Molinspiration Cheminformatics and Molecular Docking Studies of Some Natural Flavonoids: Fisetin and Its Analogs as an Anti-Breast Cancer Agents

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CHAPTER 1:

INTRODUCTION

Cancer Defined

Cancer generally refers to the multiple diseases that occur when abnormal cells of a particular organ grow, divide, and rapidly spread (metastasize) to other body organs and tissues (Davis, 2020; Garnick, 2009; Nall, 2020).

The defining abnormality preceding cancer proliferation is a result of the ongoing unregulated growth of metastatic cells. Instead of responding favorably to the biochemical factors that influence ordinary cell functioning, metastatic cells proliferate and multiply in an unregulated manner, attacking normal organs and tissues. Consequently, they spread out through the entire body. The considerable absence of proliferation control displayed by metastatic cells is the sum outcome of cumulative abnormalities experienced in the cell’s numerous regulatory mechanisms. This phenomenon is witnessed in various cell behavioral aspects that separately define normal cells from their cancerous counterparts.

At the cell level, the onset and growth of metastasis are considered a multistep progression that entails mutation and preference for cells with the probability of the most proliferative cell (Cooper, 2000). Besides, such cells should be very likely to survive, infiltrate, and metastasize. Tumor initiation is considered as the beginning step. This step is associated with the alteration of genetic mechanisms that beget abnormal individual cell proliferation. Cell growth then precedes shooting forth of a group of cloned cancerous cells. Tumor progression as the subsequent step unfolds as more genetic alterations recur inside the cancerous cells group (Cooper, 2000). Several such mutations give the cell a selective advantage for more pronounced flourishing. As a result, daughter cells coming from this line of mutation become mainstays within the group of tumor cells. The unfolding of such clonally produced tumor cells makes for continued cancer spread within the body courtesy of the clonal advantages. Tumors continue to grow rapidly as long as clonal selection is not regulated.

Therefore, a tumor is any proliferation of cells that is considered abnormal. Tumors could either be malignant or benign. Benign tumors stay restricted to their original area of genesis (Cooper, 2000). Such tumors do not attack neighboring normal tissues. They do not also escalate to distant body tissues. Conversely, malignant tumors are characterized by infiltrating neighboring normal tissues and escalating to distant body tissues (Cooper, 2000). Their spread is facilitated by the lymphatic and circulatory systems. Cancer is largely a reference to malignant tumors. This capability for infiltration and metastasis is what renders cancer so lethal. While benign tumors are easy to excise by surgical intervention, malignant tumors’ metastasis to farther body tissues usually renders them hard to terminate by such localized management.

Causes of Cancer

Cancer-inducing agents include chemicals, radiation, and viruses. Chemical carcinogens and radiation spark mutations through DNA damage. Mutations convert proto-oncogenes into oncogenes. Proto-oncogenes regulate cellular division related functions. When altered by mutation, they become oncogenes that now drive ungoverned cell division that forms tumors. Additionally, oncogenes facilitate the infiltrative metastasis of cancerous cells (Miller and Suzuki et al., 2020). Mutations can be spontaneous or environmental. Cancer initiating carcinogens are usually described as initiating factors. For example, solar ultraviolet radiation is a major precursor for skin cancers in humans, while nickel compounds and dimethylnitrosamine in tobacco chemicals are major carcinogenic precursors for lung cancers. The chemical carcinogen aflatoxin that arises from grains contaminated by mold growth is responsible for hepatocarcinoma. Well over 80 % of lung cancers have are directly related to smoking. The single largest cause of cancer-related mortality is tobacco (Sloan and Gelband, 2007). Also, of the non-communicable diseases, tobacco is the foremost cause of mortality. Adults can be encouraged to quit smoking. The long term approach has been encouraging youth to desist from beginning tobacco smoking. Nearly 5 million global deaths in 2000 were tobacco-related.

Heavy consumption of alcohol is linked to larynx, liver, esophagus, and oral cavity cancers. An estimated 5% of cancer deaths are attributed to heavy drinking (Sloan and Gelband, 2007). As physical activity and diet, the burden of disease from excessive alcohol consumption is multiple times above such other ailments as cardiovascular disease.

Exposures to carcinogens at such places of work-as family farms, factories, and cities’ streets were responsible for 2 million mortalities in 2007. According to the International Labor Organization, this was a considerable number of the global workforce of 2.7 billion (Sloan and Gelband, 2007). Over 25 chemicals of occupational application have been classified as carcinogenic. For example, the use of asbestos in manufacturing industries that employ older machinery and that have little safeguarding of workers poses a threat to occupational health.

Outdoor and indoor air pollution and soil and water are responsible for approximately 1-4% worldwide cancer cases. Most of these people are affected by involuntarily. Outdoor and indoor pollution of air mainly causes cancer of the lungs. Motor vehicle exhaust emissions cause the majority of ambient air pollution. Indoor air pollution is traced to cooked food fumes and cooking and heating fires. In developing countries where biomass and coal are the most used is smokiest. Substandard quality of drinking water is thought to cause some cancers due to chlorination by-products. Arsenic that is a natural occurrence in soils is carcinogenic and contaminates water bodies of areas where it abundant. Many contagious microbes cause cancer. However, the most prevalent microbe-linked cases of cancers gastric metastasis caused by Helicobacter pylori and hepatic metastasis caused by hepatitis B and C viruses and cervical cancer induced by human papillomaviruses.

The Difference between Normal Cells and Cancer Cells

A healthy body’s normal cells renew themselves in a reproductive cycle that eliminates the dying ones and replenish them with new cells (Healthline Editorial Team, 2017). Whereas apoptosis facilitates the replacement of normal dying cells with new ones, cancerous cells are deprived of mechanisms that instruct them to cease growing, proliferating to die (Garnick, 2009). While normal cells remain confined to their parent organs, cancer cells metastasize (spread from the original organ to any part of the body) (Garnick, 2009). A non-cancerous cell that dies is usually replaced by a new one (Nall, 2017). However, Cancerous cells continue to multiply in the body and utilize nutrition and oxygen than other cells. This way, they supersede normal cells and impair their natural functioning (American Cancer Society, 2019).

Cancers are classified by their localization in the body and the tissues where they form (Garnick, 2009). Cancer-associated mortality and morbidity result majorly from damage inflicted on organs due to localized growth or metastases. Cancer deaths can be prevented by avoiding tobacco, applying sun safety guidelines, eating healthy, physical activity, obtaining immunization against HPV, and regular cancer screening for early detection (World Health Organization, 2009). As of 2009, tobacco use worldwide was the single highest preventable cancer risk factor, causing 1.5 million deaths (World Health Organization, 2009). Well over 30% of cancer cases originate from modifiable environmental and behavioral risks. Early detection accounts for more comfortable treatment since cancer patients can very well be treated. Cancer survivors go on to live fully after treatment. These are but a mention of the over 100 types that have been identified (Garnick, 2009).

Clinical options for cancer management include chemotherapy, hormone therapy, radiation, immunotherapy, and surgery. Chemotherapy applies medications to kill rapidly proliferating tumor cells and help contract tumors (Nall, 2017). As is commonly used to manage breast and prostate cancers, hormone therapy aims to use medications that alter the production of hormones used by tumor cells to proliferate. Immunotherapy aims to boost immunity through adoptive cell transfer and such medications as checkpoint inhibitors (Nall, 2017). The immune system is boosted and encouraged to destroy the tumor cells. Radiotherapy applies intense energy beams to kill tumor cells or contract a tumor before surgery.

Stem cell transplants (SCT) are useful for lymphoma and leukemia patients. SCT involves the replacement of damaged stem cells with healthy ones after doses of radiation therapy or chemotherapy. It also entails the removal of that radiation or chemotherapy destroyed white and red blood cells. Surgery is usually a preferred intervention for removing a cancerous lymph node or (malignant) tumor (Nall, 2017).

Types of Cancer

Majority of cancers are categorized as lymphomas, leukemias, sarcomas and carcinomas. Carcinomas, make up nearly 90% of malignancy diagnoses in humans. They are predominantly epithelial cells cancers. Carcinomas may be metastatic or benign. The various forms of cancer include invasive carcinoma, metastatic carcinoma and carcinoma in situ. Invasive carcinoma’s spread to the surrounding tissue and metastatic carcinoma spread throughout the entire body (Cancer Treatment Centers of America, 2020). Some of the prevalent types of carcinomas include basal cell, squamous cell, renal cell, ductal carcinoma and invasive ductal, carcinomas. Basal cell carcinomas are the most common skin malignancy types. The malignant cells proliferate in the cells of the skin’s basal layer or the basest segment of the epidermis but rarely spread to adjacent tissues (Cancer Treatment Centers of America, 2020). Squamous cell carcinoma cells grow from the squamous cells but rarely spread further than their primary tissue. Ductal carcinoma in situ affects the breast and is the most prevalent type of breast malignancy with cancerous cells rarely spreading beyond the milk ducts. Invasive ductal carcinoma spread from the duct walls into local breast tissue and may reach other parts of the body (Cancer Treatment Centers of America, 2020). Renal cell carcinoma is most prevalent among kidney cancer types with malignant cells growing in the tubules and the resulting mass may induce obstruction.

Sarcomas are mainly soft tissue and bone sarcomas. Soft tissue sarcomas can affect any part of the body’s connective tissue from blood to skeletal muscles to cartilage (Popovich et al, 2020). The benign sarcomas are usually referred to as lipomas while the metastatic ones are identified as angiosarcomas. Sarcomas are scarce cancer types. Often, it is associated with Children and young adults.

Melanomas are primarily skin tumors that spread from tissue of primary origin. They originate from melanocytic cells. They can also originate from the meninges, conjunctiva of the eye and mucosa surfaces ( Garbe et al, 2016). They are heavily pigmented but can be without the pigment (amelanotic). Small tumors could metastasise. Of the cutaneous tumors mortalities, melanomas a are responsible for 90% of mortalities.

Lymphomas are malignant cancers that originate from growth of cancerous lymphocytes (natural killer cells, T-cells and B- cells). It represents 5% of malignancies. Lymphomas can be divided into Hodgkin lymphomas and Non-Hodgkin lymphomas (Jamir and Mukammalla, 2020). Hodgkin lymphomas specifically originate from lymphocytes while non-Hodgkin’s lymphoma is associated with the lymphatic system. Other types are marginal zone, mantle cell, Burkitt and follicular lymphomas.

Leukemias are a proliferation of the bone marrow’s hematopoietic cells. They are classified into acute myelogenous and lymphoblastic leukemias and, chronic myelogenous and lymphocytic leukemias (Davis, 2014). Often, acute lymphoblastic leukemias are associated with children and originate from the lymphocytes. The rest of the three leukemia subtypes are adults prone. Besides originating from lymphocytes, myelogenous leukemias may arise from platelets, red blood cells and other forms of white blood cells.

Importance of Breast Cancer Research

Breast cancer research gives hope to the 1 in 8 of American women who are likely to be diagnosed with the malignancy. The increasing rates of survival and previously diagnosed women continuing to live healthy lives after overcoming the disease is attributed to extensive research on breast cancer remedies. When sustained, this momentum should result in better outcomes and improved care. Continued research will help increase our understanding of the malignancy for better detection, prevention and treatment options.

A primary application of breast cancer research involves discovering new and more effective treatments. Some management models involve treatment combinations, drugs and surgical techniques Triple negative breast cancer has posed a treatment challenge. Research has attributed this aggressive malignancy to BRCA gene mutations. Findings have proposed that routine screening be conducted to provide diagnosis and treatment benefits available. Its treatment option is different from what a triple negative breast maligancy patient would receive. The Cancer Research UK has produced carboplatin as a better remedy for triple negative breast malignancies (Cancer Research UK, 2020).

Breast cancer research incentives include researching into how diet, body weight, diet and physical activity affect risk for cancer. Others include applying genetic testing for gene mutations detection, exploration of gene variations that influence cancer risks and environmental carcinogens that induce breast cancer. Hormonal and non-hormonal drugs that help minimize the risk for breast malignancy are being explored. Research helps give insight on management of unpredictable nature of breast malignancy. The elusive proliferation and invasiveness of post-surgery Ductal Carcinoma in Situ has prompted research into how computer algorithms and statistical models can be used to study this post-surgery behavior. Usually, DCIS is regarded as pre-invasive or non-invasive malignancy of the breasts. However, they can become infiltrating after surgical intervention that is not accompanied by treatment. Research birthed remedies should help to manage breast cancer recurrence, side effects, survival rates and prevention avenues.

CHAPTER 2

LITERATURE REVIEW

Breast Cancer Defined

Breast cancer is in the class of cancers that originate from surface cells of glands and epithelial surfaces (Garnick, 2009). It begins in the breasts. The majority of cancers of the breast are carcinomas. Breast cancers are usually a carcinoma type referred to as adenocarcinoma (Garnick, 2009). The carcinomas that cause breast cancer begin in the ducts or the lobules (American Cancer Society, 2018). Certain biomarkers like CD44+CD24_/low, HER2 (human epidermal growth factor receptor 2) and ER 1 (Estrogen receptor 1) distinguish breast cancer. for example expression of CD44⁺CD24^-/^low in breast metastasis stem cells is linked to drug resistance and continued metastases The applicability of hormone therapy will predictably be effective if breast cancer expresses ER-alpha markers (Colomer et al 2018). A greater than a third of all patients expressing ER-alpha usually respond objectively to hormone therapy. Over-expression of HER2 in breast tumors is predictive of very aggressive cancer subtypes (Colomer et al 2018). Other biomarkers for aggressive tumors are Ki-67 and PRS (polygenic risk scores).

Genetic mutations associated with prolonged exposure to estrogen and DNA damage induce development of breast cancer. Hereditary factors may contribute to passing down of such cancer supporting genes as BRCA 1 and BRCA2. Defective DNA may also be inherited. Such abnormal DNA would be destroyed by immune cells of a normal individual but immune cells fail to destroy such cells in patients with breast cancer or any metastasis type. Development of breast cancer is higly probable in individuals with a breast or ovarian cancer family history (Alkabban and ferguson, 2020). Cancer growth and metastasis could be managed internally if the immune system were able to induce apoptosis in cancerous cells.

Estrogen Receptors

Estrogen receptors (E.R.) alpha and beta are nuclear transcription factors involved in the regulation of many complex physiological processes in humans. They are responsible for regulating the transcription of multiple genes for cell division and mammary glands. Estrogen and Estrogen separately contain 560 and 530 amino acids, respectively. Figure 1 shows the crystalline structure of the estrogen receptor α schematically represented.

Figure 1

E.R. also facilitates similar cell proliferation and development in pregnancy. Hormone receptors are found around breast cells. The receptors stimulate the growth of both healthy and malignant cells. In patients with breast cancer, these receptors signal the cancer cells’ uncontrollable growth that forms tumors (Silberman, 2020). Estrogen receptors serve as binding sites for a hormone –ligand relationship with Estrogen and progesterone. Estrogen-receptor positive malignancies are more widespread than estrogen-negative.

Upon diagnosing cancer by biopsy, the physician usually tests cancer cells’ surface for other characteristics, the presence or absence of receptors (Silberman, 2020). ER-negative cells usually lack hormone receptors on the surfaces of the cancer cells. About 68% of breast cancer cases diagnosed in women are estrogen receptor-positive (ER-positive) (Silberman, 2020). This means that estrogen receptors found on cell surfaces attach to the hormone estrogen. ER-positive cancer of the breast means that the patient’s cells proliferate in the female hormone, Estrogen. Most women with ER-positive breast cancers usually respond appropriately when hormone therapy is administered. The prognosis is typically determined by how appropriately the body reacts to therapy and the stage of cancer at the time of diagnosis (Silberman, 2020). Breast cancers that are diagnosed as ER-positive will have a presentable outlook upon early therapy. Therapeutic drugs that treat this type of cancer alter Estrogen’s role in promoting cancer cell proliferation (Silberman, 2020). Recent declines in mortality rates caused by breast cancer have been attributed to the effectiveness of such drugs. Hormone therapy prescriptions for women with ER-positive breast cancer are contributing a healthy chunk to breast cancer patients’ recovery.

Breast Cancer Incidence

Breast malignancy accounts for the highest number of cancer-related deaths in women in the U.S. Approximately 12% of women in the U.S. are reported to experience infiltrative breast cancer during the span of their lifetime (Alkabban and Ferguson, 2020; Breastcancer.org., 2020). Comparatively, 0.1% of men in the U.S. will likely develop breast malignancy during the span of their lifetime. Most cancer cases are diagnosed and recorded in women in the U.S., and breast cancer comes second after skin cancer as the most prevalent in the sex (Centers for Disease Control, 2020). In the U.S., breast cancer is the most prevalent type after skin cancer. Lung cancer and prostate cancer come behind breast cancer.

In 2018, approximately 266,100 females were projected to develop infiltrative breast carcinoma (Alkabban and Ferguson, 2020). The comparative male sex estimate was 63,960, with slightly over 2500 of the cases being invasive. This year in the U.S., it is estimated that slightly over 276,000 and 48 500 new instances of invasive breast and in situ cancers will be diagnosed in women (Breastcancer.org., 2020). The estimated number for men stands at 2,620.

The incidence rate is uniform across white and black ladies. However, black women pass away from this disease at a rate higher than their white counterparts. Native-American, Asian, and Hispanic are at the most minimal risk of developing and passing away from cancer. Jewish women (Ashkenazi) are at a higher risk of breast cancer due to a greater gene mutation rate (Breastcancer.org., 2020). Globally, breast cancer is recorded to be the most common cancer type in women. (World Health Organization, 2020). Slightly above 2 million women become victims of this disease annually. It accounts for the highest number of cancer-associated morbidities in women, with approximately 627,000 dying from breast cancer in 2018 (WHO, 2020). Presently, slightly over 3 million living American women have a history of breast malignancy. This estimate is representative of women on therapeutic management and those previously treated. More effective cancer treatment remedies are continually being re-invented, leading to yearly drops in cancer-related deaths. Table 1 gives a summary of types of cancer by percentage prevalence in both sexes. According to the table’s statistical presentation, prostate cancer was the most diagnosed among men accounting for 21 % of cancer cases in men. Among women breast cancer accounted for the highest of diagnoses with a 30 % of all cancers in the sex. Pancreatic cancer (3%) and leukemia (3%) were the least common cancer types in men and women respectively. The leading cause of cancer associated mortalities in men and women are is cancer of the lung and bronchus at 23 and 22 % respectively. The least cancer related mortalities are those of brain and nervous system, accounting for 3% of cancer mortalities in either sexes.

Table 1

Types and Subtypes of Breast Cancer

Ductal carcinoma, the most prevalent of breast cancers, originates from the milk conveying ducts (American Cancer Society, 2019). The least common types have been associated with the cells of the connective, fat, and muscle tissues. Examples of the latter category include sarcomas, angiosarcomas, Paget’s disease, and phyllodes. Ductal carcinoma can be in situ (DCIS) or infiltrating (IDC). IDC is the most prevalent breast cancer type (American Cancer Society, 2018). Figure 2 shows the percentage prevalence of infiltrative versus non- invasive cancer.

Figure 2

It begins in any of the breast’s milk ducts and navigates through the duct wall into the breast fatty tissue where it proliferates. Once outside the duct’s confines, it can potentially metastasize to other body organs via the bloodstream or lymphatic system. Approximately 80 % of breast cancers in men are IDCs. The male breast’s size means that all breast cancers in men begin closer to the nipple and will likely get thereupon spreading (American cancer society, 2018).

Lobular cancers begin in the mammary glands that secrete milk in suckling mothers. Such types as angiosarcoma and phyllodes are categorized as the least common malignancies. Invasive lobular carcinomas (ILC) begin in the lobules and metastasize to other breast or other body organs. ILC is relatively scarce in men with 2% (American Cancer Society, 2018). This low occurrence is explained by the little, if any, lobular tissue of the breast. Lymphomas and sarcomas, which begin in other breast tissues, are not considered cancers (American Cancer Society, 2019). These breast cancer identities are typical of the extent of the spread, in situ, meaning they have not spread and infiltrative or invasive, meaning they have metastasized into immediate brain tissues. Although DCIS is considered non-invasive, it is often described as pre-invasive since some instances have been observed to proceed and become invasive (American Cancer Society, 2018). Lobular carcinomas can also be in situ with the cancerous cells proliferating in the breast lobules remaining restricted within the lobules’ walls. Therefore, LCIS is a non-invasive type of breast cancer and is scarcely found in men (American Cancer Society, 2018).

Five significant subtypes of breast cancer defined by the malignancy genes they express are luminal A, luminal B, triple-negative/basal-like, normal-like, and (human epidermal growth factor receptor 2 (HER2). Table 2 shows these types of breast cancer as immuno-histochemically defined (Lachapelle and Foulkes, 2011). These subtypes are derived from immune-histochemical assessments.

Table 2

Molecular subtype	Immunohistochemical staining^a
	E.R.	PR	HER	ck5/6b	egfrb
Luminal A	+	+	–	–	–
Luminal B	+	+	+	–	–
HER-positive	–	–	+	–	–
Basal –Like	–	–	–	+	+

Luminal A cancers being progesterone-and estrogen-receptor-positive are usually less prevalent, proliferate much slowly, and have the best prognosis. Luminal B cancer is progesterone-and estrogen-receptor-positive and varies from HER2 negative to HER2 positive (Lachapelle and Foulkes, 2011). This type has higher concentrations of the protein Ki-67. Ki-67 serves to modulate the speed of growth of malignant cells (Breast acncer.org, 2020). Luminal B cancers tend to increase relatively faster when compared to luminal A cancers. Its prognosis is considered relatively worse (Breast cancer.org, 2020). Basal-like Breast cancer is referred to as triple-negative, which means that they do not express the HER2 genes and lack Estrogen and progesterone receptors (Lachapelle and Foulkes, 2011). Triple-negative breast cancer, HER2, progesterone, and estrogen-receptor negative, is prevalent in women who experience BRCA mutations. Triple-negative cancers have a 75% prevalence rate in BRCA1 women (Lachapelle and Foulkes, 2011). Women who manifest a triple-negative breast malignancy are deemed to have begun experiencing menopause earlier than age 45, been overweight during the years preceding their menopause, and have breastfed their newborns for the least duration (Lachapelle and Foulkes, 2011). Youthful women of color and Hispanic descent have the highest recorded instances of this type of cancer (Breast cancer.org, 2020). Triple-negative aggressive and can metastasize mostly to the brain and lungs and the least to bones. Their typical survival duration is usually 3 to 5 years post-diagnosis. They are less likely to distant-recur as compared to ER-positive cancers. The patients benefit less from trastuzumab or hormone therapy and are usually treated with a more effective chemotherapy option.

The HER2-enriched type is progesterone-and estrogen-receptor negative but is HER2 positive. Malignancies enriched with HER2 comparatively proliferate faster than luminal ones. Targeted therapies like Kadcyla and Nerlynx are most effective in treating these malignancies (Breast cancer.org, 2020). Normal-like malignancies are very like luminal ones: progesterone and estrogen-receptor-positive, negative for HER2, and minimal concentrations of Ki-67, the protein that regulates malignancy proliferation speed.

Risk Factors of Breast Cancer

Generally, some of the significant risk factors for cancer widely discussed are genetic make-up, tobacco use accompanied by excessive alcohol intake, physical inactivity, harmful solar radiation, diet, environmental input, and viruses (Garnick, 2009). The specific risk factors for breast cancer are female, increasing age, an individual’s personal and family history of cancer, exposure to radiation, inherited BRCA1 and BRCA2 genes, obesity, female periods beginning at an age lower than 12 years, menopause starts at an advanced age, childbearing at an older age, and taking hormone therapy (women) (Mayo Clinic, 2020).

Breast malignancy risks and associated mortality increase considerably with age across the interracial divide in American women. An increase in age elevates one’s risk for breast malignancy. Women aged 45 years and above are the highest victims on an age-factor basis. In 2004, over 45% of breast malignancy instances and 59% related mortality occurred in this age group (Hewitt et al., 2020). Presently, over 95% of new cancer incidences are reported in 40 and above-year-old women (Alkabban and Ferguson, 2020). For every 100,000 women in the U.S., 1.5 malignancy cases were reported in women aged 20-24 years. Women between 75 and 79 years had an incidence rate of 421.3 cancer diagnoses for every 100,000 groups of women (Alkabban and Ferguson, 2020). The diagnosis of breast cancer cases in women stands at a median age of 61 years.

Diagnosis of breast malignancy varies by racial consideration, with 128.1 cases per 100,000 young white women against an African American incidence rate of 124.3 cases for every 100,000 people (Alkabban and Ferguson, 2020). However, the malignancy-related mortality rates were higher in the later population than in the former. Breast malignancy incidences are least among young women of American Indians, Latina origin, and Asian Americans standing at per case rate of 88.3 cases for every 100,000 women, 91.0 women for every 100,000, and 91.9 women for every 100,000

White women have a higher survival chance than similarly diagnosed African American women. Women of the last race are most often diagnosed with cancer when it has advanced than women of the last race. This delayed diagnosis explains the reason for minimal survival among cancer-diagnosed African American women. The lower survival rates in black women (72%) compared to whites (87% ) are caused by aggressive tumors that are first detected at advanced stages aggravated by deprived health insurance and the prevalence of other illnesses.

Symptoms of Breast Cancer

Breasts can have non-normal disease-induced lumps and diseased lumps. Diseased lumps could be caused by infection, trauma, or malignancy. The cells responsible for breast cancer are identified by the tumors they form. When a part of the breast swells or thickens, breast skin being dimpled or irritated, the nipple retreating into the breast, the nipple being in pain or producing a non-milk discharge, changing of the breast shape or size and breast localized pain (Centers for Disease Control, 2020). Breast cancer tumors are usually discerned by a lump’s feeling upon hand examination and through x-ray examination. Most lumps in the breasts are non-cancerous (benign) rather than cancerous (malignant). Although benign lumps are abnormal growths that do not metastasize beyond the breasts, they can increase women’s risk of contracting breast cancer (American Cancer Society, 2019).

A physician’s examination of the benign lump can help a lady determine if it may predispose her to future cancer risk. Certain breast cancers present as thickened or red skin. A biopsy is usually conducted to test the tumor cells for human epidermal growth factor receptor 2 (HER2) and progesterone and estrogen receptors (American Cancer Society, 2019). Laboratory tests could help establish how far cancer has progressed. Malignancy management options are dependent on the extent of the anomaly and the type of proteins identified (American Cancer Society, 2019). It is not all types of breast cancers that cause lumps in the breast. Screening mammograms are efficient at the earliest detection of breast cancers (American Cancer Society, 2019). They can detect cancer way before the onset of any symptoms or presenting of a lump. However, mammograms are limited in not being able to trace every cancer. Breast cancers may usually begin from any part of the breast.

The cancer cells can metastasize through circulating blood, and lymph fluid usually conveyed away from the breast through lymph vessels (American Cancer Society, 2019). A high prevalence of breast cancer cells in the lymph nodes is a likely indicator of the tumor cells metastasizing to other body parts. Surgical removal of one or several lymph nodes will help establish if the breast cancer has metastasized. It has been established that not all patients that bear tumor cells in lymph nodes experience metastases (American Cancer Society, 2020). In certain instances, a unit tumor could be an accumulation of different tumor types. In certain very rare instances of breast cancer, the malignant cells do not present a tumor or lump at all (American Cancer Society, 2018). Still, several individuals without tumor cells in lymph nodes have been observed to develop metastases later in life. Women need to know how their breasts normally feel and look. As much as having regular screening tests is vital, women are encouraged to note any not normal changes in their breasts and the typical cancer signs and symptoms. Besides treatment, breast cancer efforts will bear more significant fruits if early detection is emphasized through screening and early diagnosis.

Side Effects of Breast Cancer

The side effects of breast malignancy are ailments or symptoms that manifest from the treatments applied or the malignancy itself. Following radiation therapy, the under-arm and breast tissue may experience the depreciating sensation. Additional side effects include skin anomalies (radiation dermatitis) like redness, peeling, and itching in the area treated (Agrawal, 2014). Radiation therapy may spark cardiovascular toxicity that could be characterized by arrhythmias, stenosis, valvular disease, and conduction disruptions. Acute side effects associated with chemotherapy include vomiting, diarrhea, and infection (Agrawal, 2014). Chemotherapy may also spark cardiovascular toxicity. Apparent congestive heart failure, arrhythmias, endothelial dysfunction, and cardiomyopathy can develop from cardiovascular toxicity. Anthracycline and trastuzumab therapies are linked to myocardial damage (left ventricular) and cardiac dysfunction (Agrawal, 2014). An additional chemotherapy effect is an ovarian dysfunction in instances where cyclophosphamide was prescribed (Agrawal, 2014). Reduced levels of circulating antimullerian hormone, estradiol, and inhibin B are indicators of declining ovarian function. Surgical procedures normally have to deal with seroma as a side effect. Paclitaxel (figure 3) produces such side effects as hair loss, diarrhea, and aching joints and muscles. Common side effects of artemisinin (figure 4) include tremors, nausea, vomiting, and skin rash.

Figure 3

Figure 4

Long-term side effects start at the time of treatment and prevail even after treatment has ceased. Side effects that manifest late could appear after the end of treatment, which could vary from weeks to years (Johns Hopkins Medicine, 2020). The combination of the last and long term side effects is bone loss, dental challenges, heart problems, cataracts, pain and numbness, blood clots, Fatigue, new cancers, and blood clots, lymphedema, infertility, and memory loss (Johns Hopkins Medicine, 2020). Additional symptoms include sores of the mouth, loss of hair, nausea, diarrhea, the onset of menopause before age 45 in women, and a tendency to put on weight (Felson, 2020).

Flavonoids

Introduction of Flavonoids and Analogs

Plant derivatives have, for centuries, been to bear a broad spectrum of beneficial biological mechanisms. Flavonoids are a part of this category. Flavonoids have been credited with helping plant systems fight oxidative stress (Kumar and Pandey, 2013). Besides, they serve as growth regulators. They also shield plants from damaging solar radiation, modulate metabolism in plants, offer defense against herbivorous feeders and pathogens, and offer pollinators’ visual appeal (Salehi et al., 2019). They are a type of anti-oxidative substance with phenolic structures that vary within the group. They can be found in bark, fruits, flowers, roots, stems, vegetables, flowers, wine, and tea (Panche et al., 2016). People directly derive flavonoids by dieting on such predominantly plant sources like fruits, vegetables, tea, and wine. Alongside carotenoids, flavonoids give fruits and vegetables their distinguishing colors. Rutin was the first flavonoid isolated from fruits (oranges) in 1930; although it had earlier been thought of as a vitamin, it is then labeled as vitamin P (Kumar and Pandey, 2013). To date, research has seen over 4000 diverse flavonoids identified.

Microbial biotechnology has facilitated the harnessing of and mass production of their beneficial ingredients for pharmaceutical gains. Flavonoids and their components are identified for the health benefits they confer to humans. Cosmetic, pharmaceutical, medicinal, and nutraceutical industries are currently exploiting the benefits of flavonoid ingredients. Their known health benefits include hepatoprotective, anti-carcinogenic, free radical scavenging, anti-mutagenic, anti-inflammatory, anti-cancer, anti-viral, anti-carcinogenic, and anti-oxidative properties (Kumar and Pandey, 2013; Panche et al., 2016). They can also mediate enzymes’ primary cellular functions, prevent coronary heart diseases, and lower mortality rates related to cardiovascular disease. They comprise the most significant class of phytonutrients, with famous examples being kaempferol and quercetin.

The replacement of flavonoid functional groups around the structure of their nucleus and the sum count of hydroxyl groups determine their biological function and bioavailability. They are situated in the genetic material of mesophyll cells and within the centers responsible for generating reactive oxygen species (Kumar and Pandey, 2013). The health properties of flavonoids in humans have attracted much study and research.

The Classification & Structure of Flavonoids

Flavonoids comprise an extensive group of polyphenols that bear a benzo-γ-pyrone structure. The chemical structure of flavonoids is built around a 15 –carbon skeleton made of double benzene rings joined by a pyrene ring. Figure 5 shows the carbon skeleton around which flavonoids are based (Kumar and Pandey, 2013). A and B are the benzene rings in the model, while C is the linking pyrene ring.

Figure 5

Some of the classes of flavonoids include flavonols, flavones, and flavanones. Examples of flavonols, flavones, and flavanones are kaempferol, luteolin, and hesperetin (Kumar and Pandey, 2013). Species within a class are distinguished by how differently they substitute the rings A and B. Inter-class distinguishing factor is in how differently flavonoids substitute the C ring and the oxidation level (Kumar and Pandey, 2013). For purposes of this study, we shall focus on fisetin, apigenin, luteolin, and myricetin as diagrammatically represented in Figure 6.

Figure 6

Flavonoids are classified into two classes depending on the position at which the benzene ring was substituted. Substitution at 2-position and 3-position gives rise to the classes flavonoids and isoflavonoids, respectively (Kumar and Pandey, 2013). Table 4 demonstrates the role of molinspiration in determining molecular properties of apigenin, myricetin, fisetin, and luteolin.

Table 4

Using Molinspiration’s methodology, LogP was determined as a total of correction factors and contributions from fragments. LogP is the octanol/water partition coefficient. The logP values are positive. This means that the molecules are lipophilic that is their concentration in the lipid phase. Topological Polar Surface Area (PSA) is the total described as the surface total divided by the polar molecules or atoms or molecules. These atoms or molecules are majorly nitrogen and oxygen. The TPSA values of the molecules are below 140. this means that they are good at permeating plasma membranes. Molecular volume obtains such transport features of molecules as ability to penetrate blood-brain interface and intestinal absorption. The molecules bear the feasible transport characteristics. Molecular weight determines the sum the values of the atoms’s atomic weight in a molecule. In Chemistry, MW is used to measure stoichiometry in equations and chemical reactions. MW is expressed in Daltons or terms of atomic mass units. Using Molinspiration group contributions, MW is obtained when the sum of fragment contributions are fitted to actual 3D volume for a approximately 12,000 drug-like molecules. Hydrogen bond acceptors (nON) value indicate that the molecules’ electronegative atoms whose single lone pair take part in the hydrogen bond. Number of Rotatable Bonds (nrotb) determines molecular flexibility. It substantiates a candidate drug’s oral bioavailability. A non-ring bond tied to non-hydrogen atom is the very definition of rotatable bond.

Biological activities of flavonoids

The increasing research interest on flavonoids is attributed to their perceived and suggested bioactive potential traits. The bioactivity, metabolism and bioavailability of these phenolic compounds are determined by their configuration, level of polymerization, the total number of OH groups and the functional groups substituted about the core structure (Kumar and Pandey, 2018). Flavonoids’ bioactive potential in humans is demonstrated in their anti-cancer activity, ability to scavenge free radicals, their antioxidative characteristics, hepatopreotective capacity, prevention of coronary cardiac disease, anti-inflammatory and antiviral properties. Flavonoids’ antioxidant properties are mediated by their functional OH groups through which they chelate toxic metal ions and forage free radicals. Generation of free radicals non-chelated toxic metal ions normally damages free candidate biomolecules (Kumar and Pandey, 2018). Atherosclerosis is thought to result from oxidative adjustment of low density lipoprotein cholesterol. By foraging free radicals, a polyphenol isoflavan glabridin prevents oxidation of LDL (Kumar and Pandey, 2018). Extracts of the flavonoid Astragalus complanatus have demonstrated anti-hypertensive features.

The liberation of flavonoids from chewed food for absorption depends on their solubility, pka, lipophilicity, molecular size and configuration. For example, solubility majorly affects the therapeutic effect of flavonoids. Low flavonoid solubility in water presents an absorption challenge. Water soluble semi-synthetic forms of flavonoids are being developed for medicinal efficacy. A semi-synthetic example is hydroxyethylrutosides which is being studied as a micro-bleeding and hypertension remedy.

In plants, flavonoids are found in the mesophyll cells nuclei and reactive oxygen species generation centers. Plants benefit from their anti-oxidative stress properties and their growth-regulatory contribution (Kumar and Pandey, 2018). Flavonoids growth regulatory role lies in their regulation of auxins in plants. Flavonoids contribute to the stability and quality of foods through their acting as antioxidants, colorants and flavorants.

Flavonoids as anti-cancer agents

Anti-cancers efforts have linked consumption of flavonoid-rich fruits and vegetables to chemopreventive capacity. An inverse proportionality on breast, lung, prostate and stomach cancer is observed in increased dietary intake quercetin rich apples and onions (Pandey and Kumar, 2018). Quercetin has been tested for its ability to hinder the carcinogenetic activities of tyrosine kinase. The most prevalent genetically engineered abnormalities of cell cancers are caused by mutations of p53 proteins. Should p53 be inhibited, cancer cells will be arrested in the G2-M mitotic phase (Kumar and Pandey, 2018).

The proposed molecular mechanisms that sabotage carcinogenesis include hindering the cell cycle, inhibiting tyrosine kinase, suppressing mutants of the protein p53, hindering heat shock proteins, binding to ER-1 and preventing the expression of such Ras proteins as K-Ras4A and H-Ras. In breast cancer, flavonoids can diminish p53 expression to very negligible un-discernible levels. . Flavonoids that prevent the release of heat shock proteins in breast and colon cancers have been tested. Quercetin is effective in halting the progression of cancerous cell cycle in metastatic stomach ovarian, breast and lymphoid cancer cells.

Tyrosine kinases promote oncogensis by by-passing normal cell growth regulatory checks. These enzymes normally transport growth factor signals across the cell membrane to the nucleus. Anti-tumor drugs can hinder the activity of tyrosine kinase are being proposed due to ther minimal chemo-toxic side effects. Epigallocatechin gallate is a flavanol that inhibits the activity of protate cancer causative activities like lipogenesis and fatty acid synthase (kumar and Pandey, 2018). This flavonoid causes the arrest of cell cycle and cancer cell apoptosis. The anticancer activity of flavones is associated with the carbonyl function of the flavonoid nucleus. Increased dietary intake of flavonoids rich in phytoestrogens, has been linked with minimized risks for prostate cancer.

Introduction of Fisetin

Tetrahydroxyflavone, otherwise called fisetin, is a bioactive molecule of the flavonol group. Apples, cucumbers, grapes, onions, persimmon, and strawberry are fisetin rich dietary sources (Khan et al., 2013). Figure 8 shows the chemical structure of fisetin (Khan, 2013). Figure 7 shows the structural representation of fisetin. It has a chemical formula C₁₅H₁₀O_6,, a melting point and a boiling point of 330 °C and 599.4°C respectively. fisetin has a density of 233 g/mL. It is insoluble in water but soluble in ethanol and indofin. It has a LogP value of 1.915.

Figure 7

Its abundance in plant dietary sources and its antioxidant, apoptotic, and antiproliferative properties has inspired further research on its potential. Fisetin is in chemotherapeutic and chemopreventive molecules against multiple cancer (Khan et al., 2013). Its neuroprotective function is also documented. Research continues to give promise of fisetin’s antioxidant capabilities. Their ability to hunt for free radicals in the human body distinguishes their particular antioxidant properties and various biological impacts. Amino acids, lipids, and nucleic acids are normally victims of free radicals’ exposure. The oxidative stress that results from an imbalance between free radicals and antioxidants causes a mismatch between the body’s release of free radicals and the capacity to protect the body against them (Khan et al., 2013). Carcinogenesis, aging, and mutagenesis have been linked to this imbalance.

Fisetin has been demonstrated as a planar molecule that imparts a cross-conjugation influence using Trolox equivalent activity concentration. The dissociation energy of fisetin’s hydroxyl bond (O.H.) and dipole moment confirmed the molecule’s high antioxidant property (Khan et al., 2013). Studies indicate that fisetin adherently binds between phospholipid hydrophobic tail and polar head. The region of this type of fisetin binding is usually open to reactive oxygen species. The region is the interfacial region of liposomes from egg phosphatidyl-choline (Khan et al., 2013). This area is usually the site of reaction for fisetin’s antioxidant function inhibiting lipids’ oxidative degradation. Free radicals have a tendency to abstract electrons from cell membrane lipids. Continued lipid extraction can seriously damage to cell membranes. The antioxidant properties come in handy in preserving the integrity of cell membranes against free radicals. The antic-cancer properties of fisetin demonstrated in cervical cancer show that the flavonoid bars the infiltration and metastasis of cervical metastasis cells. Figure 8 shows the interaction between fisetin and the NFκB pathway that contributes to cancer development and metastasis (Chou et al., 2013). The flavonid also suppressed urokinase plasminogen activator from being expressed and from functioning.

Figure 8

Introduction of Apigenin

4′,5,7-trihydroxyflavone, also referred to as apigenin, is among the most abundant flavonoids in plants. It subscribes to the sub-class flavone. Matricaria, Asteraceae, Achillea, Artemisia, and Tanacetum are the primary sources of apigenin. The genera Genista, Sideritis, Lamiaceae, and Teucrium, is found in its genin form. Like derivatives of O-methyl ethers, glucuronides, and O-glucosides, Apigenin’s derivatives are found in the latter group of genera. Biogenetically, apigenin can be derived from tyrosine and phenylalanine as a phenylpropanoid channel (Salehi et al., 2019).

Its antioxidant, anti-apoptotic, anti-inflammatory, and anti-hyperglycemic properties have been studied and reported as a flavonoid. Apigenin’s biological effect on numerous cancer cells has been described as cytotoxic and cytostatic (Salehi et al., 2019). Apigenin’s protective and anti-atherogenic effects have been tested to manage abnormal cardiac muscles’ abnormal enlargement, high blood pressure, and cardiac myocarditis management. Such efficient means of apigenin extraction like dynamic maceration and deep eutectic solvents have helped large scale harvesting from its plant sources (Salehi et al., 2020). The molecular structure of apigenin and its derivative forms is demonstrated in Figure 10 (Salehi et al., 2019). Figure 6 demonstrates the ligand-receptor molecular docking between apigenin and progesterone (Dean et al., 2018). Figure 10 shows the structural representation of apigenin and its analogs. Apigenin (chemical formula C₁₅H₁₀O₅₎ is a yellow solid lipid molecule with a melting point of 347.5°C and a boiling point of 555.5 °C. It is hydrophobic and partially neutral. It has an enthalpy of vaporization value of 86.8 kJ/mol. Its logP value is 1.138.

Studies continue to abound in line with the molecule’s multiple chemical properties.

Figure 10

Apigenin facilitates the expression of superoxide dismutase, catalase, and glutathione synthase. The enzymes superoxide dismutase and catalase defend cells against attack from free radicals. These enzymes also mediate cellular functions against xenobiotic and oxidative stress. Glutathione synthase attaches GSH to proteins as a protective step before they are shipped out of the synthesizing cell. Apigenin has also been observed to offset the prevention of the formation of tumor nourishing blood vessels. It also prevents the spread of cancerous cells to other body parts. Apigenin has been noted to regulate the functions of interleukin 6 in macrophages. IL-6 is a cytokine that has been linked to the unfolding of inflammatory body anomalies.

Introduction of Luteolin

Luteolin, a flavonoid, has apoptosis-inducing, anti-inflammatory, antioxidant and chemopreventive properties. The molecule has a chemical formula C₁₅H₁₀O₆. It has a density and molecular mass of 239.5 g/mL and 286.24 g/mol. It has a melting and a boiling point of 330 °C and 616.1 °C respectively. It has a logP value of 1.486. It has an enthalpy of vaporization at 94.7 kJ/mol. It is partially water soluble and fully soluble in ethanol and methanol. The molecule hunts for free radicals, upon being administered, defends body cells against damage by reactive oxygen species and prompts apoptosis and momentary cell cycle seizure in malignant cells (National Center for Biotechnology Information, 2020). This action prevents cell proliferation of tumor cells and subdues metastasis. Luteolin, also referred to as flacitran and digitoflavone, subscribes to the flavonoids class of flavones. Luteolin is found in solid-state and is regarded as water-insoluble and with a neutral pH. Luteolin within the body cells is majorly localized in the cell membrane (National Center for Biotechnology Information, 2020). It is bitter and is abundant in coconut, mushroom, and oyster (National Center for Biotechnology Information, 2020). Fruits and vegetables like onion leaves, broccoli, parsley, peppers, celery, cabbage, carrots, and apple skins are luteolin rich. Luteolin is also abundant in chrysanthemum flowers (Lin et al., 2009). Figure 11 shows luteolin’s molecular structure (Lin et al., 2009), while figure 12 shows luteolin and quercetin’s binding with ribosomal protein s19 to prevent metastasis (Chen et al., 2018). The molecule’s structure has four O.H. groups found at C 3′, 4′ 5, and 7. It is studied for its role in the human body. It is thought to facilitate antioxidant, anti-inflammatory, and ant-carcinogenesis properties.

Figure 11

Figure 12

Introduction of Myricetin

The molecular structure of myricetin is similar to several studied phenols like kaempferol, morin, quercetin, and fisetin. The molecule’s chemical formula is C₁₅H₁₀O_8.It is a greenish powder with a density and molar mass of 1.912 g/mL and 318.237 g·mol⁻¹respectively. It has a melting point of 357 °C. It is hydrophobic but soluble in ethanol. Myricetin boils at 747.6 °C. It has a log P value of 2.182 and enthalpy of vaporization of 114.4 kJ/mol.

The structural similarity of myricetin to quercetin gives the former molecule its other identity, hydroxyquercetin (Semwal, 2016). It has been observed to have antioxidant and nutraceuticals properties. Studies confirm that myricetin shows many pharmacological properties such as antitumor, analgesic, anti-inflammatory, antidiabetic and hepatoprotective properties (Semwal, 2016). Myricetin is abundant in plants. Plants of the families Anacardiaceae, Pinaceae, Polygonaceae, Myricaceae, and Primulaceae are exploited for flavonoids with pharmacological potential. This type of flavonoid was first studied towards the end of 18th C Myrica nagi. This molecule is a phenol abundant in vegetables, berries, wines, and teas, all plant derivatives.

It exists both in glycosidically-bound and free forms. Some of the glycosidically-bound analogs in which it exists are myricetin-3-O-β-D-xylopyranoside and myricetin-3-O-α-L-arabinopyranoside. Figure 13 shows the molecular structure of myricetin and its derivatives. Myricetin dissolves poorly in water. When deprotonated,it dissolves in acetone, tetrahydrofuran, dimethylacetamide, and dimethylformamide (Semwal et al., 2016). However, it rapidly dissolves when deprotonated in organic solvents and alkaline aqueous media. The molecule is steady at acidic pH 2. Its dissolution is temperature and pH reliant.

Figure 13

Myricetin is among the many components of numerous human beverages and foods derived from vegetables and fruits. It is primarily recognized for the anti-cancer, antioxidant, iron-chelating anti-inflammatory properties it displays. Numerous studies have probed and confirmed these properties against multiple helicases, kinases, reverse transcriptase, DNA and RNA polymerases, and telomerase (Semwal, 2016). Studies have explored and documented their antioxidant property. It scavenges for several free radicals and ions. The cytotoxic antitumor properties of myricetin have been observed in research on managing cancers of the pancreas, skin, liver, and colon (Semwal et al., 2016).

Additionally, in hinders primary enzymes that participate in cancer onset and malignancy in these cell lines. Myricetin was observed to impart its cytotoxic properties against a malignant brain tumor, medulloblastoma, commonly found among children. It inhibited cell signaling in medulloblastoma cells and hindered the creation of actin-rich membrane surfaces (Semwal et al., 2016).

In human lung carcinoma, myricetin caused metastasis, adhesion, and invasion of malignant and benign cells. It has been shown to produce zero cytotoxicity towards normal cells (Semwal et al., 2016). It demonstrated in vitro cytotoxicity against pancreatic cancer cells. In the metastatic and primary cells, it induced the death of the cancerous cells through apoptosis. Simultaneously, it lowered the activity of the enzyme PI3 kinase.

CHAPTER 3

METHODS

Research Aims

The following aims will help understand Molinspiration Cheminformatics and Molecular Docking Studies of Some Natural Flavonoids: Fisetin and Its Analogs as an Anti-Breast Cancer Agents. The study’s objectives and their respective hypotheses are:

Determine molecular properties and predict bioactivity scores of fisetin, apigenin, luteolin and myricetin using molinsipiration chemoinformatics. This aim attempts to validate and build upon past studies that have investigated the chemotherapaeutic value of flavonoids in the hope of proposing their drugability in cancer management. The oral activity potential of the flavonoids shall be determined from a Lipinski’s rule of five perspective. We hypothesize that the flavonoids will demonstrate relative anti-cancer properties.
Molecular docking analysis of fisetin, apigenin, luteolin and myricetin against a class of malignancy participating receptors. Scientists have for a while associated flavonoids found in vegetables and fruits with anti-cancer characteristics. Reports suggest that people who consume more vegetables and fruits record the least risk of developing cancer. The flavonoids shall be the ligands in our study.

Introduction of Lipinski’s Rules

Christopher Lipinski and the team have been credited with the rule of 5 proposed in 1997. The rule was a culmination of the studies they conducted on the physicochemical properties of certified drugs already in circulation and pre-certified clinical samples then (Doak, 2014). Pre-certified clinical compounds analyzed had successfully passed the first phase into the second phase of clinical trials (Pollastri, 2010). Lipinski tallied the molecules’ physicochemical features to their oral bioavailability, aqueous solubility, and how absorbent they were. The rule was designed to help drug discovery medicinal chemists promptly assess the molecules (Pollastri, 2010). The rule became a guiding predictor of whether a pharmaceutical molecule would become orally active or not. The oral route of drug administration is desirable where a drug’s systemic exposure is needed. Some of the other advantages of the oral route are heightened dependence of exposure and capacity to avail of a bigger dosage (Doak, 2014). The solid forms of oral drugs can be stored with greater stability than suspensions and liquid drugs. This study focused on molecules with masses under 500 Daltons, a water distribution coefficient below 5, less than 5 or equivalent of 5 H-bond donors, and less than or equal to 10 H-bond acceptors (Mullard, 2018). Red flagged molecules whose parameters were out of range by more than one were also examined. The rule predicted that exceeding these limits would translate to dismal permeation or absorption for the drug (Benet et al., 2017). This rule would only apply as long as the compounds did not serve as nourishment for active transporters. However, well beyond 90% of molecular compounds compiled in the Available Chemicals Directory were compliant to the rule of 5 (Benet et al., 2017).

Michael Shultz of Norvatis argued later that this rule of thumb for drug assessment had passed its time (Mullard, 2018). In his analysis of certain post-1997 FDA approved molecules, the physicochemical composition of a number of them indicated that their molecular mass had traversed the Lipinski rule barrier. His analysis into 2016 and 2017 approved samples confirmed that the FDA had approved pharmaceutical molecules of an average molecular mass beyond the 500 Dalton Lipinski advisory (Mullard, 2018). A decade long pharmaceuticals production and approval window have seen orally administered molecules of as much as 600 Daltons approved. Schlutz reports that the H-bond acceptors had begun surpassing the Lipinski advisory limit. The hydrogen bond threshold acceptors increase substantially. Shultz suggested that drug developers reconsider their application of Lipinski’s rule in defining oral drugs. With such new modalities as constrained peptides and targeted protein degraders being brought on board, caution is advised against their use in assessing drug-likeness (Mullard, 2018). Lipinski’s rule stands to be as relevant today in classifying therapeutically significant pharmacokinetic molecules as it was when first proposed. However, the rule fails to separate non-drugs from drugs (Benet et al., 2017). Lipinski’s rules have guided drug discoveries into focusing on molecules with a promising chance of being highly absorbed when administered orally. However, the rule has been seen to correlate with current FDA approved drugs administered by non-oral means (Choy and Praustniz, 2011). Table 2 below shows five modified rules for delivery routes (Choy and Praustniz, 2011).

Table 2

Administration route	M.W. (Da)	# of H donors	# of H acceptors	log P
Ophthalmic	500 ^a	3	8	4.2
Inhalation	500 ^a	4	10 ^a	3.4
Transdermal	335	2	5	5.0 ^a

However, strict dependence on the rules is thought to have denied drug discovery the opportunity to dwell on the difficult but high potential targets of molecular weight above 500 Daltons (Doak et al., 2014). Studies have suggested that drug bioavailability could be enhanced by reconsidering the strict reliance on the rule of 5. Some orally administered drugs exist outside the scope of Ro5, and such properties as formulations, dosage, and macrocyclization can be complementary to Ro5 (Doak, 2014). Examples include natural compounds discerned from peptidic clues that defy the rule but are fundamental reservoirs for oral drugs (Doak, 2014). This class of natural compounds comprises the difficult targets group that is still pharmaceutically indispensable.

Molinspiration Cheminformatics

The values of drug scores signify a compound’s general potential to function as a drug candidate. As web-based software, Mol inspiration can be used to forecast apigenin’s, mricetin’s , luteolin’s and fistein’s bioactivity values. The score shall be juxtaposed against human breast receptors ER -α and ER-β. For instance, previous studies have investigated the role of sigma receptors in proliferation of lung cancer.

Molinspiration Chemoinformatics Analysis: Analyses of the molecular properties, bioactivity and structure the sample flavonoids were conducted. The driving objective was to determine the flavonoids’ molecular lipophilicity potential, polar surface area, hydrogen bonding and molecular weight in line with “Lipinski Rule of Five.

Molecular modeling- Optimization of receptor structure: The 3-D structure of alpha ligand-binding domain, human estrogen receptor, combined with diethylstilbestrol was downloaded as a PDB file from the RCSB Protein Data Bank (PDB ID: 3ERD). A UCSF Chimera version 1.13.1 was used to open the estrogen receptor’s PDB file accompanied by the extraction of non-standard atoms and bonds. Upon opening the protein in AutoDock vina, the co-ordinates of the binding pocket were obtained using the grid box functionality. The size of the grid box was at 25.5446 × 27.2512 × 23.3912 points with the grid box center setting at 18.9547 × 15.8184 × 109.135. The dock prep functionality was used to eliminate water molecules and allot partial charges. The functionality aided with the addition of hydrogen to the estrogen receptor. The protein was then saved in the Mol 2 format primed for the docking stage.

Molecular Modeling – Building the ligand structure: The flavonoid derivatives (fisetin, myricetin, luteolin and apigenin) were drawn, optimized, and saved in a mol2 format with the aid of ACD Lab Chemsketch. The canonical SMILES of the flavonoid molecules were entered into UCSF Chimera using the build structure tool. Separate addition of hydrogen through the “structure editing function” to the flavonoid molecules readied them and moved the system towards energy minimization using the “minimize structure tool”. The ligand (fisetin, myricetin, apigenin and luteolin molecules) were named and saved in the Mol2 format ready for the docking step.

iv) Molecular Docking: On a new UCSF Chimera session, firstly, the estrogen recpetor Mol2 file was opened followed by the ligand (flavonoid molecules). Using the surface/binding analysis tool, the AutoDock vina function was done. The estrogen receptor PDB format was selected as the receptor and the target flavonoid molecule SMILES was selected as the ligand. An output file was created and the binding pocket co-ordinates were entered. All ligand and receptor options were set as true and the exhaustiveness of search was set at the maximum of 8. Furthermore, the binding modes number was selected as 9. Vina.exe via a local path was selected as the executable location and docking was done.
v) Docking out-put analysis: visualization of the docking poses and analysis using PyMol is underway.

Molinspiration Chemoinformatics is a software tool that enhances the manipulation of molecules and their processing. Additionally, the software can generate tautomers, fragment molecules, compute molecules’ properties, design of drugs, modeling of molecules, determining similarities, and normalizing molecules (Molinspiration Cheminformatics, 2020). In attempting to determine flavonoid pharmacokinetic properties, the software can calculate rotatable bonds number, polar surface area, drug score and likeness, solubility, lipophilicity and, hydrogen bond acceptor and donor numbers (Molinspiration Cheminformatics, 2020). The software will virtually screen flavonoid molecule fragments, predict bioactivity, and facilitate data visualization. Given that they are decoded in Java, the tools can practically be used across all computer platforms provided Java run times are available. For example, molecules encoded as SDfile or SMILES are easily visualized using 2D representation. It can display related data, molecule selection, and search for internal substructure. Mib engine conducts processing of molecules, calculates vital molecular features, converts SDfiles and SMILES, depicts SMILES, and generates molecular images. The misearch engine conducts searches on pharmacophore-based similarity and supporting an adaptable molecular database. Through the miscreen engine, molecular libraries of large sizes can be screened (figure 11). It also facilitates pharmacophore models’ production. Galaxy generates 3-dimensional structures.

Drug score values indicate overall potential of a compound to be a drug candidate. Mol inspiration is a web-based tool used to predict the bioactivity score of the synthesized compounds against regular human receptors. A virtual screening process in molinspiration the disintegrates a molecule to be screeneed into substructure fragments. The molecule’s bioactivity score is then obtained when the Bayesian statistical model is employed to compute the bioactivity contribution of each fragment (Molinspiration, 2020). A total of the bioactivity contribution of all fragments gives us the bioactivity score of the flavonoid molecule. Molinspiration virtual screening can help obtain bioactivity scores of up to 100, 000 molecules as fast as in an hour’s time.

Explanation of Molecular Properties

The four properties of molecular compounds are boiling points, melting points, vaporization, and Low enthalpies of fusion, thermal and electrical conductivity, and brittle or soft solid (Clark, 2000). The intermolecular forces of attraction between molecules are comparatively feeble and are why molecular compounds are usually low melting point solids, liquids, and gases. There are no covalent bonds that need breaking while melting or boiling molecular compounds. The value of the M.P. or B.P. is determined by how strong intermolecular forces are and if any hydrogen bonds are present (Clark, 2000). Larger molecules have more numerous van der Waals forces, which could also require extra energy to break.

Water solubility. The majority of molecular compounds are either water-insoluble or sparingly water-soluble. The water-soluble molecular substances either react with the water or formulating hydrogen bonds (Clark, 2000). For example, methane is water-insoluble because, as a gas, its molecules are farther apart. The hydrogen bonds between water molecules would demand an energy supply to find its way between the water molecules. The possible weak van der Waals attraction between water and gases is energy-wise non-profitable. Molecular compounds usually dissolve in organic solvents (Clark, 2000). These solvents are equally molecular. The dissolving compound and the solvent will probably have molecular attraction through van der Waals’ forces. The van der Waals attractions usually are disrupted upon dissolving, but similar interests are replaced in their place.

The absence of ions and delocalized imply that molecular compounds are poor electrical conductors. Even if the electrons were to be delocalized, within a specific molecule, the lack of adequate inter-molecular contact would limit electrons’ movement in the entire solid or liquid (Clark, 2000).

Molecular Docking Studies

Molecular docking studies entail examining the interaction of two or more molecular compounds with reasonable accuracy (Ferreira, 2015). Examples could involve enzyme and drug and enzyme interaction. In simpler terms, a prediction of how micro molecules like ligands link with proteins and enzymes. It is applied in lead optimization, hit identification, predicting biological activity, identifying binding sites, receptor de-orphaning, the study of functions of structures, and protein-protein binding (Moman, 2000). Molecular docking approaches employ molecular structures to understand molecular recognition in a bid to hypothesize how a complex resulting from two or more component molecules would be bonded. Through molecular docking, molecules are placed in configurations that will facilitate their interaction with receptors. This fitting process occurs naturally in cells and takes seconds to materialize. Over time, biochemists have formulated several models that can explain the significant constituents of molecular recognition. Table 3 summarizes the molecular docking models.

Table 6

Year	Model	Author(s)
1890	Lock and key	Emil Fischer
1958	Induced fit	Daniel Koshland
2003	Conformation ensemble	Buyong Ma et al

As proposed by Emil Fischer, the lock and key model (Figure 14) implied that biological systems worked so that substrates inserted into active macromolecule sites were very like key and a lock fitting.

Figure 14

Daniel Koshland’s induced fit model implied that both target and ligand mutually adjusted to adapt and conform to each other until a suitable fit was realized during molecular recognition. The conformation ensemble model complimented Koshland’s model by proposing that besides the miniature induced-fit adjustment, the plasticity of proteins enabled them to alternate between states. The conformational flexibility of proteins is the informing principle behind this latter model. Proteins are considered a pre-available assemblage of conformational forms. The plasticity of the protein allows it to switch from one state to another.

Experimental means of examining molecular docking have enabled biochemists to obtain 3-dimensional images of biomolecules. Examples include electron microscopy, immune-precipitation, site-directed mutagenesis, X-ray crystallography, as well as nuclear magnetic resonance. Drug discoveries employ molecular docking to comprehend drug-receptor relationships (Ferreira et al., 2015). The protein-ligand relationship is exploited by medicinal chemists to predict binding between the target protein and the drug candidates. This way, the activity and affinity of the molecule in the design are expected.

Steps of Molecular Docking

Molecular docking is a two-step process that involves: forecasting the conformation, orientation, and position of ligands and determining molecules’ binding affinity. For step one to be actualized efficiently, the binding site must be identified. In most instances, the binding site is located in prior. The six degrees of rotational and translational freedom offer many potential modes of ligand-protein binding (Meng et al., 2012). The ligand-protein conformational degrees of freedom offer an equivalent advantage. Molecular shape and chemical composition dependent matching algorithms map a drug into a target protein’s active site. Matching algorithms are fast enough and can enrich active molecules from vast libraries. DOCK applies chemical and geometrical matching algorithms to conduct molecule-receptor site docking (Meng et al., 2012). Figure 15 demonstrates docking in fisetin with the enzymes acetylcholinesterase, beta-secretase 1, and Aβ-binding alcohol dehydrogenase (Dash et al., 2014). Through its procedure of clique detection, the receptor and ligand are identified as sets of spheres.

Figure 15

Figure 16 shows a 3-D representation of the progression of molecular bonding involving a ligand (A) and a receptor (B) (Ferreira et al., 2015). The possible conformations are explored in C, and in D, the possible intermolecular conformations are seen.

Figure 16

The most probable binding conformations are identified in a two-step process: conformations: (i) a conformational space that is a representative of possible fitting modes is identified (ii) interaction needed for the projected binding conformations is accurately determined (Ferreira et al., 2015). Programs used in molecular docking conduct these duties in a cyclical procedure where particular scoring functions are used to evaluate ligand conformations. The first of the two-step mentioned above process incrementally alter the structural properties of the ligands. This entails adjustments to the rotational, translational, and torsional degrees of freedom in the ligand (Ferreira et al., 2015). Conformational search algorithms use stochastic and systematic methods to execute this step. Figure 17 below demonstrates this kind of search method (Ferreira et al., 2015).

Figure 17

In (A), the two dihedrals are used to define the molecule’s conformation. (B) is the one-dimensional energy landscape for Φ2. (C) The Stochastic search’s energy landscape is surveying the conformational space.

In molecular docking, programs apply scoring functions in predicting energetics of binding the likely ligand-receptor formations (Ferreira et al., 2015). The formation of the complex produces a variation in energy issued by the Gibbs free energy and the binding constant. Desolvation, intermolecular fittings, and entropic effects are used to estimate the binding energy. The scoring function’s accuracy is enhanced by the use of a larger number of physicochemical properties. However, this comes at the cost of increasing and prohibitive computational charges. This shortcoming minimizes the efficiency of the docking algorithm (Ferreira et al., 2015). However, large ligand samples will call for a balance between speed and accuracy.

Covalent drugs are pharmaceutical resurgence. They have been tested for diabetes, cancer, neurologic, cardiovascular, and infectious diseases. Covalent ligands permanently inactivate their protein targets. To recover the biological function that had been inhibited, the target protein has to be re-synthesized.

Studies Protein Structure from PDB

Proteins are molecular compounds that act as the building blocks of all cells. They maintain life, facilitate replication, defend cells, and facilitate reproduction. Twenty natural amino acids constitute the proteins or polypeptide chains when grouped in ways that are differently influenced by the genetic code. According to the Gene Ontology Project, the 12 molecular functions of proteins include; nucleic acid modification and replication, signaling within cells, metabolism, cellular processes, and intercellular communication. Protein structures normally have tens of thousands of atoms. Protein structures are classified from primary to secondary, tertiary to quaternary.

The primary protein structure is the series of amino acids for every chain of a polypeptide that makes up the protein. The twenty natural amino acids make up the polypeptide chains. However, they are arranged differently depending on the encoding genes. In extraordinary circumstances, stop codons have been seen to incorporate pyrrolysine and selenocysteine (Breda et al., 2007). Figure 18 shows the α-amino acids (a) and the alternating L-and D- amino acid structures (b). Amino acids are made up of a carboxylic acid and amino groups, and H joined the α carbon. It is the side chain (R group) that fluctuates from one amino acid to another. The R group differs in terms of polarity, size, polarity, charge, hydrophobicity, volume, and shape (Breda et al., 2007).

Figure 18

The α-carbon is a typical tetrahedral. The carbon is joined to four chemical groups that are all different. The C is a chiral head (asymmetric) with two distinct D and L enantiomers.

An exception to the rule in natural amino acids is glycine. The amino acid has hydrogen for an R-group. Subsequently, its carbon is symmetric (Breda et al., 2007).

Secondary protein structure is the regular recurring composition, usually in the space of close to residues of amino acids in a polypeptide chain. The H-bonds maintain the structure between the peptide’s carbonyl oxygen molecules and amide hydrogens. The major secondary structures are the β-structures, and α-helices are the primary, secondary structures. Figure 19 demonstrates the protein’s secondary structure (Breda et al., 2007). The β-sheets architecture scarcely forms parallel coils. Its common forms are anti-parallel. The rest of the forms it takes are hyperbolic paraboloids, coiled and parallel barrels of 8-strands. (Breda et al., 2007).

Figure 19

Results

1) Molinspiration Cheminformatics:

Results of molecular properties

Table 3 demonstrates molecular properties of apigenin, myricetin, fisetin and luteolin obtained using molinspiration.

Table 4

Using Molinspiration’s methodology, LogP is the calculation of octanol-water partition co-efficient. Through logP, molecular hydrophobicity can be measured. It was determined as a total of correction factors and contributions from flavonoid fragments. LogP as the octanol/water partition coefficient helps determine if a drug can be absorbed, become bioavailable, toxicity and its interaction with receptors (Molinspiration, 2020). The logP values obtained are positive ;fisetin 1.97, apigenin 2.24, myricetin 2.27 and luteolin 2.27. This means that the flavonoid molecules are lipophilic that is their concentration in the lipid phase.

Topological Polar Surface Area (PSA) is the total described as the surface total divided by the polar molecules or atoms or molecules. These atoms or molecules are majorly nitrogen and oxygen. The PSA is the area surface related to polar H atoms and hetero-atoms phosphorous, nitrogen and atoms. The TPSA values are fisetin 1.112, apigenin 90.89, myricetin 90.89 and luteolin 70.67. The TPSA values of the flavonoid molecules are below 140.

Molecular volume obtains such transport features of molecules as ability to penetrate blood-brain interface and intestinal absorption. The molecules bear the feasible transport characteristics. The flavonoid molecules’ transport characteristics like penetration of the blood-brain interface and intestinal absorption can be predicted using molecular volume values. The molecular volume values are: fisetin 237.07, apigenin 224.05, myricetin 224.05 and luteolin 254.24.

Molecular weight determines the sum the values of the atoms’s atomic weight in a molecule. In Chemistry, MW is used to measure stoichiometry in equations and chemical reactions. MW is expressed in Daltons or terms of atomic mass units. Using Molinspiration group contributions, MW is obtained when the sum of flavonoid fragment contributions was fitted to actual 3D volume for a approximately 12,000 drug-like molecules. The MW values range between 286.24 and 254.24.

Hydrogen bond acceptors (nON) value indicate that the molecules’ electronegative atoms whose single lone pair take part in the hydrogen bond. Number of Rotatable Bonds (nrotb) determines molecular flexibility. It substantiates a candidate drug’s oral bioavailability. A non-ring bond tied to non-hydrogen atom is the very definition of rotatable bond. The nrotb values are 1 for each of the flavonoids.

The nON (number of H-bond acceptors) values are fisetin 6, apigenin 5, myricetin 5 and luteolin 4. These values are in line with Lipinski’s rule of 5 recommendation of nON ≤ 10.

The nOHNH (number of H bond donors) values were: fisetin 4, apigenin 3, myricetin 3 and luteolin 2. The nOHNH value for the molecules are below 5 in line with Lipinski’s rule of 5 drug-likeness recommendation of nOHNH < 5. The nViolation values for the flavonoid molecules were 0 for each.

Results of Bioactivity scores

Table 4 shows bioactivity scores of luteolin, myricetin, apigenin, and myricetin.

Table 4

Bioactivity scores of the flavonoid molecules predicted against kinase inhibition, ion channel modulation, protesase inhibition, GPCRs, nuclear receptor ligand, kinase inhibition, and enzyme inhibition. This helps estimate drug-likeness indicators. fisetin, apigenin, myricetin and luteolin values. Molecules that bear the highest scores demonstrate that they can be highly active.

GPCR ligand’s highest bioactivity score was -0.07 followed by -0.08. That for the activity of ion channel modulator lay between -0.13 and -0.27 which imply their moderate capacity for interaction. Similarly, the kinase inhibitor values ranged between 0.18 and 0.08. The positive scores for the nuclear receptor ligand, protease inhibitor and enzyme inhibition was found to be in the range of 0.09- 0.22, -.0.34 to 0.38 and 0.16 to 0.21respectively. They predict that the molecules are highly bioactive. The most bioactive molecule was Apigenin (compound 2) whose figures were -0.34, 0.24 and 0.21. The figures respectively represent protease inhibitor, nuclear receptor ligand, and enzyme inhibition.

2) Molecular docking studies

The flavonoid ligand molecules and metal receptors were examined for molecular docking. The programs Auto Dock version 2 and Auto Dock Tools (version 1.5.6) were used to nderstand their molecular interactions.

Discussion

Molecular Properties

The positive logP values obtained: fisetin 1.97, apigenin 2.24, myricetin 2.27 and luteolin 2.27 imply that the flavonoid molecules are lipophilic. This means that their concentration in the lipid phase. Myricetin and luteolin have the highest drug like-ness going by their logP values 2.27 followed by apigenin and fisetin. Their positive figures ascertain their desirable drug-like properties. The TPSA values of fisetin 1.112, apigenin 90.89, myricetin 90.89 and luteolin 70.67 lie below 140. This means that they are good at permeating plasma membranes. Luteolin has the least TPSA value at 70.67 hence the most suitable in this category. The molecular volume values ( fisetin 237.07, apigenin 224.05, myricetin 224.05 and luteolin 254.24).

The molecular volume shows the flavonoid molecules’ transport characteristics like penetration of the blood-brain interface and intestinal absorption can be predicted using molecular volume values. The molecular volume values (fisetin 237.07, apigenin 224.05, myricetin 224.05 and luteolin 254.24), indicate their drug likeness properties. The MW values obtained (fisetin 286.24, apigenin 270.24, myricetin 270.24 and luteolin 254.24. This is in line with Lipinki’s rule of thumb recommendation of MW ≤ 500. The nON (number of H-bond acceptors) values are fisetin 6, apigenin 5, myricetin 5 and luteolin 4. These values are in line with Lipinski’s rule of 5 recommendation of nON ≤ 10.

The nOHNH (number of H bond donors) values were: fisetin 4, apigenin 3, myricetin 3 and luteolin 2. The nOHNH value for the molecules are below 5 in line with Lipinski’s rule of 5 drug-likeness recommendation of nOHNH < 5. (nrotb ) number of rotatable bonds values are 1 for each of the flavonoids in line with the recommended nrotb ≤10.

The molecules fiestin apigenin, myricetin and luteolin demonstrate their drug-likeness properties in line with Lipinski’s rule of 5. Their values: TPSA ≤ 140 Å, nrotb ≤10, nON ≤ 10, MW ≤ 500, nOHNH < 5 and logP≤ 5 agree with the rule of 5 (Lipinski et al, 1997; Veber et al, 2002). The nViolation values for the flavonoid molecules were 0 for each. This is in line with Lipinski’s rule of five which stipulate that if a molecule deviates with two or more properties (MW, logP, H-bond acceptors and H-bond donors), then a poor permeability or absorption is likely.

Bioactivity scores

The flavonoids’ bioactivity scores determined based can be interpreted to be active. Active and moderate bioactivity are determined by activity scores of > 0 and -5.0-0.0 values. Scores of less than 5.0 indicate inactivity (Molinspiration, 2020). The least bioactivity value was -0.38 for protease inhibitor in luteolin and the highest score was 0.24 of apigenin’s nuclear receptor ligand. Collectivel the flavonoid molecules bioactive scores lie between -5.0 – 0.0. The flavonoids are classified as being moderately active.

Molecular Docking

The computational docking software programs used predicted the flavonoids’ active site surface and their ability to interact with the targets. Estimated inhibition constants and binding energies of the flavonoid ligand molecules were used to summarize this view. The bonding between the flavonoid molecules and target active sites could be attributed to non-covalent bonding likely to van der waals and hydrophobic forces and, hydrogen bonding.

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