Structure-based lead identification of ATP-competitive MK2 inhibitors

Bioorganic and Medicinal Chemistry Letters

Volumn 21, Issue 12, 2011, Pages 3818-3822

  Barf, Tjeerd ^a   Kaptein, Allard ^a   De Wilde, Sander ^a   Van Der Heijden, Ruud ^a   Van Someren, Richard ^a   Demont, Dennis ^a   Schultz Fademrecht, Carsten ^a   Versteegh, Judith ^a   Van Zeeland, Mario ^a   Seegers, Nicole ^a   Kazemier, Bert ^a   Van De Kar, Bas ^a   Van Hoek, Maaike ^a   De Roos, Jeroen ^a   Klop, Henri ^a   Smeets, Ruben ^a   Hofstra, Claudia ^a   Hornberg, Jorrit ^a   Oubrie, Arthur ^a  

^a MERCK RESEARCH LABORATORIES   (United States)

Author keywords

Anti TNF ; MAPKAPK2; MK2; Rheumatoid arthritis; SBDD

Indexed keywords

MAPK ACTIVATED PROTEIN KINASE 2 INHIBITOR; PHOSPHOTRANSFERASE INHIBITOR; UNCLASSIFIED DRUG;

ANIMAL EXPERIMENT; ARTICLE; CONTROLLED STUDY; DRUG BIOAVAILABILITY; DRUG DESIGN; DRUG EFFICACY; DRUG STRUCTURE; ENZYME ACTIVITY; ENZYME INHIBITION; IN VIVO STUDY; NONHUMAN; RAT; X RAY CRYSTALLOGRAPHY;

ADENOSINE TRIPHOSPHATE; ANIMALS; BINDING, COMPETITIVE; CACO-2 CELLS; CRYSTALLOGRAPHY, X-RAY; DISEASE MODELS, ANIMAL; DRUG DESIGN; HUMANS; INHIBITORY CONCENTRATION 50; INTRACELLULAR SIGNALING PEPTIDES AND PROTEINS; MODELS, MOLECULAR; MOLECULAR STRUCTURE; PROTEIN KINASE INHIBITORS; PROTEIN-SERINE-THREONINE KINASES; RATS; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 79957833356     PISSN: 0960894X     EISSN: 14643405     Source Type: Journal    
DOI: 10.1016/j.bmcl.2011.04.018     Document Type: Article

References (19)

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10. Structures of MK2:1 and MK2:2 were determined in collaboration with Sareum and obtained using a construct comprising residues 41-364 with a C-terminal His-tag. This construct was expressed in Escherichia coli and purified using immobilized metal affinity, size exclusion, and ion exchange chromatography. The protein was crystallized in space group P 4132 using malonate as precipitant. Data sets with 1 and 2 were collected at 3.0 and 3.2 Å, respectively. The coordinates have been deposited with the Protein Data Bank at Rutgers, the respective codes are 3R2Y and 3R3O.
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13. MK2 enzyme activity is measured using an IMAP assay (Immobilized Metal Assay for Phosphochemicals-based coupled assay). Test compounds in DMSO (final concentration is assay is 0.1% DMSO) and MK2 enzyme (peptide 46-end (Millipore) 25 mU/mL final concentration in assay) are pre-incubated 30 min at room temperature, before adding Fluorescin labeled substrate peptide (FluobetaA-11A NeoMPS, final substrate peptide concentration is 50 nM). Kinase assay is started by adding ATP (final ATP concentration is 1 μM (=Km ATP)). Following incubation for 2 h at room temperature the enzyme reaction is stopped by adding IMAP Progressive Binding Solution (according to suppliers (Molecular Devices) protocol). After 60 min incubation at room temperature in the dark the FP signal is read. Each concentration is tested in two independent experiments with duplicates in each experiment.
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17. 1 with 12 molecules per asymmetric unit. The structure of MK2:5b was determined at 2.9 Å resolution. MK3 protein was purchased from Evotec and crystallized with 1 using protocols described in Ref. 12. MK3:1 crystals were soaked and exchanged with 5b. The structure of MK3:5b was determined at 2.1 Å resolution. The coordinates have been deposited with the Rutgers Protein Data Bank, with accession codes 3R2B and 3R1N.
18. 2 at 37 °C in humidified atmosphere, culture medium is collected for the measurement of TNFα. The amount of TNFα in the culture medium is quantified by ELISA using HuTNFα assay from Cytoset according to protocol.
19. Rats are treated with different compound doses. Control animals are treated with placebo. 1 h after compound treatment, rats are intraperitoneally injected with LPS (0.5 mg/kg). 1.5 h after LPS injection, serum is collected for the measurement of TNFα level. The amount of TNFα in the serum is quantified by ELISA.