes (Baxter Healthcare, Deerfield, IL) and assayed for LPS-stimulated cytokine production as described (Burnette et al., 2009). In brief, PF-3644022 was added to human whole blood (HWB) ex vivo 1 h before stimulation with 100 ng/ml LPS at 37°C. TNF was measured 4 h poststimulation by assaying plasma with a human TNF MSD kit. TNF , IL-1, IL-6, and IL-8 in plasma was also measured after 16-h LPS stimulation of HWB by using a four-plex human cytokine MSD plate (MesoScale Discovery). MK2 activity in HWB was measured by monitoring phospho-Ser82 HSP27 and total HSP27 levels after a 30-min stimulation of 100 ng/ml LPS by using a dissociation-enhanced lanthanide fluorescent immunoassay (PerkinElmer Life and Analytical Sciences, Waltham, MA) as described (Burnette et al., 2009). Measurement of PF-3644022 in Plasma. Plasma was analyzed for total PF-3644022 by high-performance liquid chromatography. In brief, calibration standards ranging from 0.27 nM to 13 M were prepared by fortifying appropriate amounts of PF-3644022 to blank control plasma by a series of dilutions. Samples and calibration standards were briefly vortex-mixed, and 0.025-ml aliquots were transferred from the vials into corresponding 96-well plates. Internal standard working solution (0.25 M tolbutamide in 97.5% methanol/ 2.5% acetonitrile containing 1% formic acid) was then added as a 0.225-ml aliquot to all samples. The plates were centrifuged at approximately 3800 rpm for 5 min. A total of 90 l of supernatant were transferred to a new 96-well Quadra 96–320 automatic sample handling system (Tomtec, Hamden, CA). The samples (5 l) were injected onto the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for analysis. Samples were chromatographed with a Rheos pump (Thermo Fisher Scientific, Waltham, MA) and an Extend C18 (20 2.1 mm, 5m particle size) column (Agilent Technologies, Santa Clara, CA) connected to a HTS-PAL autosampler from LEAP Technologies (Carrboro, NC). The mobile phases were 10 mM ammonium acetate in 95% water/5% methanol (A) and 10 mM ammonium acetate in methanol (B). The running condition was 70% A for 0.5 min isocratically, ramped to 100% B in 1 min, holding for 0.9 min followed by dropping to 30% B in 0.1 min, and holding at 30% B another 0.5 min before the next injection. The total run time was 3 min. The high-performance liquid chromatography flow rate was maintained at 0.4 ml/min for the entire analysis. An API 4000 triple quadruple mass spectrometer (AB/MDS-Sciex, Concord, Canada) with a turbo-ionspray interface operated in positive ionization mode was used for the multiple reaction monitoring LCMS/MS analyses. The mass spectrometric conditions were optimized for detection of PF-3644022 and tolbutamide. The following precursor product ion transitions were used for multiple reaction monitoring: PF-3644022: m/z 375 3 291; tolbutamide, m/z 271 3 155. LPS-Induced TNF Production in Rats. All rat in vivo studies were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee according to guidelines sanctioned by the Association for Assessment and Accreditation of Laboratory Animal Care International. In the LPS-induced acute endotoxemia inflammation model, adult male Lewis rats (225–250 g; Harlan, Indianapolis, IN) were fasted 18 h before oral dosing and allowed free access to water. PF-3644022 was prepared as a suspension in a vehicle consisting of 0.5% methylcellulose (Sigma-Aldrich) and 0.025% Tween 20 (SigmaBenzothiophene MK2 Kinase Inhibitors Are Anti-Inflammatory 799 at A PE T Jornals on O cber 5, 2017 jpet.asjournals.org D ow nladed from Aldrich) in water. PF-3644022 or vehicle was orally administered in a volume of 1 ml by using an 18-gauge gavage needle 4 h before LPS challenge. LPS was administered by injection into the penile vein at 1 mg/kg in 0.5 ml of sterile saline, and blood was collected 90 min later by cardiac puncture. Serum TNF levels were measured by rat TNF enzyme-linked immunosorbent assay (Burnette et al., 2009), and PF-3644022 levels were determined by LC-MS/MS. Streptococcal Cell Wall-Induced Arthritis in Rats. Arthritis was induced in 125to 140-g female Lewis rats (Harlan) by a single intraperitoneal injection of peptidoglycan–polysaccharide complexes from group A SCW purchased from Lee Laboratories (Grayson, GA) as described (Hope et al., 2009). The disease course was biphasic with an acute inflammatory non-T cell-dependent phase on days 1 to 3 followed by a chronic T cell-dependent inflammatory-erosive arthritis developing over days 14 to 28. Animals developing the acute inflammatory phase were pooled into groups of seven to eight animals per group and dosed with PF-3644022 or methylcelluloseTween 20 vehicle by oral gavage (1 ml) twice a day for days 10 to 21. Hind paw swelling volumes were measured on day 21 with a displacement plethysmometer. After the last PF-3644022 dose on day 21, plasma was collected at various times up to 11 h to determine compound exposure parameters. Determination of In Vivo Rat Pharmacokinetic Parameters of PF-3644022. Male Sprague-Dawley rats weighing 275 to 300 g were purchased from Charles River Laboratories Inc. (Wilmington, MA) and acclimated to their surroundings for approximately 1 week with food and water provided ad libitum. A minimum of 1 day before study, animals were anesthetized with isoflurane (to effect) and then implanted with Culex (BASi, West Lafayette, IN) vascular catheters in the carotid artery. Animals were acclimated in Culex cages overnight before dosing. Patency of the carotid artery catheter was maintained by using the “tend” function of Culex ABS. PF-3644022 was administered dissolved in 70% normal saline/20% polyethylene glycol-400/10% ethanol (intravenously) or suspended in 0.5% hydroxypropylmethylcellulose/0.1% Tween 80 in distilled water (by mouth). Blood collections were obtained from the carotid artery and performed by the Culex at 2 (intravenous only), 5, 15, 30 min and 1, 2, 4, 6, 8, 12, 18, and 24 h. Plasma was separated and frozen for