PHA-680626 Is an Effective Inhibitor of the Interaction between Aurora-A and N-Myc

Abstract

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcription factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation by directly binding to a highly conserved N-Myc region. As a result, elevated levels of N-Myc are observed. During recent years, it has been demonstrated that some ATP competitive inhibitors of AURKA also cause essential conformational changes in the structure of the activation loop of the kinase that prevents N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complexes with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results identify PHA-680626 as an amphosteric inhibitor both in vitro and in MYCN overexpressing cell lines, thus expanding the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex and confirming that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

Keywords: Aurora-A; N-Myc; PHA-680626; amphosteric inhibitors; neuroblastoma.

Figure 1

Structures of human AURKA catalytic domain in complex with the investigated inhibitors. The activation loop is shown in orange. The angle between the N- and C- lobes, measured between α-carbons of V324, E308, and A172, is also shown. The chemical structures of the compounds investigated are also represented.

Figure 2

SPR analyses of ligands binding to AURKA. Full kinetic analysis of the AURKA inhibitors CD532 (A), MLN8054 (B), PHA-680626 (C), RPM1722 (D), Alisertib (E), and N-Myc (F). Determined binding parameters are listed in Table 2. Kinetic data for N-Myc: K_on, 6.3 ± 1 × 10⁴ M⁻¹s⁻¹; K_off, 0.0659 ± 0.0008 s⁻¹; K_d, 990 nM; Res SD 2.17. The interaction between AURKA and the ligands is indicated by the increase in Resonance Units (RUs) on the vertical axis compared to the baseline.

Figure 3

SPR analyses of N-Myc binding to AURKA in the presence of inhibitors. Sensorgrams of competition experiments between the inhibitors CD532 (A), MLN8054 (B), PHA-680626 (C), RPM1722 (D), Alisertib (E), MK8754 (F), ZM447439 (G), and N-Myc. The experiments were carried out by injecting Myc-AIR at different concentrations (125 nM, 250 nM, 500 nM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM) and at a constant flow rate (150 μL/min) of running buffer. Myc-AIR was previously diluted in running buffer containing a saturating concentration of the inhibitor (at least 10 times higher than its K_d). Apparent K_d values of Myc-AIR are shown in Table 2. Apparent K_d values for the negative controls MK8754 and ZM447439 are 690 nM and 4 μM, respectively.

Figure 4

PHA-680626 is able to decrease AURKA/N-Myc interaction. (A) Schematization of the combined synchronization and treatment protocol. (B) Western blot analysis from U2OS and U2OS MYCN cell lines treated with 1 μM of each compound, or DMSO as control, following the protocol described in (A), without MG-132. Lamin B1 was used as loading control. The mean and SD of the quantification of N-Myc signal from three independent experiments are indicated. (C) in situ Proximity Ligation Assays to visualize the AURKA/N-Myc complex formation. The fluorescence panels show examples of the negative (U2OS that do not express N-Myc) and the positive (U2OS MYC dox-induced) reference cultures. The histograms on the right show the distribution (%) of cells in classes, defined by isPLA spot values. Values represent isPLA spots per nucleus, normalized to the average value in control cells (at least 1400 measured cells per condition, from four independent experiments); mean and SDare shown. Statistical analysis by 2-way ANOVA-Tukey’s multiple comparison, *: p < 0.05; ***: p < 0.001. Scale bar: 10 µm.

Figure 5

PHA-680626 causes N-Myc protein decrease and cellular stress in neuroblastoma cells. (A) Western blot analysis from IMR-32 neuroblastoma cell line treated with 1 μM of each inhibitor, or DMSO for control, for 48 h. Both full-length and cleaved PARP-1 signals are shown, as indicated. The mean and SD of the quantification of N-Myc signal from three independent experiments are indicated. (B) In situ Proximity Ligation Assay of AURKA/N-Myc complex in IMR-32 cells treated with 1 μM PHA-680626 and MG-132 for 4 h. The graph shows the number of spots of interactions per nucleus from three independent experiments (at least 207 cells per condition were analyzed). Statistical analysis by Mann Whitney test, *** p < 0.0001, scale bar 10 μm. (C) FACS analysis of IMR-32 cells treated as in (A) and stained with PI. The region used to evaluate the fraction of cells displaying sub-G1 DNA content is indicated. The percentage of vital cells distributed in each phase of the cell cycle, as well as the percentage of cells with sub-G1 DNA content calculated on the total acquired events, are shown (one experiment representative of three independent experiments). (D) Morphological appearance of IMR-32 cultures by bright-field microscopy (20× magnification), treated as in (A). Scale bar: 100 μm (E) Morphological appearance of SH-SY5Y neuroblastoma cultures by bright-field microscopy (20× magnification), treated as in (A). Scale bar: 100 μm. (F) Western blot analysis from SH-SY5Y and IMR-32 cells treated as in (A).

Figure 6

Scheme of the conformational disruption of AURKA/N-Myc interaction. CD inhibitors can dissociate N-Myc by adopting two distinct strategies: wide-opening the N-terminal lobe of the kinase to create an ideal cleft for A-loop closure or stabilizing the A-loop in a closed conformation via formation of direct contacts. (A) CD532 does not engage any stabilizing contact with the A-loop. Instead, the bulky moiety of CD532 wide-opens the N-terminal lobe of the kinase, creating a large cleft in which the A-loop is stabilized in an inactive orientation. (B) The thiophene moiety of PHA-680626 is able to stabilize the A-loop in a close conformation that is unproductive for N-Myc binding, thanks to a stacking interaction with His280 of the A-loop.