Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2

The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.

. JNJ does not significantly alter the protein--peptide binding and was used in the assays after HTS to displace any ATP--site binders. e) The Z--Factor of the assay, z = 0.67, demonstrating a good assay. f) Calculated dynamic range of the FP assay 1 , constrained by inhibitor solubility at the low--inhibitor--affinity end and TAMRA--TPX2 1--43 -Aurora A K d at the high--inhibitor-affinity end. The ideal working dynamic range range was determined to be between mM and 100 nM.   AurkinA. c) The thermodynamic signature of binding between AurkinA and AURKA. The association was thalpy--driven, and enthalpically unfavourable. The large and opposite enthalpy and entropy terms ggest an induction of significant conformation changes upon binding.

Preparation of human Aurora--A Kinase Expression Plasmids
Human Aurora--A kinase was produced using λPP co--expression method described for mouse Aurora--A 3 . Enterbacterial phage lambda phosphatase (λPP) gene (GenBank ID: 270346) was amplified by PCR using cloning primers 1 and 2 that included XhoI and HindIII restriction sites as well as the Shine--Dalgarno ribosome binding sequence. Digested and purified PCR product was ligated into pBAT4 and pHAT4 expression plasmids 4 linearized with XhoI and HindIII restriction endonucleases resulting in plasmids pBAT4--λPP and pHAT4--λPP, respectively.
Human Aurora--A kinase gene fragment encoding residues 126--390 was amplified by PCR using cloning primers 3 and 4, digested with NcoI and XhoI restriction endonucleases and ligated into the NcoI and XhoI linearised pBAT4--λPP plasmid upstream the λPP gene resulting in Aurora--A expression construct termed AurA G--126--390 .
AurA G--126--390 T287A mutant was generated by PCR overlap extension method using cloning primers 3 and 4 and mutation primers 5 and 6.
The Aurora--A kinase expression construct used for FA assay termed AurA 123--403 , was produced differently. The Human Aurora--A kinase gene fragment encoding residues 123--403 was amplified by PCR using cloning primers 9 and 10. It was then digested by ClaI and XhoI restriction endonucleases and ligated into the ClaI and XhoI linearised pBAD--HisA vector (Invitrogen), prepared in SCS110 E.coli to prevent methylation of ClaI site. AurA 123--403 expression construct encoded an N--terminal region carrying 6xHis--tag as well as EK recognition site (sequence: MGGSHHHHHHGMASMTGGQQM GRDLYDDDDKDR) in addition to Aurora--A kinase residues 123--403.
Primer Sequence mCherry--TPX2 1--43 was sub--cloned from the previously generated pCDNA3.1 (--) construct (see above) into pcDNA5/FRT/TO vector at PmeI site. The clones generated were tested by colony PCR and the directionality of insert was established using EcoRI / XbaI restriction digestion. A construct with desired orientation were verified by sequencing and used for generating the stable cell line.
Pooled clones were sorted by a cell sorter (BD Biosciences) into 96--well plate with one cell per well. The medium was changed regularly and when cells had expanded these were replica plated and tested for the expression of mCherry--TPX2 by high content microscopy as described below. A clone with high expression and low background was selected, expanded and used for further experiments.

Preparation of Supplementary Video
The video depicts the transition between the Aurora A crystals structure containing ATP (AurA G--126--390 _T287A_ATP (5DRD)) and crystals structure of Aurora A with bound ATP and AurkinA (AurkinA_AurA G--126--390 _T287A_ATP (5DOS)). The video was prepared by 'morphing' from the apo structure to the AurkinA complex and back, using UCSF chimera package 5 .

Chemical Synthesis of AURKA--TPX2 inhibitor analogues
General procedure: Pfitzinger reaction According to the procedure described by Giardina et al. 6 , isatin (1.0 eq), acetophenone (1.2 eq) and KOH (3.0 eq) were dissolved in EtOH. The reaction mixture was heated under reflux for 48--72 h and the solvent was removed under reduced pressure. The residue was dissolved in H 2 O and washed with equal volume of Et 2 O twice. The aqueous layer was cooled to 0 °C, acidified to pH 2 with (conc.) HCl and the precipitate was collected as a crude product, which was then triturated or recrystallised.

methyl 2--(2--phenylquinoline--4--carboxamido)benzoate
Methyl anthranilate (0.30 mL, 2.41 mmol), cinchophen (500 mg, 2.01 mmol) and DIPEA (0.70 mL, 4.01 mmol) were added to oven--dried round--bottomed flask containing dry EtOAc (10 mL). The mixture was cooled to 0 °C and T3P (50% in EtOAc, 1.8 mL, 3.01 mmol) was added dropwise and the reaction mixture was stirred at 0 °C for 30 min. The mixture was allowed to warm up to RT and after overnight stirring, the reaction was quenched by addition of H 2 O. The mixture was diluted with EtOAc and the organic layer was washed with (sat.) NaHCO 3 , brine, dried (MgSO 4 ), filtered and the solvent was removed under reduced pressure. The crude product was purified by flash column