Withaferin A: a potential therapeutic agent against COVID-19 infection

doi:10.21203/rs.3.rs-37637/v1

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Withaferin A: a potential therapeutic agent against COVID-19 infection

https://doi.org/10.21203/rs.3.rs-37637/v1

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published 19 Jul, 2020

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The outbreak and continued spread of the novel coronavirus disease 2019 (COVID-19) is a preeminent global health threat that has resulted in the infection of over 6 million people worldwide. In addition, the pandemic has claimed the lives of over 350,000 people worldwide. Age and the presence of underlying comorbid conditions have been found to be key determinants of patient mortality. One such comorbidity is the presence of an oncological malignancy, with cancer patients exhibiting an approximate two-fold increase in mortality rate. Due to a lack of data, no consensus has been reached about the best practices for the diagnosis and treatment of cancer patients. Interestingly, two independent research groups have discovered that Withaferin A (WFA), a steroidal lactone with anti-inflammatory and anti-tumorigenic properties, may bind to the viral spike (S-) protein of SARS-CoV-2. Further, preliminary data from our research group has demonstrated that WFA does not alter expression of ACE2 in the lungs of tumor-bearing female mice. Downregulation of ACE2 has recently been demonstrated to increase the severity of COVID-19. Therefore, WFA demonstrates real potential as a therapeutic agent to treat or prevent the spread of COVID-19 due to the reported interference in viral S-protein to host receptor binding and its lack of effect on ACE2 expression in the lungs.

Cancer Biology

Coronavirus

Pandemic

Withanolides

Ashwagandha

Cancer

The novel coronavirus disease 2019 (COVID-19) has rapidly spread around the world since it was first reported in December 2019 within Wuhan, China as a pneumonia of unknown etiology [1]. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), termed by the World Health Organization (WHO), represents the third large-scale epidemic related to coronaviruses [1]. Although the disease was first reported within China, a retrospective study has subsequently found evidence that SARS-CoV-2 was spreading within France four days before it was reported in Wuhan, China and one month before the first official case in the country [2]. Since its initial discovery, SARS-CoV-2 has spread worldwide, infecting over 6.0 million people and led to the death of more than 350,000 people [3]. The severity of the disease widely ranges from an asymptomatic disease-state to patients exhibiting acute respiratory distress syndrome (ARDS), necessitating critical medical intervention to attempt to prevent patient death [4]. It was subsequently discovered that Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the SARS-CoV-2 spike (S-) protein, allowing the virus to enter cells [5]. ACE2 is a potent negative regulator of the renin angiotensin system (RAS), which is critical for maintaining the homeostasis of RAS.

The ACE2 gene is composed of 805 amino acids and is a type I integral membrane glycoprotein. ACE2 degrades angiotensin (Ang)-II, a potent vasoconstrictor (that is also pro-inflammatory and promotes fibrosis), and converts it into Ang (1-7) [6]. Ang (1-7) is a vasodilator, that also inhibits proliferation and apoptosis [6]. Beside the systemic effect on blood pressure regulation, ACE2 has local regulatory effects in the pathological changes of several organs, including the heart, kidney, and lungs [7]. ACE2 is highly expressed in lung alveolar cells, providing the main entry site for the virus into human host [8]. In addition to expression of ACE2 in lung alveolar cells, it is also expressed in various tissues, including: the vascular system (endothelial cells, migratory angiogenic cells and vascular smooth muscle cells), heart (cardiofibroblasts, cardiomyocytes, endothelial cells, pericytes, and epicardial adipose cells) and kidneys (glomerular endothelial cells, podocytes and proximal tubule epithelial cells), liver (cholangiocytes and hepatocytes), retina (pigmented epithelial cells, rod and cone photoreceptor cells, and Müller glial cells), enterocytes of the intestines, circumventricular organs of the central nervous system, and the upper airway (goblet and ciliated epithelial cells) [9].

There are two subunits of the SARS-CoV-2 S-protein: the S1 subunit has a receptor binding domain that engages with the host cell receptor ACE2, and the S2 subunit is involved in regulating fusion between the viral and the host membrane [10]. It has been reported that SARS-CoV-2 has a ten times higher affinity to ACE2 compared to SARS-CoV, which is consistent with the higher efficiency of infection of SARS-CoV-2 [11]. While no cure has currently been found, several clinical trials are being performed to determine what the most efficacious treatment regimen is for COVID-19, with an extensive list of potential therapies detailed in a review by Gosain et al. [12]. Currently, patient management involves supportive treatment and measures to prevent further spread of the virus [13]. Despite differences in patient population characteristics between Europe and China, two of the main determinants of patient mortality risk that were found in both groups are age and the presence of underlying comorbid conditions [14, 15]. One such underlying condition associated with an increase in COVID-19 patient mortality is the presence of cancer [16].

Due to their potentially immune-compromised status, the proper treatment of cancer patients is a real and serious problem being faced by oncologists, regardless of if the patient is experiencing a SARS-CoV-2 infection [16]. Data from four SARS-CoV-2 hot spots (the United States, Italy, Spain and China) has shown that cancer patients infected with the novel coronavirus have a significantly increased risk of admission to an intensive care unit (ICU) and/or requiring mechanical ventilation, as well as an increase in patient mortality [15, 17-19]. In a retrospective study, the fatality rate for cancer patients in China infected with COVID-19 was found to be approximately 28% [20], compared to the overall symptomatic fatality rate of 1.4% or the crude mortality rate of 4.5% in China [21]. Cancer patients and their oncologists are currently facing the dilemma as to whether or not the patient should begin or continue therapy for their primary disease state due to the associated risks of contracting SARS-CoV-2 and the reduction in resources available to healthcare workers [22]. Currently, there are no globally accepted guidelines to address cancer patient management in the settings of a pandemic due to a lacking of data available [23]. Recently, an international collaboration has proposed a series of practical approaches for the diagnosis and treatment of cancer patients [23]. However, until more information or an effective therapeutic regimen against SARS-CoV-2 become available, cancer patients will continue to remain at a very high-risk of mortality due to the COVID-19 epidemic [23].

Withaferin A (WFA) is a steroidal lactone isolated from the plant Withania somnifera, also known as Ashwagandha [24]. It is known for its anti-inflammatory properties, as well as its anti-tumorigenic properties [25-27]. Recent work has demonstrated that COVID-19 infections have a large immune component and can result in the development of cytokine storm, a potentially life-threatening immune reaction in which the body release too many cytokines into the blood at a rapid rate [28]. Interestingly, at least three independent research groups have suggested that phytochemicals found in the plant Withania somnifera could be developed as a therapeutic agent against COVID-19 infection using molecular docking approaches [29-31]. Two of the groups reported that various Withanolides, such as WFA, should be able to bind to the viral S-protein receptor binding domain, thereby blocking or reducing interactions with host ACE2 receptor [29, 30]. The third group reported that WFA and a separate withanolide, Withanone, are predicted to interact with the main protease of SARS-CoV-2, although WFA is predicted to have less of a binding affinity than an established N3 protease inhibitor used for baseline docking scores [31]. In an unrelated study, our group has been investigating WFA as a potential therapeutic to treat cancer, including the targeting of cancer stem cells and cancer-induced cachexia (a muscle wasting disorder).

As Ang-II signaling is a known mediator of skeletal muscle atrophy [32], we investigated the effect of WFA on Ang-II signaling as it pertains to cachexia. Unpublished data from our lab has indicated that WFA treatment can reduce circulating levels of Angiotensin II in an experimental model of cancer-induced cachexia. Further, we found that WFA treatment reduced the relative mRNA expression of AT1R (Angiotensin II Receptor Type 1) compared to the vehicle-treated group in tumor samples. Based upon our findings and the independently reported molecular docking studies, we investigated whether or not WFA treatment would alter ACE2 expression in the lungs under tumor-free and tumor-bearing conditions. Interestingly, we found no significant differences (NSD) in relative mRNA expression of ACE2 in response to WFA treatment (Fig. 1). However, it was recently reported that, as a byproduct of SARS-CoV-2 infection, ACE2 expression is decreased as part of the disease process, which in turn facilitates the development of multiorgan damage [33]. Due to this effect, others have suggested that blocking the binding of SARS-CoV-2 to the ACE2 receptor may be a more beneficial strategy to combat the virus than augmenting ACE2 expression, due to its antagonistic effect on AT1R signaling [9]. In line with this rationale, it is within the realm of possibility that WFA can block or impede COVID-19 through interactions with the viral S-protein based upon the molecular docking studies [29, 30], without affecting ACE2 expression (as reported in our data) leading to a worsening of the pathological state.

The COVID-19 outbreak has become a significant clinical threat worldwide to both the general population and healthcare workers. Additionally, cancer patients and the elderly remain a very high-risk subpopulation that are more susceptible to disease-related fatality. While knowledge about this virus remains limited, over 100 clinical trials are currently being performed to help find a means to combat this epidemic. Due to their potentially immune-compromised position and associated rates of mortality, it would seem that special considerations should be taken in developing a potential therapeutic regimen for cancer patients and patients with other high-risk comorbidities. Withaferin A could be developed into an attractive therapeutic agent for both the general population and cancer patients due to its anti-tumorigenic properties and the preliminary studies showing that it is capable of binding to the S-protein of SARS-CoV-2, thereby potentially inhibiting infection and/or spread of the disease.

ACE2 = Angiotensin-converting enzyme-2; Ang = Angiotensin; ANOVA = Analysis of variance; AT1R = Angiotensin II Receptor Type 1; ARDS = Acute respiratory distress syndrome; COVID-19 = Coronavirus disease 2019; ICU = Intensive care unit; SARS-CoV-2 = Severe acute respiratory syndrome coronavirus-2; NSD = No significant differences; RAS = Renin angiotensin system; S-protein = Spike protein; WFA = Withaferin A; WHO = World Health Organization;

Ethical Approval: All procedures involving the usage of mice were carried out in strict accordance to the standards of the National Institute of Health guide for the care and use of laboratory animals. The Institutional Animal Care and Use Committee (IACUC, protocol # 15405) and Institutional Biosafety Committee (IBC, protocol # 18-208) of the University of Louisville approved all experimental protocols in mice in advance. No human data or tissue was used in this study.

Conflict of Interest: All authors declare that they have no competing interests.

Funding: This work was supported by NIH grants T32 HL134644 and R25GM133328 to SSK.

Authors’ Contributions: SSK and ARS conceived the concept for the manuscript. ARS wrote the initial draft of the manuscript. All authors edited the manuscript. All authors read and approved the final manuscript.

Bogoch, II, Watts A, Thomas-Bachli A, Huber C, Kraemer MUG, Khan K: Pneumonia of unknown aetiology in Wuhan, China: potential for international spread via commercial air travel. J Travel Med 2020, 27(2).
Deslandes A, Berti V, Tandjaoui-Lambotte Y, Alloui C, Carbonnelle E, Zahar JR, Brichler S, Cohen Y: SARS-CoV-2 was already spreading in France in late December 2019. Int J Antimicrob Agents 2020:106006.
COVID-19 Coronavirus Pandemic [https://www.worldometers.info/coronavirus/]
Hassan SA, Sheikh FN, Jamal S, Ezeh JK, Akhtar A: Coronavirus (COVID-19): A Review of Clinical Features, Diagnosis, and Treatment. Cureus 2020, 12(3):e7355.
Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC et al: Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003, 426(6965):450-454.
Hamming I, Cooper ME, Haagmans BL, Hooper NM, Korstanje R, Osterhaus AD, Timens W, Turner AJ, Navis G, van Goor H: The emerging role of ACE2 in physiology and disease. J Pathol 2007, 212(1):1-11.
Kuba K, Imai Y, Ohto-Nakanishi T, Penninger JM: Trilogy of ACE2: a peptidase in the renin-angiotensin system, a SARS receptor, and a partner for amino acid transporters. Pharmacol Ther 2010, 128(1):119-128.
Ge XY, Li JL, Yang XL, Chmura AA, Zhu G, Epstein JH, Mazet JK, Hu B, Zhang W, Peng C et al: Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 2013, 503(7477):535-538.
Gheblawi M, Wang K, Viveiros A, Nguyen Q, Zhong JC, Turner AJ, Raizada MK, Grant MB, Oudit GY: Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2. Circ Res 2020, 126(10):1456-1474.
Du L, He Y, Zhou Y, Liu S, Zheng BJ, Jiang S: The spike protein of SARS-CoV--a target for vaccine and therapeutic development. Nat Rev Microbiol 2009, 7(3):226-236.
Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS: Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020, 367(6483):1260-1263.
Gosain R, Abdou Y, Singh A, Rana N, Puzanov I, Ernstoff MS: COVID-19 and Cancer: a Comprehensive Review. Curr Oncol Rep 2020, 22(5):53.
Clinical management of severe acute respiratory infection (SARI) when COVID-19 disease is suspected
[The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID-19) in China]. Zhonghua Liu Xing Bing Xue Za Zhi 2020, 41(2):145-151.
Fratino L, Procopio G, Di Maio M, Cinieri S, Leo S, Beretta G: Coronavirus: Older Persons With Cancer in Italy in the COVID-19 Pandemic. Front Oncol 2020, 10:648.
Brunetti O, Derakhshani A, Baradaran B, Galvano A, Russo A, Silvestris N: COVID-19 Infection in Cancer Patients: How Can Oncologists Deal With These Patients? Front Oncol 2020, 10:734.
Dai M, Liu D, Liu M, Zhou F, Li G, Chen Z, Zhang Z, You H, Wu M, Zheng Q et al: Patients with Cancer Appear More Vulnerable to SARS-COV-2: A Multicenter Study during the COVID-19 Outbreak. Cancer Discov 2020.
Liang W, Guan W, Chen R, Wang W, Li J, Xu K, Li C, Ai Q, Lu W, Liang H et al: Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China. Lancet Oncol 2020, 21(3):335-337.
Mehta V, Goel S, Kabarriti R, Cole D, Goldfinger M, Acuna-Villaorduna A, Pradhan K, Thota R, Reissman S, Sparano JA et al: Case Fatality Rate of Cancer Patients with COVID-19 in a New York Hospital System. Cancer Discov 2020.
Zhang L, Zhu F, Xie L, Wang C, Wang J, Chen R, Jia P, Guan HQ, Peng L, Chen Y et al: Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three hospitals within Wuhan, China. Ann Oncol 2020.
Wu JT, Leung K, Bushman M, Kishore N, Niehus R, de Salazar PM, Cowling BJ, Lipsitch M, Leung GM: Estimating clinical severity of COVID-19 from the transmission dynamics in Wuhan, China. Nat Med 2020, 26(4):506-510.
Disruptions in Cancer Care in the Era of COVID-19 [https://www.medscape.com/viewarticle/927215]
Al-Shamsi HO, Alhazzani W, Alhuraiji A, Coomes EA, Chemaly RF, Almuhanna M, Wolff RA, Ibrahim NK, Chua MLK, Hotte SJ et al: A Practical Approach to the Management of Cancer Patients During the Novel Coronavirus Disease 2019 (COVID-19) Pandemic: An International Collaborative Group. Oncologist 2020.
Dutta R, Khalil R, Green R, Mohapatra SS, Mohapatra S: Withania Somnifera (Ashwagandha) and Withaferin A: Potential in Integrative Oncology. Int J Mol Sci 2019, 20(21).
Fugner A: INHIBITION OF IMMUNOLOGICALLY INDUCED INFLAMMATION BY PLANT STEROID WITHAFERIN-A. Arzneimittel-Forschung/Drug Research 1973, 23(7):932-935.
Kakar SS, Parte S, Carter K, Joshua IG, Worth C, Rameshwar P, Ratajczak MZ: Withaferin A (WFA) inhibits tumor growth and metastasis by targeting ovarian cancer stem cells. Oncotarget 2017, 8(43):74494-74505.
Straughn AR, Kakar SS: Withaferin A ameliorates ovarian cancer-induced cachexia and proinflammatory signaling. Journal of ovarian research 2019, 12(1):115-115.
Ye Q, Wang B, Mao J: The pathogenesis and treatment of the `Cytokine Storm' in COVID-19. J Infect 2020, 80(6):607-613.
Balkrishna A, Pokhrel S, Singh J, Varshney A: Withanone from Withania somnifera may inhibit novel coronavirus (COVID-19) entry by disrupting interactions between viral S-protein receptor binding domain and host ACE2 receptor. In. Research Square; 2020.
Maurya DK, Sharma D: Evaluation of traditional ayurvedic preparation for prevention and management of the novel Coronavirus (SARS-CoV-2) using molecular docking approach. In. ChemRxiv; 2020.
Kumar V, Dhanjal JK, Kaul SC, Wadhwa R, Sundar D: Withanone and Caffeic Acid Phenethyl Ester are Predicted to Interact with Main Protease (M(pro)) of SARS-CoV-2 and Inhibit its Activity. J Biomol Struct Dyn 2020:1-17.
Yoshida T, Tabony AM, Galvez S, Mitch WE, Higashi Y, Sukhanov S, Delafontaine P: Molecular mechanisms and signaling pathways of angiotensin II-induced muscle wasting: potential therapeutic targets for cardiac cachexia. The international journal of biochemistry & cell biology 2013, 45(10):2322-2332.
Wang K, Gheblawi M, Oudit GY: Angiotensin Converting Enzyme 2: A Double-Edged Sword. Circulation 2020.

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Journal Publication

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Withaferin A: a potential therapeutic agent against COVID-19 infection

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Abstract

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Introduction

Cancer Patients And The Covid-19 Epidemic

Withaferin A As A Prospective Treatment

Conclusion

Abbreviations

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References

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