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

Abstract

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.

Figure 1. Identification of AurkinA, inhibitor of Aurora A-TPX2 complex.

(a) The binding of AURKA_123–403 to TAMRA-TPX2_1–43 in fluorescence anisotropy assay. K_d = 83 nM (pK_d = 7.07 ± 0.07). (b) Displacement of TAMRA-TPX2_1–43 by inhibitors in fluorescence anisotropy assay. (c) Synthetic strategy: deconstruction and optimisation of compound 1 identified from the HTS screen. K_i value was derived from FA competition assay using free-concentration corrected Cheng-Prusov equations. K_d values were obtained using isothermal titration calorimetry. Ligand efficiency (L_eff) was calculated using K_i or K_d values. Error bars represent standard deviation (n = 3).

Figure 2. AurkinA triggers conformational changes in AURKA protein.

(a) Crystal structure of AURKA_126–390 liganded with Mg²⁺ -ATP and AurkinA (5DT4,gray), overlayed with TPX2_1–43 (1OL5, orange). AurkinA (blue) is bound to the pocket defined by αC and αB helices, a binding site of the YSY motif of TPX2. The hydrophobic Y-pocket sits above the ATP-site. (b) The detail of AurkinA (blue) and TPX2_8–11 (orange) binding in the Y-pocket. (c) Binding pose of AurkinA in the Y-pocket. Carboxylic acid of AurkinA interacts with amine of K166, which is stabilised by H201. 2Fo-Fc map (blue) is countered at 1σ, anomalous map (pink) is contoured at 5σ. (d) The conformational change of Mg²⁺ -ATP liganded structure (5DT3, orange) upon AurkinA binding (5DT4, gray). The binding induces an anticlockwise rotation of αC helix by interacting with R179 and the hydrophobic floor. The shift of αB helix causes changes of hydrogen bonding pattern in glycine-rich loop, breaking of Q168-K143 hydrogen bond and movement of F144. (e) The overlay of AurkinA (magenta, 5DPV) and its analogues AA29 (green, 5DR9) and AA30 (cyan, 5DR6) bound to the Y-pocket. The related compounds bind with a consistent binding mode and the conformations of the amino acids lining the pocket are very similart between the liganded crystal structures. (f) The structures of the Y-pocket binders: AA29, AA30 and AurkinA; and JNJ-7706621, a potent ATP-competitive AURKA inhibitor.

Figure 3. AurkinA causes allosteric inihibition of the kinase activity of AURKA.

(a) AurkinA inhibits the kinase activity of pT287/pT288 AURKA_126–390 in homogeneous time-resolved fluorescence assay (HTRF, KinEASY, Cisbio) in absence of TPX2. The volume of enzymatic reaction was 10 μL, [AURKA] = 5 ng/well, excess propriatory peptide substrate (1 μM) and ATP (100 μM). The reaction was incubated for 10 min. MLN8237 is an ATP-competitive inhibitor used as a positive control. AurkinA excess did not cause a complete ablation of AURKA kinase activity. (b) In presence of AurkinA, the maximum rate of AURKA kinase activity (V_max) decreased, but K_m for ATP remained unchanged. Such a kinetic profile is consistent with a non-ATP competitive inhibition, suggesting that AurkinA inhibited the kinase activity of AURKA allosterically. (c) AurkinA slowed down the in vitro autophosphorylation of AURKA_{GSMGS-126-390} with uncleaved His-tag (30 μM), as detected by anti-pT288 antibody. [ATP] = 100 μM, J* = JNJ-7706621. Error bars represent standard deviation (n = 3).

Figure 4. AurkinA mislocalises AURKA from the bipolar spindle in mitotic cells.

(a) Representative immunofluorescence images of HeLa cells expressing mCherry-TPX2_1–43 fusion protein under doxycycline (DOX) control. mCherry-TPX_21–43 fusion overexpression as well as AurkinA mislocalise of AURKA from the spindle. (b) Quantitative analysis of AURKA mislocalisation from the spindle, as defined by % cells with below threshold AURKA intensity under TPX2 mask. AurkinA mislocalises AURKA from the spindle in a dose dependent manner with EC₅₀ = 85 μM. (c) AurkinA deactivates AURKA, as measured by increase of % of cells with below threshold phosohorylated T288 (pT288). EC₅₀ = 135 μM. The error bars represent standard deviation (n = 5).