key: cord-1055030-fwqmdenn
authors: Gupta, Ankur; Pradhan, Anish; Maurya, Vimal K; Kumar, Swatantra; Theengh, Angila; Puri, Bipin; Saxena, Shailendra K
title: Therapeutic Approaches for SARS-CoV-2 Infection
date: 2021-05-05
journal: Methods
DOI: 10.1016/j.ymeth.2021.04.026
sha: 021f19ce043e44c2036175a6055f0de429b87b1c
doc_id: 1055030
cord_uid: fwqmdenn

Therapeutic approaches to COVID-19 treatment leverage on crucial target proteins of SARS-CoV-2 replication machinery. It’s been approximately 12 months of pandemic and yet there is no known specific drugs are available. However, research is progressing with time in terms of high throughput virtual screening (HTVS) and rational design of repurposed, novel synthetic and natural products drug discovery by understanding the viral life cycle, immuno-pathological and clinical outcomes in patients based on host’s nutritional, metabolic and lifestyle status. Further, complementary and integrative health approaches (CIHA) have also shown to improve the resiliency and immune responses among people. In this article, we summarize about all the therapeutic approaches for COVID-19 antiviral drug discovery including computer aided virtual screening, repurposed drugs, immunomodulators, vaccines, plasma therapy, miscellaneous adjunct therapies and phage technology to unravel insightful mechanistic pathways of targeting SARS-CoV-2 and host’s intrinsic innate immunity at various checkpoints that aid in the containment of the disease.

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December 2019 outbreak of SARS-CoV-2 from China has infected vast population of 5 216 countries by now with millions of deaths and yet the pandemic infection is growing 6 exponentially without any specific prophylaxis or treatment modalities. Parallelly, the 7 collective scientific community has been trying to explore and target the key proteins of 8 SARS-CoV-2 mutant's pathogenesis including after infection human immuno- 9 pathophysiology. Subsequently, a lot of data has been reported in terms of review articles that 10 describe hosts responses, clinical features, proposed treatment interventions and their 11 mechanism of action (MOA) associated with SARS-CoV-2 infection or COVID-19 [1] [2] [3] [4] [5] [6] [7] . 12 Nevertheless, research on SARS-CoV-2 and COVID-19 is progressing with time in terms of 13 rational design of novel drugs, natural products drug discovery, immuno-pathological and 14 clinical outcomes based on host nutritional, metabolic and lifestyle status, role of herbo- 15 mineral formulations in innate/adaptive immunity modulation to contain the disease. Hence, 16 it is necessary to review the current data for critical therapeutic options and therefore, is the 17 highlights of this article. 18 SARS-CoV-2 is a single stranded positive-sense RNA virus of ~30kb in length [8] 19 with a diameter of approximately 125 nm [9] and manipulates the host cell environment [10] 20 depending on host's nutritional, metabolic and lifestyle status [11] [12] [13] to alter the host gene 21 expression and immune responses [14] leading to long incubation period [15] and a large 22 number of asymptomatic [16] but transmissible infection preventing effective containment 23 and mitigation of the disease [17] . SARS-CoV-2 has four structural proteins namely; the 24 homo-trimer spike (S) protein, the membrane (M) protein, the envelope (E) protein, and the 25 nucleocapsid (N) protein [ Figure 1 ]. The S protein mediates host cell attachment whereas M 26 protein aids in envelope formation and viral entry [8, 18, 19] . The other lifecycle processes 8 1 are also prone to microbial infection which further promotes the disease progression and 2 severity leading to viral sepsis and inflammation-induced lung injury. Further, the disease 3 progresses to acute respiratory distress syndrome (ARDS), respiratory failure, shock, 4 gastrointestinal (GIT) issues, multiple organ failure [ Figure 3 ], and potentially death [54] . 5 Hence, a safe "multicomponent-multitarget -multichannel therapeutic approach" along with 6 respiratory and physical exercises has to be considered specially to avoid the disease 7 progression towards severity. 8 9 3. Potential therapeutic approaches 10 3.1. Antiviral approaches 11 12 First step in the infection process is that the SARS-CoV-2 Spike (S) glycoprotein 13 utilize its receptor binding domain (RDB) to recognise, interact and attach itself with host cell 14 receptor angiotensin converting enzyme 2 (ACE2). S-protein has two extracellular subunits 15 S1 and S2 [55] . The RBD of S1 subunit after binding to ACE2 gets cleaved at the interface of 16 S1 and S2 by host cell derived transmembrane proteases serine 2 (TMPRSS2) enzyme, 17 cathepsin L, and furin. Further, the fusion protein (FP) of S2 subunit triggers viral fusion 18 process [56] . Therefore, S-protein becomes the principle target for vaccines and therapeutic 19 drugs to inhibit viral entry [21] . 20 Structural understanding of the RBD-ACE2 interface [57] is a crucial step for 21 inhibitor design. Besides peptides, monoclonal antibodies (mAb) and small molecule 22 inhibitors are still the preferred intervention modality in terms of cost, dosage, stability, 23 pharmacokinetics and logistics. To identify small molecule inhibitors against RBD, virtual 24 insilico screening of 1582 FDA-approved drugs was carried out which showed that 25 Simeprevir and Lumacaftor bind RDB with high affinity and prevent ACE2 interaction. 9 1 Further, virtual screening and in vitro studies of the same drugs suggested that Lumacaftor 2 and Simeprevir are also SARS-CoV-2 Mpro inhibitors showcasing the concept of multi- 3 target drugs that inhibit several proteins simultaneously [58] . Similarly, various other studies 4 have also been reported where small molecule libraries of natural products are screened 5 against RBD of SARS-CoV-2. Few molecules such as Nimbin, Curcumin, Withaferin A, 6 Mangiferin, Piperine, Thebaine, Andrographolide, and Berberine were found effective in 7 inhibiting the interaction of spike glycoprotein with its receptor ACE2 [59] . Nevertheless, 8 few other molecules such as Eufoliatorin, Amarogentin, Caesalpinins, α-Amyrin, Kutkin, β- 9 Sitosterol, and Belladonnine [60] showed the high affinity towards both the S-protein RBD 10 and ACE2. ACE2 is a functional receptor required for SARS-CoV-2 attachment and 11 internalization. In this context, Chloroquine, an antimalarial repurposed drug, was reported to 12 block SARS-CoV-2 virus infection, with an IC 50 value of 1.13 μM and a CC 50 > 100 μM in 13 Vero E6 cells. Chloroquine is believed to inhibit terminal glycosylation of ACE2 along with 14 increased endosomal pH required for fusion leading to reduced affinity of SARS-CoV-2 to 15 ACE2. Apart from its antiviral activity, chloroquine is also shown to synergistically enhance 16 its antiviral effect through immunomodulation [61] . Another analogue of chloroquine, 17 namely, Hydroxychloroquine exhibited much safer and better in vitro results than 18 chloroquine [62] . Nevertheless, these repurposed drugs are also reported to cause ventricular 19 arrhythmias, QT prolongation, and other cardiac-related toxicities in severely ill patients [63] . 20 Regardless of the availability of ACE2 inhibitors, its inhibition is not a viable therapeutic 21 approach as it plays important physiological roles including lung injury protective role in 22 ARDS [64] and its attenuation may aggravate oxidative inflammatory responses [65] . 23 Clinically approved TMPRSS2 inhibitors are safe and effective drugs considered to 24 contribute in the containment of the disease by inhibiting host cell entry. Few TMPRSS2 25 inhibitors such as Camostat, Nafamostat and Aprotinin have shown to effectively decrease 10 1 the rate of infection and replication of the virus in Calu-3 lung cell lines. Camostat is an FDA 2 approved drug for the treatment of pancreatitis and was found effective in reducing airway 3 virus replication by inhibiting S-protein initiated fusion. Similarly, Nafamostat, an FDA 4 approved anticoagulant drug in Japan for continuous renal replacement, was recently reported 5 to show 15 folds higher inhibitory potency than Camostat with 50% effective concentration 6 [EC 50 ] in the low-nanomolar range against SARS-CoV-2 fusion [66] [67] [68] . In comparison, 7 Gabexate mesylate is least active in inhibiting SARS-CoV-2 S-driven host cell entry [69] . 8 The suitability of these TMPRSS2 inhibitors including Bicalutamide to block TMPRSS2 for 9 treatment of COVID-19 is currently being evaluated under clinical trial [70] [71] [72] . Further, in 10 silico approaches using homology modelling, docking and ADME/T (absorption, distribution, 11 metabolism, excretion, toxicity) studies for the identification of high affinity interaction and 12 potent antagonists of TMPRSS2 have been reported. The study revealed that, six amino acid 13 residues are essential which act as an active site of TMPRSS2 where three residues His296, 14 Asp345, Ser441 present at the catalytic site and three residues Asp435, Ser460, Gly462 15 present at the substrate binding site. The results unravelled various natural and synthetic 16 molecules including columbin, meloxicam, proanthocyanidin A2, ganodermanontriol, 17 myricetin, jatrorrhizine and baicalein and should be proceeded for wet-lab evaluations [73, 18 74]. 19 Further, various studies have also demonstrated that low endosomal pH environment 20 activates pH sensitive proteases such as cathepsins L. Hence, few potent cathepsin L 21 inhibitors, namely, MDL28170, EST, dec-RVKR-CMK, 5705213, K11777, oxocarbazate, 22 and SSAA09E1 has been reported. However, due to concern over their unwanted side effects, 23 FDA approved drugs that exhibit cathepsin L inhibitory activity including antimicrobials, 24 immunomodulators, antimalarials, anti-tuberculous, anti-HIV, antioxidant, etc were 11 1 considered to be repurposed. Nevertheless, these drugs have their own unwanted side effects 2 in patients [75] . 3 Additionally, an abelson non-receptor tyrosine kinase (Abl) promotes cathepsin L 4 secretion which indicate that drugs inhibiting Abl tyrosine kinases might indirectly serve as 5 cathepsin secretion inhibitors and inhibit entry/fusion of SARS-CoV-2 [76]. Subsequently, 6 imatinib, has been shown to inhibit SARS-CoV-2 in an invitro study [77] . Similarly, several 7 kinase inhibitors as anti-inflammatory immunomodulators for cytokine suppression are 8 proposed as potential therapeutic approach to contain COVID-19 [78]. 9 Apart from these host-based, cell surface and endosomal proteases inhibitors, fusion 10 inhibition is an attractive strategy to block viral entry through inhibition of a heptad repeat 11 region HR1 of S-protein [79] . EK1 (optimized analogue of OC43-HR2 peptide) was found to 12 be highly potent (IC50 = 0.19 μM) in shutting down S-protein mediated cell-cell fusion 13 through hydrophobic interactions for SARS-CoV-2. EK1 was further optimized to EK1C4 14 which showed higher effectiveness than EK1 against SARS-CoV-2 S protein-mediated 18 Inflammatory signalling pathways directed by distinct ubiquitin signals which are 19 regulated by complex mechanisms in human cells. Viral proteases generally regulate innate 20 immune pathways through antagonising ubiquitin and interferon-stimulated gene 15 19 SARS-CoV-2 RNA encodes for two large polyproteins, pp1a and pp1ab, which are 20 inactive until the viral chymotrypsin-like cysteine protease enzyme (3CL Mpro or Nsp5) 21 cleaves them into 12 non-structural proteins (Nsp4-Nsp16) including RdRp (Nsp12) and 22 helicase (Nsp13). Inhibition of Mpro would prevent the virus from replication. Hence, 23 making it an attractive drug target for SARS-CoV-2 [90, 91] . In the absence of targeted 24 therapeutic drugs, the only option for identification and discovery of lead compounds is 25 through the application of computer aided structure-based high-throughput virtual screening 13 1 (HTVS) of approved or clinical candidates. Therefore, utilising the same HTVS route, a 2 mechanism-based inhibitor (N3) was identified and crystal structure of Mpro complexed with 3 N3 was determined. Further, HTVS was carried out using 10,000 known compounds 4 including approved drugs, natural products and drug candidates in clinical trials. The primary 5 hits were seven compounds namely; Ebselen, Disulfiram, Tideglusib, Carmofur, Shikonin, 6 PX-12 and TDZD-8 having IC 50 ranging from 0. inhibitor that targets the viral RNA replication [110] . Glecaprevir has also been evaluated 11 insilico for its Mpro inhibition [109] which suggested that Glecaprevir is a highly potential 12 inhibitor of SARS-CoV-2 Mpro. Likewise, Lopinavir, a small peptidomimetic antiretroviral 13 aspartate protease inhibitor, was assumed to inhibit the Mpro of the SARS-CoV-2 as well, 17 Coronavirus replication and transcription is mediated through a multisubunit complex 18 of viral nonstructural proteins (nsps) including the core component, nsp12 (RdRp), and 19 accessory cofactors, nsp7 and nsp8, that increase RdRp template binding and processivity 20 [120]. Hence, RdRp is a crucial target for inhibition of viral replication and is also known to 21 be inhibited by a class of antivirals called as "nucleotide analogs" such as Remdesivir [120].

Remdesivir (GS-5734) is an adenosine monophosphate antiviral prodrug which gets 23 metabolized to its pharmacologically active nucleoside triphosphate metabolite. The 24 triphosphate metabolite acts as a competitive inhibitor of RdRp [ Figure 5 ] which leads to 25 termination of chain elongation and ceases the viral RNA replication process [121]. 16 1 Remdesivir was observed to be active in vitro against Vero E6 cells infected with SARS- In contrast, a pyrazine-carboxamide derivative, favipiravir is currently in use against 12 mild to moderate COVID-19 infections in various countries including China, Italy, Japan, 13 Russia, Ukraine, Uzbekistan, Moldova, Kazakhstan, Saudi Arabia, UAE, Turkey, 14 Bangladesh, Egypt and India. Favipiravir is a broad-spectrum antiviral drug which gets 15 converted to its potentially active form favipiravir-ribose-5'-triphosphate, in host-infected Protopine was observed to be the most potential inhibitor ligand. 2 To combat SARS-CoV-2 infection, apart from physical barriers, host's immune 

Similarly, Vitamin D also plays a crucial role in immune-modulation during viral 25 infections. Vitamin D production in human body takes place through subcutaneous sunlight 19 1 exposure or is made available through external dietary sources such as dairy products, fish 2 liver oil or cholecalciferol pills. After absorption, vitamin D binds to intracellular nuclear 7 A preliminary report by researchers announcing that synthetic glucocorticoid 8 dexamethasone (6mg once daily) is a potential treatment option, to reduce mortality in 9 severely ill patients, filled the scientific community with excitement. The study trial reported 10 that, after initiation of immunosuppressive dexamethasone therapy, the mortality rate of 11 patients who were on ventilator support was dropped by one-third, whereas, the mortality rate 12 was reduced by one-fifth among patients who were on oxygen support without ventilation. 13 However, patients who were not receiving any respiratory support did not see any positive 

Interleukin-6 (IL-6) is a major factor responsible for cytokine storm and ARDS in Interleukin-1β (IL-1β), a proinflammatory cytokine, along with its natural interleukin- 2 The ongoing viral pandemic has resulted in scaling up of pooled plasma from 

The SARS-CoV-2 outbreak as a pandemic has lasted for almost a year now and it is 3 likely that the infection will remain in humans until clinically approved vaccines are made 4 available through out the world. Nevertheless, mutation and cross species jump among 5 coronaviruses remain the matter of concern and needs to be kept under scientific surveillance 6 for future pandemics. Currently, the best way to prevent SARS-CoV-2 infection, indeed, are 7 personal preventive measures such as social distancing, washing hands regularly, and 8 wearing face mask. However, high nutritional, metabolic and lifestyle status has a major role 9 to play in activating host's innate and adaptive immune system and keeping the disease L., Xiong, L., Guo, C., Tian, J., Luo, J., Yao, J., Pang, R., Shen, H., Peng, C., Liu, T., 28 Zhang, Q., Wu, J., Xu, L., Lu, S., Wang, B., Weng, Z., Han, C., Zhu, H., Zhou, R., 29 Zhou, H., Chen, X., Ye, P., Zhu, B., Wang, L., Zhou, W., He, S., He, Y., Jie, S., Wei, Peng, C., Duan, Y., Yu, J., Wang, L., Yang, K., Liu, F., Jiang, R., Yang, X., You, T., 13 Liu, X., Yang, X., Bai, F., Liu, H., Liu, X., Guddat, L. W., Xu, W., Xiao, G., Qin, C., 14 Shi, Z., Jiang, H., Rao, Z., Yang, H. (2020) . Structure of Mpro from SARS-CoV-2 15 and discovery of its inhibitors. Nature. 582(7811), 289-293. Y., Shen, C., Li, X., Peng, L., Huang, D., Zhang, J., Zhang, S., Wang, F., Liu, J., 23 Chen, L., Chen, S., Wang, Z., Zhang, Z., Cao, R., Zhong, W., Liu, Y., and Liu, L. 

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-14

CoV-2 and its 3C-like protease in vitro

Discovery of Baicalin and Baicalein as Novel, Natural Product 19 Inhibitors of SARS-CoV-2 3CL Protease

A Trial of Lopinavir-Ritonavir in Adults Hospitalized 15 with Severe Covid-19

Lack of antiviral activity of darunavir against 19

Revisiting the old and 12 learning the new of zinc in immunity

Zinc metabolism in patients with the syndrome of iron 16 deficiency anemia, hepatosplenomegaly, dwarfism, and hypogonadism

Dysregulation in Alzheimer's Disease

Insight to physiology and 23 pathology of zinc(II) ions and their actions in breast and prostate carcinoma

The role of zinc in 26 antiviral immunity

Zinc and COVID-19: Basis of

The potential impact of zinc 2 supplementation on COVID-19 pathogenesis

The key role of zinc in elderly immunity: A 5 possible approach in the COVID-19 crisis

Micronutrient status of COVID-19 patients: a critical 8 consideration

Dexamethasone in Hospitalized Patients 14 with Covid-19 -Preliminary Report

Advance online publication

After 62 years of regulating immunity, 18 dexamethasone meets COVID-19

Alarmins and immunity

Antiviral activities of type I interferons to SARS-CoV-2 infection

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated

Antiviral activity of type I, II, and III interferons 11 counterbalances ACE2 inducibility and restricts SARS-CoV-2

Inhibition of SARS-CoV-2 by type I and type 15 III interferons

Type I 21 and Type III interferons restrict SARS-CoV-2 infection of human airway epithelial 22

Complex Immune 9

Dysregulation in COVID-19 Patients with Severe Respiratory Failure

Intravenous Anakinra for Macrophage Activation 12

Syndrome May Hold Lessons for Treatment of Cytokine Storm in the Setting of 13

Continuous Intravenous

Anakinra Infusion to Calm the Cytokine Storm in Macrophage Activation Syndrome

Should we stimulate or suppress immune responses 20 in COVID-19? Cytokine and anti-cytokine interventions

COVID-19: benefits and risks of passive immunotherapeutics

Immunological considerations for COVID-19 vaccine strategies

A systematic 29 review of SARS-CoV-2 vaccine candidates

Prospects for a safe 7 COVID-19 vaccine

Emerging Concepts 10 and Technologies in Vaccine Development

What defines an efficacious COVID-19 vaccine? A review of the challenges 14 assessing the clinical efficacy of vaccines against SARS-CoV-2

The race for a 17 COVID-19 vaccine: Current trials, novel technologies, and future directions

Vaccinomics and adversomics in the era 21 of precision medicine: A review based on HBV, MMR, HPV, and COVID-19 22 vaccines

Monitor for COVID-19 vaccine resistance 24 evolution during clinical trials

Why does drug resistance readily evolve but 27 vaccine resistance does not?

Complete Mapping of Mutations to the SARS-CoV-2

Spike Receptor-Binding Domain that Escape Antibody Recognition

Recombinant vaccines for COVID-19

Physiological resiliency in diving 8 mammals: Insights on hypoxia protection using the Krogh principle to understand 9 COVID-19 symptoms

Statins and SARS-CoV-2 12 disease: Current concepts and possible benefits

The use of renin-angiotensin-aldosterone system (RAAS) inhibitors is 16 associated with a lower risk of mortality in hypertensive COVID-19 patients: A 17 systematic review and meta-analysis

Immunomodulatory and antiviral activity of metformin and its potential implications 21 in treating Coronavirus Disease 2019 and lung injury

Italian Society for Vascular Investigation and the Italian Society of Vascular 25

Coagulopathy, thromboembolic complications, and the use of 26 heparin in COVID-19 pneumonia

The Impact of COVID-19 Disease on platelets 29 and coagulation

COVID-19 as part of the hyperferritinemic syndromes: the role of 56 1 iron depletion therapy

Hyperbaric oxygen therapy in preventing mechanical ventilation in COVID-19 5 patients: a retrospective case series

Mesenchymal stem cell 8 therapy for COVID-19: present or future

What are complementary 11 and integrative health approaches? NCCIH's Funding Priorities and Research Focus | 12 NCCIH (nih.gov) (Accessed on

AYUSH | National Health Portal of India (nhp.gov.in) (Accessed on

Principles and treatment strategies for the use of Chinese 16 herbal medicine in patients at different stages of coronavirus infection

Traditional herbal medicine candidates as complementary treatments for COVID-19: 20 A review of their mechanisms, pros and cons. Evid Based Complement Alternat Med

Public health 23 approach of ayurveda and yoga for COVID-19 prophylaxis

Immunity Against 26 COVID-19: Potential Role of Ayush Kwath

GUIDELINES for SIDDHA PRACTITIONERS for COVID 19. Ministry of AYUSH. 30 siddhaGuidlines_v4a.cdr (ayush.gov.in) (Accessed on

GUIDELINES for HOMOEOPATHIC PRACTITIONERS for COVID 19. Ministry 2 of AYUSH

Synergistic 5 immunomodulatory effect of complex polysaccharides from seven herbs and their 6 major active fractions

Insight into the molecular mechanism of a herbal injection by integrating 10 network pharmacology and in vitro

Bacteriophages could be a potential game changer in the 15 trajectory of coronavirus disease (COVID-19)

The brain and immune system prompt energy shortage in 18 chronic inflammation and ageing

Phylogeny of fever