Figure 2.

Surface plasmon resonance binding kinetics of selected hits from MMV400 library binding to ezrin. (A) Chemical structures are given for NSC305787 and nine antimalarial compounds identified through SPR screen from MMV400 library. (B) A representative sensorgram showing binding of MMV020243 to immobilized ezrin at seven concentrations in triplicates ranging from 0.78 to 50.0 μM is presented with the steady-state affinity curve given in the right panel. Black ∗ represent binding levels for each concentration that were used to calculate the steady state affinity from the curve in the right panel. The final K_D value calculated from n=6 separate experiments is given in Table 1. C. Inhibition of cell migration in osteosarcoma cells by the known anti-ezrin molecule NSC305787 and selected hits from MMV400 library. Upper Panel: Immunoblotting showed that highly metastatic K7M2 cells express high levels of ezrin relative to the low metastatic K12 cells. Lower Panel: Cell migration experiments were performed using xCELLigence electric cell impedance system in CIM-Plates 16 with 10% serum serving as the chemoattractant in the lower chamber. Cell migration was monitored in real-time for a period of 21 h. Data was normalized to control treatment (DMSO), which was taken as 100%. NSC305787 and the selected hits from SPR screening except MMV020549 and MMV396680 showed greater inhibition of K7M2 motility compared to its less aggressive counterpart K12 cells. K7M2 and K12 cells were treated with 5.0 μM of MMV667492, MMV666069, MMV665977, MMV020549, MMV020243, MMV666103 and MMV396680 and 3.0 μm of NSC305787 and MMV006172 for 21 hour. Values are presented as the means ± standard deviations of n separate experiments as given in Supplemental Figure 4. (*; p<0.05, ns; not significant, compared to control; using a Student’s unpaired t test).