## 2 Results and Discussion
### In Vitro Biological Evaluation
#### 2.2.2 Antibioticfilm Activity  
Many microorganisms are able to form biofilms. The cells in biofilms are much less sensitive to antimicrobial agents than planktonic ones. It is known that staphylococci and fungi of the genus _Candida_ are able to form robust biofilms [41; 42]. With regard to the high activity of a number of the synthesized compounds against _S. aureus_ and _C. albicans_, we tested their ability to kill cells within mature biofilms and to affect the biofilm formation process. Minimal biofilm eradication concentrations (MBECs) against _S. aureus_ and _C. albicans_ were determined for the most active compounds **3 aa**, **3 ad**, **3 ao**, and **3 aq**. All tested compounds were able to effectively kill cells in biofilms of staphylococci and fungi of the genus _Candida_. The most effective against staphylococci was indolylbenzimidazole **3 aq** (Table 2). The study of the effect of compounds **3 aa**, **3 ad**, **3 ao**, and **3 aq** on biofilm formation showed that they effectively prevented the biofilm formation of _S. aureus_ ATCC 21923 and MRSA by reducing the number of living cells in the planktonic culture (Figure 2). The activity of the synthesized substances against staphylococcal biofilms significantly exceeded the effect of the control antibiotic amikacin (see Supplementary Materials). The sublethal concentrations of 5-bromo-2-(5-bromo-1\(H\)-indol-3-yl)-1\(H\)-benzo[\(d\)]imidazole (**3 ao**) stimulated staphylococci biofilm formation. This effect may be caused by the stress action of the antibacterial agent, since it is known that biofilm formation can be one of the components of bacterial cell stress response. However, in this case, the regulatory effect of sublethal concentrations of benzimidazole **3 ao** on genes encoding metabolites involved in the process of bacteria biofilm formation cannot be completely excluded (Figure 2). In Silico Characterization of Benzimidazole Derivatives Binding (p)ppGpp Synthetases/Hydrolases, Pyruvate Kinases, and FtsZ Proteins Molecular Docking of Benzimidazole Derivatives  
In this study, we employed in silico molecular docking calculations to unravel the possible mechanisms of action and potential targets of the investigated compounds. Initially, we conducted virtual screening on (p)ppGpp synthetase/hydrolases: _E. coli_ RelA, _S. aureus_ \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Compounds**} & _C. albicans ATCC_ & \multirow{2}{*}{**S. aureus ATCC**} & \multirow{2}{*}{**MRSA**} \\  & **10231** & **25923** & \\ \hline
**3 aa** & 125 & 250 & 250 \\
**3 ad** & 62.5 & 125 & 125 \\
**3 ao** & 125 & 125 & 125 \\
**3 aq** & 62.5 & 62.5 & 125 \\ \hline \hline \end{tabular}  
* Minimal biofilm eradication concentration; \({}^{\mathrm{b}}\) the table presents the mode values from 3–4 independent experiments.  
\end{table} 
Table 2: Antibiotic matrix data (MBEC \({}^{\mathrm{a}}\), \(\mu\)g/mL) for the selected indolylbenzo[\(d\)]imidazoles \({}^{\mathrm{b}}\). Figure 2: Influence of sublethal concentrations of 7-bromo-5-chloro-2-(1\(H\)-indol-3-yl)-1\(H\)-benzo[\(d\)]imidazole (**3 aa**), 5,7-dibromo-2-(1\(H\)-indol-3-yl)-1\(H\)-benzo[\(d\)]imidazole (**3 ad**), 5-bromo-2-(5-bromo-1\(H\)-indol-3-yl)-1\(H\)-benzo[\(d\)]imidazole (**3 ao**), and 2-(5-bromo-1\(H\)-indol-3-yl)-6,7-dimethyl-1\(H\)-benzo[\(d\)]imidazole (**3 aq**) on the _S. aureus_ ATCC 43300 (MRSA) (**a–d**) and _S. aureus_ ATCC 25923 (**e–h**) biofilm biomass (OD570) and the number of colony-forming units (CFU) in plankton. Diagrams show the mean values (\(\pm\)SE) of three to five experiments. Rel, and _M. smegmatis_ RelMsm. The respective predicted 3D structures (P0 AG20, P0A0F0, A0 QWJ6) were obtained from the AlphaFold database [43]. To identify the active site of the synthetase domain, we utilized the RelSeq crystal structure (IVJ7), which included a co-crystallized GDP substrate. It is worth noting that (p)ppGpp synthetase/hydrolases belong to non-essential bacterial pathways and their inhibition may not necessarily correlate with strong antibacterial activity.