Crystal structure of an Aurora-A mutant that mimics Aurora-B bound to MLN8054: insights into selectivity and drug design

Abstract

The production of selective protein kinase inhibitors is often frustrated by the similarity of the enzyme active sites. For this reason, it is challenging to design inhibitors that discriminate between the three Aurora kinases, which are important targets in cancer drug discovery. We have used a triple-point mutant of Aurora-A (AurAx3) which mimics the active site of Aurora-B to investigate the structural basis of MLN8054 selectivity. The bias toward Aurora-A inhibition by MLN8054 is fully recapitulated by AurAx3 in vitro. X-ray crystal structures of the complex suggest that the basis for the discrimination is electrostatic repulsion due to the T217E substitution, which we have confirmed using a single-point mutant. The activation loop of Aurora-A in the AurAx3-MLN8054 complex exhibits an unusual conformation in which Asp274 and Phe275 side chains point into the interior of the protein. There is to our knowledge no documented precedent for this conformation, which we have termed DFG-up. The sequence requirements of the DFG-up conformation suggest that it might be accessible to only a fraction of kinases. MLN8054 thus circumvents the problem of highly homologous active sites. Binding of MLN8054 to Aurora-A switches the character of a pocket within the active site from polar to a hydrophobic pocket, similar to what is observed in the structure of Aurora-A bound to a compound that induces DFG-out. We propose that targeting this pocket may be a productive route in the design of selective kinase inhibitors and describe the structural basis for the rational design of these compounds.

Dynamic Equilibrium of the Aurora A Kinase Activation Loop Revealed by Single‐Molecule Spectroscopy

“…This is a physiologically relevant scenario at the mitotic spindle, and kinetic studies have shown that the presence of TPX2 increases the K i  value of MLN8054 by a factor of greater than four 5b. Similar changes have been observed for VX680 and GSK623906A 4d.…”

“…MLN8054 and CD532 are both nanomolar inhibitors of Aurora A, and X‐ray structures show that each binds in an inactive T‐loop conformation (Figure S1 c, d) 5b,5d,5g. Although both are referred to as type II inhibitors, neither extends into the allosteric hydrophobic pocket, and neither has been captured binding the kinase with a canonical DFG‐out motif.…”

“…Although both are referred to as type II inhibitors, neither extends into the allosteric hydrophobic pocket, and neither has been captured binding the kinase with a canonical DFG‐out motif. Aurora A bound to MLN8054 adopts an unusual DFG conformation previously termed DFG‐up,5b and Aurora A bound to CD532 is DFG‐in 5d…”

“…To determine whether this result represents a mixture of binary Aurora A–MLN8054 and Aurora A–TPX2 complexes or population of an Aurora A–TPX2–MLN8054 triple complex, we calculated the expected experimental result for the mixture of binary complexes based on the published affinities of the two ligands4b, 5b (see the Supporting Information). A mixture of binary complexes would result in an inactive T‐loop population of 43 %, which is inconsistent with the experimental result (15 %; Table 1).…”

“…Three PDB structures (2WTV, 3H10, and 2X81) show Aurora bound to MLN8054 or a similar compound in an inactive T‐loop conformation, while in a fourth, low‐resolution structure (2WTW), the crystal packing is incompatible with an inactive T‐loop, and the MLN8054‐bound kinase adopts an active T‐loop conformation 5b. All four Aurora–MLN8054 structures superpose within the N and C lobes of the kinase, varying only in the orientation of the activation loop and in the exact angle between the two lobes.…”