Differential selectivity of JAK2 inhibitors in enzymatic and cellular settings
2013; Elsevier BV; Volume: 41; Issue: 5 Linguagem: Inglês
10.1016/j.exphem.2013.01.005
ISSN1873-2399
AutoresVioleta Yu, Jeanne Pistillo, Ivonne Archibeque, Josie Han Lee, Bee-Chun Sun, Laurie B. Schenkel, Stephanie Geuns-Meyer, Liqin Liu, Renee Emkey,
Tópico(s)Chronic Myeloid Leukemia Treatments
ResumoSmall molecule inhibitors of Janus kinase (JAK) family members (JAK1, JAK2, JAK3, and Tyk2) are currently being pursued as potential new modes of therapy for a variety of diseases, including the inhibition of JAK2 for the treatment of myeloproliferative disorders. Selective inhibition within the JAK family can be beneficial in avoiding undesirable side effects (e.g., immunosuppression) caused by parallel inhibition of other JAK members. In an effort to design an assay paradigm for the development of JAK2 selective inhibitors, we investigated whether compound selectivity differed between cellular and purified enzyme environments. A set of JAK2 inhibitors was tested in a high-throughput JAK family cell assay suite and in corresponding purified enzyme assays. The high-throughput JAK cell assay suite comprises Ba/F3 cells individually expressing translocated ETS leukemia (TEL) fusions of each JAK family member (TEL-JAK Ba/F3) and an AlphaScreen phosphorylated-STAT5 (pSTAT5) immunoassay. Compound potencies from the TEL-JAK Ba/F3 pSTAT5 assays were similar to those determined in downstream cell proliferation measurements and more physiologically relevant cytokine-induced pSTAT5 PBMC assays. However, compound selectivity data between cell and purified enzyme assays were discrepant because of different potency shifts between cell and purified enzyme values for each JAK family member. For any JAK small molecule development program, our results suggest that relying solely on enzyme potency and selectivity data may be misleading. Adopting the high-throughput TEL-JAK Ba/F3 pSTAT5 cell assay suite in lead development paradigms should provide a more meaningful understanding of selectivity and facilitate the development of more selective JAK inhibitors. Small molecule inhibitors of Janus kinase (JAK) family members (JAK1, JAK2, JAK3, and Tyk2) are currently being pursued as potential new modes of therapy for a variety of diseases, including the inhibition of JAK2 for the treatment of myeloproliferative disorders. Selective inhibition within the JAK family can be beneficial in avoiding undesirable side effects (e.g., immunosuppression) caused by parallel inhibition of other JAK members. In an effort to design an assay paradigm for the development of JAK2 selective inhibitors, we investigated whether compound selectivity differed between cellular and purified enzyme environments. A set of JAK2 inhibitors was tested in a high-throughput JAK family cell assay suite and in corresponding purified enzyme assays. The high-throughput JAK cell assay suite comprises Ba/F3 cells individually expressing translocated ETS leukemia (TEL) fusions of each JAK family member (TEL-JAK Ba/F3) and an AlphaScreen phosphorylated-STAT5 (pSTAT5) immunoassay. Compound potencies from the TEL-JAK Ba/F3 pSTAT5 assays were similar to those determined in downstream cell proliferation measurements and more physiologically relevant cytokine-induced pSTAT5 PBMC assays. However, compound selectivity data between cell and purified enzyme assays were discrepant because of different potency shifts between cell and purified enzyme values for each JAK family member. For any JAK small molecule development program, our results suggest that relying solely on enzyme potency and selectivity data may be misleading. Adopting the high-throughput TEL-JAK Ba/F3 pSTAT5 cell assay suite in lead development paradigms should provide a more meaningful understanding of selectivity and facilitate the development of more selective JAK inhibitors. The kinase family represents a large class of drug discovery opportunities for which selectivity among its members is a well-recognized challenge [1Cohen P. Protein kinases—the major drug targets of the twenty-first century?.Nat Rev Drug Discov. 2002; 1: 309-315Crossref PubMed Scopus (1808) Google Scholar, 2Fedorov O. Müller S. Knapp S. The (un)targeted cancer kinome.Nat Chem Biol. 2010; 6: 166-169Crossref PubMed Scopus (230) Google Scholar]. The potential for promiscuous inhibition is generally due to the combination of highly conserved adenosine triphosphate (ATP)-binding sites and the ATP-competitive mechanism of most kinase inhibitors [3Davies S.P. Reddy H. Caivano M. Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors.Biochem J. 2000; 351: 95-105Crossref PubMed Scopus (3910) Google Scholar, 4Zhang J. Yang P.L. Gray N.S. Targeting cancer with small molecule kinase inhibitors.Nat Rev Cancer. 2009; 9: 28-39Crossref PubMed Scopus (1995) Google Scholar, 5Liu Y. Gray N.S. Rational design of inhibitors that bind to inactive kinase conformations.Nat Chem Biol. 2006; 2: 358-364Crossref PubMed Scopus (858) Google Scholar, 6Zuccotto F. Ardini E. Casale E. Angiolini M. Through the "gatekeeper door": exploiting the active kinase conformation.J Med Chem. 2010; 53: 2681-2694Crossref PubMed Scopus (372) Google Scholar]. To avoid unwanted side effects from the parallel inhibition of untargeted kinases, it is thus beneficial to test compound activity against as many kinases as possible. Selectivity profiling can also be useful for understanding target patient populations and for exploring potentially favorable multitargeted approaches. In recent years, selectivity profiling has been simplified by the availability of outsourced profiling and high-throughput commercial prevalidated assay options for a broad variety of recombinant purified kinases [7Karaman M.W. Herrgard S. Treiber D.K. et al.A quantitative analysis of kinase inhibitor selectivity.Nat Biotechnol. 2008; 26: 127-132Crossref PubMed Scopus (1885) Google Scholar, 8Goldstien D.M. Gray N.S. Zarrinkar P.P. High-throughput kinase profiling as a platform for drug discovery.Nat Rev Drug Discov. 2008; 7: 391-397Crossref PubMed Scopus (175) Google Scholar, 9Ma H. Deacon S. Horiuchi K. The challenge of selecting protein kinase assays for lead discovery optimization.Expert Opin Drug Dis. 2008; 3: 607-621Crossref PubMed Scopus (146) Google Scholar, 10Card A. Caldwell C. Min H. et al.High-throughput biochemical kinase selectivity assays: Panel development and screening.J Biomol Screen. 2009; 14: 31-42Crossref PubMed Scopus (39) Google Scholar]. However, without the contextual and biological relevance of a cellular environment, purified enzyme selectivity data alone may be insufficient. The Janus kinase (JAK) family is an example in which both selectivity and large enzyme to cell potency shifts have historically been challenges [11Wernig G. Kharas M.G. Okabe R. et al.Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera.Cancer Cell. 2008; 13: 311-320Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 12Quintás-Cardama A. Vaddi K. Liu P. et al.Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms.Blood. 2010; 115: 3109-3117Crossref PubMed Scopus (571) Google Scholar, 13Verstovsek S. Therapeutic potential of JAK2 inhibitors.Hematology Am Soc Hematol Educ Program. 2009; : 636-642Crossref PubMed Scopus (95) Google Scholar, 14Wrobleski S.T. Pitts W.J. Advances in the discovery of small molecule JAK3 inhibitors.in: Macor J.E. Annual reports in medicinal chemistry volume 44. San Diego: Academic Press, 2009: 247-264Crossref Scopus (23) Google Scholar, 15Pesu M. Laurence A. Kishore N. et al.Therapeutic targeting of Janus kinases.Immunol Rev. 2008; 223: 132-142Crossref PubMed Scopus (195) Google Scholar]. The JAK family comprises JAK1, JAK2, JAK3, and Tyk2. JAKs are cytoplasmic tyrosine kinases that are associated with cytokine receptors and are normally dependent on ligand-induced receptor oligomerization for their activity. JAK activation leads to a series of events that result in JAK-catalyzed phosphorylation of signal transducers and activators of transcription (STAT) substrates. The phosphorylated STATs dimerize and translocate into the nucleus to mediate the transcription of specific genes. Through this relay that converts external cytokine signals into the expression of target genes, JAKs are essential for hematopoiesis, inflammation, and immune responses [16Rodig S.J. Meraz M.A. White J.M. et al.Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses.Cell. 1998; 93: 373-383Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 17Neubauer H. Cumano A. Müller M. et al.Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis.Cell. 1998; 93: 397-409Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, 18Parganas E. Wang D. Stravopodis D. et al.Jak2 is essential for signaling through a variety of cytokine receptors.Cell. 1998; 93: 385-395Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar, 19Thomis D.C. Gurniak C.B. Tivol E. et al.Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3.Science. 1995; 270: 794-797Crossref PubMed Scopus (470) Google Scholar, 20Nosaka T. van Deursen J.M.A. Tripp R.A. et al.Defective lymphoid development in mice lacking Jak3.Science. 1995; 270: 800-802Crossref PubMed Scopus (568) Google Scholar, 21Karaghiosoff M. Neubauer H. Lassnig C. et al.Partial impairment of cytokine responses in Tyk2-deficient mice.Immunity. 2000; 13: 549-560Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 22Stoiber D. Kovacic B. Schuster C. et al.TYK2 is a key regulator of the surveillance of B lymphoid tumors.J Clin Invest. 2004; 114: 1650-1658Crossref PubMed Scopus (56) Google Scholar]. Dysregulation of JAK pathways has been implicated in a variety of disorders that include rheumatoid arthritis (JAK1, JAK3, Tyk2), asthma (JAK3, Tyk2), graft-versus-host rejection (JAK3), and cancer (JAK1, JAK2, JAK3) [11Wernig G. Kharas M.G. Okabe R. et al.Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera.Cancer Cell. 2008; 13: 311-320Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 12Quintás-Cardama A. Vaddi K. Liu P. et al.Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms.Blood. 2010; 115: 3109-3117Crossref PubMed Scopus (571) Google Scholar, 13Verstovsek S. Therapeutic potential of JAK2 inhibitors.Hematology Am Soc Hematol Educ Program. 2009; : 636-642Crossref PubMed Scopus (95) Google Scholar, 14Wrobleski S.T. Pitts W.J. Advances in the discovery of small molecule JAK3 inhibitors.in: Macor J.E. Annual reports in medicinal chemistry volume 44. San Diego: Academic Press, 2009: 247-264Crossref Scopus (23) Google Scholar, 15Pesu M. Laurence A. Kishore N. et al.Therapeutic targeting of Janus kinases.Immunol Rev. 2008; 223: 132-142Crossref PubMed Scopus (195) Google Scholar, 23Wilks A.F. The JAK kinases: not just another kinase drug discovery target.Semin Cell Dev Biol. 2008; 19: 319-328Crossref PubMed Scopus (70) Google Scholar, 24Pardanani A. Lasho T. Smith G. et al.CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.Leukemia. 2009; 23: 1441-1445Crossref PubMed Scopus (149) Google Scholar]. JAK2 has been implicated as a target for myeloproliferative disorders [11Wernig G. Kharas M.G. Okabe R. et al.Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera.Cancer Cell. 2008; 13: 311-320Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 14Wrobleski S.T. Pitts W.J. Advances in the discovery of small molecule JAK3 inhibitors.in: Macor J.E. Annual reports in medicinal chemistry volume 44. San Diego: Academic Press, 2009: 247-264Crossref Scopus (23) Google Scholar, 23Wilks A.F. The JAK kinases: not just another kinase drug discovery target.Semin Cell Dev Biol. 2008; 19: 319-328Crossref PubMed Scopus (70) Google Scholar, 24Pardanani A. Lasho T. Smith G. et al.CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.Leukemia. 2009; 23: 1441-1445Crossref PubMed Scopus (149) Google Scholar]. Patients with myeloproliferative disorders, such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, have been shown to carry a mutation in the pseudokinase domain of JAK2 (V617F), which renders the kinase constitutively active [11Wernig G. Kharas M.G. Okabe R. et al.Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera.Cancer Cell. 2008; 13: 311-320Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar]. These discoveries have garnered interest in the pharmacologic inhibition of specific JAK family members. However, the essential physiologic roles of the individual JAK kinases may dictate the need for selective compound therapies to understand and avoid undesirable side effects, especially in chronic settings. To facilitate the measurement of cellular JAK selectivity, we used commercially available engineered Ba/F3 cell lines that individually overexpress fusions of the translocated ETS leukemia (TEL) protein and the kinase domain of each JAK member. JAKs have seven defined regions of homology called Janus homology domain 1–7 (JH1-7). JH1 is the kinase domain, and JH2 is a pseudokinase domain. JH2 may be involved in regulating the activity of JH1. The TEL-JAK Ba/F3 cells used in this study contained a construct that consisted of only the JH1 kinase domain of JAK, with TEL fused to the N-terminal end. TEL-fusion BaF3 cells are widely used for kinase drug discovery, because they have been used to determine the cellular potency of compounds against more than 90 different kinases [25Warmuth M. Kim S. Gu X.J. et al.Ba/F3 cells and their use in kinase drug discovery.Curr Opin Oncol. 2007; 19: 55-60Crossref PubMed Scopus (146) Google Scholar]. In these genetically engineered cells, constitutive kinase activity is conferred through TEL-domain–mediated oligomerization, which provides growth factor independence to the otherwise interleukin (IL) 3–dependent Ba/F3 murine pro–B cell line. The ability to impart dependency of unstimulated Ba/F3 cells on the heterologously expressed kinase has made them an ideal system to test the selectivity of drug candidates, with the added benefit of studying these activities in the same cellular background to allow a more controlled comparison. In the TEL-JAK Ba/F3 cells, constitutive kinase activity leads to persistent phosphorylated-STAT5 (pSTAT5) [26Lacronique V. Boureux A. Monni R. et al.Transforming properties of chimeric TEL-JAK proteins in Ba/F3 cells.Blood. 2000; 95: 2076-2083Crossref PubMed Google Scholar]. To enable the rapid determination of compound potencies, pSTAT5 was measured in a homogeneous 384-well format with an AlphaScreen SureFire kit (PerkinElmer, Waltham, MA). The novel combination of TEL-JAK Ba/F3 cells and SureFire pSTAT5 AlphaScreen technology led to a facile, proximal, and high-throughput JAK cell assay suite. The high-throughput TEL-JAK Ba/F3 pSTAT5 assay suite enabled in vitro cellular and purified enzyme inhibitory concentration of 50% (IC50) determinations to be performed in parallel. In an effort to establish an assay scheme that could facilitate the generation of JAK2 selective compounds, we used the TEL-JAK Ba/F3 pSTAT5 assay suite to investigate whether compound selectivity measurements differed between cell and purified enzyme assays. We also tested a subset of compounds in an orthogonal TEL-JAK Ba/F3 proliferation assay, which serves as a functional downstream measurement of JAK activity. Finally, compound activity was examined in a more physiologically relevant setting by monitoring pSTAT5 in cytokine-stimulated peripheral blood mononuclear cells (PBMCs). Our findings changed the way we assessed JAK2 selectivity. The methods disclosed here can also be applied to other programs targeting the development of selective kinase inhibitors within the JAK family. The JAK3 inhibitor reference compound, CP-690550, was purchased from a commercial supplier (Sequoia Research Products, Pangebourne, UK). All other JAK inhibitor compounds were synthesized at Amgen (Thousand Oaks, CA, USA). The JH1 (catalytic kinase) domain of each human JAK (JAK1, JAK2, JAK3, and Tyk2) was expressed in Sf-9 cells as an N-terminal GST fusion. Kinase activity of the purified recombinant Jaks was assessed using a Lance TR-FRET assay that measured the phosphorylation of a Tyk2-based peptide substrate (Biotin-LC-EQEDEPEGDYFEWLE; Biopeptide, San Diego, CA, USA). Kinase assays were performed in black 384-well assay plates (Costar) using a reaction buffer that contained 5 mmol/L MgCl2, 50 mmol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (pH 7.5), 4 mmol/L Dithiothreitol (DTT), and 0.05% bovine serum albumin (BSA). The enzyme reactions (40 μL) contained 0.5 μmol/L of peptide, apparent Km concentrations of ATP (19 μmol/L for JAK1, 10 μmol/L for JAK2, 1.8 μmol/L for JAK3, and 15 μmol/L for Tyk2), and either 625 pmol/L JAK1, 4 pmol/L JAK2, 4 pmol/L JAK3, or 75 pmol/L Tyk2. Reactions were incubated for 90 min at room temperature with compound (in a 22-point [1:2] titration series with final concentrations ranging 62.5 μmol/L to 29.8 pmol/L). Two columns of wells (consisting of 16 wells per column) in each assay plate were reserved for a control group. One column was used for the high control samples and consisted of enzyme reactions that were exposed to vehicle only (1.23% dimethyl sulfoxide [DMSO] final). The other column was used for the low control samples and contained vehicle, substrate, and no enzyme. Reactions were stopped with 40 μL of a detection mixture containing 400 pmol/L Lance Eu-PT66 Antibody (Perkin Elmer, Downer's Grove, IL, USA), 6.6 μg/mL SA-APC (Perkin Elmer), 40 mmol/L ethylenediaminetetraacetic acid, 100 mM HEPES (pH 7.5), 100 mmol/L NaCl, 0.1% BSA, and 0.5% Tween 20. Plates were incubated for 30 min at room temperature before reading the TR-FRET signal on the RUBYstar (BMG Labtek, Cary, NC, USA) instrument using an excitation setting of 320 nm and an emission collection at 615 and 665 nm. Phosphorylation of the peptide resulted in an increased emission at 665 nm. The ratio of signal generated at 665 nm divided by the signal generated at 615 nm was used in calculations. Automated liquid handling was accomplished with a Mulitdrop 384 bulk liquid dispenser equipped with a 160-plate Titan stacker (Thermo Electron Corporation, Waltham, MA, USA) for all additions (enzyme, substrate, and detection mix). The TEL-JAK family Ba/F3 cell lines were purchased from Advanced Cellular Dynamics (San Diego, CA, USA). Cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1x penicillin-streptomycin-glutamine (Gibco–Invitrogen, Carlsbad, CA, USA), and 0.5 μg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained at 37°C in a humidified 5% CO2 incubator and at a density less than 2 × 106 cells/mL. For pSTAT5 measurement, TEL-JAK Ba/F3 cells were washed once with assay buffer (Hank's balanced salt solution, 0.1% BSA, 5 mmol/L HEPES, pH 7.0) and then resuspended to a density of 5 × 106 cells/mL (7.2 × 106 cells/mL for TEL-JAK2 Ba/F3). Three microliters of cell-suspension were added to a 384-well, low-volume, white-walled polystyrene ProxiPlate (Perkin Elmer) that contained 1 μL of compound (in a 22-point [1:2] titration series with final concentrations ranging from 25.0 μmol/L to 11.9 pmol/L) in 2% DMSO (98% Assay Buffer) per well. Two columns of wells (consisting of 16 wells per column) in each assay plate were reserved for control samples. One column was used for the high control samples and contained cells that were exposed to vehicle in buffer only (0.5% DMSO in assay buffer). The other column was used for the low control samples and contained cells that were exposed to a final concentration of 10 μmol/L pan-JAK standard inhibitor (at least 10-fold the cellular IC50 of each Jak). Cells were incubated with compound (final DMSO concentration of 0.5%) at room temperature for 60 min before proceeding with pSTAT5 detection. Measurement of pSTAT5 was accomplished with the SureFire® pSTAT5 Assay kit (Perkin Elmer). Cells were lysed with 1μL of 5x Lysis Buffer and incubated at room temperature for 10 min. Next, 4.3 μL of a solution was added, containing reaction buffer, activation buffer, and Acceptor beads prepared per the manufacturer's protocol. Plates were incubated overnight in the dark at room temperature before adding 1.8 μL of a mixture containing dilution buffer and donor beads also prepared per the manufacturer's protocol. After a final incubation of 1 hour at room temperature, TEL-JAK plates were read on the EnVision (Perkin Elmer) multilabel reader using the AlphaScreen assay setting. Automation of cell, lysis, and detection reagent additions was performed with a FlexDrop (Perkin Elmer) bulk liquid dispenser. Peripheral blood samples were obtained from healthy donors, and PBMCs were isolated via density gradient separation using Ficoll-Paque Premium density gradient medium (GE Healthcare, Uppsala, Sweden). PBMCs (300,000–400,000 per well) were dispensed in 36 μL of Dulbecco's phosphate-buffered saline (DPBS) into a 96-well (deep well, V bottom) polypropylene plate (Costar, Corning, NY, USA) containing 4 μL of 10x compound (in a 10-point [1:3] titration series with final concentrations ranging from 25.0 μmol/L to 1.27 nmol/L) in 10% DMSO (90% DPBS) per well. Cells were pretreated with compound for 30 min at 37°C followed by stimulation with EC90 concentrations of 0.1 ng/mL granulocyte macrophage colony-stimulating factor, 10 ng/mL thrombopoietin, or 10 ng/mL IL-2 for an additional 15 min at 37°C (R&D Systems, Minneapolis, MN, USA). Two columns of wells (consisting of eight wells per column) in each assay plate were reserved for control samples. One column was used for the high control samples and contained cytokine-stimulated cells that were exposed to vehicle in buffer only (1% DMSO in DPBS). The other column was used for the low control samples and contained unstimulated cells that were exposed to vehicle in buffer only. PBMCs were fixed with 800 μL of Lyse Fix buffer (Becton Dickinson, Franklin Lakes, NJ, USA) for 15 min at 37°C. Next, cells were pelleted, resuspended, and permeabilized with 600 μL of 100% MeOH (JT Baker, Phillipsburg, NJ, USA) and stored at −40 ° C in MeOH for at least 1 hour before staining. For intracellular pSTAT5 staining, permeabilized cells were centrifuged and resuspended in 200 μL blocking buffer (1% mouse serum [Sigma-Aldrich] in stain buffer [Becton Dickinson]) and incubated for 20 min at room temperature. After removing the blocking buffer by centrifugation followed by aspiration, cells were stained with Alexa Fluor 647 conjugated mouse anti-phospho-STAT5 (Y694; Becton Dickinson Biosciences, San Jose, CA) according to the manufacturer's suggestion and incubated at 37°C for 30 min. Finally, cells were washed and resuspended in 150 μL of wash buffer (0.5% BSA in DPBS). Events were acquired on the FACS Calibur flow cytometer (Becton Dickinson Biosciences) equipped with a Cytek Development AMS 96-well plate loader using side and forward scatter properties. More than 10,000 events were captured for each sample. TEL-JAK Ba/F3 cells were washed and resuspended in fresh assay media (RPMI 1640, 10% fetal bovine serum, 1% L-glutamine) before plating of 10,000 cells/well (100 μL) in a white-walled 96-well plate (Costar). Cells were treated with 1 μL compound (prepared in 100% DMSO in a 10-point [1:3] titration series with final concentrations ranging from 25.0 μmol/L to 1.27 nmol/L) and incubated at 37°C in a humidified 5% CO2 incubator for 48 hours. Two columns of wells (consisting of eight wells per column) in each assay plate were reserved for control samples. One column was used for the high control samples and contained cells that were exposed to vehicle in buffer only (1% DMSO in assay media). The other column was used for the low control samples and contained cells that were exposed to a pan-JAK standard inhibitor at a final concentration of 100 μmol/L (which is at least 10-fold its cellular IC50 against each Jak). To measure proliferation, 100 μL of Cell Titer-Glo reagent (Promega, Madison, WI) was added (prepared per the manufacturer's suggestion). Plates were shaken for 1 min and incubated for 10 min at room temperature before measuring of the luminescence on an LMax instrument (Molecular Devices, Sunnyvale, CA, USA). The amount of signal generated in the presence of compounds versus that in the presence of DMSO vehicle alone (high control sample) was calculated using the formula: % Control sample = (Compound – Average low) / (Average high – Average low) × 100. For IC50 determination, data were fitted to a four-parameter equation: y = A + ([B – A] / [1 + {(x / C)D}]), where A is the minimum y (% control sample) value, B is the maximum y (% control sample), C is the x (compound concentration) at the point of inflection, and D is the slope factor. AlphaScreen and TR-FRET data was analyzed with the mFit nonlinear regression algorithm and the Screener data analysis software (Genedata, Basel, Switzerland) [27Fomenko I. Durst M. Balaban D. Robust regression for high throughput drug screening.Comput Meth Prog Bio. 2006; 82: 31-37Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. For Phosflow data, offline analysis was performed using FCS Express software (Becton Dickinson Biosciences). A monocyte–granulocyte gate and a lymphocyte gate were created based on the side and forward scatter. For the monocyte–granulocyte gate, the geometric mean fluorescence intensity was analyzed. For the lymphocyte gate, the percent gated was analyzed. Phosflow and proliferation IC50 determination was performed using a Levenburg-Marquardt nonlinear regression algorithm and with Activity base Global fit software (ID Business Solution Limited, Emeryville, CA, USA). Correlation (Pearson r), linear regression analysis plots, and goodness of fit (R2) values were determined with Prism version 5 (GraphPad Software, San Diego, CA, USA). For data quality monitoring, the raw values from the high and low control samples were used to calculate plate Z', and data from only the plates with a Z' value of equal to or greater than 0.5 were accepted. In addition, reference compounds were included with each assay run and data from only the assay runs where reference compound IC50 values were within threefold of the expected values were accepted, regardless of the Z' values. A set of 120 ATP-competitive JAK2 small molecule kinase inhibitors, composed of compounds from our internal JAK program and an external reference compound (Pfizer's CP-690550, which is a JAK3 inhibitor currently in the clinic), was tested in the JAK family enzyme and TEL-JAK Ba/F3 pSTAT5 panels (Table 1). CP-690550 was chosen as a reference because it was commercially available, is associated with published in vitro and in vivo data, is structurally different from our internal compounds, and produced reliable IC50 values in all enzyme and cell selectivity assays. Resulting IC50 values for all compounds tested were plotted to visualize the intra-JAK family potency distribution of the JAK2 inhibitors in the enzyme and cell assay panels (Fig. 1). Distribution of the median enzyme IC50 values revealed that the JAK2 inhibitors had more than 10-fold selectivity compared with the other JAK members. Based on the purified enzyme data, the JAK2 compounds appeared to have the least selectivity over JAK3. In contrast, distribution of the median cell IC50 values demonstrated less than threefold selectivity compared with the other JAK members, with the least selectivity over JAK1.Table 1JAK family cell-based and purified enzyme assay summaryAssay typeAssay detailJAK1JAK2JAK3Tyk2EnzymeConstructGST-JH1 JAK1 (844-1142aa)GST-JH1 JAK2 (837-1132aa)GST-JH1 JAK3 (802-1124 aa)GST-JH1 Tyk2 (887-1187aa)AnalyteTyk2 − peptide phosphorylationTyk2 − peptide phosphorylationTyk2 − peptide phosphorylationTyk2 − peptide phosphorylationTechnologyLANCE®LANCE®LANCE®LANCE®Engineered cellCell lineBa/F3Ba/F3Ba/F3Ba/F3StimuliNoneNoneNoneNoneConstructTEL-JH1 JAK1TEL-JH1 JAK2TEL-JH1 JAK3TEL-JH1 Tyk2AnalytepSTAT5pSTAT5pSTAT5pSTAT5TechnologyAlphaScreen®AlphaScreen®AlphaScreen®AlphaScreen®PBMCPBMC typeLymphocyteMonocyte or PlateletLymphocyte-StimulusIL-2GM-CSF or TPOIL-2-AnalytepSTAT5pSTAT5pSTAT5-TechnologyPhosflowPhosflowPhosflow-Key details of the enzyme, engineered cell, and primary PBMC assays for determining selectivity amongst JAK1, JAK2, JAK3, and Tyk2. Open table in a new tab Key details of the enzyme, engineered cell, and primary PBMC assays for determining selectivity amongst JAK1, JAK2, JAK3, and Tyk2. To determine whether differences in IC50 values between enzyme and cell assays caused the discrepant selectivity profiles, correlation (Pearson r) and linear regression (R2) analysis were performed (Fig. 2). Comparison of the compound potency data between enzyme and cell assays resulted in Pearson r values ranging from 0.69 to 0.84, and R2 values between 0.48 and 0.70 (Table 2). JAK1 was associated with the lowest Pearson r and R2 values, and JAK2 was associated with the highest Pearson r and R2 values.Table 2IC50 comparison of TEL-JAK Ba/F3 pSTAT5 versus enzyme and PBMC pSTAT5 versus TEL-JAK Ba/F3 pSTAT5Assay pairComparisonJAK1JAK2JAK3Tyk2TEL-JAK Ba/F3 pSTAT5 vs. JAK enzymePearson r0.690.840.770.72R20.480.700.590.52XY pairs50874444PBMC pSTAT5 vs. TEL
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