# N-Heterocyclic Carbene-Gold(I) Complexes Targeting Actin Polymerization
## 3 Results
### AuL4 and AuL7 Interface with the Normal Intracellular Actin Organization
*AuL4** and **AuL7** Block the In Vitro Actin Polymerization Reaction but Do Not Accelerate the Depolymerization Reaction  
Once established that the two metal complexes provoked a dramatic change of the cytoskeleton actin structure, we wondered whether the observed effect was due to a direct action toward the actin or through indirect effects, given that these compounds have already been proved as tubulin polymerization inhibitors. Thus, we adopted an in vitro actin assay that exploits a fluorescent-labeled purified rabbit actin, which polymerization/depolymerization reactions can be easily followed using a fluorimeter, in order to confirm the immunofluorescence results, and establish if **AuL4** and **AuL7** could act as actin polymerization inhibitors and/or accelerate the filamentous actin depolymerization. In this assay, we employed two reference molecules, namely the LA and CB. Particularly, LA can bind and sequester G-actin monomers, hampering the actin polymerization and, at the same time, accelerate the rate of the actin subunits dissociation from the assembled filaments. As a result, LA is able to block the F-actin formation, one way or another. Conversely, CB inhibits the actin polymerization, avoiding the monomers addition to the \"barbed\" end of the actin filaments, but it cannot increase the depolymerization reaction. The results from this assay were plotted and, as visible in Figure 6, in the control reaction (DMSO), the G-actin polymerizes very fast; indeed, the reaction curve reached a value of about 37,000 RFU (see experimental section for details), and kept the plateau until the end of the experiment. Conversely, the two reference molecules, LA and CB, used at a concentration of 5 uM, blocked the actin polymerization; indeed, the initial rapid curve growth, seen in the control reaction, was absent. In more detail, the LA curve decreased until a value of about 12,000 RFU after 13-15 min and reached a value of about 10,000 RFU  
Figure 5: Immunofluorescence analysis. MDA-MB-231 cells were treated with DMSO (CTRL, negative control) 0.1 uM Latrunculin A (LA, positive control) or **AuL7** (at its IC\({}_{50}\) value) for 24 h. After treatment, the cells were further processed (see Materials and Methods section) and imaged under the inverted fluorescence microscope at 20\(\times\) magnification (see Materials and Methods section). In the CTRL cells, the actin filaments exhibited a normal organization in the cells cytoplasm. Actin filaments in MDA-MB-231 cells treated with LA and **AuL7**, look very bright and arranged in dot-like structures: cells appear round and others with a thread-like structure (see white arrows). (**Panels a**): nuclear stain with DAPI (\(\lambda\)ex/\(\lambda\)em = 350/460 nm); (**Panels b**): \(\beta\)-actin (Alexa Fluor® 568; \(\lambda\)ex/\(\lambda\)em = 644/665 nm); (**Panels c**): show the merge. Representative fields are shown. at the end of the experiment. The exposure of actin monomers to CB produced a curve with a little increase (about 22,000 RFU) in the first 4-5 min, then decreased, and ended at an RFU value of 22,000, which is equal to the initial value. Finally, **AuL4** and **AuL7**, at a concentration equal to 5 \(\upmu\)M, exhibited both an inhibiting activity similar to that of CB, and to a lesser extent, with respect to LA. Specifically, the **AuL4** curve was lower than that of CB in the first 5 min and then rose, maintaining RFU values ranging from 24,000 to 26,000, and ended at a final value of about 27,000 RFU. The **AuL7** curve trend was similar to that of **AuL4**, with the exception of the first 5 min, when it decreased to values lesser than LA, ending with a final value of 24,000 RFU. Overall, the inhibitory activity of **AuL7** seems better than **AuL4** but, in both cases, this assay demonstrates a direct effect on actin polymerization reaction, supporting the immunofluorescence assays.