Sequencing:Article Title: Structure-Based Design of Orally Bioavailable 1 H -Pyrrolo[3,2- c ]pyridine Inhibitors of Mitotic Kinase Monopolar Spindle 1 (MPS1)
Article Snippet: The percent inhibition at each concentration was then fitted to a four-parameter logistic fit using the Studies package (Dotmatics, Bishops Stortford, UK): y = ( a + (( b – a )/(1 + (( c / x d )))), where a = asym min, b = asym max, c = IC 50 , and d = Hill coefficient. .. The enzyme reactions (10 μL total volume) were carried out in black 384-well low-volume plates containing full-length AurB/INCENP complex (1 nM, Carna Biosciences, Japan) or N-terminal HIS-tagged AurA (5 nM, in-house), fluorescent-labeled peptide [FL-Peptide 21, PerkinElmer, sequence: 5FAM-LRRASLG-CONH 2 ] (1.5 μM), ATP (15 or 20 μM), either 1% (v/v) DMSO or the test compound (in the range from 0.25 nM to 100 μM in 1% (v/v) DMSO), and assay buffer (50 mM Tris, pH 7.4, 200 mM NaCl, 5 mM MgCl 2 , 2 mM DTT, and 0.1% (v/v) Tween 20). .. The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μL) containing 20 mM EDTA and 0.05% (v/v) Brij-35 in 0.1 M HEPES-buffered saline (Free acid, Sigma, UK).
Article Title: Aurora Isoform Selectivity: Design and Synthesis of Imidazo[4,5-b]pyridine Derivatives as Highly Selective Inhibitors of Aurora-A Kinase in Cells
Article Snippet: For the Aurora-A kinase the enzyme reaction (total volume 10 μL) was carried out with 5 nM N-terminal HIS-tagged AurA, which was expressed and purified as previously described,46 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAM-LRRASLG-CONH2), 20 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and the test compound or DMSO control. .. For the Aurora-B kinase the enzyme reaction (total volume 10 μL) was carried out with 1 nM full-length AurB/INCENP complex (no. 05- 102, Carna Biosciences, Inc., Kobe, Japan), 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAMLRRASLG-CONH2), 15 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and either the test compound or a DMSO control. ..
Article Title: 6-[5-amino-6-(2-ethoxyethoxy)-imidazo[4,5-B]pyridin-3-yl]-nicotinonitrile derivatives and their use as IRAK inhibitors
Article Snippet: .. TABLE VI Conditions for human kinase off-targets inhibition assays Kinase, Substrate, Incubation [Kinase] [Substrate] ATP Assay buffer time ABL PolyGT (Sigma- 0.5 μM ATP + 50 mM Tris pH 7.7 60 min (Life Technologies, Aldrich, P0275), 0.25 μCi/25 μL 0.03% Triton X-100 P3049), 40 ng/mL 5 μg/mL [γ-33P]ATP 1 mM DTT 25 mM MgCl2 Aurora B (Carna Histone H3 peptide 1.3 μM ATP + 25 mM Tris pH 7.7 90 min BioSciences, (SEQ ID2), 0.5 μM 0.25 μCi/25 μL 0.01% Triton X-100 05-102), 10 ng/mL [γ-33P]ATP 5 mM MgCl2 5 mM DTT CDK2 (Carna Histone H1-derived 0.1 μM ATP + 8 mM MOPS pH 7.0 60 min Biosciences, peptide (SEQ ID3), 0.25 μCi/25 μL 0.01% Brij-35 04-103), 30 ng/mL 0.36 μM [γ-33P]ATP 1 mM DTT 5 mM MnCl2 CDK9 (Millipore, PDKtide (SEQ 0.25 μM ATP + 20 mM MOPS pH 7.0 60 min 14-685), ID4), 0.5 μM 0.125 μCi/25 μL 0.01% Triton X-100 230 ng/mL [γ-33P]ATP 5 mM MnCl2 c-KIT (Millipore, PolyGT (Sigma- 3 μM ATP + 16 mM Tris pH 7.0 90 min 14-559), Aldrich, P0275), 0.25 μCi/25 μL 500 μM EDTA 0.01 mU/μL 0.1 mg/mL [γ-33P]ATP 0.01% Triton X-100 10 mM MnCl2 1 mM DTT 10 mM MgOAc GSK3b (Carna Phospho glycogen 1.5 μM ATP + 50 mM Tris pH 8.0 90 min Biosciences, synthase peptide2 0.25 μCi/25 μL 0.01% Brij-35 04-141), 20 ng/mL (Millipore, 12-241), [γ-33P]ATP 1 mM DTT 1.25 μM 5 mM MgOAc c-SRC (Carna PolyGT (Sigma- 0.25 μM ATP + 25 mM MOPS pH 7.0 60 min Biosciences, Aldrich, P0275), 0.125 μCi/25 μL 10 mM MnCl2 08-173), 8 ng/mL 2 μg/mL [γ-33P]ATP 2.5 mM DTT 0.01% Brij35 TABLE VII Peptide substrates used in human kinase off-targets inhibition assays SEQ ID Substrate No. Sequence Provider RIP140 1 CYGVASSHLKTLLKKSKVKDQ Almac Group Ltd. peptide 20 Seagoe Industrial Estate Craigavon BT63 5QD UK Histone H3 2 ARTKQTARKSTGGKAPRKQLC AnaSpec Inc. peptide 34801 Campus Drive Fremont, CA 94555 USA Histone 3 GGGPATPKKAKKL AnaSpec Inc. H1-derived 34801 Campus Drive peptide Fremont, CA 94555 USA PDKtide 4 KTFCGTPEYLAPEVRREPRILSEEEQE Thermo Fisher Scientific Inc. MFRDFDYIADWC 81 Wyman St. Waltham, MA 02451 USA TABLE VIII In vitro human kinase off-targets IC50 of illustrative compounds of the invention ABL Aurora B CDK2 CDK9 GSK3b c-SRC IC50 IC50 IC50 IC50 IC50 IC50 Cpd# (nM) (nM) (nM) (nM) (nM) (nM) 1 — — >6670 >1330 >6670 >1330 2 — — >1330 712 >1330 >1330 5 2690 — >4000 2290 >4000 >2940 6 656 — 2670 1030 >4000 754 9 633 — >4000 639 3250 286 11 — — >1330 834 >1330 >1330 12 138 >1880 944 1150 >1330 477 14 — — 1380 1390 2240 319 ..
Article Title: Structure-Based Design of Orally Bioavailable 1H-Pyrrolo[3,2-c]pyridine Inhibitors of Mitotic Kinase Monopolar Spindle 1 (MPS1)
Article Snippet: .. The enzyme reactions (10 μL total volume) were carried out in black 384-well low-volume plates containing full-length AurB/INCENP complex (1 nM, Carna Biosciences, Japan) or N-terminal HIS-tagged AurA (5 nM, in-house), fluorescent-labeled peptide [FL-Peptide 21, PerkinElmer, sequence: 5FAM-LRRASLG-CONH2] (1.5 μM), ATP (15 or 20 μM), either 1% (v/v) DMSO or the test compound (in the range from 0.25 nM to 100 μM in 1% (v/v) DMSO), and assay buffer (50 mM Tris, pH 7.4, 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, and 0.1% (v/v) Tween 20). .. The reaction was carried out for 60 min at room temperature and stopped by the addition of buffer (10 μL) containing 20 mM EDTA and 0.05% (v/v) Brij-35 in 0.1 M HEPES-buffered saline (Free acid, Sigma, UK).
Article Title: Aurora isoform selectivity: design and synthesis of imidazo[4,5-b]pyridine derivatives as highly selective inhibitors of Aurora-A kinase in cells.
Article Snippet: For the Aurora-A kinase the enzyme reaction (total volume 10 μL) was carried out with 5 nM N-terminal HIS-tagged AurA, which was expressed and purified as previously described,46 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAM-LRRASLG-CONH2), 20 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and the test compound or DMSO control. .. For the Aurora-B kinase the enzyme reaction (total volume 10 μL) was carried out with 1 nM full-length AurB/INCENP complex (no. 05- 102, Carna Biosciences, Inc., Kobe, Japan), 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAMLRRASLG-CONH2), 15 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and either the test compound or a DMSO control. ..
Labeling:Article Title: Aurora Isoform Selectivity: Design and Synthesis of Imidazo[4,5-b]pyridine Derivatives as Highly Selective Inhibitors of Aurora-A Kinase in Cells
Article Snippet: For the Aurora-A kinase the enzyme reaction (total volume 10 μL) was carried out with 5 nM N-terminal HIS-tagged AurA, which was expressed and purified as previously described,46 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAM-LRRASLG-CONH2), 20 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and the test compound or DMSO control. .. For the Aurora-B kinase the enzyme reaction (total volume 10 μL) was carried out with 1 nM full-length AurB/INCENP complex (no. 05- 102, Carna Biosciences, Inc., Kobe, Japan), 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAMLRRASLG-CONH2), 15 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and either the test compound or a DMSO control. ..
Article Title: Aurora isoform selectivity: design and synthesis of imidazo[4,5-b]pyridine derivatives as highly selective inhibitors of Aurora-A kinase in cells.
Article Snippet: For the Aurora-A kinase the enzyme reaction (total volume 10 μL) was carried out with 5 nM N-terminal HIS-tagged AurA, which was expressed and purified as previously described,46 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAM-LRRASLG-CONH2), 20 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and the test compound or DMSO control. .. For the Aurora-B kinase the enzyme reaction (total volume 10 μL) was carried out with 1 nM full-length AurB/INCENP complex (no. 05- 102, Carna Biosciences, Inc., Kobe, Japan), 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAMLRRASLG-CONH2), 15 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and either the test compound or a DMSO control. ..
Control:Article Title: Aurora Isoform Selectivity: Design and Synthesis of Imidazo[4,5-b]pyridine Derivatives as Highly Selective Inhibitors of Aurora-A Kinase in Cells
Article Snippet: For the Aurora-A kinase the enzyme reaction (total volume 10 μL) was carried out with 5 nM N-terminal HIS-tagged AurA, which was expressed and purified as previously described,46 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAM-LRRASLG-CONH2), 20 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and the test compound or DMSO control. .. For the Aurora-B kinase the enzyme reaction (total volume 10 μL) was carried out with 1 nM full-length AurB/INCENP complex (no. 05- 102, Carna Biosciences, Inc., Kobe, Japan), 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAMLRRASLG-CONH2), 15 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and either the test compound or a DMSO control. ..
Article Title: Aurora isoform selectivity: design and synthesis of imidazo[4,5-b]pyridine derivatives as highly selective inhibitors of Aurora-A kinase in cells.
Article Snippet: For the Aurora-A kinase the enzyme reaction (total volume 10 μL) was carried out with 5 nM N-terminal HIS-tagged AurA, which was expressed and purified as previously described,46 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAM-LRRASLG-CONH2), 20 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and the test compound or DMSO control. .. For the Aurora-B kinase the enzyme reaction (total volume 10 μL) was carried out with 1 nM full-length AurB/INCENP complex (no. 05- 102, Carna Biosciences, Inc., Kobe, Japan), 1.5 μM fluorescently labeled peptide (no. 760365, FL-Peptide 21, PerkinElmer, sequence 5FAMLRRASLG-CONH2), 15 μM ATP in assay buffer (50 mM Tris (pH 7.4), 200 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1% Tween 20), and either the test compound or a DMSO control. ..
Recombinant:Article Title: The Greatwall kinase safeguards the genome integrity by affecting the kinome activity in mitosis
Article Snippet: .. Kinase assays with recombinant proteins were performed in EB buffer (containing 15mM Mg 2+ ) by incubating 1.25 μg of GST purified recombinant proteins with 35–100 ng of active recombinant kinases (CDK1/cycB1 [homemade or Carna Biosciences 04-102], CDK1/cycA2 [homemade], AURKA/TPX2 [Carna Biosciences 05-186], AURKB/Incenp [Carna Biosciences 05-102], TTK/MPS1 [Carna Biosciences 05-169], NEK2 [Carna Biosciences 05-226]) or with immunoprecipitated MASTL (WT or KD) or WT PLK1. .. Incubation was carried at room temperature (23 °C) under constant shaking (1200 rpm) for 30 min in presence of 50-μM ATP (Sigma; A2609) and 2 μCi of [gamma- 32 P] ATP (PerkinElmer; BLUE502A).
Purification:Article Title: The Greatwall kinase safeguards the genome integrity by affecting the kinome activity in mitosis
Article Snippet: .. Kinase assays with recombinant proteins were performed in EB buffer (containing 15mM Mg 2+ ) by incubating 1.25 μg of GST purified recombinant proteins with 35–100 ng of active recombinant kinases (CDK1/cycB1 [homemade or Carna Biosciences 04-102], CDK1/cycA2 [homemade], AURKA/TPX2 [Carna Biosciences 05-186], AURKB/Incenp [Carna Biosciences 05-102], TTK/MPS1 [Carna Biosciences 05-169], NEK2 [Carna Biosciences 05-226]) or with immunoprecipitated MASTL (WT or KD) or WT PLK1. .. Incubation was carried at room temperature (23 °C) under constant shaking (1200 rpm) for 30 min in presence of 50-μM ATP (Sigma; A2609) and 2 μCi of [gamma- 32 P] ATP (PerkinElmer; BLUE502A).
Immunoprecipitation:Article Title: The Greatwall kinase safeguards the genome integrity by affecting the kinome activity in mitosis
Article Snippet: .. Kinase assays with recombinant proteins were performed in EB buffer (containing 15mM Mg 2+ ) by incubating 1.25 μg of GST purified recombinant proteins with 35–100 ng of active recombinant kinases (CDK1/cycB1 [homemade or Carna Biosciences 04-102], CDK1/cycA2 [homemade], AURKA/TPX2 [Carna Biosciences 05-186], AURKB/Incenp [Carna Biosciences 05-102], TTK/MPS1 [Carna Biosciences 05-169], NEK2 [Carna Biosciences 05-226]) or with immunoprecipitated MASTL (WT or KD) or WT PLK1. .. Incubation was carried at room temperature (23 °C) under constant shaking (1200 rpm) for 30 min in presence of 50-μM ATP (Sigma; A2609) and 2 μCi of [gamma- 32 P] ATP (PerkinElmer; BLUE502A).
Inhibition:Article Title: 6-[5-amino-6-(2-ethoxyethoxy)-imidazo[4,5-B]pyridin-3-yl]-nicotinonitrile derivatives and their use as IRAK inhibitors
Article Snippet: .. TABLE VI Conditions for human kinase off-targets inhibition assays Kinase, Substrate, Incubation [Kinase] [Substrate] ATP Assay buffer time ABL PolyGT (Sigma- 0.5 μM ATP + 50 mM Tris pH 7.7 60 min (Life Technologies, Aldrich, P0275), 0.25 μCi/25 μL 0.03% Triton X-100 P3049), 40 ng/mL 5 μg/mL [γ-33P]ATP 1 mM DTT 25 mM MgCl2 Aurora B (Carna Histone H3 peptide 1.3 μM ATP + 25 mM Tris pH 7.7 90 min BioSciences, (SEQ ID2), 0.5 μM 0.25 μCi/25 μL 0.01% Triton X-100 05-102), 10 ng/mL [γ-33P]ATP 5 mM MgCl2 5 mM DTT CDK2 (Carna Histone H1-derived 0.1 μM ATP + 8 mM MOPS pH 7.0 60 min Biosciences, peptide (SEQ ID3), 0.25 μCi/25 μL 0.01% Brij-35 04-103), 30 ng/mL 0.36 μM [γ-33P]ATP 1 mM DTT 5 mM MnCl2 CDK9 (Millipore, PDKtide (SEQ 0.25 μM ATP + 20 mM MOPS pH 7.0 60 min 14-685), ID4), 0.5 μM 0.125 μCi/25 μL 0.01% Triton X-100 230 ng/mL [γ-33P]ATP 5 mM MnCl2 c-KIT (Millipore, PolyGT (Sigma- 3 μM ATP + 16 mM Tris pH 7.0 90 min 14-559), Aldrich, P0275), 0.25 μCi/25 μL 500 μM EDTA 0.01 mU/μL 0.1 mg/mL [γ-33P]ATP 0.01% Triton X-100 10 mM MnCl2 1 mM DTT 10 mM MgOAc GSK3b (Carna Phospho glycogen 1.5 μM ATP + 50 mM Tris pH 8.0 90 min Biosciences, synthase peptide2 0.25 μCi/25 μL 0.01% Brij-35 04-141), 20 ng/mL (Millipore, 12-241), [γ-33P]ATP 1 mM DTT 1.25 μM 5 mM MgOAc c-SRC (Carna PolyGT (Sigma- 0.25 μM ATP + 25 mM MOPS pH 7.0 60 min Biosciences, Aldrich, P0275), 0.125 μCi/25 μL 10 mM MnCl2 08-173), 8 ng/mL 2 μg/mL [γ-33P]ATP 2.5 mM DTT 0.01% Brij35 TABLE VII Peptide substrates used in human kinase off-targets inhibition assays SEQ ID Substrate No. Sequence Provider RIP140 1 CYGVASSHLKTLLKKSKVKDQ Almac Group Ltd. peptide 20 Seagoe Industrial Estate Craigavon BT63 5QD UK Histone H3 2 ARTKQTARKSTGGKAPRKQLC AnaSpec Inc. peptide 34801 Campus Drive Fremont, CA 94555 USA Histone 3 GGGPATPKKAKKL AnaSpec Inc. H1-derived 34801 Campus Drive peptide Fremont, CA 94555 USA PDKtide 4 KTFCGTPEYLAPEVRREPRILSEEEQE Thermo Fisher Scientific Inc. MFRDFDYIADWC 81 Wyman St. Waltham, MA 02451 USA TABLE VIII In vitro human kinase off-targets IC50 of illustrative compounds of the invention ABL Aurora B CDK2 CDK9 GSK3b c-SRC IC50 IC50 IC50 IC50 IC50 IC50 Cpd# (nM) (nM) (nM) (nM) (nM) (nM) 1 — — >6670 >1330 >6670 >1330 2 — — >1330 712 >1330 >1330 5 2690 — >4000 2290 >4000 >2940 6 656 — 2670 1030 >4000 754 9 633 — >4000 639 3250 286 11 — — >1330 834 >1330 >1330 12 138 >1880 944 1150 >1330 477 14 — — 1380 1390 2240 319 ..
Incubation:Article Title: 6-[5-amino-6-(2-ethoxyethoxy)-imidazo[4,5-B]pyridin-3-yl]-nicotinonitrile derivatives and their use as IRAK inhibitors
Article Snippet: .. TABLE VI Conditions for human kinase off-targets inhibition assays Kinase, Substrate, Incubation [Kinase] [Substrate] ATP Assay buffer time ABL PolyGT (Sigma- 0.5 μM ATP + 50 mM Tris pH 7.7 60 min (Life Technologies, Aldrich, P0275), 0.25 μCi/25 μL 0.03% Triton X-100 P3049), 40 ng/mL 5 μg/mL [γ-33P]ATP 1 mM DTT 25 mM MgCl2 Aurora B (Carna Histone H3 peptide 1.3 μM ATP + 25 mM Tris pH 7.7 90 min BioSciences, (SEQ ID2), 0.5 μM 0.25 μCi/25 μL 0.01% Triton X-100 05-102), 10 ng/mL [γ-33P]ATP 5 mM MgCl2 5 mM DTT CDK2 (Carna Histone H1-derived 0.1 μM ATP + 8 mM MOPS pH 7.0 60 min Biosciences, peptide (SEQ ID3), 0.25 μCi/25 μL 0.01% Brij-35 04-103), 30 ng/mL 0.36 μM [γ-33P]ATP 1 mM DTT 5 mM MnCl2 CDK9 (Millipore, PDKtide (SEQ 0.25 μM ATP + 20 mM MOPS pH 7.0 60 min 14-685), ID4), 0.5 μM 0.125 μCi/25 μL 0.01% Triton X-100 230 ng/mL [γ-33P]ATP 5 mM MnCl2 c-KIT (Millipore, PolyGT (Sigma- 3 μM ATP + 16 mM Tris pH 7.0 90 min 14-559), Aldrich, P0275), 0.25 μCi/25 μL 500 μM EDTA 0.01 mU/μL 0.1 mg/mL [γ-33P]ATP 0.01% Triton X-100 10 mM MnCl2 1 mM DTT 10 mM MgOAc GSK3b (Carna Phospho glycogen 1.5 μM ATP + 50 mM Tris pH 8.0 90 min Biosciences, synthase peptide2 0.25 μCi/25 μL 0.01% Brij-35 04-141), 20 ng/mL (Millipore, 12-241), [γ-33P]ATP 1 mM DTT 1.25 μM 5 mM MgOAc c-SRC (Carna PolyGT (Sigma- 0.25 μM ATP + 25 mM MOPS pH 7.0 60 min Biosciences, Aldrich, P0275), 0.125 μCi/25 μL 10 mM MnCl2 08-173), 8 ng/mL 2 μg/mL [γ-33P]ATP 2.5 mM DTT 0.01% Brij35 TABLE VII Peptide substrates used in human kinase off-targets inhibition assays SEQ ID Substrate No. Sequence Provider RIP140 1 CYGVASSHLKTLLKKSKVKDQ Almac Group Ltd. peptide 20 Seagoe Industrial Estate Craigavon BT63 5QD UK Histone H3 2 ARTKQTARKSTGGKAPRKQLC AnaSpec Inc. peptide 34801 Campus Drive Fremont, CA 94555 USA Histone 3 GGGPATPKKAKKL AnaSpec Inc. H1-derived 34801 Campus Drive peptide Fremont, CA 94555 USA PDKtide 4 KTFCGTPEYLAPEVRREPRILSEEEQE Thermo Fisher Scientific Inc. MFRDFDYIADWC 81 Wyman St. Waltham, MA 02451 USA TABLE VIII In vitro human kinase off-targets IC50 of illustrative compounds of the invention ABL Aurora B CDK2 CDK9 GSK3b c-SRC IC50 IC50 IC50 IC50 IC50 IC50 Cpd# (nM) (nM) (nM) (nM) (nM) (nM) 1 — — >6670 >1330 >6670 >1330 2 — — >1330 712 >1330 >1330 5 2690 — >4000 2290 >4000 >2940 6 656 — 2670 1030 >4000 754 9 633 — >4000 639 3250 286 11 — — >1330 834 >1330 >1330 12 138 >1880 944 1150 >1330 477 14 — — 1380 1390 2240 319 ..
ATP Assay:Article Title: 6-[5-amino-6-(2-ethoxyethoxy)-imidazo[4,5-B]pyridin-3-yl]-nicotinonitrile derivatives and their use as IRAK inhibitors
Article Snippet: .. TABLE VI Conditions for human kinase off-targets inhibition assays Kinase, Substrate, Incubation [Kinase] [Substrate] ATP Assay buffer time ABL PolyGT (Sigma- 0.5 μM ATP + 50 mM Tris pH 7.7 60 min (Life Technologies, Aldrich, P0275), 0.25 μCi/25 μL 0.03% Triton X-100 P3049), 40 ng/mL 5 μg/mL [γ-33P]ATP 1 mM DTT 25 mM MgCl2 Aurora B (Carna Histone H3 peptide 1.3 μM ATP + 25 mM Tris pH 7.7 90 min BioSciences, (SEQ ID2), 0.5 μM 0.25 μCi/25 μL 0.01% Triton X-100 05-102), 10 ng/mL [γ-33P]ATP 5 mM MgCl2 5 mM DTT CDK2 (Carna Histone H1-derived 0.1 μM ATP + 8 mM MOPS pH 7.0 60 min Biosciences, peptide (SEQ ID3), 0.25 μCi/25 μL 0.01% Brij-35 04-103), 30 ng/mL 0.36 μM [γ-33P]ATP 1 mM DTT 5 mM MnCl2 CDK9 (Millipore, PDKtide (SEQ 0.25 μM ATP + 20 mM MOPS pH 7.0 60 min 14-685), ID4), 0.5 μM 0.125 μCi/25 μL 0.01% Triton X-100 230 ng/mL [γ-33P]ATP 5 mM MnCl2 c-KIT (Millipore, PolyGT (Sigma- 3 μM ATP + 16 mM Tris pH 7.0 90 min 14-559), Aldrich, P0275), 0.25 μCi/25 μL 500 μM EDTA 0.01 mU/μL 0.1 mg/mL [γ-33P]ATP 0.01% Triton X-100 10 mM MnCl2 1 mM DTT 10 mM MgOAc GSK3b (Carna Phospho glycogen 1.5 μM ATP + 50 mM Tris pH 8.0 90 min Biosciences, synthase peptide2 0.25 μCi/25 μL 0.01% Brij-35 04-141), 20 ng/mL (Millipore, 12-241), [γ-33P]ATP 1 mM DTT 1.25 μM 5 mM MgOAc c-SRC (Carna PolyGT (Sigma- 0.25 μM ATP + 25 mM MOPS pH 7.0 60 min Biosciences, Aldrich, P0275), 0.125 μCi/25 μL 10 mM MnCl2 08-173), 8 ng/mL 2 μg/mL [γ-33P]ATP 2.5 mM DTT 0.01% Brij35 TABLE VII Peptide substrates used in human kinase off-targets inhibition assays SEQ ID Substrate No. Sequence Provider RIP140 1 CYGVASSHLKTLLKKSKVKDQ Almac Group Ltd. peptide 20 Seagoe Industrial Estate Craigavon BT63 5QD UK Histone H3 2 ARTKQTARKSTGGKAPRKQLC AnaSpec Inc. peptide 34801 Campus Drive Fremont, CA 94555 USA Histone 3 GGGPATPKKAKKL AnaSpec Inc. H1-derived 34801 Campus Drive peptide Fremont, CA 94555 USA PDKtide 4 KTFCGTPEYLAPEVRREPRILSEEEQE Thermo Fisher Scientific Inc. MFRDFDYIADWC 81 Wyman St. Waltham, MA 02451 USA TABLE VIII In vitro human kinase off-targets IC50 of illustrative compounds of the invention ABL Aurora B CDK2 CDK9 GSK3b c-SRC IC50 IC50 IC50 IC50 IC50 IC50 Cpd# (nM) (nM) (nM) (nM) (nM) (nM) 1 — — >6670 >1330 >6670 >1330 2 — — >1330 712 >1330 >1330 5 2690 — >4000 2290 >4000 >2940 6 656 — 2670 1030 >4000 754 9 633 — >4000 639 3250 286 11 — — >1330 834 >1330 >1330 12 138 >1880 944 1150 >1330 477 14 — — 1380 1390 2240 319 ..
In Vitro:Article Title: 6-[5-amino-6-(2-ethoxyethoxy)-imidazo[4,5-B]pyridin-3-yl]-nicotinonitrile derivatives and their use as IRAK inhibitors
Article Snippet: .. TABLE VI Conditions for human kinase off-targets inhibition assays Kinase, Substrate, Incubation [Kinase] [Substrate] ATP Assay buffer time ABL PolyGT (Sigma- 0.5 μM ATP + 50 mM Tris pH 7.7 60 min (Life Technologies, Aldrich, P0275), 0.25 μCi/25 μL 0.03% Triton X-100 P3049), 40 ng/mL 5 μg/mL [γ-33P]ATP 1 mM DTT 25 mM MgCl2 Aurora B (Carna Histone H3 peptide 1.3 μM ATP + 25 mM Tris pH 7.7 90 min BioSciences, (SEQ ID2), 0.5 μM 0.25 μCi/25 μL 0.01% Triton X-100 05-102), 10 ng/mL [γ-33P]ATP 5 mM MgCl2 5 mM DTT CDK2 (Carna Histone H1-derived 0.1 μM ATP + 8 mM MOPS pH 7.0 60 min Biosciences, peptide (SEQ ID3), 0.25 μCi/25 μL 0.01% Brij-35 04-103), 30 ng/mL 0.36 μM [γ-33P]ATP 1 mM DTT 5 mM MnCl2 CDK9 (Millipore, PDKtide (SEQ 0.25 μM ATP + 20 mM MOPS pH 7.0 60 min 14-685), ID4), 0.5 μM 0.125 μCi/25 μL 0.01% Triton X-100 230 ng/mL [γ-33P]ATP 5 mM MnCl2 c-KIT (Millipore, PolyGT (Sigma- 3 μM ATP + 16 mM Tris pH 7.0 90 min 14-559), Aldrich, P0275), 0.25 μCi/25 μL 500 μM EDTA 0.01 mU/μL 0.1 mg/mL [γ-33P]ATP 0.01% Triton X-100 10 mM MnCl2 1 mM DTT 10 mM MgOAc GSK3b (Carna Phospho glycogen 1.5 μM ATP + 50 mM Tris pH 8.0 90 min Biosciences, synthase peptide2 0.25 μCi/25 μL 0.01% Brij-35 04-141), 20 ng/mL (Millipore, 12-241), [γ-33P]ATP 1 mM DTT 1.25 μM 5 mM MgOAc c-SRC (Carna PolyGT (Sigma- 0.25 μM ATP + 25 mM MOPS pH 7.0 60 min Biosciences, Aldrich, P0275), 0.125 μCi/25 μL 10 mM MnCl2 08-173), 8 ng/mL 2 μg/mL [γ-33P]ATP 2.5 mM DTT 0.01% Brij35 TABLE VII Peptide substrates used in human kinase off-targets inhibition assays SEQ ID Substrate No. Sequence Provider RIP140 1 CYGVASSHLKTLLKKSKVKDQ Almac Group Ltd. peptide 20 Seagoe Industrial Estate Craigavon BT63 5QD UK Histone H3 2 ARTKQTARKSTGGKAPRKQLC AnaSpec Inc. peptide 34801 Campus Drive Fremont, CA 94555 USA Histone 3 GGGPATPKKAKKL AnaSpec Inc. H1-derived 34801 Campus Drive peptide Fremont, CA 94555 USA PDKtide 4 KTFCGTPEYLAPEVRREPRILSEEEQE Thermo Fisher Scientific Inc. MFRDFDYIADWC 81 Wyman St. Waltham, MA 02451 USA TABLE VIII In vitro human kinase off-targets IC50 of illustrative compounds of the invention ABL Aurora B CDK2 CDK9 GSK3b c-SRC IC50 IC50 IC50 IC50 IC50 IC50 Cpd# (nM) (nM) (nM) (nM) (nM) (nM) 1 — — >6670 >1330 >6670 >1330 2 — — >1330 712 >1330 >1330 5 2690 — >4000 2290 >4000 >2940 6 656 — 2670 1030 >4000 754 9 633 — >4000 639 3250 286 11 — — >1330 834 >1330 >1330 12 138 >1880 944 1150 >1330 477 14 — — 1380 1390 2240 319 ..
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