## 3 Results and Discussion
### Derivative **2** Interfaces with Actin Polymerization and Depolymerization  
A multitarget therapy is considered a successful approach to obtain compounds with higher therapeutic effectiveness and fewer side effects, thus we evaluated whether the derivative **2** could interfere with another important cytoskeleton component, i.e., actin, as already supposed by docking simulations. The actin cytoskeleton is a dynamic network strictly implicated in migration and invasion processes [6], and cancer cells can exploit the actin system to escape from primary tumors and spread to distant organs. Thus, targeting the actin cytoskeleton represents a valid strategy in cancer treatment [7]. For this purpose, MCF-7 and MDA-MB-231 cells were treated for 24 h with the derivative **2** (used at 25 \(\upmu\)M) or with vehicle (DMSO, negative control). The IF results, shown in Figure 6, demonstrate that the derivative **2** also interferes with the normal organization of the actin system in both breast cancer cells. Indeed, contrarily to the DMSO-treated cells (CTRL), which showed a normal arrangement of the actin filaments within the cell cytoplasm (Figure 6, Panels B, CTRL, MCF-7, and MDA-MB-231), both the breast cancer cells exposed to the derivative **2** exhibited a strong disorganization of the actin system (Figure 6, Panels B, derivative **2**, MCF-7, and MDA-MB-231). In particular, cancer cells lost their normal morphology, appearing with a smaller size and round. Moreover, the actin system resulted squeezed in a dot-like structure and not well distributed in the cytoplasm and the actin filaments are more stocked around the cell nuclei, appearing also brighter (see white arrows in Figure 6). A parallel experiment has been conducted on the mouse fibroblast 3T3-L1 cells, demonstrating that the derivative **2** had no effect on the actin cytoskeleton of the normal cells (Figure 6,Panels B, derivative **2**, 3T3-L1). Finally, the effect of RSV on the actin cytoskeleton of all the used cell lines was also assessed. The results demonstrated that MCF-7, MDA-MB-231 and 3T3-L1 cells exposed to RSV showed a regularly organized actin cytoskeleton (Figure 6, Panels B, RSV, MCF-7, MDA-MB-231, and 3T3-L1). 3T3-L1 cells treated with the derivative **2** (25 \(\upmu\)M), RSV (25 \(\upmu\)M), or with the vehicle (CTRL, DMSO) for 24 h, were incubated with a mouse anti-\(\beta\)-tubulin primary antibody and then with the mouse secondary antibody. CTRL cells and RSV-treated cells showed a regular cytoskeleton organization in all the cell lines used; 3T3-L1 cells treated with the derivative **2** also showed a normal arrangement of tubulin microtubules; contrarily, the derivative **2** induced a strong microtubules disorganization in both the breast cancer cells (see white arrows). Cells were monitored using the inverted fluorescence microscope at 40\(\times\) magnification. (**A**) DAPI (\(\lambda_{\text{ex}}\)/\(\lambda_{\text{em}}\) = 350/460 nm); (**B**) \(\beta\)-tubulin (Alexa Fluor\({}^{\text{\tiny\textregisteredregistered}}\) 568 (\(\lambda_{\text{ex}}\)/\(\lambda_{\text{em}}\) = 644/665 nm); (**C**) overlay channel. The images are representative fields. Further, we performed specific in vitro actin polymerization and depolymerization assays, using a fluorescent-labeled rabbit muscle actin reconstituted and Latrunculin A (LA), and Cytochalasin B (CB) as positive controls (5 \(\upmu\)M). In the actin polymerization assay (Figure 7A), the plotted relative fluorescence indicated that the derivative **2** (used at the concentration of 10 \(\upmu\)M) strongly impeded the actin polymerization, with a similar effectiveness to LA and CB. Indeed, the control reaction (DMSO) showed a very fast actin polymerization as the reaction curve reached a value of about 25,000 relative fluorescence units (RFU) after 7/8 min, sustaining the plateau until the end. On the contrary, the LA curve, starting from 14,000 RFU, rapidly decreased after 5 min reaching an RFU of 11,000.