Tyrosine Kinase Inhibition: Ligand binding and conformational change in c-Kit and c-Abl Eamonn F. Healy*†, Charles R. Hauser,§ Skylar Johnson† andPeter J. King‡. †
Department of Chemistry, St. Edward’s University, Austin, TX 78704, USA; ‡Department of
Biology, St. Edward’s University, Austin, TX 78704, USA and §Department of Bioinformatics, St. Edward’s University, Austin, TX 78704, *Corresponding author. Tel: (512) 448 8467; fax: (512)448 8492 ; E-mail address:
[email protected]
METHODOLOGY Kinase Assay A fluorescence-based tyrosine kinase assay was utilized to determine the 50% inhibitory concentrations (IC50) of 9-hydroxy-elipticine hydrochloride and 9-methoxy-2-methyellipticinium acetate against purified N-terminal His6-tagged recombinant human Abl kinase expressed by baculovirus in Sf21 insect cells (US Biological, Swampscott, MA). The assay chosen (Antibody BeaconTM Tyrosine Kinase Assay, Molecular Probes, Eugene, OR) represents a simple but robust assay for measuring the activity of tyrosine kinases, their inhibitors and modulators. The assay is based on detection of a complex that comprises a small-molecule tracer ligand (Oregon Green 488) that exhibits quenched fluorescence when bound to anti-phosphotyrosine antibodies. In the presence of phosphotyrosine-containing peptides, the ligand is rapidly displaced. Kinase activity is observed as an increase in fluorescence. Inhibitory activity of putative inhibitors can be measured as decreases in fluorescence when compared to maximal fluorescence controls. Briefly, 50 µL kinase reactions were assembled in triplicate in 96-well plates in the presence of
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increasing concentrations of either inhibitor (0 – 1µΜ final). Each reaction contained kinase buffer (50mM Tris-HCl, 10mM MgCl2, 1mM EGTA, 0.01% Brij 35, pH 7.5), 0.72mM DTT, 0.025µM Oregon Green, 200µg/mL tyrosine kinase substrate peptide (poly(Glu:Ala:Tyr)) and 50nM anti-phosphotyrosine antibody. Reactions were started by the addition of ATP (0.5mM final) and incubated at 37oC for 1 hour. Following incubation, fluorescence (Excitation/Emission = 492/517 nm) was measured using a fluorescence-capable microtiter plate reader. Positive controls (no anti-phosphotyrosine antibody representing maximum fluorescence) and negative controls (no kinase representing background fluorescence) were also performed in triplicate. Kinase activity as measured by fluorescence was calculated as [(mean fluorescence per triplicate) – (mean background fluorescence)] / (mean fluorescence of maximal fluorescence controls).
Docking Crystal structures for imatinib mesylate bound to c-Kit (1T46) , ligand-bound c-Abl (1OPJ) as well as for the inactive (1T45) and active (1PKG) are available from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data bank (www.rcsb.org). After adding hydrogens, the proteins were subjected to a short energy minimization using the CHARMm force field as implemented in the Discovery Studio program suite. The ligand was docked using CDOCKER, a CHARMm-based molecular dynamics docking algorithm that keeps the protein rigid but treats the ligand as fully flexible. Refining the poses in a final minimization step resulted in docked solutions that reproduced the X-ray finding with a rootmean-square deviation (rmsd) of between 1.0 and 1.1 Å . To evaluate ligand fit a consensus scoring scheme was utilized, generating five scores for each of the docking solutions. LigScore 1 and LigScore 2 are two related empirical scoring functions, CDOCKER is a CharmM-based
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energetic function, and PMF and PMF04 are knowledge-based potentials of mean force derived from the structures of known protein-ligand complexes. For the docking of ellipticine and its methylated derivative an assembled form of c-Abl was utilized, a structure of the kinase with the SH3 and SH2 regulatory domains present (1OPK). In the absence of a ligand-bound complex a shape-directed docking methodology was employed to identify potential c-Abl binding sites. The LigandFit protocol, as implemented in the Discovery Studio program suite, combines a cavity detection algorithm with a Monte Carlobased conformational search to generate ligand poses consistent with the active site shape. The estimation of free energy of non-covalent complex formation from a protein and a ligand is based on the assumption that ΔGbinding = Gcomplex – Gprotein - Gligand, where GX stands for the free energy of species X in aqueous solution at the standard concentration. GX is further decomposed into the solute gas phase energy, solvation free energy Gsolvation, and the solute entropy contributions, i.e., GX = Egas + Gsolvation - TSsolute. Egas is drived directly from energy calculations in vacuum. Gsolvation is decomposed into polar and non-polar terms i.e., Gsolvation = Gpolar + Gnonpolar where Gpolar is computed by continuum electrostatics, and Gnonpolar is estimated based on the solvent accessible surface area of the solute. For this calculation the Poisson Boltzmann SurfaceArea (PBSA) has been utilized as the implicit solvent model, as implemented in the Discovery Studio program suite. In this protocol, only the gas phase translational and rotational contributions are included in the calculation of ΔGbinding. To allow for side chain rotation in the region of the DFG motif a flexible docking scheme was used.
Molecular Dynamics Molecular dynamic (MD) simulations were performed starting from the crystal structure of the wild-type kinase complexed with imatinib mesylate , (1T46). The initial structure was immersed in a truncated orthorhombic cell of TIP3P explicit water molecules, with the water molecules -3-
extending 7 Å from the surface of the protein and Na+ and Cl– ions added to neutralize the system. The system was minimized by steepest descent and conjugate gradient and then heated to 300K. Equilibriation was performed for 6 ns, employing periodic boundary. The SHAKE algorithm was employed to keep bonds involving hydrogen atoms at their equilibrium length, allowing the use of a 2 fs time step . The resulting structures were the starting point of the production 1 ns MD simulations in an NPT ensemble using the Berendsen weak coupling method. Movies for the MD simulation with (www.cs.stedwards.edu/keck/WithL.mpg) and without (www.cs.stedwards.edu/keck/WoutL.mpg) bound ligand are available for download.
Quantum Mechanics A structure for the peptide sequence corresponding to residues 650 to 655 of the β4 loop of c-Kit was generated from the coordinates of PDB entry 1T46. After first fixing the Cα atoms of the terminal His650 and Asn655 residues, the structure was optimized using the AM1 Hamiltonian. A potential energy surface was then generated for rotation around the Cα-Cβ bond of the Ile653 residue (Figure 3b). Rotations around selected bonds of the side chains of the other residues were also used to generate a structure of the protein from the ligand-bound complex with the molecular brake on (“T46 brake on” in Table 1). All calculations were performed with water as solvent, as implemented in the conductor-like screening model (COSMO).
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Homology Human protein tyrosine kinase sequences, c-Kit (P10721), c-Abl (P00519) and FGFR2 (P21802), were obtained from the UniProt database. Multiple sequence alignments of the catalytic kinase domains were created using ProbCons, a progressive protein multiple sequence alignment program based on probabilistic consistency. Proteins were aligned and superimposed using the MODELER protocol as implemented in the Discovery Studio program suite from Accelrys Inc.
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