**2. Materials and methods**

### **2.1 Computer hardware and software**

The molecular docking simulation was performed on the Lenovo Precision workstation 6.1.7600 running Intel® Core™ i5 Duo Processor, 4.0GB RAM, 436 GB hard disk and AMD Radeon graphics card (Lenovo PC HK Limited, China). The 3D structures of the small molecules were obtained from the National Centre for Biotechnology Information, Pubchem database www.ncbi.nlm. nih.gov/ pccompound) in SDF format and prepared with Maestro, using ligprep version 3.6 (LigPrep 2015). The solution x-ray crystal structure of the human ERα (3UUD, 1.60 Å resolution) was retrieved from the protein databank (**www.rcsb.org**) using Discovery Studio visualizer 4.5 (Accelryls, USA). Protein-ligand docking simulation was performed using the Schrodinger molecular docking suite version 2018-4.

#### **2.2 Preparation of ligands and protein**

The ligands were prepared using LigPrep, a utility of Schrodinger software suit that combines tools for generating 3D structures from 1D (Smiles) and 2D (SDF) representation. Molecular mechanics force fields, optimized potentials for liquid simulations-2005 (OPLS\_2005) with default settings were employed for the ligand minimization and the ligands were thereafter filtered for computational studies.

The crystal structure of hERα (3UUD) was prepared using Schrodinger protein preparation wizard tool (Glide), which performed the following steps: assigning of bond orders, addition of hydrogens, optimization of hydrogen bonds by flipping amino side chains, correction of charges and minimization of the protein complex. All the bound water molecules, ligands and cofactors were removed (preprocess) from the protein and the output file was saved in maestro format. The idle side chains were neutralized before restrained minimization of co-crystallized complex, which reoriented side chain hydroxyl groups and alleviated potential steric clashes. The complex obtained was minimized using OPLS\_2005 force field with Polack-Ribiere Conjugate Gradient (PRCG) algorithm. The minimization was terminated when the energy gradient converged below 0.05 kcal/mol [14].

## **2.3 Prediction of pharmacokinetic properties and drug-likeness**

The molecular weight, number of hydrogen bond donor, number of hydrogen bond acceptor and octanol–water partition coefficients were used to verify the compounds adherence to Lipinski's rule of five which qualifies their drug-likeness. To nominate drug candidates, certain pharmacokinetics descriptors that portray their drug-likeness [15] were investigated using the QikProp module of the Schrodinger Suite, a program designed by Professor William L. Jorgensen [16]. In addition to predicting physically significant and pharmaceutically relevant molecular descriptors, QikProp also provides ranges for comparing predicted descriptors of each compound with those of 95% of drugs known for oral use. The pharmacokinetic descriptors evaluated were: molecular weight(Mwt), total solvent accessible surface area (SASA), Donor hydrogen bond (DonorHB), number of acceptable hydrogen bond (Accept HB), predicted octanol/water partition coefficient (QPlogPo/w), predicted aqueous solubility (QPlogS), predicted apparent Caco-2 cell permeability (QPPCaco), predicted brain/blood partition coefficient (QPlogBB), number of likely metabolic reactions(#metab), human oral absorption, van der Waals surface area of polar nitrogen and oxygen atoms (PSA) and prediction of plasma protein binding(Khsa). Cytochrome P450 inhibitory promiscuity and inhibition of the human either-a-go-go-ralted gene was also accessed via admerSAR web server. The analysis in the present study was run on QikProp at the normal processing mode with default settings (QikProp 2018). The prepared ligands were used as input structures and their pharmacokinetics profiles with respect to properties shared by 95% of drugs known for oral use were evaluated. Compliance or deviant of the tested potential drug candidates to the Lipinski's rule of five was also examined before they were considered drug-like [17].

### **2.4 Docking studies**

Docking studies were carried out using Glide XP of the Schrodinger Suite (Maestro Version 11.8 and Glide version 8.0, 2018-4) docking program following the reported standard procedures [18]. Each ligand was individually docked onto the LBD of the hERα using Glide extra precision (XP) mode. In the course of the docking, several binding poses were generated for each ligand and the best binding pose was selected at the end of the docking process.

### **2.5 Calculation of ligand free energy of binding with the hERα using the MM-GBSA approach prime energy analysis**

The Prime MM-GBSA or 'molecular mechanics energies combined with the generalized Born and surface area continuum solvation' approach was used in the *Small Molecules Inhibit Extranuclear Signaling by Estrogen: A Promising Strategy to Halt Breast… DOI: http://dx.doi.org/10.5772/intechopen.94052*

post-assessment of free energy of binding of ligands-hERα complex [19]. This approach uses the OPLS\_2005 all-atom force field for protein residues, ligands and cofactors [20, 21]. The input structures for these calculations were taken from a pose viewer file Glide output after the docking study.

The following descriptors were generated by the prime MM-GBSA approach:

1.MM-GBSA\_∆G\_bind (ligand binding energy ( ∆*Gbind* ))

2.MM-GBSA\_E\_complex (energy of the complex ( *Gcomplex* ))

3.MM-GBSA\_E\_protein (energy of the receptor without the ligand ( *Gprotein* )) and

4.MM-GBSA\_E\_ligand (energy of the unbound ligand ( *Gligand* )).

The total free energy ( ∆*G*bind) of binding is expressed as:

$$
\Delta \mathbf{G}\_{bind} = \mathbf{G}\_{complex} - \left( \mathbf{G}\_{proticu} + \mathbf{G}\_{ligand} \right) \tag{1}
$$

The other parameters for the complex were:

1.Prime Coulomb energy ( ∆*Gbind* coulomb)

2.Prime Van der Waals energy ( ∆*Gbind* vdW)

3.Prime Hydrogen Bond ( ∆*Gbind* H-bond)

The MM-GBSA scoring and experimental binding affinity data of the binding site for the molecules on hERα were recorded.

#### **3. Results**

#### **3.1 The structures of the protein and studied ligands**

The 3D structures of the studied ligands are as shown in **Figure 1c**. Complete X-ray structure of of the hERα (**Figure 2A**) and its binding amino acids depicted with green sticks are as shown in **Figure 2B** above.

#### **Figure 2.**

*(A) Complete X-ray structure of hER*α *shown as ribbon (B) active amino acids shown in green sticks at the catalytic site of hER*α*.*


**Table 1.**

*Pharmacokinetic properties of studied ligands.*

*Small Molecules Inhibit Extranuclear Signaling by Estrogen: A Promising Strategy to Halt Breast… DOI: http://dx.doi.org/10.5772/intechopen.94052*
