**2. Homology model**

The sequence of the human form of the AhR was downloaded from the NCBI website (access code: NP\_001612.1). Since only the ligand-binding PAS-B domain was of interest to our study, the sequence was appropriately truncated before Pro275 and after Lys397. A search in the modeling suite MOE's structural database for suitable templates returned the structures of 4F3L [14], 3RTY [15], and 2KDK [16] as the best matches. Of these, only 4F3L, a murine transcriptional activator complex, provided complete coverage of the PAS-B domain with a sequence identity of 24.4% and a sequence similarity of 48.0% (**Figure 4**). Only three indels were noted in the

**Figure 4.**

*Alignment of the target sequence of the human form of the AhR (NP\_00161) with the sequence of a murine transcriptional activator complex, 4F3L.*

alignment—deletions of positions 361 and 362 in the target sequence and an insertion in position 308. The absence of major gaps in the alignment is favorable for the development of homology models as it reduces the need for loop modeling and grafting, which can be challenging [17]. Model development based on the alignment in **Figure 4** was performed using MOE's default settings.

#### **Figure 5.**

*(A) Homology model for the AhR colored by secondary structure. (B) Comparison of backbone traces of homology models obtained by using the MOE modeling suite (template 4F3L) and the automated SWISS-MODEL server (template 5SY7). Coloring is according to RMSD between the two structures (green—yellow—red, in order of increasing deviation), showing very good agreement between the two models. The model obtained from the SWISS-MODEL server had a somewhat longer sequence, resulting in the gray loops at the termini that have no counterpart in the MOE model.*

#### **Figure 6.**

*Ramachandran diagram for the homology model for the AhR. Green ( ) symbols represent torsion angles in favored regions, whereas yellow ( ) symbols represent angles in allowed regions. No entries are present in the "forbidden" areas.*

**11**

**Figure 7.**

*Binding of Chlorinated Phenylacrylonitriles to the Aryl Hydrocarbon Receptor…*

The resulting homology model of the AhR (**Figure 5A**) was subjected to a number of quality tests, such as an analysis of the Ramachandran diagram (**Figure 6**) and an inspection of observed bond lengths, bond energies, and torsion angles. No abnormalities that would have questioned the quality of the model were detected. An additional check of the model's reliability was carried out by submitting the PAS-B sequence to the automated server SWISS-MODEL [18]. The returned homology model was superimposed to the model obtained from MOE. Even though the new model was derived using a different template (5SY7, an NPAS3-ARNT complex) [19], a very good agreement between the backbones of the two structures was observed (**Figure 5B**), which further instilled confidence in the accuracy of the model.

Before ligands could be docked into the homology model of the AhR, the exact location of the binding site had to be identified. We subjected the homology model to a binding site search, a feature implemented in MOE that screens the surface of a protein for concave areas capable of binding small molecules. Two areas large enough to accommodate a typical AhR ligand were detected: one on the surface and another one in the core of the receptor. To decide which of these two sites was more realistic, the crystal structures of the ligand/receptor complexes 3F1O [20], 3H7W [21], and 3H82 [21], all of which are proteins related to the AhR, were superimposed onto the homology model. As shown in **Figure 7**, all three ligands were found in an area equivalent to the binding site located at the center of the protein (**Figure 5**). To facilitate a convenient designation of the binding site for the subsequent docking runs, the ligand of 3F1O—N-[2-nitro-4-(trifluoromethyl)phenyl]morpholin-4-amine (**5**)—was copied into the file of the homology model as a point of reference.

*(A) Superposition of protein/ligand complexes related to the AhR onto the homology model of the AhR. Spheres delineate the putative binding site predicted by MOE that coincides with the position of the ligands seen in the crystal structures. (B) A closeup view of the ligand 5, overlaid with the spheres depicting the binding site predicted by MOE. (C) The chemical structure of the ligand of 3F1O—N-[2-nitro-4-(trifluoromethyl)phenyl]morpholin-4-amine (5).*

*DOI: http://dx.doi.org/10.5772/intechopen.84818*

**3. Computational ligand docking**

*Binding of Chlorinated Phenylacrylonitriles to the Aryl Hydrocarbon Receptor… DOI: http://dx.doi.org/10.5772/intechopen.84818*

The resulting homology model of the AhR (**Figure 5A**) was subjected to a number of quality tests, such as an analysis of the Ramachandran diagram (**Figure 6**) and an inspection of observed bond lengths, bond energies, and torsion angles. No abnormalities that would have questioned the quality of the model were detected.

An additional check of the model's reliability was carried out by submitting the PAS-B sequence to the automated server SWISS-MODEL [18]. The returned homology model was superimposed to the model obtained from MOE. Even though the new model was derived using a different template (5SY7, an NPAS3-ARNT complex) [19], a very good agreement between the backbones of the two structures was observed (**Figure 5B**), which further instilled confidence in the accuracy of the model.
