key: cord-0953908-b5jlf6i6
authors: Kumar, Mukesh; Madan, Jitender; Sodhi, Rupinder Kaur; Singh, Shashi Bala; Katyal, Anju
title: Decoding the silent walk of COVID-19: Halting its spread using old bullets
date: 2020-10-19
journal: Biomed Pharmacother
DOI: 10.1016/j.biopha.2020.110891
sha: b42012920fa8b8794030a2dcd59e260cbcdf3de3
doc_id: 953908
cord_uid: b5jlf6i6

Severe acute respiratory syndrome (SARS) develops within 3-14 days when CoV2 invades epithelial, myeloid cells in the nasopharynx and pneumocytes in the respiratory tract through angiotensin converting enzyme (ACE2). Infection swiftly disseminates to gastrointestinal, cardiovascular, renal organs as well as immune system to deregulate their normal functioning through unique and distinct mechanisms. The health system and economy has been intensely thwarted by the rapid spread and exorbitant mortality caused by COVID-19 disease across the globe. The acute progression of the disease and high infection rate pose an enormous challenge for its therapeutic management and critical care. The viral structure, genome and proteome have been deciphered which yielded cues for targeting already available therapeutic entities. More than 200 compounds have been screened and till date approximately 69 therapeutic agents are undergoing clinical trials across the world. Among these, remedesivir (RMD), chloroquine (CQ), hydroxychloroquine (HCQ), noscapine (NOS) and heparin have demonstrated fairly promising results in preclinical and clinical studies. Recently, RMD has been approved by USFDA for the management of COVID 19. However, intense research is going on to screen and ace the ‘magic bullets’ for the management of SARS-CoV2 infection worldwide. The current review illustrates the plausible therapeutic targets in SARS-CoV2 important for inhibition of virus cycle. In addition, the role of RMD, CQ, HCQ, NOS and heparin in combating infection has been addressed. The importance of vitamin C and D supplements as adjunct therapies in the prevention of SARS-CoV2 virus infection have also been summarized.

Severe acute respiratory distress and immune system impairment following Severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) infection further escalate multi-organ failure in almost 5-20% cases. The disease can be grossly asymptomatic (80% asymptomatic carriers) or demonstrates mild to severe devastating effects in corona virus disease (COVID19) patients [1] . SARS-CoV-2 was initially traced on December 31, 2019 in Wuhan city, located in Hubei province of China. Since its spread to 215 countries, more than 31 million infection cases have been recorded worldwide with 965 K deaths till date. The catastrophic spread of disease has brought extreme personal, societal, economic, medical and industrial impact. COVID19 has forced the human civilizations to think, innovate, develop and prioritize all possible strategies to combat the infection in last 9 months. Therefore, World Health Organization (WHO) in coordination with global expert network is working rigorously to analyze the available epidemiological data, clinical histories and devising treatment strategies to form guidelines for controlling the infection.

SARS-CoV-2, the etiological agent for COVID19 pandemic was initially isolated from a Chinese patient and its genome sequence was provided to researcher by January 7, 2020 [2] .

It suggested that SARS-CoV2 is an enveloped positive sense RNA virus belonging to βcoronavirus subfamily. Bats have been found to be the potential primary source of SARS-CoV2 along with some of the bat predators which express virus receptors on their respiratory cells and serve as intermediate/reservoir host for virus transmission. It has been indicated that J o u r n a l P r e -p r o o f the first human transmission of novel corona virus was indicated from Pangolin, the key reservoir of CoV_RaTG13 [3] . Interestingly, SARS-CoV2 and CoV_RaTG13 had identical genomic regions owing to 96 .2% homology w.r.t Spike (S) protein [4] [5] [6] [7] . Previously, bats were also identified to be the natural reservoirs for a wide variety of COVs including SARS-COV and Middle East respiratory syndrome coronavirus (MERS-COV) [8] [9] [10] . The origin of current pandemic of COVID-19 has been traced in a Chinese sea food market located in Wuhan city [11] . Owing to the high stability of SARS-CoV-2 viruses in air/fomites as well as on inanimate surfaces and human bodies, the probability of transmission is high either through inhalation, inadvertent contact with body fluids or other contaminated sources [12] [13] . Respiratory air droplets and fomites are the main mode of human-to-human transmission. The virus can be present in aerosol for 3 h and infect upto 10 meters in closed rooms [12] .

The best clinical management strategies employed in the hospital settings across the world could not show decrement in the mortality rate. In a desperate need to manage the disease repurposing of clinically approved drugs is the sole option available with clinic's armory at present. Moreover, the tested drug combinations have demonstrated mixed non-uniform results in the affected patients. Therefore, a need to extend the repertoire of drugs or adjunct therapeutics to deal with COVID19 is warranted. A number of reviews have already been published on repurposing of drugs to combat SARS-COV-2; however, none of them have [13] . Upon entry into human host, the virus primarily infects the epithelial cells of salivary gland, upper respiratory tract and then further transgresses to infect the lower respiratory tract, liver, kidney, heart etc. The entry of virus in the mammalian cells is maneuvered by a coordinated interplay between viral proteins, recipient cell membrane proteins and the cellular proteases.

Angiotension converting enzyme 2 (ACE2) present on type I and type II pneumocytes towards the apical surface acts as receptor for attachment of SARS-CoV2 virus [14] [15] . The viral spike protein (S protein) performs the pivotal role of binding to the extracellular domain of ACE2 [16] . The peptidase domain in ACE2 and S1 domain in S protein are essential for mediating direct interaction (Figure 1 ). S2 domain adjoining S1 in the native S protein masks ACE2 binding region of S1. Consequently, proteolytic cleavage between S1 and S2 is prerequisite for liberating S1 domain to facilitate its interaction with the peptidase domain of ACE2, while S2 domain still remains attached to the complex in a pre-fusion state.

Interestingly, SARS-CoV2 has been found to acquire a unique cleavage site between S1 and S2 which is recognized by the host furin protease [17] . Widespread expression of furin protease in various tissues [18] [19] is proposed to be a key factor in conferring tissue specific viral tropism. The interference in the viral attachment machinery through development/identification of lead molecules which prominently target the S1 domain and ACE2 peptidase domain or their interaction, can be a wise pharmaceutical approach to prevent SARS-CoV2 infection. SARS-CoV2 interaction with ACE2 results in internalization of the viral-ACE2 complex through receptor mediated endocytosis. The internalization of the virus can also take place alternatively/additionally through yet to be deciphered process, independent of clathrin and caveolin (Figure 2 ) [20] . Whether such process is active in J o u r n a l P r e -p r o o f SARS-CoV2 cellular entry is still a matter of debate. Further, during the sojourn of virus laden vesicle prior to its envelope fusion with endosome, the virus primes itself and acquires the functionality to exit the endosome. In the process, TMPRSS2, a trans-membrane host serine-protease trims S2 domain of spike protein [21] . An additional cleavage in S2 domain is facilitated by Cathepsin-L in the endosomal compartment which finally unmasks the endosomal membrane fusion domain located in S2 to mediate its endosomal escape [22] .

Although no study has been conducted so far for SARS-CoV2 entry through this process, but evolutionary conservation with SARS-CoV signifies the existence of similar mechanism. Therefore, inhibition of TMPRSS2 and Cathepsin-L may also be vital therapeutic targets to halt endosomal escape of SARS-CoV2.

Endosomal escape results in release of viral RNA in the cytoplasm, where N protein can also be a key mediator in preventing the activation of antiviral response of host cells. This feature is embodied in the RNA binding properties of N protein. 200-400 copies of N protein are found to adorn the complete RNA genome of the virus. It serves as primary barrier in preventing interaction of the 'exogenous RNA' sensing mechanism of the host cells. One such mechanism is Dicer-RISC mediated anti-viral defense. In eukaryotic cells, under normal healthy conditions, the entry of the foreign double stranded RNA (dsRNA) triggers its recognition by Dicer which processes it to 20-25 bp dsRNAs by cleavage phenomenon [23] .

The thermodynamically unstable strand of this 20-25bp dsRNA, then gets incorporated in RISC complex [24] . Using this strand, RISC complex recognizes the invading RNA by complementary base pairing and performs a cleavage reaction in the foreign RNA to render it non-functional. In case of SARS-COV, N protein is found to possess unique functionality of preventing action of DICER-RISC machinery through direct binding to their substrate at the stage of pre-DICER or post DICER processing stage [25] . In light of the established facts, pharmacological interventions interfering with N protein-RNA genome interaction thus may J o u r n a l P r e -p r o o f serve as important method to increase the susceptibility of RNA genome for recognition by RNA sensing machinery to mediate its clearance.

The downstream viral replication process is tightly regulated by complex interplay between viral machinery and the host cell proteome. The process is mediated through formation of a transcription complex constituted mainly by NSP1-16 associated with proteolytic digests of polyprotein complexes (pp1a, pp1ab). The proteolytic digestion of these polyprotein complexes takes place through two proteases viz. main protease (M pro , also called as 3CL pro ) and papain like protease (PP pro ) [26, 27] . Unique mutations gained by SARS-CoV2 have been found to enhance the catalytic efficiency of M pro [28] . This can be one of the contributing factors in accentuating the viral pathogenicity. Further to initiate negative RNA strand synthesis, NSP12 (RNA dependent RNA polymerase, RdRP) utilizes its intrinsic polymerase activity and recruits various regulatory factors for this process. RdRP interaction with NSP7 and NSP8 enhance its polymerase activity [29] . The helicase activity of NSP13 required for RNA synthesis also gets pronounced upon association with RdRP [30] . Moreover, NSP10 interaction with NSP14 and NSP16 has been found to be an essential event for activating NSP14 exoribonuclease activity and NSP-16 Mtase (methyltransferase) activity which are important determinants of viral RNA synthesis [31] . Other accessory mediators such as NSP1, NSP2, NSP4, NSP6, NSP9, and NSP11 are generally not essential for virus viability but aid in the viral replication/transcription process [32, 33] . Therefore, targeting the essential machinery of viral replication is an interesting avenue to combat COVID-19 spread.

The cumulative response of the replication/transcription complex results in production of a large RNA genome (~30 kb) as well as synthesis of sub-genomic RNA (sgRNA). sgRNA then gets translated to yield various structural and non-structural proteins for virion packaging. The synthesized protein further undergoes trafficking along the boundaries of cytosol to their targeted destination. Microtubule based transport system can play a key role J o u r n a l P r e -p r o o f in facilitating the directional trafficking of newly synthesized viral proteins; hence any defect in such transportation may compromise the virus assembly processes [34] . A similar observation has been noticed in case of porcine transmissible gastroenteritis virus (TGEV). It is reported that upon treatment with a β-tubulin depolymerising agent (Nocadazole), the S protein got dispersed throughout the cytosol. The scattered distribution of S protein was further correlated with decreased incorporation of S protein in virion and subsequently a low viral titer in nocadazole treated cells [35] . If S protein of SARS-CoV2 follows identical pattern, the pharmaceutical interventions targeting tubulin polymerization may interfere with viral assembly thereby decreasing the viral load. Curtailing S-protein incorporation in virion can also decrease its binding probability on host cells and thus can help in decreasing its pathogenicity.

The synthesized protein further undergoes processing in endoplasmic reticulum and golgi compartments. S-glycosylation of the protein is one such modification that can regulate protease accessibility as well as modify their functions. Seven glycosylation sites have been found to be located in ACE2 extracellular domain [16] . Although, inhibition of glycosylation machinery did not facilitate enhanced viral clearance but it could decrease the viral load [36] .

Thus targeting glycosylation machinery can also be another avenue to exploit for increasing the efficacy of the pharmaceuticals. Furthermore, the unique properties of individual viral protein/s or their subdomains may play an essential function in posttranslational processing in addition to their classical role in viral assembly. In case of SARS-COV, the carboxy end domain of N protein (Residues 366-422) mediates interaction with TRIM25 and thus prevents its further interaction with RIG-1. This leads to inhibition of TRIM25 mediated ubiquitination of RIG-1, which prevents downstream activation of RIG-1, associated type-1 interferon response [37] [38] . In case of SARS-CoV2, a 78.33% protein level homology in this interacting domain suggests retention of the same anti-viral response. The effectiveness of the J o u r n a l P r e -p r o o f IFN based therapies in combating the COVID-19 disease further substantiate the possible involvement of SARS-CoV2 N protein in down-regulating type I IFN response in the affected patients [39] . Therefore, pharmaceutical strategies that can induce IFN response can be helpful in management of COVID19.The viral proteins become ready to be packaged in a complete virion following completion of post-translational processing. The trans-golgi intermediate compartment (ERGIC) was found to be an imperative site for the virus assembly and budding [40, 41] . At this site RNA genome complexation occurs with N protein which further interacts with other structural proteins of SARS-CoV2. Among them, the interaction of cytoplamic domain of M protein with S protein is quite important for RNA genome encapsulation in the virion. Further interaction of M protein with N protein through carboxy terminal domain also aids in its assembly into virion structure [42, 43] .

Another structural protein that plays an essential function is envelope protein (E). E protein forms homoligomeric structures, in which homopentameric structure seems to have the property to behave as mild cation (Ca 2+ , K + , Na + ) selective ion channel [44] [45] [46] . The formation of such cation selective ion channel in the host plasma membrane, Trans ER-Golgi complex (ERGIC) can induce flux of ions across the membranes and thus can alter the ionic balance of the cellular and sub-cellular compartments. The Ca 2+ ionic imbalance across the ERGIC due to ion channel activity of E protein results in activation of NLRP3 inflammasome mediated IL-1β production [46] . Furthermore, E protein expression has been found to be associated with increase in NF-κβ stimulatory pathway [47] . Therefore, E protein seems to act as a mediator resulting in the exacerbated inflammatory responses through NF-κβ and IL-1β thus aiding in increased virus infectivity. The virus lacking E protein has been found to be severely compromised in replication. Therefore ,the association of E protein expression not only relates to viral pathogenesis but also to its unique function to alter the ionic composition of sub-cellular compartments of lysosome and endoplamic reticulum which in J o u r n a l P r e -p r o o f turn can correlate well with the change in the cellular pH. Therefore, it is likely that E protein also alters the pH of cellular compartment to a level that is conducive for viral assembly. Additionally, a low pH of the endosomal compartments is required for resident protease functionality and dissociation of virus-receptor complexes [48] . Therefore, pharmacological strategies modulating the pH of these sub-cellular compartments can provide unique advantages to not only prevent viral endosomal escape but can also help to alter viral packaging in ERGIC.

Finally, assembled virus particles exit from the primary infected cells through exocytosis, enter interstitial fluid, infect the cells in the vicinity and then transiently enter the systemic circulation. The virus may undergo further maturation in tissue fluid or blood through a novel mechanism that may increase its infectivity. One such role is thought to be played by the blood kallikerin system. Kallikerin 13 (KL13), the component of kallikerin system is found to play an essential role in coronovirus (HCoV-HKU1) priming by mediating cleavage at S1/S2 boundary [49] . Similar involvement of host KL13 in SARS-CoV2 priming is a matter of investigation. Normally, kallikerin acts on kininogens (High molecular weight kininogens, HMWK and Low molecular weight kininogens, LMWK) directing the synthesis of bradykinin which in turn exerts its vasodilatory response through bradykinin receptor 2 (B2).

Excess of the bradykinin is associated with blood vessels leakage, tissue swelling and inflammation. In case of COVID-19, hypofunction of ACE2 dependent negative regulatory loop may further cause stimulation of bradykinin dependent downstream signalling [50] [51] .

A significant homology of bradykinin motif (GFSPFRSSRIG) has been found in SARS-COV S protein whose role in the viral pathogenesis is still undefined. A probable role of bradykinin in aiding the viral spread can be assumed since the antibody response to SARS-CoV S protein is found to cross-react with bradykinin protein [52] . Therefore, it is probable that antibody response generated against S protein motif during SARS-CoV2 infection might be J o u r n a l P r e -p r o o f neutralized by bradykinin in the cellular system. This may aid in viral evasion manoeuvring during cellular spread. Thus, any alteration in kallikerin system functionality can influence the SARS-CoV2 propagation and can amplify the inflammatory responses. Consequently, pharmacological interventions targeting KL13 or its associated molecular signalling can be effective in dealing the current COVID-19 crisis.

Further propagation of the viral spread is associated with hypercoagulative response in severe cases of COVID-19 as indicated by an increased level of D-dimer (>1µg/ml) [53] . The increase in D-dimer level does not correlate with thrombolysis observed in COVID patients at late stages therefore; mechanism that might be responsible for activation of this coagulative response needs to be explored further. Interestingly, dipyridamole, a FDA approved anti-coagulant was effective in the functional recovery of few severe COVID-19

cases. Other than anti-coagulation properties, its proposed interaction with M pro protease of SARS-CoV2 may also be responsible for curative effects observed in affected patients [54] .

Moreover, heparin was also found to possess affinity for S1 domain of S protein [55] , its use can not only interfere with the viral spread but also prevent the hypercoagulation response.

Thus, any intervention having the ability to restore the homeostasis of the coagulative machinery may act as a supportive therapy for COVID patients.

The desperate need of therapeutic entities for COVID19 has drawn the attention of scientists and clinicians world over. Since vaccine development for SARS-CoV2 is still in pipeline, the repurposing of drugs already in the armor is the best plausible approach. A plethora of anti- patients is presented in Table 1 .

A number of antiviral nucleotide analogues such as favipir (FAV), ribavirin and remdesivir (RMD) have gained importance for treatment of SARS-CoV2 and undergoing clinical trials.

FAV (originally designed for swine flu) has RNA-dependent RNA polymerase activity (RdRp) against nCoV2, has been recommended strictly for emergency use in china as it has teratogenic potential [56] .

Similarly, ribavirin is being tried in combination with other antiviral agents [57] . RMD an investigational nucleotide analog prodrug, originally developed by Gilead against Ebola virus, effectively inhibits viral RNA-dependent RNA polymerase (RdRp) activity (Figure 3) .

There was no consensus data for safety, adverse events and its effect on viral loads or dosedosage regimens. Its Phase I and II studies have been completed recently [58] [59] RMD was first used for the treatment of COVID19 patient in United States in January 2020.

Thereafter, Gilead in collaboration with Chinese partners (since February 2020) conducted phase 3 randomized, double-blinded, placebo-controlled, multicenter trials to determine the safety and efficacy of RMD in hospitalized patients with severe COVID-19 [58] . The treatment was designed as 200 mg of RMD on day 1 followed by 100 mg/day for either 5 or 10 days in two separate trials (NCT04257656 &NCT04252664). A double-blind, randomized, placebo-controlled trial of intravenous RMD administered to hospitalized adult COVID-19 patients with the evidence of lower respiratory tract involvement was performed.

Patients were randomly assigned to receive either RMD (200 mg loading dose on day 1, followed by 100 mg daily for up to 9 additional days) or placebo for up to 10 days. RMD was superior to placebo in shortening the time to recovery in this group of COVID-19 patients with comparatively reduced infection load in lower respiratory tract [60] .

The clinical trial indicated 62% recovery of the patients treated early as compared to 49% of the patients who were treated late during the infection course. Gilead is scaling up production of RMD but its wide usage to treat COVID19 patients in future will depend on its safe use in co-morbid patients, adverse effects and the treatment regimens. Although RMD showed promising results against SARS-CoV2 with acceptable side-effects, scientific fraternity is still waiting for the results of clinical trial (NCT04252664).

Chloroquine, CQ and hydroxychloroquine, HCQ (Figure 3 ) have been used extensively in the treatment of malaria for more than 70 years. CQ/HCQ reduces the infection load by J o u r n a l P r e -p r o o f blocking the entry of viruses into mammalian cell systems [61] . Recently, it has been described to be effective in reducing the viral loads in COVID-19 patients. The virus enters the intracellular milieu owing to receptor mediated endocytosis. Once in the endosome, the viral particles experience a drop in pH which in turn facilitates release of viral genome into the cytoplasm. However, treatment of infected cells with CQ raises the endosomal pH which prevents viral release and blocks viral propagation [62, 63] . The reproduction cycle/s of a number of viruses are known to be inhibited by CQ [64] [65] . SARS-CoV-1 reproduction cycle was found to be inhibited following CQ treatment in vitro [66] [67] . On similar lines, HCQ Therefore, combination of HCQ with azithromycin was found to be more effective in reducing viral load than HCQ alone [68] . It has been found that the primary action of HCQ or CQ is because of their ability to increase the pH of the vesicles from pH 4 to 6. In its basic form, HCQ can readily pass through the plasma membrane and get accumulated in the endocytic vesicles. Due to low pH of the vesicles, it creates suitable environment for protonation of the basic form. In its protonated form (B + H + ), HCQ becomes polar and hence its ability to cross the plasma membrane decreases. Owing to this event, HCQ concentration in the vesicle continuously accumulates until a steady state concentration is achieved accompanied by rise in pH of the vesicles by 1 to 2 pH units. Under such modified conditions, the functionality of the vesicles' acid proteases gets altered which may lead to a variety of consequences. Therefore, the efficacy of HCQ/CQ in treatment the COVID-19 cases can be mechanistically related to rise in vesicular pH. Support for this can be derived J o u r n a l P r e -p r o o f from the essential role played by the TMPRSS2 and Cathepsin-L in the escape of the SARS-COV from the endosome into the cytoplasm [16] . SARS-CoV2 also requires TMPRSS2 for its pathogenicity, hence similar mechanism might be utilised for its endosomal escape.

Therefore, HCQ mediated alteration in endosome pH might be the key interfering factor for SARS-CoV2 endosome escape pathways which needs further elucidation. A recent study has identified the endoplasmic reticulum and Golgi apparatus as site for the glycosylation of SARS-CoV2 S-protein [68] . The glycosylation event is thought to be essential for virus attachment to ACE2 and its priming in the endosome for the membrane fusion required for its cytoplasmic entry. Inhibition of the ACE2 glycosylation by CQ can successfully decrease the SARS CoV2 viral load similar to an earlier observation in case of SARS CoV infection [66] .

CQ treatment may modulate ACE2 and S protein interaction that ultimately interferes with virus internalization and virus spread. Efficacy of the HCQ in treating COVID patients at the secondary stage of infection involving brain manifestations is still questionable. Since, HCQ can cross the blood brain barrier, therefore, its usage can also be extended to the patient's diagnose with the neurotransmission of the SARS-CoV2. The classical role of CQ and HCQ in inhibiting the reproduction cycle of the virus to its wide usage in Systemic lupus erythematosus (SLE) and other autoimmune diseases suggests a multipronged effect of CQ/HCQ on COVID-19 patients [69] . CQ can also inhibit complement system which has not been taken into consideration in case of COVID-19 infections. A previous study has also indicated its effect on complement pathway inhibition [70] . Therefore additives have also been recommended to improve the efficacy of CQ and HCQ against SARS-CoV2. HCQ could be considered for the patients who cannot be administered RMD or when RMD is not available but with caution [71] . In conclusion, based on the results of ongoing /completed (Table 2 and 3) clinical trials (151 trials) stringent parameters need to be considered to further support the usage of CQ and HCQ in COVID-19 patients. Our laboratory is actively engaged in elucidating the molecular pathways of therapeutic modalities used in malaria [62, [112] [113] [114] [115] . The effective nanoformulations of HCQ/CQ can help in reducing the side-effects of these wonder cost effective drugs for use in future dosedosage regimens for COVID-19.

NOS, (Figure 3 ) an opium alkaloid classically used as antitussive agent has been repositioned for its anti-tumor effect as it strongly binds to tubulin, arrests the cells in metaphase, induces apoptosis in the dividing human cells and has the advantage of large therapeutic window [116] [117] [118] [119] [120] [121] [122] . The antitussive effect of NOS is documented through suppression of bradykinin production or inhibition of its function through inhibition of B2 receptor thus preventing bronchoconstriction, mucous production and histamine release [123] [124] . Bradykinin release has already been associated with concomitant increase in intracellular calcium and activation of cAMP signaling pathway to down regulate the protein abundance and TJ localization of Claudin 5, occludin and ZO-1. It is well documented that increased level of bradykinin is associated with inflammation and is a putative reason for dry cough response observed in patients [124] . Such imbalances in the bradykinin cellular level might be attributed to alteration in the functionality of its regulatory loop. ACE2 is a part of one such regulatory axis that reduces bradykinin's downstream metabolite viz. des-

Arg bradykinin (DABK) by mediating its cleavage under normal cellular conditions [125] . A decrease in ACE2 expression has also been associated with increased DABK signaling. Thus, any alteration in ACE2 functionality can flare up bradykinin-DABK signaling axis associated responses [126] . Surprisingly, in COVID-19, a similar decrease in ACE2 functionality in infected cells has been observed that may be attributed to ACE2 shedding during virus spread [127] . Thus, subsequent loss of DABK regulatory loop may increase DABK cellular functionality and result in a pronounced bradykinin-DABK signaling associated inflammatory responses. Therefore, regulation of bradykinin signaling may be essential components of pharmaceuticals formulation to counter SARS-CoV2 virulence and pathogenesis.

Bradykinin, a nine aminoacid peptide is produced from HWMK when cleaved by plasma kallikrein. Any interruption in this process can result in alteration in bradykinin production in the cellular system. In lines with this, an interesting observation regarding NOS binding to HMWK has been speculated (Unpublished lab data). Formation of such NOS-HMWK complex may thus interfere with its processing through KL13 axis to produce bradykinin and DABK. Furthermore, HMWK also hinders contact activation of Factor XII [128] which is vital for hydrolysis of pre-kallikerin to product KL [129] . Therefore, NOS induced inhibition of HMWK processing into bradykinin might raise the physiological level of HMWK. It can be expected to decrease Factor-XII activation and which shall subsequently lead to defect in KL synthesis from prekallikerin, hence decreasing bradykinin synthesis. The restrained factor XII activation will decrease the coagulation responses and thus may be beneficial in improving the conditions of COVID-19 patients. Thus, NOS action in modulating the bradykinin-DABK may be an important factor for its success in dealing with SARS-CoV2 pathogenesis.

Additionally, the binding affinity of NOS and its analogues with protease of COVID-19 complex has recently been evaluated through MD simulations at different temperatures. It has been speculated NOS may exert its antiviral effects by inhibiting viral protein synthesis [130] . Moreover, the well-versed role of NOS to disturb microtubule dynamics (anti-tubulin polymerisation activity) might also serve as important mode for meddling with the virus assembly [131] . Therefore, we propose that NOS may disperse S protein of SARS-CoV2 throughout the cytosol by interfering with the tubulin associated S protein transport. This event may attenuate incorporation of S protein in virion that can subsequently interfere with viral assembly and eventually viral load may decrease. Bradykinin is an important participant in thrombotic, inflammatory and renin angiotensin system. NOS has a substantial direct /indirect effect on all three systems in addition to its effect on microtubule dynamics and viral spread. Therefore, NOS can be an important drug candidate to combat COVID-19

for prophylactic and therapeutic interventions in near future.

Heparin is a polydispersible, heterogenous natural product widely used as blood thinner.

Unfractionated heparin, low molecular weight heparins and heparinoids are commonly employed clinically as anticoagulants due to excellent safety, bioavailability, stability and biopharmaceutical attributes. Several heparin analogues possessing antiviral activity are in clinical trials for coronaviridae and SARS associated coronavirus strain HSR1 [132] , flaviviruses [133] , herpes [123, 134] , influenza [135] and HIV [136] [137] . Furthermore, heparan sulfate (HS), a glycosaminoglycan (GAG) member, binds to CoV surface proteins used by coronavirus for its attachment to target cells. Hence, binding of SARS-CoV-2 S1 RBD to heparin was studied using spectroscopic techniques and molecular modeling. A significant structural alteration is induced by heparin upon binding to SARS-CoV-2 S1 RBD.

Further, heparin binding domains (moieties of basic amino acid residues) are solvent J o u r n a l P r e -p r o o f accessible on the SARS-CoV-2 S1 RBD surface and constitute a nonstop patch for convenient binding to heparin. Heparin is reported to inhibit SARS-associated coronavirus strain HSR1 cell invasion [133] and this supports the utilization of pharmaceutical heparins against SARS-associated coronavirus. Surface plasma resonance (SPR) studies elucidated that SARS-CoV-2 spike protein binds with higher affinity to heparin (KD~55 nM) as compared to the receptor binding domain (RBD, KD~1 µM) alone. An octasaccharide composed of IdoA2S-GlcNS6S could inhibit spike-heparin interaction with an IC50 of 38 nM [138] .

Therefore, heparin may prove beneficial in patients with COVID-19, however; accurate adjustment of heparin dose is a matter of concern for this drug. In an uncontrolled retrospective study, Tang and colleagues recently described that LMWH diminished mortality in patients with COVID-19 with coagulopathy when compared to those who did not receive heparin. Although the molecular mechanism for this benefit is not completely understood, it seems likely that the anti-inflammatory effects of heparin are contributory.

However, the most efficacious ratio of anticoagulant to anti-inflammatory activity of heparin is still to be determined [139] [140] .

Vitamin C modulates the susceptibility to a variety of bacterial and viral infections, influences the immune system and at high concentrations was found to protect lung epithelium [141] [142] . Owing to virus internalization and spread, the cytokine cascade is activated, and infilterating neutrophils in the lungs destroy alveolar capillaries. Vitamin C indirectly regulates the alveolar fluid accumulation by attenuating the activation and prevention of neutrophils infilteration. In addition, it is postulated that vitamin C prevents the formation of neutrophil extracellular traps, a biological event of neutrophil associated J o u r n a l P r e -p r o o f vascular injury. Therefore, vitamin C should be investigated under controlled randomized clinical trials for the prevention of viral pneumonia in COVID-19 [143] .

Apart from vitamin C, vitamin D supplementation is a useful approach to reduce risk by inducing cathelicidins and defensins. Vitamin D lowers viral replication rates and also controls the pro-inflammatory cytokines production responsible for lung injuries.

Observational studies and clinical trials have reported that vitamin D supplementation can manage the risk of influenza [144] . The 1,25-dihydroxyvitamin D, an active metabolite of vitamin D exhibited the inhibitory effect in human nasal epithelial cells infected with SARS-CoV-2 [145] .

Middle East respiratory syndrome coronavirus (MERS-CoV) employed its S proteins as an adhesion molecule to augment host entry through a receptor called dipeptidyl peptidase-4 (DPP4). Dipeptidyl peptidase-4 (DPP4) is considered a key player in the transmission of signals and triggering of innate and adaptive immune responses. Hence, designing monoclonal antibodies may be a great and prolific approach as compared to vaccine development to exhibit the immediate protective effect. It has been demonstrated that m336, a human monoclonal antibody extracted from the phage display library binds to the receptorbinding region of S protein of MES coronavirus and exhibits strong neutralization intensity to MERS-CoV [146] . Furthermore, m336 also decreases the viral RNA titer in lungs by 40,000-90,000 folds [147] . Injections of monoclonal antibody m336 or hyperimmune plasma against MERS-CoV when administered to infected monkeys resulted in considerable reduction in symptoms of clinical disease however; viral load in lungs was reduced considerably only in hyperimmune plasma group. Both hyperimmune plasma and m336 therapy exhibited promising results in terms of mitigating the symptoms however; none of them completely prevented them [148] . H Mab m336 significantly decreased the viral RNA titers in addition J o u r n a l P r e -p r o o f to viral-associated pathological alterations in lung tissue of rabbit [149] . S nanoparticles in mice yielded high-level neutralizing antibodies against homologous viruses [150] . Similar to the concept of using neutralising Ab, a plasma therapy (using plasma of recovered COVID-19 patients) based approach is also gaining momentum and is already approved for treating COVID-19 patients in some cases [151] .

The exponential surge in COVID-19 cases has put forth numerous challenges for every affected nation but has also demonstrated some promises. The knowledge gained by the scientific community during first SARS-COV pandemic in 2003 has been quite useful to deal with the current pandemic. RMD, an antiviral drug previously developed for Ebola disease has shown a great promise in treating COVID-19 patients. Although approximately 2/3 of patients treated with this drug were cured, well designed studies are required to confirm its suitability in management of COVID-19. Repeated use of RMD may lead to development of resistance in SARS-CoV2 strain which can be of prime concern in near future. HCQ is already being recommended for clinical use in selective patients. However the side-effects associated with its use are alarming and require further investigation. This creates further opportunities to identify additional therapeutic approaches that can be helpful in such scenarios. As proposed, NOS is one such drug which can be effective in managing the viral spread. Additionally, combinatorial approaches can be of use to decrease the toxicity related issues. Most importantly, to prevent the viral spread, immunotherapy options are also depicting great potential. However, one of biggest problem associated with this is the rapidly mutating nature of the virus. It can be seen that the virus is rapidly evolving and mutating at an unprecedented rate. This may render the vaccination approaches non-functional in near future. Therefore, alternative therapeutic strategies in addition to the classical drug modalities will play important role to deal resurgence of epidemic in times to come. 

None of the authors has conflict of interest with this submission. SARS-CoV2 enters the cell using its glycosylated S protein and host ACE2 interaction, its voyage in endocytic vesicle, endocytic priming and the endocytic escape occurs through a well orchestrated maneuvering of host enzymes followed by smart use of host transcriptional machinery and post translational mechanisms for a complete virus assembly. Finally the externalization of viral particles to enter the cells in the vicinity is shown as graphics. LOP-RTO+ Atomized INF α-2b and thymosin α1

No severe cases was seen and the clinical symptoms (such as fever, cough) were quickly improved, and the curative effect was remarkable [94] LOP-RTO, ARB, LOP-RTO+ ARB Insignificant difference in viral nucleic acid negative conversion rate, symptom improvement rate and CT improvement rate was observed between test group and control group, but deterioration rate was higher in treatment group

Test groups have shown adverse reactions than the control group.

[95]

LOP-RTO M o r t a l i t y, clinical improvement duration and virus negative conversion rate andbetween the test group and the control group was not significantly different Adverse drug rections were common among trial group [96] LOP-RTO Virus negative rate was shorter than control group [97] LOP-RTO Effectiveness rate was higher in test group as compare to control group.

[98]

LOP-RTO Stability of body temperature and blood biochemistry profile was better in experimental groups as compate to control groupThe changes of body temperature, blood routine and blood biochemistry in the experimental group were better than those in the control group Adverse drug reactions were not observed [99] LOP-RTO Moderate drug reactions occurred in 29 cases (72.5%), 2 or more adverse reactions occurred [100] J o u r n a l P r e -p r o o f in 13 cases (32.5%), and severe reactions were seen in 4 cases (10%). Elevated triglyceride (50%), nausea (17.5%), diarrhoea (17.5%) were recorded as adverse drug reactions. Female and male groups were indistinguishable for adverse drug reactions. LOP-RTO Diarrhea as adverse drug reactions increased to 45.45% tested patients. 14 cases were cured and 1 case continued to be observed.

[101]

LOP-RTO Five patients among 7 tested had shown adverse drug reaction i.e abnormal liver function, diarrhea, decreased heart rate, hypertriglyceridemia etc.

[102]

LOP-RTO No difference in virus negative rate was seen between test and control group.

[103]

Favipiravir+ INF-α Virus clearance rate was higher in test group than control group A low level of adverse drug reactions were seen in test group [104] ARB+INF-α The absorption rate of pneumonia in the test group was faster than that in the control group [105] ARB+ Diammonium Glycyrrhizinate Symptoms, lymphocytes, IL-6, albumin content and ALT were improved, and the overall cure rate and overall effective rate were higher.

ARB combined with diammonium glycyrrhizinate in the treatment of COVID-19 has less adverse reactions and high safety.

[106]

ARB+ LOP-RTO CT improvement rate and virus negative conversion rate was faster in test group [107] ARB A lower infection rate was seen in test group [108] J o u r n a l P r e -p r o o f

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Abbreviation: RMD: Remdesivir; CQ: Chloroquine; HCQ: Hydroxychloroquine; Arbidol: ARB; Azithromycin: AZT; Lopinavir: LOP; Ritonavir: RTO

Favipiravir: FAV; QTc: corrected QT-interval

Coronavirus Disease

We 

RMD Oxygen therapy requirements proportion was decreased in patients.[75]HCQ+AZT Good clinical results and low mortality was seen in patients [76] HCQ+AZT Hospitalization time got lowered [77] HCQ+AZT QTc interval was prolonged especially in high transaminase level group [78] HCQ+AZT/ HCQ alone Insignificant difference in the mortality was seen in test v/s control group [79] HCQ+ AZT/ HCQ alone QTc prolongation was severe in 12% of the patients treated with combination therapywhich was higher than that caused by any single drug [80] CQ, HCQ, AZT, LOP-RTO Adverse cardiac reactions in 103 cases were reported to be related only to HCQ (86%) or to AZT (60% No adverse reactions seen [109] ARB + Lianhuaqingwen capsules Body temperature recovery time improved, respiratory symptoms were cured, inflammation was lowered and hospitalisation time got shortened Nausea, diarrhoea and dizziness were not seen in all cases [110] ARB An effective rate of 75% was recorded. It was significantly effective in 45%, effective in 30%) and was ineffective in 25%