Figure 6

(a) Superimposition of Nek2 (PDB ID: 2JAV), EGFR (PDB ID: 2JIV) and HER2 (PDB ID: 3PP0) kinases. Nek2, EGFR and HER2 are shown as yellow, orange and green colored ribbon models, respectively. Key conserved residues are also shown as stick model with color scheme by element type as implemented in Maestro. (b) An analysis of the residues present within 5 Å distance from the bound pyrroleindolinone inhibitor in the co-crystal structure of hNek2 (PDB ID: 2JAV), and the corresponding residues at the equivalent positions from the in silico docked complex of EGFR and HER2 kinases. A total of 26 residues are compared; red color, identical residues; pink color, similar residues; black color, no homology; *Met86; Thr86 in wild type; Nek2/EGFR: 12 identical, 3 similar, 11 with no homology; Nek2/HER2: 11 identical, 3 similar, 12 with no homology. (c) Binding model of neratinib within the active site of human Nek2 kinase (left). Important amino acids are depicted as sticks with the atoms colored as carbon—green, hydrogen—white, nitrogen—blue, oxygen—red, sulfur—yellow. Neratinib is shown as ball and stick model with the same color scheme as above except carbon atoms are represented in orange and chloro atom as green. Anilino ring centroid is displayed as green sphere. Dotted black line indicates hydrogen bonding interaction, whereas dotted red line indicates possible hydrogen bonding and ionic interactions with distances in Å. (d) Inhibitory screening profile of selected small molecule library compounds against wild-type (wt) human Nek2 kinase. The screening was performed at 10 μM inhibitory concentrations. (e) Dose–response curves for neratinib- (red circle) and pelitinib-mediated (green square) inhibition of wt human Nek2 kinase. The points are experimental and the line joining them is the fit to obtain the IC-50 values for neratinib as 247 nM and pelitinib as 661 nM, respectively. (f) Nek2-driven cell migratory phenotype (distant seeding; foci in GFP alone compared with Nek2GFP flies fed with DMSO) is suppressed upon treatment with pelitinib in two transgenic fly lines (#1 and #6) and upon treatment with neratinib in one fly line (#1). Distant seeding quantitation was performed as in Figure 5. Asterisks indicate P-values <0.05, in a two-tailed students t-test.