**3.4 Post docking prime analysis of studied ligands**

From the prime energy calculations, the quantity of free energy of binding, ∆*Gbind* calculated from Eq. (1) was in the following order: estradiol>myricetin>cate chin>gelagin>pinobankskin>pinocembrin>pinostrobin (**Figure 6**). Other components which contributed to the electrostatic interaction like the quantity of prime coulomb energy of the complex ( ∆*Gbind* coulomb), prime van der Waals energy of the complex interaction ( ∆*Gbind* vdW), the quantity of prime hydrogen bonding interaction are as presented in **Table 2**.

#### **Figure 5.**

*Molecular interaction of tamoxifen (f) and estradiol (g) with crucial amino acids at the ligand binding domain of hER*α*.*

**Figure 6.** *The free energy of binding* Δ*Gbind (kcal/Mol) for the studied ligands with the hER*α *binding site.*


**Table 2.**

*Output properties from a prime MM-GBSA calculation for the studied ligands.*

#### **4. Discussion**

Estrogen, a major sex hormone plays an important role in mammary gland development and in the initiation and progression of breast cancer. The activities of estrogen are being mediated via its unique receptors, ERα and ERβ. The former (ERα), is the major ER subtype in the mammary epithelium. Upon activation of the hERα following its occupation, the receptor translocates to the nucleus, where it interacts with the target gene promoters of estrogen response element to mediate nuclear as well as extranuclear signaling [8, 22]. This results in the regulation of numerous critical cellular processes and is implicated in the dilemma of breast cancer. Research reveals that ERα extranuclear pathways, which are incited by undue activation of hERα have the potential to activate gene transcription, modulate cytoskeleton, and promote tumor cell proliferation, survival, as well as metastasis. The expression of extranuclear components ERα is deregulated in tumors, thus, serving as an important target for tumorigenic and metastatic control. Resistance to available endocrine therapies provokes metastasis and frustrates the management of this disease, thus, reducing the survival rate of patients bearing such tumors [23, 24]. This study evaluated the inhibitory potential of the reported small molecules from nature against hERα in a bid to suppress its downstream signaling.

The high attrition rate of new chemical entities has been attributed majorly to poor pharmacokinetic profile [25]. The reported ligands have been shown to be drug-like, having satisfied the Lipinski's rule of five [17] and also possess pharmaceutically relevant properties when compared to 95% of orally available drugs [16]. Hence, these compounds are fit in their current state to be developed into drugs without any modification/optimization except myricetin. Myricetin showed poor human absorption when examining its caco-2 permeability and human oral bioavailability. This observed poor absorption of myricetin will retard how quickly and how much of it will reach its intended target (site) of action. Hence, this phytoconstituent will require optimization to prevent its failure in the market [26].

Aqueous solubilities and human oral absorption are critical for oral dosage formulation and their *in silico* prediction had been reported to correlate well with *in vivo* bioavailabilities [27]. According to Bergström 2005 [28], aqueous solubility and intestinal permeability are the two rate-limiting barriers for oral drug absorption while the therapeutic potential of a drug is dependent on its bioavailabilty [29]. All the tested compounds showed positive human intestinal absorption.

Following absorption, the drug or intended drug molecule will circulate through the body, permeating different tissues at varying speed, depending on its ability to cross membranes. Some drugs migrate very slowly from the bloodstream because

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

they get tightly bound to proteins circulating in the blood. Others quickly leave the bloodstream and enter other tissues because they are less tightly bound to blood proteins. There are also possibilities for virtually all molecules of a drug to bind tightly to blood proteins. It is worthy of note that irrespective of how promising a drug molecule is, its efficacy will be lost if its maximum concentration gets bound to plasma proteins. This will eventually result in the decrease of effective concentration at the site of action in the tissues, as only unbound drugs can be available for pharmacological activity [14]. To predict the distribution of the studied ligands, their plasma protein binding and blood brain barrier penetration was investigated. Unlike all compounds which showed good distribution, tamoxifen, however, did not comply within the range, indicating its high potential binding to albumin. This observation was in line with previous report on the high binding affinity of tamoxifen to serum albumin [30, 31]. Considering blood brain barrier permeation, studied compounds showed no tendency of crossing it. This can be explained by the lipophobicity of the studied ligands. It therefore means that they will not provoke any significant central effect that will result in subsequent toxicity.

Another key parameter is metabolism, which is responsible for the elimination of drugs from the body. Through metabolism, drug molecules can also be converted into pharmacologically more active substrates. In this study, the molecules were investigated to predict their possible number of biotransformation which could point to potential toxicities [32, 33]. From the results, all the compounds complied with the range of metabolic reactions displayed by 95% of orally available drugs. Some of the phytoligands were predicted to possess cytochrome P450 (CYP 450) inhibitory promiscuity, revealing their capacity to bind to and decrease or diminish the activity of multiple different CYP 450 isoform enzymes [34]. It should be noted that all the molecular descriptors predicted by qikprop are exclusively for drugs intended for oral delivery. This route of drug administration is still the most preferred route for new chemical entities (NCEs) in spite of advances in drug delivery methods. Oral mode of drug delivery is convenient, cheap and has high patient compliance. Using the *in silico* prediction of pharmacokinetics-related profile of intended drug molecules helps to reduce the rate of attrition of new chemical entities in clinical trial and reduces the cost of bringing a candidate drug to the market.

In modern drug discovery, molecular docking has been gainfully employed in the screening and selection of potent inhibitors [35] especially when the anticancer target has been identified. In this study, structurally similar phytochemicals from plant origin were used to probe for binding interaction with the human estrogen receptor α. As a prerequisite, the molecular docking protocol was validated. From the results, the redocked binding pose of the native ligand was correctly reproduced within the root mean square tolerance of 2 Å. This distance is an indication of an appropriate reproducibility for a docking experiment [36, 37]. This reproducibility of the redocked native ligand was similar to the poses of the docking control reported by Hocker *et al.* (2013) [38].

Inhibiting hERα is a valid approach in ameliorating the progression of breast cancer. This study revealed strong binding affinities of the investigated compounds to the hERα. This interaction which was depicted in their free energy of binding was greatly influenced by the vicinal diol groups. The residues of estrogen receptor which partook in this binding interaction was earlier reported to play critical roles in the inhibition of the ligand binding domain of the hERα [39, 40]. The interaction of these compounds with the binding domain of hERα, created a conformational change in the receptor, inducing its dimerization, thus, interrupting its downstream signaling as well as crosstalks [41]. The conformational change observed in the estrogen receptor upon interaction with the studied ligands will also impair phosphorylation on hERα specific residues, impairing ligand-independent estrogen

receptor activation. It is promising that these interactions with the sex hormone's receptor, hERα with provoke breast cancer cell death, thus halting its progression to immortality [42].
