key: cord-1036947-1h0oncwn
authors: Niemeyer, Brian F.; Benam, Kambez H.
title: Untapping host-targeting cross-protective efficacy of anticoagulants against SARS-CoV-2
date: 2021-10-28
journal: Pharmacol Ther
DOI: 10.1016/j.pharmthera.2021.108027
sha: 3b1bd32c68dcdf0d0d9992978414a654a9303926
doc_id: 1036947
cord_uid: 1h0oncwn

Responding quickly to emerging respiratory viruses, such as SARS-CoV-2 the causative agent of coronavirus disease 2019 (COVID-19) pandemic, is essential to stop uncontrolled spread of these pathogens and mitigate their socio-economic impact globally. This can be achieved through drug repurposing, which tackles inherent time- and resource-consuming processes associated with conventional drug discovery and development. In this review, we examine key preclinical and clinical therapeutic and prophylactic approaches that have been applied for treatment of SARS-CoV-2 infection. We break these strategies down into virus- versus host-targeting and discuss their reported efficacy, advantages, and disadvantages. Importantly, we highlight emerging evidence on application of host serine protease-inhibiting anticoagulants, such as nafamostat mesylate, as a potentially powerful therapy to inhibit virus activation and offer cross-protection against multiple strains of coronavirus, lower inflammatory response independent of its antiviral effect, and modulate clotting problems seen in COVID-19 pneumonia.

The continued emergence of respiratory viruses with pandemic potential highlights a dire need for evolved targeted antiviral therapies. Over the past 20 years zoonotic transmission has led to the appearance of highly pathogenic strains of coronaviruses (CoVs) in human populations, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and most recently severe acute respiratory syndrome coronavirus 2 (SARS-COV-2)the etiological agent of coronavirus disease 2019 . In a little over a year, SARS-CoV-2 alone has impacted tens of millions of people worldwide with a fatality rate of ~1-2% manifesting diversely ranging from asymptomatic infection to long-lasting respiratory impairment and organ failure (Chen, Liu, & Guo, 2020;  With the rise of drug-resistant microbes, there has been a growing interest and drive to identify alternative strategies to conventional antiviral therapeutics. One promising avenue has been targeting the host. Unlike pathogen-targeting antivirals, host-based therapies do not inhibit virus infection by targeting viral components directly, but rather block host factors that are either key for viruses to be infective or lead to unregulated, often excessive, immune activation. We describe both strategies below.

Unlike virus-directed therapies, targeting of host factors that are essential for virus activation may potentially lead to cross-protection against emerging and reemerging viruses, regardless of their strain, and reduce risk of drug-resistant variants formation (Dolgin, 2021). Most of high-and low-pathogenic strains of CoVs converge on the same class of host molecular machinery for virus entry, replication, and release (Wong & Damania, 2021) . For instance, SARS-CoV, MERS-CoV, and SARS-CoV-2 utilize host angiotensin-converting enzyme 2 (ACE2) as a receptor to attach to host cells (Flerlage, Boyd, Meliopoulos, Thomas, & Schultz-Cherry, 2021; Hatmal, et al., 2020) . By targeting host ACE2, rather than its ligand which is virus spike (S) proteinone of the key proteins covering surface envelop of coronaviruses, therapeutic proteases for S activation to be infective. The key host proteases suggested to mediate activation of CoV S are Type II Transmembrane Serine Proteases (TTSPs; TMPRSS), in particular TMPRSS2 (Bertram, et al., 2013; Glowacka, et al., 2011; Hoffmann, Kleine-Weber, et al., 2020; Iwata-Yoshikawa, et al., 2019; Matsuyama, et al., 2020) , although other surface bound proteases, such as furin, may also contribute to SARS-CoV-2 activation and entry (Tharappel, Samrat, Li, & Li, 2020; Wu, Zheng, et al., 2020) .

Bioinformatic analysis has revealed furin cleavage sites located in the S protein of SARS-CoV-2 (Wu, Zheng, et al., 2020) . Computational screening uncovered a library of 4,000 compounds with predicted interactions with furin, including the anti-parasitic drug diminazene (Wu, Zheng, et al., 2020) . In vitro studies have shown that diminazene inhibits furin with half maximal inhibitory concentrations (IC 50 ) of 5.42 ± 0.11 µM (Wu, Zheng, et al., 2020) . Given their importance in coronavirus infections, host proteases represent key druggable targets that can be exploited for therapeutic purposes (Tharappel, et al., 2020) .

Nafamostat mesylate is a low-molecular-weight synthetic broad-spectrum serine protease inhibitor that has been approved clinically as anticoagulant during hemodialysis and continuous renal replacement therapy as well as a treatment for pancreatitis in Japan and South Korea for over three decades (Choi, et al., 2015; Pak, et al., 1988; Park, et al., 2011; Yoo, et al., 2011) . It also has inhibitory effects on plasmin activity and plasminogen activators (Ji, Wagener, Ness, & Zhao, 2021; K. Okajima, Uchiba, & Murakami, 1995; Uchiba, Okajima, Abe, Okabe, & Takatsuki, 1994) . In a high-throughput evaluation of almost 1,000 clinically approved drugs in vitro, Yamamoto et al. identified nafamostat as a potent inhibitor of MERS-CoV S protein-mediated J o u r n a l P r e -p r o o f Journal Pre-proof membrane fusion using reporter cell lines. Moreover, the authors reported that camostat mesylatean earlier analogue of nafamostat, was also effective in blocking MERS-CoV S protein activation, although at much lower potency (IC 50 of 0.1 µM and 1 µM for nafamostat and camostat, respectively) (Yamamoto, et al., 2016) . With the emergence of SARS-CoV-2, several groups expanded upon these findings to demonstrate nafamostat and camostat are able to limit viral entry of SARS-CoV-2 using vesicular stomatitis virus (VSV) pseudotyped virus expressing SARS-CoV-2 spike protein in human Calu-3 lung cell line (Hoffmann, et al., 2021; Hoffmann, Schroeder, et al., 2020; Yamamoto, et al., 2020) 

CoV-2 infection model that utilizes polarized mucociliated primary human bronchiolar epithelia (Niemeyer, et al., 2021) . Using this model system and authentic viral particles (rather than pseudotyped virions) for infection, they observed that nafamostat inhibits apical virus shedding from infected epithelia reconstituted using cells derived from healthy non-smokers, smokers and subjects with chronic obstructive pulmonary disease (COPD; a COVID-19 comorbidity). Importantly, Niemeyer and colleagues revealed nafamostat has antiviral-independent anti-inflammatory properties by lowering homeostatic secretion of pro-inflammatory cytokines from human airway epithelia in the absence of viral challenge, and that this compound exhibits considerable antiviral efficacy against two seasonal human coronaviruses (hCoV-229E and hCoV-NL63) (Niemeyer, et al., 2021) . These findings illustrate cross-protective and multi-beneficial effects of (preclinical) application of nafamostat as a prime example of host-targeting strategy in treatment of SARS-CoV-2 and other coronaviral infections.

From a clinical perspective, two major concerns may arise when considering administration of nafamostat for COVID-19: its safety profile, and its undesired anticoagulatory and off-target effects. Specifically, one may argue nafamostat use may be associated with increased likelihood of cytotoxicity and disruption of homeostatic physiology, since it modulates host biological processes. While a valid concern, in vitro studies have revealed that localized delivery of nafamostat to well-differentiated (i.e., mucociliated) airway epithelial cells neither yields discernable cytotoxicity no induces cellular stress at its effective antiviral doses (Niemeyer, et al., 2021) . Additionally, transgenic animals lacking TTSPs mostly exhibit normal physiological functioning and survival rates comparable to wild-type controls. For instance, TMPRSS2 -/mice develop normally, survive to adulthood and have no defect in fertility or survival (Kim, Heinlein, Hackman, & Nelson, 2006; Sakai, et al., 2014) . Similarly, other in vivo studies with mice have shown that TMPRSS11A and TMPRSS11D are dispensable for development and health (Sales, et al., 2011) . Such observations imply that inhibition of host TTSPs may not be detrimental to host, particularly when treatment with a serine protease-blocking compound such as nafamostat is temporary. Furthermore, clinical data show nafamostat to be a safe and well-tolerated drug as an anticoagulant (Breining, et al., 2021; Maruyama, et al., 2011; Sawada, et al., 2016) . Thus, it is not surprising that as of July 2021 there are at least seven clinical trials using nafamostat for treatment of CVOID-19 underway (NCT04352400; NCT04418128; NCT04390594; NCT04628143; NCT04623021; NCT04473053; NCT04483960).

Avoiding undesired off-target effects of nafamostat requires thorough validation of TTSP family member(s) responsible for SARS-CoV-2 activation so that new J o u r n a l P r e -p r o o f Journal Pre-proof derivatives of nafamostat with increased specificity to these target(s) can be developed.

Currently, nafamostat similar to camostat is primarily thought to act through inhibition of TMPRSS2 (Hoffmann, et al., 2021; Hoffmann, Kleine-Weber, et al., 2020; Niemeyer, et al., 2021; Sonawane, et al., 2021) . While it is likely that inhibition of TMPRSS2 by these drugs confers a degree of protection from SARS-CoV-2 infection, more research is needed to fully understand which TTSPs are essential for their antiviral (and antiinflammatory) efficacy as several proteases other than TMPRSS2, such as TMPRSS4, TMPRSS11D, and TMPRSS13, have been shown to activate SARS-CoV-2 and related viruses (Kishimoto, et al., 2021; Laporte, et al., 2021; Zang, et al., 2020) . As such, comprehensive studies ideally using primary (not lines or immortalized) human (not animal)-derived cells are required where homeostatic surface protein expression of TTSPs in target tissues are well-characterized, and the protease expression can be modulated through knock-down systems (e.g., as performed via CRISPR by (Niemeyer, et al., 2021) ) (rather than overexpression) to identify serine proteases essential for SARS-CoV-2 activation. However, such efforts are partially hampered by absence of adequate bioreagents and poor understanding into biology of all TTSPs. For instance, not all members of TTSP family are well-characterized and limited specific, validated antibodies and chemical inhibitors are available to the scientific community.

Interestingly, early during the COVID-19 pandemic it was found that SARS-CoV-2 infection, particularly in severe cases, often becomes complicated with coagulopathies like stroke, thrombophilia, pulmonary embolisms, and disseminated intravascular coagulation Connors & Levy, 2020; Merkler, et al., 2020; Poissy, et al., 2020; Tang, et al., 2020) . These pathologies are thought to arise from a mix of virus-J o u r n a l P r e -p r o o f Journal Pre-proof associated factors including elevated inflammation, lung injury, and endothelial damage and dysfunction rather than direct coagulation via the virus (Carfora, et al., 2021;  Connors & Levy, 2020). Therefore, one may suggest another beneficial effect of nafamostat, besides its antiviral and anti-inflammatory properties, could be its ability to inhibit coagulation and plasmin activity (as well as plasminogen activators). In support of this, in a recent case report it has been shown that nafamostat and heparin combinatorial therapy has dramatic efficacy on a patient with COVID-19 pneumonia (Takahashi, et al., 2021) . Similarly, Jang et al. reported three cases of elderly patients with COVID-19 pneumonia whose disease progressed while taking antivirals and needing supplementary oxygen therapy but improved after receiving nafamostat (Jang & Rhee, 2020) . Additionally, another small case study found that combination treatment of antiviral favipiravir and nafamostat may be effective (lowering mortality rate) for critically ill Covid-19 patients (Doi, et al., 2020) . Based on the initial limited number of clinical studies, it appears that hyperkalemia may be the major adverse event when treating COVID-19 patients with nafamostat (M. Okajima, Takahashi, Kaji, Ogawa, & Mouri, 2020) . However, larger sample size and randomized controlled trials are required to detect adverse effects of nafamostat in COVID-19 more accurately and identify optimal dose and route(s) of administration (e.g., inhaled vs. oral vs. systemic).

Moreover, timing (Takahashi, et al., 2021) of nafamostat treatment needs to be identified (1) as this drug can theoretically be applied prophylactically for pre-emptive protection (in addition to its therapeutic efficacy), and (2) to minimize unnecessary prolonged delivery and/or undesired drug effects.

Immune modulators represent another class of host-targeting therapeutics currently being applied in treatment of COVID-19, which primarily focus on either boosting antiviral immunity or dampening exaggerated excessive immune activation such as cytokine storm (Rizk, et al., 2020; Song, Li, Xie, Hou, & You, 2020) . Interferon alpha-2a and -2b are immune modulators which have been used to treat both hepatitis B and C infections as well as SARS-CoV and MERS-CoV (Maughan & Ogbuagu, 2018; Woo, Kwok, & Ahmed, 2017; Zeng, et al., 2020) . Interferon alpha has been shown to limit SARS-CoV-2 replication and early reports indicate its use significantly improved clinical outcomes in COVID-19 patients (Lokugamage, et al., 2020; Pandit, et al., 2021) .

Colchicine, an anti-inflammatory approved for treatment of gout in 2009, has also received much attention as a COVID-19 therapeutic agent (Reyes, et al., 2020; Vitiello & Ferrara, 2021) . Early reports suggest that colchicine treatment of COVID-19 patients results in reduced clinical deterioration and mortality, this is despite observing limited reduction in inflammatory biomarkers (Deftereos, et al., 2020; Vrachatis, et al., 2021) .

Immune modulation may also be achieved through pharmacological activation of host factors with direct and indirect anti-inflammatory activities, such as nuclear factor erythroid 2 p45-related factor 2 (Nrf2). Nrf2 is a transcription factor which heterodimerizes with numerous other factors to regulate antioxidant response elements, redox homeostasis, damage repair, and cellular redox homeostasis and can control inflammation by repressing interleukin 6 (IL6) and interleukin 1 beta (IL1B) expression (Cuadrado, et al., 2020; Kobayashi, et al., 2016) . Apart from their anti-inflammatory properties, some Nrf2 activators, such as bardoxolone and bardoxolone methyl, have been shown to directly limit SARS-CoV-2 replication by inhibition of its main protease J o u r n a l P r e -p r o o f Journal Pre-proof (Sun, et al., 2021) . Similar in principle to the anti-inflammatory effects of Nrf2 activation, direct IL6 antagonists, including monoclonal antibodies tocilizumab and sarilumab are predicted to have therapeutic value in treating COVID-19 and cytokine storm associated with virus infection (Castelnovo, et al., 2021; Lu, Chen, Lee, & Chang, 2020; Zhang, Zhong, Pan, & Dong, 2020) . Clinical studies suggest that for patients with medium-tosevere forms of COVID-19 pneumonia, early intervention with tocilizumab and sarilumab is indeed associated with positive therapeutic outcomes including increased survival; however, treatment must be administered very early during infection (Castelnovo, et al., 2021; Remap-Cap Investigators, et al., 2021) .

Besides conventional drugs, cell-based therapeutic interventions utilizing mesenchymal stem cell (MSC) delivery to patients with COVID-19 represents a unique immune-modulating treatment modality. MSCs are capable of suppressing activated immune cells though direct cell-cell contacts via programmed death-ligand 1 (PD-L1) and Fas ligand (FasL), and can reduce proinflammatory cytokine and chemokines in the lung during influenza infection (Kavianpour, Saleh, & Verdi, 2020) . Additionally, MSCs are known to promote repair of lung injury during COPD, asthma, pneumonia, and idiopathic pulmonary fibrosis (Harrell, et al., 2019) . In the context of COVID-19, multiple clinical trials are underway and initial findings indicate that MSC treatment can reduce lung injury and improve patient outcome (Shi, et al., 2021) .

Finally, hormones such as vitamin D can play key a role in regulating host immune response during virus infections. At the airway epithelium, vitamin D promotes protection from pathogens both through production of antimicrobial β-defensins and cathelicidin and maintenance of epithelial junctions (Banerjee, et al., 2021) . Moreover, J o u r n a l P r e -p r o o f vitamin D3 has been shown to limit respiratory virus replication and alter expression of interleukin 8 and interferons (Banerjee, et al., 2021; Telcian, et al., 2017) . In the context of COVID-19, lower levels of vitamin D3 have been associated with more severe disease including mortality and pro-inflammatory cytokines in the serum of patients (Banerjee, et al., 2021) . Further studies found that vitamin D3 supplementation reduced the disease severity, diminished admittance to the intensive care, and lowered mortality rates among patients with COVID-19 (Banerjee, et al., 2021; Entrenas Castillo, et al., 2020; Ling, et al., 2020) . Altogether, immune modulation exhibits great potential in treating COVID-19, yet truly effective application of this class of drugs, cell therapies and vitamin supplements requires a much clearer understanding of the disease pathogenesis and particularly disease kinetics as timing, dosing, and patient selection are vital to therapeutic efficacy (Snow, Singer, & Arulkumaran, 2020) .

The rise of SARS-CoV-2 and consequent COVID-19 pandemic has placed the global health communities in a precarious position. We are in dire need of effective therapies to treat existing and emerging strains of SARS-CoV-2; yet we lack the time needed to develop new targeted drugs. Thus, repurposing therapeutics that are already approved for other indications may provide an untapped resource for treatment of SARS-CoV-2 infection and associated pathologies. Host-targeting therapeutic strategies, and in particular serine protease-inhibiting anticoagulants such as nafamostat, provide a strong alternative to conventional drugs. Specifically, nafamostat exerts antiviral, anti-inflammatory and anticoagulatory effects which are complementary and may be highly beneficial to COVID-19 patients. Importantly, nafamostat, by acting J o u r n a l P r e -p r o o f on host molecular machinery that SARS-CoV-2 and several seasonal human coronaviruses need to hijack to their advantage to cause infection, offers crossprotection against multiple CoV strains and could be deployed rapidly as new viral variants emerge. However, moving forward more clinical insight into optimal dose and route(s) of administration, prophylactic vs. therapeutic use, timing of application and adverse effects of nafamostat are needed. Randomized controlled trials with large number of participants would provide valuable information on this end. In addition, rigorous comprehensive preclinical studies are needed to thoroughly validate exact target of SARS-CoV-2, its mutant variants and other CoVs in primary human cells for new compound development for future. Such approach would allow minimizing adverse and off-target effects of nafamostat. Lastly, while we present a strong case for use of host-targeting therapeutics over traditional antivirals for treatment of COVID-19 and other coronaviral infections, we do not propose that this should entirely replace virustargeting drugs. Rather, these two distinct strategies complement one another to help mitigate the burden of current and future CoV outbreaks.

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