**1. Introduction**

The aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix/ Per-ARNT-SIM (bHLH/PAS) transcription factor family [1–4]. In its inactive state, the AhR resides in the cytosol of the cell as a complex with a number of other proteins. This complex ensures the stability of the AhR in a high-affinity ligandbinding form and prevents the premature translocation of the receptor. Upon binding of a ligand, it dissociates from these proteins and travels to the cell nucleus, where it binds to DNA xenobiotic response elements (XREs). This in turn induces the expression of several cytochrome P450 enzymes and a sulfotransferase (typically SULT1A1) that contain XREs in their promotor sequence. These enzymes then initiate the oxidative breakdown of the offending compound.

The AhR pathway has a number of roles, including as a modulator of viral immunity and the correct functioning of the female reproductive system. Its most well-known role is a mechanism by which cells defend themselves against the toxic effects of polycyclic and polyhalogenated aromatic hydrocarbons, such as the Seveso toxin dioxin (**1**) (**Figure 1**) [5, 6].

Hijacking of the pathway is based on the use of compounds capable of activating the pathway and then converting into highly reactive species such as nitrenes once being targeted by the metabolic enzymes. This process ultimately leads to DNA damage and the death of the affected cell (**Figure 2**) [7].

It has been noted that the AhR detoxification process involves the active transport of a ligand, e.g., **1**–**4**, but not the inhibition of the AhR, which would result in a buildup of toxic materials within the cell. This hijacking of the AhR signaling pathway has been proposed as a novel strategy for designing a new class of drugs against breast cancer [1, 8]. Several compound classes, such as the aromatic acrylonitriles, have shown promise in cell-based assays, displaying remarkable potency and selectivity for breast cancer cells [9, 10]. Two reported AhR ligands, Aminoflavone (**2**) and Phortress (**3**) (**Figure 3**), have progressed to clinical trials, demonstrating the clinical applicability of this approach [11, 12]. Based on this, we have postulated that the AhR is a promising target in the development of breast cancer-specific drugs. In particular, our early studies have demonstrated activity against triple negative breast cancer cell lines [9, 10, 13]. This makes AhR ligands, including the aromatic acrylonitriles, promising candidates for further development into novel agents against breast cancer, that act by a hitherto unexploited mechanism of action.

Here, we demonstrate the use of computational tools for the elucidation of the interactions between the AhR and a targeted selection of chlorinated phenylacrylonitriles. The methods employed include homology modeling, molecular docking, and molecular dynamics (MD) simulations to model the structure of the ligand-binding domain of the AhR, identify its ligand-binding site, characterize critical ligand/

**Figure 1.** *The aryl hydrocarbon receptor ligand 2,2,6,6-tetrachlorodioxin (1).*

#### **Figure 2.**

*The AhR pathway showing ligand binding, nuclear translocation, CYP1 activation, metabolism, and cell death. AF = Aminoflavone (2), Phort = Phortress (3), and ANI-7 = (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl) acrylonitrile (4) (see Figure 3 for details).*

**9**

**Figure 4.**

*transcriptional activator complex, 4F3L.*

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

receptor interactions, and study the time-dependent behavior of a ligand bound to the AhR. The results illustrate the value of computational tools for revealing the potential binding mechanism of these compounds to their target and for guiding the

*The known AhR ligands, Aminoflavone (2) and Phortress (3), that have proceeded to clinical trials for the treatment* 

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

*Alignment of the target sequence of the human form of the AhR (NP\_00161) with the sequence of a murine* 

synthesis of novel compounds with improved properties.

*of cancer and our recently reported lead AhR ligand, ANI-7 (4) [13].*

**2. Homology model**

**Figure 3.**

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

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

**Figure 3.**

*Molecular Docking and Molecular Dynamics*

Seveso toxin dioxin (**1**) (**Figure 1**) [5, 6].

where it binds to DNA xenobiotic response elements (XREs). This in turn induces the expression of several cytochrome P450 enzymes and a sulfotransferase (typically SULT1A1) that contain XREs in their promotor sequence. These enzymes then

The AhR pathway has a number of roles, including as a modulator of viral immunity and the correct functioning of the female reproductive system. Its most well-known role is a mechanism by which cells defend themselves against the toxic effects of polycyclic and polyhalogenated aromatic hydrocarbons, such as the

Hijacking of the pathway is based on the use of compounds capable of activating the pathway and then converting into highly reactive species such as nitrenes once being targeted by the metabolic enzymes. This process ultimately leads to DNA

It has been noted that the AhR detoxification process involves the active transport

of a ligand, e.g., **1**–**4**, but not the inhibition of the AhR, which would result in a buildup of toxic materials within the cell. This hijacking of the AhR signaling pathway has been proposed as a novel strategy for designing a new class of drugs against breast cancer [1, 8]. Several compound classes, such as the aromatic acrylonitriles, have shown promise in cell-based assays, displaying remarkable potency and selectivity for breast cancer cells [9, 10]. Two reported AhR ligands, Aminoflavone (**2**) and Phortress (**3**) (**Figure 3**), have progressed to clinical trials, demonstrating the clinical applicability of this approach [11, 12]. Based on this, we have postulated that the AhR is a promising target in the development of breast cancer-specific drugs. In particular, our early studies have demonstrated activity against triple negative breast cancer cell lines [9, 10, 13]. This makes AhR ligands, including the aromatic acrylonitriles, promising candidates for further development into novel agents against breast

Here, we demonstrate the use of computational tools for the elucidation of the interactions between the AhR and a targeted selection of chlorinated phenylacrylonitriles. The methods employed include homology modeling, molecular docking, and molecular dynamics (MD) simulations to model the structure of the ligand-binding domain of the AhR, identify its ligand-binding site, characterize critical ligand/

*The AhR pathway showing ligand binding, nuclear translocation, CYP1 activation, metabolism, and cell death. AF = Aminoflavone (2), Phort = Phortress (3), and ANI-7 = (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)*

initiate the oxidative breakdown of the offending compound.

damage and the death of the affected cell (**Figure 2**) [7].

cancer, that act by a hitherto unexploited mechanism of action.

*The aryl hydrocarbon receptor ligand 2,2,6,6-tetrachlorodioxin (1).*

**8**

**Figure 2.**

**Figure 1.**

*acrylonitrile (4) (see Figure 3 for details).*

*The known AhR ligands, Aminoflavone (2) and Phortress (3), that have proceeded to clinical trials for the treatment of cancer and our recently reported lead AhR ligand, ANI-7 (4) [13].*

receptor interactions, and study the time-dependent behavior of a ligand bound to the AhR. The results illustrate the value of computational tools for revealing the potential binding mechanism of these compounds to their target and for guiding the synthesis of novel compounds with improved properties.
