## 2 Results and Discussion
### LiARG In Silico Binding Mode Analysis  
Molecular docking studies were performed to determine the binding modes of curcuminoids and turmerones **1-6** with _Li_ARG. After the validation of the docking protocol (Supplementary Material, Figure S3), the protein and the main compounds were considered in the ionized state at pH 9.5 (Figure 1) according to the literature and experimental studies [34]. The overall analysis of the complexes obtained between _Li_ARG and curcuminoids **1-3** indicated that these ligands were positioned between protein loops at the entry of the active site and showed similar binding mode values of \(-3.43\) kcal/mol, \(-4.14\) kcal/mol, and \(-3.99\) kcal/mol, respectively. \begin{table}
\begin{tabular}{l c} \hline \hline  & _Li_ARG \\  & **IC\({}_{\text{50}}\)** \(\pm\) **SE [ng/mL]** \\ \hline HEXURC & 14.4 \(\pm\) 2.4 a \\ DCCURC & 10.04 \(\pm\) 1.5 a \\ ETOHCURC & 52.64 \(\pm\) 0.3 b \\ Curcumin (**1**) & 17.55 \(\pm\) 0.4 a \\ Quercetin (**7**) * & \textless{}6.25 \\ \hline \hline \end{tabular} Results are expressed as the mean \(\pm\) standard error from three independent experiments. Means with different letters indicate significant differences (One-way ANOVA, [PERSON] post hoc test, \(p<0.05\)). HEXCURC, _C. longa_ hexane extract; DCCURC, _C. longa_ dichloromethane extract; and ETOHCURC, _C. longa_ ethanol extract. * 7 was used as reference inhibitor of _Li_ARG [7].  
\end{table} 
Table 1: Half-maximum (IC\({}_{\text{50}}\)) values of _C. longa_ extracts and curcumin (**1**) against _Li_ARG. Figure 1: Chemical structures of curcumin (**1**), demethoxycurcumin (**2**), bisdemethoxycurcumin (**3**), and turmerones (**4–6**) in alkaline media. Curcumionids have similar chemical structures except for the number of methoxy groups on each of the two phenyl moieties, which affect their binding modes. Binding mode analysis of _Li_ARG:**1** and _Li_ARG:**3** complexes indicated that both inhibitors adopted similar orientations and interactions within the binding site. Indeed, their phenolate moieties play an important role in the complex formation. One phenolate may interact with Ser150 through hydrogen bonds with its hydroxyl (OH--O- distances of 2.7 A) and main chain (NH--O distances of 2.9 and 2.6 A, respectively). Moreover, the second phenolate interacts with Arg260 through hydrogen bonds with its side chain (NH--O- distances of 2.6 and 2.8 A, respectively). Due to the presence of the methoxyl group, **1** establishes an additional hydrogen bond as acceptor with Val149 (NH--O distance of 3.2 A). Van der Waals interactions with His139, Asn143, Thr148, Gly151, Ala192, Asp194, Thr257, and Pro285 residues also contributed to the formation of both complexes. Gly151 and Gly256 residues also participate in _Li_ARG:**1** and Val259 residues and in _Li_ARG:**3** complexes (Figure 2a,b). For the complex _Li_ARG:**2**, the monosubstituted aromatic ring maintained the hydrogen bond interaction with Arg260 (NH--O distance of 3.0 A), while the disubstituted aromatic ring is oriented to the Lys198 residue for an ionic interaction through its deprotonated hydroxyl (NH--O distance of 2.4 A). In the same way as the two earlier complexes, van der Waals interactions with His139, Ala192, Thr257, Pro258, and Val259, in addition to Arg191 and Val193, also contributed to the formation of _Li_ARG:**2** (Figure 2c). Possibly due to the long chain spacer connecting the phenolate extremities, the curcuminoids interact with external loops of the _Li_ARG impacted in the estimated binding energy. Even so, residues as His139 and Pro258 that participated in the interactions with curcuminoids were also found before by our group to interact with rosmarinic acid [10]. Additionally, a hydrogen bond with Arg260 was among the interactions displayed by the chalcone derivative identified as effective against promastigotes and amastiogr forms of _L. infantum_[12].