Published: February 09, 2011 r2011 American Chemical Society 3895 dx.doi.org/10.1021/jp108286r | J. Phys. Chem. A 2011, 115, 3895–3904 ARTICLE pubs.acs.org/JPCA VX680 Binding in Aurora A: π-π Interactions Involving the Conserved Aromatic Amino Acid of the Flexible Glycine-Rich Loop Taian  a M. Oliveira, Rafi Ahmad, and Richard A. Engh* NORSTRUCT, Department of Chemistry, University of Tromsø, 9037 Tromsø, Norway ABSTRACT: The regulation of protein kinases requires flexibility, espec- ially near the ATP binding site. The cancer drug target Aurora A is inhibited by the ATP site inhibitor VX680, and published crystal structures show two distinct conformations. In one, a refolded glycine-rich loop creates a stacked π-π interaction between the conserved aromatic residue of the glycine-rich loop hairpin turn (F144) and the inhibitor. This refolding, associated with binding to a peptide derived from the cofactor TPX2, is absent in the other structure. We use surface plasmon resonance to measure VX680 binding to native and mutant F144A Aurora A kinase domains, with and without the TPX2 peptide. Results show that the F144 aromatic side chain contributes 2 kcal/mol to the VX680 binding energy, independent of the TPX2 peptide. This indicates that distinct VX680 bound conformations of Aurora A cannot be simply correlated with TPX2 binding and that Aurora A retains flexibility when inhibitor-bound. Molecular dynamics simulations show that alternate geometries for the π-π interactions are feasible in the absence of the rigidifying packing interactions seen in the crystal lattice. ’ INTRODUCTION The importance of ligand-induced structural modifications of drug targets has long been recognized, 1-3 and the unpredict- ability of the changes is one of the major obstacles to in silico methods in drug discovery. 4,5 This is particularly true for protein kinase inhibitors that target the ATP binding pocket. 6,7 Formed by the juxtaposition of the two folding subunits of the catalytic domain, the ATP site exhibits wide-ranging flexibility, including large-scale motions of the individual subunits, local refolding, especially of the activation and glycine-rich loops, and side-chain reorientations. These flexibilities are greater than those for many other enzyme classes because they arise from the mechanisms for strict control of protein kinase activity and substrate recognition, which in turn are required for signaling fidelity. 8,9 The hundreds of crystal structures that are now available for protein kinases 10 show several conformers and point out that some moieties which are highly connected to regulatory control seem to be especially pliable; these include the activation loop, the C-helix, and the glycine-rich loop. Protein kinases constitute the primary mechanism of cell signaling processes and are thus involved in virtually all aspects of cell function. Consequently, dysregulation of protein kinase activity is a common cause of disease, and protein kinases now constitute the major class of targets for new cancer drugs. 11,12 The Aurora kinases A, B, and C are among the prioritized targets; these serine/threonine protein kinases are pivotal regulators of mammalian cell division. Dysregulated Aurora protein kinase activity can lead to defects in chromatid segregation, genetic instability, and tumorigenesis, and overexpression has been linked to poor prognosis of cancer patients. 13,14 Of the many Aurora kinase inhibitors now published, 15 VX680 (also called MK-0457) was the first Aurora inhibitor in clinical trials. It is a nanomolar potent pan-Aurora kinase inhibitor, with cross reactivities that notably include inhibition of imatinib resistant Bcr-ABL kinase variants. 16 Although clinical trials with VX680 have been suspended, related inhibitors including VE465 are in active preclinical trials. 17 Two crystal structures of VX680 in complex with the kinase domain of Aurora A have been reported. In the first, VX680 binds to an inactive conformation of Aurora A, in which the activation loop segment DFGW accommodates the inhibitor via contacts with the two aromatic residues. 18 In the second, VX680 binds to Aurora A, which in turn is complexed to the 43 residue TPX2 activator peptide. This structure differs from the former, espe- cially due to a refolding of the glycine-rich loop, which positions the conserved GxGxFG aromatic residue (Phe144) to make a stacking interaction with the phenyl moiety of VX680. 19 The conserved glycine-rich loop (consensus motif GxGxxG) is near the N-terminus of the kinase domain and controls ATP binding, activity, and ADP release via solvent exclusion and Special Issue: Graham R. Fleming Festschrift Received: August 31, 2010 Revised: January 4, 2011