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• W2069809502 abstract "In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton′s tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 × 8 × 9 × 17 Å, and the thickness of the pocket is approximately 7 Å. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a K i value of 1.4 μm, inhibited human BTK in vitro with an IC50 value of 17.2 ± 0.8 μm. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 μm. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 μg/ml (∼278 μm), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK. In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton′s tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 × 8 × 9 × 17 Å, and the thickness of the pocket is approximately 7 Å. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a K i value of 1.4 μm, inhibited human BTK in vitro with an IC50 value of 17.2 ± 0.8 μm. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 μm. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 μg/ml (∼278 μm), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK. Bruton′s tyrosine kinase leflunomide metabolite EGF receptor insulin receptor kinase protein tyrosine kinase pleckstrin homology Tec homology fibroblast growth factor receptor PhosphorImager units densitometric scanning units mass spectroscopy Src homology propidium iodide acute lymphoblastic leukemia glutathione S-transferase hematopoietic cell kinase Apoptosis is a common mode of eukaryotic cell death that is triggered by an inducible cascade of biochemical events leading to activation of endonucleases that cleave the nuclear DNA into oligonucleosome length fragments (1Whyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-305Crossref PubMed Scopus (6725) Google Scholar, 2Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2431) Google Scholar, 3Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 4Korsmeyer S.J. Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (618) Google Scholar). Several of the biochemical events that contribute to apoptotic cell death as well as both positive and negative regulators of apoptosis have recently been identified (1Whyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-305Crossref PubMed Scopus (6725) Google Scholar, 2Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2431) Google Scholar, 3Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 4Korsmeyer S.J. Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (618) Google Scholar). Apoptosis plays a pivotal role in the development and maintenance of a functional immune system by ensuring the timely self-destruction of autoreactive immature and mature lymphocytes as well as any emerging target neoplastic cells by cytotoxic T cells (1Whyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-305Crossref PubMed Scopus (6725) Google Scholar, 2Steller H. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2431) Google Scholar, 3Fraser A. Evan G. Cell. 1996; 85: 781-784Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 4Korsmeyer S.J. Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (618) Google Scholar, 5Cohen J.J. Duke R.C. Fadok V.A. Sellins K.S. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1107) Google Scholar, 6Linette G.P. Korsmeyer S.J. Curr. Opin. Cell Biol. 1994; 6: 809-815Crossref PubMed Scopus (53) Google Scholar, 7Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6191) Google Scholar). Inappropriate apoptosis may contribute to the development as well as chemotherapy resistance of human leukemias and lymphomas (5Cohen J.J. Duke R.C. Fadok V.A. Sellins K.S. Annu. Rev. Immunol. 1992; 10: 267-293Crossref PubMed Scopus (1107) Google Scholar, 6Linette G.P. Korsmeyer S.J. Curr. Opin. Cell Biol. 1994; 6: 809-815Crossref PubMed Scopus (53) Google Scholar, 7Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6191) Google Scholar). Therefore, an improved understanding of the molecular basis of apoptosis and the pro-apoptotic versus anti-apoptotic regulatory signals may provide further insights into the pathogenesis of human lymphoid malignancies and have important implications for treatment of leukemias and lymphomas. Bruton's tyrosine kinase (BTK),1 a member of the BTK/Tec family of protein tyrosine kinases (PTKs), is a cytoplasmic PTK involved in signal transduction pathways regulating growth and differentiation of B-lineage lymphoid cells (8Rawlings D.J. Witte O.N. Immunol. Rev. 1994; 138: 105-119Crossref PubMed Scopus (102) Google Scholar, 9Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar, 10Uckun F.M. Biochem. Pharmacol. 1998; 56: 683-691Crossref PubMed Scopus (71) Google Scholar). BTK participates in signal transduction pathways initiated by the binding of a variety of extracellular ligands to their cell-surface receptors; following ligation of B cell antigen receptors, BTK activation by the concerted actions of the PTKs Lyn and Syk (9Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar) is required for induction of phospholipase C-γ2 mediated calcium mobilization (9Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar). Mutations in the human BTK gene are the cause of X-linked agammaglobulinemia, a male immune deficiency disorder characterized by a lack of mature, immunoglobulin-producing, peripheral B cells (11Tsukada S. Saffran D.C. Rawlings D.J. Parolini O. Allen R.C. Klisa K.I. Sparkes R.S. Kubagawa H. Mohandas T. Quan S. Cell. 1993; 72: 279-290Abstract Full Text PDF PubMed Scopus (1157) Google Scholar,12Vetrie D. Vorechovsky I. Sideras P. Holland J. Davies A. Flinter F. Hammerstrom L. Kinnon C. Levinsky R. Bobrow M.E.A. Nature. 1993; 361: 226-233Crossref PubMed Scopus (1253) Google Scholar). In mice, mutations in the BTK gene have been identified as the cause of murine X-linked immune deficiency (13Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O.N. Science. 1993; 261: 358-361Crossref PubMed Scopus (777) Google Scholar). BTK has an N-terminal region consisting of a 140-residue pleckstrin homology (PH) domain followed by an 80-residue proline-rich Tec homology (TH) domain. The PH domain is the site of activation by phosphatidylinositol phosphates and G-protein βγ subunits and inhibition by protein kinase C (14Tsukada S. Simon M.I. Witte O.N. Katz A. Prog. Natl. Acad. Sci. U. S. A. 1994; 91: 11256-11260Crossref PubMed Scopus (238) Google Scholar). The remaining portion of BTK contains Src homology (SH) domains SH3 (49 residues), followed by SH2 (96 residues), and a 250-residue C-terminal SH1 kinase domain. The SH2 domain mediates binding to tyrosine-phosphorylated peptide motifs on other molecules, and the SH3 domain mediates binding to proline-rich motifs. BTK is activated by transphosphorylation of Tyr551in the SH1 domain, followed by autophosphorylation of Tyr223 in the SH3 domain (9Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar). Phosphorylation of Tyr223 may function to disrupt an intramolecular TH-SH3 domain interaction, allowing BTK TH domain binding with SH3 domains in the Src family kinases FYN, LYN, and HCK, and BTK SH3 domain binding with a proline-rich region of Cbl (9Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar, 15Mahajan S. Fargnoli J. Burkhardt A.L. Kut S.A. Saouaf S.J. Bolen J.B. Mol. Cell. Biol. 1995; 15: 5304-5311Crossref PubMed Scopus (135) Google Scholar, 16Afar D.E. Park H. Howelle B.W. Rawlings D.J. Cooper J. Witte O.H. Mol. Cell. Biol. 1996; 16: 3465-3471Crossref PubMed Scopus (106) Google Scholar). Mutations in the catalytic domain, SH2 domain, as well as PH domain of the BTK have been found to lead to maturational blocks at early stages of B cell ontogeny in human X-linked agammaglobulinemia (17Vihinen M. Cooper M.D. de Saint Basile G. Fischer A. Good R.A. Hendriks R.W. Kinnon C. Kwan S.P. Litman G.W. Notarangelo L.D. et al.Immunol. Today. 1995; 16: 460-465Abstract Full Text PDF PubMed Scopus (66) Google Scholar). BTK-deficient mice generated by introducing PH domain or catalytic domain mutations in embryonic stem cells showed defective B cell development and function (18Kerner J.D. Appleby M.W. Mohr R.N. Chien S. Rawlings D.J. Maliszewski C.R. Witte O.N. Perlmutter R.M. Immunity. 1995; 3: 301-312Abstract Full Text PDF PubMed Scopus (274) Google Scholar). Thus, different regions of BTK are important for its physiologic functions. In murine B cells, BTK has been shown to act as an anti-apoptotic protein upstream of Bcl-xL in the B cell antigen receptor (but not the CD40 receptor) activation pathway (19Anderson J.S. Teutsch M. Dong Z. Wortis H.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10966-10971Crossref PubMed Scopus (116) Google Scholar). Our recent studies provided biochemical and genetic evidence that BTK is an inhibitor of the Fas/APO-1 death-inducing signaling complex in B-lineage lymphoid cells (20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar). Furthermore, we found that BTK also prevents ceramide- and vincristine-induced apoptosis (present study). The fate of leukemia/lymphoma cells may reside in the balance between the opposing proapoptotic effects of caspases activated by the death-inducing signaling complex and an upstream anti-apoptotic regulatory mechanism involving BTK and/or its substrates (20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar). Therefore, inhibitors of BTK are likely to enhance the chemosensitivity of leukemia/lymphoma cells. In a systematic effort to design potent inhibitors of BTK as anti-leukemic agents with apoptosis-promoting properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. Here, we first report the identification of the LFM analog α-cyano-β-hydroxy-β-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13) as a potent and specific inhibitor of BTK.LFM-A13 inhibited recombinant BTK with an IC50value of 2.5 μm, but it did not affect the enzymatic activity of other protein tyrosine kinases, including Janus kinases JAK1 and JAK2, Src family kinase HCK, and receptor family tyrosine kinases EGF-receptor kinase (EGFR) and insulin receptor kinase (IRK), at concentrations as high as 278 μm. LFM-A13enhanced the chemosensitivity of BTK-positive B-lineage leukemia cells to vincristine and ceramide. The leflunomide metabolite (LFM) and two of its analogs (LFM-A12 and LFM-A13) were crystallized using various solvents by evaporation or liquid-liquid diffusion. X-ray data from single crystals were collected using a SMART CCD area detector (Bruker Analytical X-ray Systems, Madison, WI) with MoKα radiation (λ = 0.7107 Å). The space group was determined based on systematic absence and intensity statistics. A direct methods solution provided most of the non-hydrogen atoms from the electron density map. Several full-matrix least squares/difference Fourier cycles were performed to locate the remaining non-hydrogen atoms. All non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were placed in ideal positions and refined as riding atoms with relative isotropic temperature factors. The structure was refined using full matrix least squares on F 2. Crystal structure calculations were performed using a Silicon Graphics INDY R4400-SC computer (Silicon Graphics Inc., Mountain View, CA) or a Pentium computer using the SHELXTL V 5.0 suite of programs (21Sheldrick G.M. SHELXTL-Plus Reference Manual, Version 5.0. Bruker Analytical X-Ray Systems, Madison, WI1996Google Scholar). We have constructed a homology model of BTK using crystal structures of homologous kinase domains of protein kinases HCK (22Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1045) Google Scholar), FGFR (23Mohammadi M. McMahon G. Sun L. Tang C. Hirth P. Yeh B.K. Hubbard S.R. Schlessinger J. Science. 1997; 276: 955-960Crossref PubMed Scopus (1012) Google Scholar), IRK (24Hubbard S.R. EMBO J. 1997; 16: 5572-5581Crossref PubMed Scopus (784) Google Scholar), and cAMP-dependent protein kinase (25Zheng J. Trafny E.A. Knighton D.R. Xuong N.-H. Taylor S.S. Ten Eyck L.F. Sowadski J.M. Acta Crystallogr. D. 1993; 49: 362-365Crossref PubMed Google Scholar) since no experimentally determined three-dimensional coordinates have been reported for the BTK kinase domain. The homology modeling of BTK was carried out by first obtaining the protein sequence of BTK (Swiss-Prot accession number Q06187, University of Geneva, Geneva, Switzerland) from GenBankTM (National Center for Biotechnology Information, Bethesda, MD). Next, the most reasonable sequence alignment between the BTK kinase and a coordinate template was determined. This was done by first superimposing the C-α coordinates of the kinase domains of HCK, FGFR, IRK, and cAMP-dependent protein kinase using the InsightII program (26.Insight II (1996) Molecular Simulations Inc. San Diego, CA.Google Scholar) to provide the best overall structural comparison. All four sequences were then aligned based on the superimposition of their structures (amino acid sequences were aligned together if their C-α positions were spatially related to each other). The sequence alignment accommodated such features as loops in a protein that differed from the other protein sequences. The structural superimposition was done using the homology module of the InsightII (26.Insight II (1996) Molecular Simulations Inc. San Diego, CA.Google Scholar) program and a Silicon Graphics INDIGO2 computer (Silicon Graphics Inc., Mountain View, CA). The sequence alignment was manually adjusted based on the previously mentioned considerations and produced a sequence variation profile for each superimposed C-α position. The sequence variation profile served as a basis for the next procedure, which was sequence alignment of all four proteins with BTK kinase. In this procedure, the sequence of BTK kinase was read into the program and manually aligned with the four known kinase proteins based on the sequence variation profile described previously. Next a set of three-dimensional coordinates was assigned to the BTK kinase sequence using the three-dimensional coordinates of HCK as a template, which employed the Homology module within the InsightII program (26.Insight II (1996) Molecular Simulations Inc. San Diego, CA.Google Scholar). The coordinates for a loop region where a sequence insertion occurs (relative to HCK without the loop) was chosen from a limited number of possibilities automatically generated by the program and manually adjusted to a more ideal geometry using the program CHAIN (27Sack J.S. CHAIN-A Crystallographic Modeling Program, J. Mol. Graphics. 1988; 6: 244-245Crossref Google Scholar). Finally, the constructed model of BTK was subjected to energy minimization using the X-plor program (28Brünger A.T. X-PLOR, Version 3.1. Yale University Press, New Haven1992Google Scholar) so that any steric strain introduced during the model-building process could be relieved. The model was screened for unfavorable steric contacts, and if necessary such side chains were remodeled either by using a rotamer library data base or by manually rotating the respective side chains. The final homology model of the BTK kinase domain had a root mean square deviation of 0.01 Å from ideal bond lengths and 2.2° from ideal bond angles after energy minimization. The homology model of BTK was then used, in conjunction with model coordinates of LFM and its analogs (which were later compared with crystal structures), for our modeling studies of the BTK·inhibitor complexes. Modeling of the BTK·LFM analog complexes was done using the Docking module within the program INSIGHTII (26.Insight II (1996) Molecular Simulations Inc. San Diego, CA.Google Scholar) and using the Affinity suite of programs for automatically docking a ligand to the receptor. Energy-minimized coordinates for each LFM molecule were generated and interactively docked into the ATP-binding site of BTK based on the position of quercetin in the HCK/quercetin crystal structure (22Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1045) Google Scholar). The hydrogen atoms on the kinase domain of BTK were generated, and potentials were assigned to both receptor and ligand prior to the start of the docking procedure. The docking method in the InsightII (26.Insight II (1996) Molecular Simulations Inc. San Diego, CA.Google Scholar) program used the CVFF force field and a Monte Carlo search strategy to search for and evaluate docked structures. While the coordinates for the bulk of the receptor were kept fixed, a defined region of the binding site was allowed to relax, thereby allowing the protein to adjust to the binding of different inhibitors. A binding set was defined within a distance of 5 Å from the inhibitor, allowing residues within this distance to shift and/or rotate to energetically favorable positions to accommodate the ligand. An assembly was defined consisting of the receptor and inhibitor molecule, and docking was performed using the fixed docking mode. Calculations approximating hydrophobic and hydrophilic interactions were used to determine the 10 best docking positions of each LFM analog in the BTK catalytic site. The various docked positions of each LFM analog was qualitatively evaluated using Ludi (29Bohm H.J. J. Comput. Aided Mol. Des. 1992; 6: 593-606Crossref PubMed Scopus (460) Google Scholar, 30Bohm H.J. J. Comput. Aided Mol. Des. 1994; 8: 243-256Crossref PubMed Scopus (987) Google Scholar) in INSIGHTII (26.Insight II (1996) Molecular Simulations Inc. San Diego, CA.Google Scholar) which was then used to estimate a binding constant (K i) for each compound in order to rank their relative binding capabilities and predicted inhibition of BTK. The K i trends for the LFM analogs were compared with the trend of the experimentally determined tyrosine kinase inhibition IC50 values for the compounds, in order to elucidate the structure-activity relationships determining the potency of LFM analogs. Sf21 (IPLB-SF21-AE) cells (20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar), derived from the ovarian tissue of the fall armyworm Spodotera frugiperda, were obtained from Invitrogen and maintained at 26–28 °C in Grace's insect cell medium supplemented with 10% fetal bovine serum and 1.0% antibiotic/antimycotic (Life Technologies, Inc.). Stock cells were maintained in suspension at 0.2–1.6 × 106/ml in 600-ml total culture volume in 1-liter Bellco spinner flasks at 60–90 rpm. Cell viability was maintained at 95–100% as determined by trypan blue dye exclusion. Recombinant baculovirus containing the murine BTK gene was constructed as described (20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar). In brief, the gene encoding BTK was excised from pBluescript SKII+ vector (Stratagene) by digestion with BamHI, and this fragment was then ligated into pFastBac1 (Life Technologies, Inc.). The resulting vector, pFastBac1-BTK, was then used to generate the recombinant baculovirus by site-specific transposition in Escherichia coli DH10Bac cells (Life Technologies, Inc.) which harbor a baculovirus shuttle vector (bacmid), bMON14272. The resulting recombinant bacmid DNA was introduced into insect cells by transfection with the standard liposome-mediated method using Cellfectin reagent (Life Technologies, Inc.). Four days later, transfection supernatants were harvested for subsequent plaque purification and analyzed as above. Kinase-dead BTK was generated as described (20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar) and cloned into the baculovirus expression vector as described above for wild-type BTK. Baculovirus expression vectors for JAK1 and JAK3 kinases were constructed and introduced into insect cells, as previously reported (31Goodman P.A. Niehoff L.B. Uckun F.M. J. Biol. Chem. 1998; 273: 17742-17748Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Sf21 cells were infected with a baculovirus expression vector for BTK, JAK1, or JAK3 as indicated in the figure legends. Cells were harvested and lysed (10 mm Tris, pH 7.6, 100 mm NaCl, 1% Nonidet P-40, 10% glycerol, 50 mm NaF, 100 μmNa3VO4, 50 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml aprotonin, 10 μg/ml leupeptin), and the kinases were immunoprecipitated from the lysates, as reported (20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar). Antibodies used for immunoprecipitations from insect cells are as follows: polyclonal rabbit anti-BTK serum (15Mahajan S. Fargnoli J. Burkhardt A.L. Kut S.A. Saouaf S.J. Bolen J.B. Mol. Cell. Biol. 1995; 15: 5304-5311Crossref PubMed Scopus (135) Google Scholar), polyclonal rabbit anti-JAK1 (HR-785), catalog number sc-277, rabbit polyclonal IgG affinity purified, 0.1 mg/ml, Santa Cruz Biotechnology, and polyclonal rabbit anti-JAK3 (C-21, catalog number sc-513, rabbit polyclonal IgG affinity purified, 0.2 mg/ml, Santa Cruz Biotechnology). Kinase assays were performed following a 1-h exposure of the immunoprecipitated tyrosine kinases to the test compounds, as described in detail elsewhere (15Mahajan S. Fargnoli J. Burkhardt A.L. Kut S.A. Saouaf S.J. Bolen J.B. Mol. Cell. Biol. 1995; 15: 5304-5311Crossref PubMed Scopus (135) Google Scholar,32Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 22: 1096-1100Crossref Scopus (166) Google Scholar). The immunoprecipitates were subjected to Western blot analysis as described previously (20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar). The establishment and characterization of DT40 lymphoma B cell line as well as BTK-deficient DT40 and its derivatives reconstituted with wild-type or mutant human BTK have been previously reported (32Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 22: 1096-1100Crossref Scopus (166) Google Scholar). Equal amounts of BTK protein were detected by Western blot analysis in all of the BTK-deficient DT40 clones transfected with wild-type or mutated humanBTK genes, but no BTK protein was detectable in the untransfected BTK-deficient DT40 cells (32Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 22: 1096-1100Crossref Scopus (166) Google Scholar). All cell lines derived from the chicken B cell line DT40 were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% heat-inactivated chicken serum, 2 mm glutamine, penicillin, and streptomycin. Cells were grown at 37 °C in a 5% CO2water-saturated atmosphere. The BTK positive human B-lineage leukemia cell lines NALM-6 and ALL-1 were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (33Uckun F.M. Evans W.E. Forsyth C.J. Waddick K.G. Ahlgren L.T. Chelstrom L.M. Burkhardt A. Bolen J. Myers D.E. Science. 1995; 267: 886-891Crossref PubMed Scopus (262) Google Scholar). COS-7 simian kidney cell line and HepG2 human hepatoma cell line were obtained from ATCC. Antibodies directed against BTK, JAK1, JAK3, and HCK have been described previously (15Mahajan S. Fargnoli J. Burkhardt A.L. Kut S.A. Saouaf S.J. Bolen J.B. Mol. Cell. Biol. 1995; 15: 5304-5311Crossref PubMed Scopus (135) Google Scholar, 20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar, 31Goodman P.A. Niehoff L.B. Uckun F.M. J. Biol. Chem. 1998; 273: 17742-17748Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 32Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 22: 1096-1100Crossref Scopus (166) Google Scholar). Polyclonal antibodies to BTK were generated by immunization of rabbits with glutathioneS-transferase (GST) fusion proteins (Amersham Pharmacia Biotech) containing the first 150 amino acids of BTK. The monoclonal anti-Fas antibody (F22120) was obtained from the Transduction Laboratories, Inc. (Lexington, KY). Immunoprecipitations, immune complex protein kinase assays, and immunoblotting using the ECL chemiluminescence detection system (Amersham Pharmacia Biotech) were conducted as described previously (15Mahajan S. Fargnoli J. Burkhardt A.L. Kut S.A. Saouaf S.J. Bolen J.B. Mol. Cell. Biol. 1995; 15: 5304-5311Crossref PubMed Scopus (135) Google Scholar, 20Vassilev A. Ozer Z. Navara C. Mahajan S. Uckun F.M. J. Biol. Chem. 1998; 274: 1646-1656Abstract Full Text Full Text PDF Scopus (122) Google Scholar, 31Goodman P.A. Niehoff L.B. Uckun F.M. J. Biol. Chem. 1998; 273: 17742-17748Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 32Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 22: 1096-1100Crossref Scopus (166) Google Scholar). Following electrophoresis, kinase gels were dried onto Whatman 3M filter paper and subjected to phosphorimaging on a Molecular Imager (Bio-Rad) as well as autoradiography on film. Similarly, all chemiluminescent BTK Western blots were subjected to three-dimensional densitometric scanning using the Molecular Imager and Imaging Densitometer using the Molecular Analyst/Macintosh version 2.1 software following the specifications of the manufacturer (Bio-Rad). For each drug concentration, a BTK kinase activity index was determined by comparing the ratios of the kinase activity in PhosphorImager units (PIU) and density of the protein bands in densitometric scanning units (DSU) to those of the base-line sample and using the formula: activity index = [PIU of kinase band/DSU of BTK protein band]test sample:[PIU of kinase band/DSU of BTK" @default.
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• W2069809502 title "Rational Design and Synthesis of a Novel Anti-leukemic Agent Targeting Bruton′s Tyrosine Kinase (BTK), LFM-A13 [α-Cyano-β-Hydroxy-β-Methyl-N-(2,5-Dibromophenyl)Propenamide]" @default.
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