Target fishing applied to acridine derivatives
The virtual screening approach resulted in 550 predicted targets. The results were classified according to free energy. The best targets for acridine derivatives are presented in Supplementary Table S1. We found 10 chitinase targets with a score between − 10.9 to -9.1 kJ/mol. The best interaction was observed for A. fumigatus chitinase (AfChiB), with − 10.9 kJ/mol. The other interactions were observed for Serratia marcescens presented in 7 different PDB IDs, between − 10.4 to -9.2 kI/mol. Other species was observed, such as the moth Ostrinia furnacalis and the bacterium Vibrio harveyi, with − 9.4 kJ/mol and − 9.3 kJ/mol, respectively.
For A. fumigatus the position of the acridine ring overlaps with the aromatic ring of the PDB inhibitor (caffeine) (Fig. 1). The amino acid Tyr29 showed π-π interactions with acridine fragments, since Gly322, Asp246 and Tyr299 showed H-bond with the ligand. This conformation may indicate a possible activity of functional mimicry [29]. In regard to chitinases of S. Marcescens, we observed the 3D overlay of structures, thus presenting the same protein structure. Also, the acridine derivative presents similar interactions with PDB inhibitors [30, 31]. The π-π interactions were observed between Trp97 and Trp403 with acridine fragments. The H-bond may indicate greater stability of the compound at the site, indicated by Asp215, Tyr292, Gln407 and Asp334.
Despite the structural differences, the active site for these enzymes is conserved, which helps to explain the score values for the IVS. In addition to the DXDXE domain in the active site (Fig. S1), the presence of a nonpolar region with aromatic amino acids, and the presence of polar amino acids, stands out. This feature assists in the positioning of the acridine compound, promoting the intercalations of aromatic rings and H-bond formation. Such characteristics have been shown to be crucial to ensure the positioning of the derivative in the active site.
Molecular docking simulation and consensus analysis
Additionally, molecular docking studies were applied to chitinase isoforms. Results indicate that acridine derivatives interact with amino acid residues in the active site (Table S2-S3). Using AutoDockTools v. 1.5.7, the Root Mean Standard Deviation (RMSD) of the redocking of A. fumigatus inhibitor was calculated, with an RMSD of 1 Å (Fig. S3). The RMSD calculation helps to compare the position of a crystallized ligand with the position of a simulated ligand in the active site of the same protein. With this analysis, known as redocking, it is possible to indicate that the simulation presented a conformation similar to that of the crystallizing ligand, which could serve as a validation of the docking procedure. A value between 0–2 Å could validate docking simulation [32].
All acridine derivatives interact with the DXDXE motif, which is involved in the catalysis and loss of catalytic activity of chitinase [8]. The acridine derivatives showed better energy value with chitinases isoforms from A. fumigatus and T. harzianum, when compared to the plumieridine compound [12]. Among acridine derivative classes, it is possible to note that the spiro-acridine derivatives showed a stronger interaction when compared to the thiophene-acrinide and acridine-thiosemicarbazides. To perform a consensus analysis between molecular docking, it was observed that compounds 5, 7 and 9 were shown to be the top hit compounds. These compounds were selected for in vitro activity on the T. harzianum enzyme, the model species for chitinase assay.
As already highlighted, the DXDXE domain, responsible for the catalytic activity, is present in the studied isoforms. It is observed that the compounds interacted well with this domain, demonstrated by the Asp, Asp and Glu residues with H-bond. In regard to A. fumigatus, it was observed that the active site has a more hydrophobic and polar region. The score values showed compound 5 was more potent (-10.9 kJ/mol), followed by compound 9 (-10.6 kJ/mol), with compound 7 having the lowest potency (-10.1 kJ/mol). When comparing the score values with the PDB inhibitor, caffeine (-6.3 kJ/mol), it is observed that our acridine derivatives show stronger interactions with the active site (Table S2).
Compounds 5 and 9 have similar conformations in the active site. Thus, the formation of stacked π-π interactions with the acridine fragment, exchanged by Trp384 and Trp137, stands out. On the other hand, there is a more polar region at the site, composed of the amino acid residues Glu177, Asp246, Thr138. This region, where the protein domain is inserted, favored the presence of H-bond with the aromatic portions of the acridine derivatives (Fig. 2A). However, compound 7 presents a different conformation in the active site, being highlighted by the H-bond of Asp246 with the acridine ring. The π-π stacked bonds were observed for the aromatic portions, highlighted by residues Phe247, Phe251, Trp137 (Fig. 2A).
The T. harzianum species have two chitinases, known as Chitinase 33 (Chit33) and Chitinase 42 (Chit42) [33]. In regard to T. harzianum chitinase, it is observed that the acridine portion interacts positively with the active site region of two isoforms. There is a difference between the active site of the isoforms: for Chit33 the site presents more polar amino acids, while Chit 42 favors the presence of aromatic and polar amino acids. Independent of the isoform, it is observed that the compounds present interactions with the catalytic domain of chitinase. Due to the structure of acridine derivatives presenting a high prevalence of aromatic portions and H-bond, the score values were higher for Chitinase 42, indicating greater interaction in this complex (Fig. 2B).
For the Chit33 isoform compound 5 was more potent (-7.8 kJ/mol), followed by compound 9 (-7.4 kJ/mol), and compound 7 having the lowest potency (-5.9 kJ/mol) (Table S3). Compounds 5 and 9 present similar conformation and interaction, such as H-bond with amino acid residues Asn225, Gln222, Glu167, Asp117 to acridine and pyrrolidine groups. Also, Trp301 showed the π-π bonds with the acridine fragment, being crucial for the interaction of the complex. However, the bromobenzene portion of compound 5 formed an H-bond with Gln35, while the nitrobenzene group of compound 9 formed a H-bond with Ser18 (Fig. 2B). Compound 7 was divergently positioned at the active site of the chitinase 33 isoform (Fig. 2B). The acridine fragment performed H-bond with Ala38 and Ser18, while the pyrrolidine fragment ensured stability with residues Asn73 and Gln35. The benzene ring, on the other hand, formed π-π bonds with the residue Trp301, indicating that this group favors the interactions of the complex.
The simulation showed compound 5 as the most potent (-9.8 kJ/mol), followed by compound 9 (-9.2 kJ/mol), and compound 7 had the lowest potency (-8.7 kJ/mol) (Table S3). In the active site there is a high occurrence of aromatic amino acids that favored the presence of π-π interactions. Additionally, the active site integrates more polar regions with inhibitors, being exchanged by water molecules, stabilizing the structure in this region (Fig. 2C).
For the Chit42, compounds 5 and 9 showed similar positioning in the active site. Interactions were mostly driven by the presence of carboxyl and nitrobenzene groups when forming H-bond with Trp131 and Ser244, respectively. These compounds also aligned the acridine group with the amino acid residue Trp378 and Tyr293, forming π-π interactions. The H-bonds also demonstrated the high stability of the compounds in the active site, of which the residues Asp240, Tyr293 and Thr132 stand out. However, the presence of bromobenzene in compound 5 favored the presence of π-stacked bonds with the Trp131 residue, with a smaller distance, which may be an indication of greater stability and activity of compound 5 (Fig. 2C).
For compound 7, on the other hand, it is observed that the presence of the dimethylaniline group influenced the positioning of the compound, failing to create an H-bond with residues 244 and Asp240, as observed for the other compounds. Instead, interactions with residues Thr132 and Glu316 appeared, favoring their positioning in this region. However, the presence of benzene, once again, interacted with the Trp131 residue, indicating that its presence is essential to allow π-stacked interactions to happen. It was also observed that the acridine core interacted with π-stacked bonds with Trp358 residues and a new Trp47 bond. These factors may be crucial for the positioning of the compound at the active site (Fig. 2C).