key: cord-0822558-oysw7eo1
authors: AbdelMassih, Antoine Fakhry; Ye, Jianping; Kamel, Aya; Mishriky, Fady; Ismail, Habiba-Allah; Ragab, Heba Amin; El Qadi, Layla; Malak, Lauris; Abdu, Mariam; El-Husseiny, Miral; Ashraf, Mirette; Hafez, Nada; AlShehry, Nada; El-Husseiny, Nadine; AbdelRaouf, Nora; Shebl, Noura; Hafez, Nouran; Youssef, Nourhan; Afdal, Peter; Hozaien, Rafeef; Menshawey, Rahma; Saeed, Rana; Fouda, Raghda
title: A multicenter consensus: A role of Furin in the endothelial tropism in obese patients with COVID-19 infection
date: 2020-07-15
journal: Obes Med
DOI: 10.1016/j.obmed.2020.100281
sha: 500feb76ae56583e8a7bbdc2f5bc344f8e6f5a80
doc_id: 822558
cord_uid: oysw7eo1

Furin, a cleavage enzyme, is increasingly recognized in the pathogenesis of metabolic syndrome. Its cleavage action is an essential activation step for the endothelial pathogenicity of several viruses including SARS-CoV-2. This Furin-mediated endothelial tropism seems to underlie the multi-organ system involvement of COVID-19; which is a feature that was not recognized in the older versions of coronaviridae. Obese and diabetic patients, males, and the elderly, have increased serum levels of Furin, with its increased cellular activity; this might explain why these subgroups are at an increased risk of COVID-19 related complications and deaths. In contrast, smoking decreases cellular levels of Furin, this finding may be at the origin of the decreased severity of COVID-19 in smokers. Chinese herbal derived luteolin is suggested to be putative Furin inhibitor, with previous success against Dengue Fever. Additionally, Furin intracellular levels are largely dependent on concentration of intracellular ions, notably sodium, potassium, and magnesium. Consequently, the use of ion channel inhibitors, such as Calcium Channel blockers or Potassium Channel blockers, can prevent cellular transfection early in the course of the illness. Nicotine patches and Colchicine have also been suggested as potential therapies due to Furin mediated inhibition of COVID-19.

Furin, a cleavage enzyme, is increasingly recognized in the pathogenesis of metabolic syndrome.

Its cleavage action is an essential activation step for the endothelial pathogenicity of several viruses including SARS-CoV-2. This Furin-mediated endothelial tropism seems to underlie the multi-organ system involvement of COVID-19; which is a feature that was not recognized in the older versions of coronaviridae. Obese and diabetic patients, males, and the elderly, have increased serum levels of Furin, with its increased cellular activity; this might explain why these subgroups are at an increased risk of COVID-19 related complications and deaths. In contrast, smoking decreases cellular levels of Furin, this finding may be at the origin of the decreased severity of COVID-19 in smokers. Chinese herbal derived luteolin is suggested to be putative Furin inhibitor, with previous success against Dengue Fever. Additionally, Furin intracellular levels are largely dependent on concentration of intracellular ions, notably sodium, potassium, and magnesium. Consequently, the use of ion channel inhibitors, such as Calcium Channel blockers or Potassium Channel blockers, can prevent cellular transfection early in the course of the illness. Nicotine patches and Colchicine have also been suggested as potential therapies due to Furin mediated inhibition of COVID-19. The Coronaviridae family of viruses incorporates an assortment of pathogens that are linked to a zoonotic origin. This is one of the fundamental elements causing former outbreaks of the severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV), (Phan et al. 2018) as well as the current pandemic severe acute respiratory syndrome coronavirus However, COVID-19 has shown exponential dissemination in the community, faster than MERS and SARS (Petrosillo et al. 2020) . This is supported by confirmed metrics on the total number of COVID-19 cases (as of June 2nd 2020) to be 6.06 million (Velavan and Meyer 2020) , compared to SARS-CoV-1 and MERS that were 8,439 and 2,519 (Aguanno et al. 2018) respectively. Furthermore, the clinical picture of COVID-19, which is due to multi-organ system damage on account of microvascular injury, seems to be slightly different than that of SARS-CoV-1 and MERS, mainly targeting the lungs. (AbdelMassih et al. 2020a (AbdelMassih et al. , 2020b (AbdelMassih et al. , 2020c (AbdelMassih et al. , 2020d 

FURIN is an enzyme that belongs to the PCSK (pro-protein convertase subtilizing/kexin) family, a type 1 membrane-bound protease that processes latent precursor proteins into their biologically active products. Furin is also utilized by several pathogens, where cleavage by Furin is needed for their functionality. This includes bacterial toxins, viral envelopes such as those of Human Immunodeficiency Virus (HIV), Ebola, and Marburg virus, as well as the spike protein of the SARS-Cov-2. Targeting host cell Furin is therefore a potential novel therapeutic option against these Furin-dependent infections. Of note, viruses develop high pathogenicity once they become reactive with Furin and other PC. (Solovyeva et al. 2017) A notable feature of SARS-CoV-2 is a polybasic cleavage site at the junction of S1 and S2, the two subunits of the spike. This allows effective cleavage by Furin, and has a role in determining viral infectivity and host range. These efficient polybasic cleavage sites have not been observed in related lineages of beta-coronaviruses. The acquisition of such polybasic cleavage site might have resulted from a point mutation, or from exchange of genetic material with another bat virus such as one of the filoviruses that share this common feature and a common intermediate host, namely bats. (Hoffmann et al. 2020) The subtilisin-like proteases, such as Furin, require polybasic cleavage sites rather than monobasic cleavage sites to activate respective viruses. A good example for this is, the difference in pathogenicity between highly pathogenic influenza strains, and those with lesser pathogenicity. (Braun and Sauter 2019) Furin upregulation in relation to metabolic syndrome, aging and androgen levels Several demographic factors as well as disease states have been linked to increased COVID-19 complications, notably metabolic syndrome including diabetes and obesity (Abbas and Kamel 2020; Belančić et al. 2020; Mediouni et al. 2020 ; Rahmati-Ahmadabad and Hosseini 2020).

Aging and male gender have been also linked to augmented profile of COVID-19 complications.

Could there be a relationship between Furin and COVID-19 severity in such risk factors? (Amel et al. 2020; Lithander et al. 2020; Rahmati-Ahmadabad and Hosseini 2020; Wambier et al. 2020) Although Furin is a membrane protein, earlier work indicates the existence of a secreted form of Furin. It has been previously shown that cells transfected with a recombinant Furin gene secrete a soluble truncated Furin protein, with similar enzymatic activity as the membrane-bound protein. Furthermore, it is established that Furin is partially shed from most cells.

Plasma Furin levels were associated with future DM (Diabetes Mellitus) and obesity, independently of all traditional DM risk factors. Regarding potential mechanisms, as Furin is responsible for the maturation of the insulin pro-receptor; one could speculate that more Furin in the circulation reflects a compensatory mechanism to increase the synthesis of active insulin receptors (Fernandez et al. 2018; He et al. 2020) . Another possible mechanism of action of Furin in DM development may be via pancreatic beta cells, as Furin has been demonstrated to control the proliferation and differentiation of pancreatic beta cell lines, as well as involvement in the maturation of insulin secretory granules. (Kayo et al. 1997) 

Furthermore, circulating Furin cleavage of PCSK9 (proprotein convertases subtilisin/kexin 9) implies a regulatory role on LDL (low-density lipoproteins) receptors and cholesterol levels.

There appears to be a sort of cholesterol dependent priming of SARS-Cov-2 viral entry, where cellular loading with cholesterol using apolipoprotein E, showed enhancement of endocytic entry of SARS-Cov-2. Twice the normal entry points for SARS-Cov-2 were found in the setting of increased cholesterol. Cholesterol is further implicated in trafficking of ACE2 (Angiotensin Converting Enzyme 2), the known receptor and entry point of the SARS-Cov-2 virus; cholesterol increased receptor binding domains. A cholesterol dependent theory for COVID-19 mortality is suggested; as those with increased cholesterol, i.e. obese, diabetic, and elderly, are at increased risk owing to Furin priming, and increased viral entry points. (Wei et al. 2020) 

Kappert et al underlined the importance of intracellular Furin in the pathogenesis of obesity, and related cardiometabolic syndrome. Furin has been found to play a role in redirecting lipid deposition in white adipose tissue. Moreover, Furin was found in abundance in mononuclear cells leading to increased interaction and cell signaling between adipocytes and mononuclear inflammatory cells. The latter paracrine mechanism induces vascular remodeling, and atherosclerosis through lipid deposition and chronic inflammation. (Kappert et al. 2013) The increased cellular and serum levels in diabetes, hypercholesterolemia, and obesity might explain the increased profile of COVID-19 complication in such disease states.

The only proven relationship between PCs including furin and androgen, has been proven in the context of androgen dependent prostatic cancer. Couture et al found that proprotein convertase activity is upregulated in prostate cancer. This might be mediated through increased androgen levels, or through increased activity of androgen receptors that act as protein chaperones to several types of proprotein convertases. This linear relationship between PCs and androgen might explain why COVID-19 disease burden is higher in males compared to females. (Couture et al. 2012; Wadman 2020) 

There is little data about Furin upregulation in aging. To our knowledge the only study to date to outline this relationship was conducted by Grobelna and colleagues. Grobelna et al proved that Furin is implicated in vascular aging; and proved as well that the serum levels of Furin are positively correlated with age dependent atherosclerosis. (Grobelna et al. 2019) The above relationship might explain the increased deaths from COVID-19 in the elderly In view of the above, serum Furin levels might serve as an independent predictor of COVID-19 complications in at risk groups.

Interestingly, Ranta and colleagues found an interesting relationship between smoking and shed serum Furin levels. Their study explored whether serum Furin levels can be used as a biomarker for septicemia, and results showed that that serum Furin levels were reduced in smokers compared to non-smokers. (Ranta et al. 2015) The latter finding might explain why smokers had lesser COVID-19 complications. (Israel et al. 2020) 

Furin is highly expressed in vascular endothelial cells. Mayer and colleagues succeeded in revealing the exact endothelial location of Furin. They demonstrated by electron microscopy that Furin is located over the plasma membrane of endothelial cells of the continuous, fenestrated, and discontinuous capillaries. This location goes in agreement with several previous studies relating Furin to the endothelial permeability of many molecules, as well as some organisms. (Mayer et al. 2004) Moreover, Furin-processed proteins, such as cadherins, vascular endothelial growth factor, factor IX, and von Willebrand factor, are known to be involved in vascular permeability. These facts constitute strong evidence that Furin plays a major role in viral tropism to the endothelium. As discussed earlier, several viruses harbor a polybasic cleavage site that allows easier and canonical binding to Furin, allowing them to exploit Furin to regulate cell entry and infectivity. (Schlokat et al. 1996) An important theory that can explain the endothelial pathogenicity of those viruses is the "vascular zip code" theory. Endothelial penetrability of drugs and other molecules have been the focus of many onco-pharmacology studies. The high vascularity of tumors makes targeting the endothelium an important selective strategy for treatment. Such studies have revealed that coupling drugs or nanoparticles with basic amino-acid residues such as lysine or arginine, renders those compounds highly tropic to the endothelium. SARS-CoV-2, filoviruses, HIV, and highly pathogenic strains of influenza, harbor a polybasic amino-acid sequence that not only make them readily activated by Furin, but might explain their ability to transfect the endothelial cells. (Enbäck and Laakkonen 2007; Gilbert et al. 2008; Han et al. 2006; Ruoslahti 2004) 

Furin inhibition has been tried against viruses with polybasic Furin cleavage sites with debatable results. Luteolin, a molecule derived from traditional Chinese herbal products, has been found to decrease Dengue Fever severity and viral load through selective inhibition of Furin. (Peng et al. 2017) Further evidence to the role of Furin inhibitors as a potential therapeutic target, is in their use in the treatment of HIV. Hallenberger et al confirmed the role of an old generation of Furin inhibitors, peptidyl-chloromethyl ketones, in interruption of formation of infectious viral particles. A major setback is the high toxicity of these compounds. (Hallenberger et al. 1992) Gagnon et al concluded that the use of a nanomolar particle stabilized by Aza-Beta-amino-acid could effectively inhibit H5N1 influenza. (Gagnon et al. 2014) As mentioned previously, COVID-19 related deaths and complications are paradoxically reduced in smokers, smoking might operate through reducing endothelial penetration of COVID-19 by inhibition of Furin. (Ranta et al. 2015) Israel et al proved in his large observational study that COVID-19 complications were lesser insmokers (Israel et al. 2020 );a finding that was also confirmed by Gonzalez-Rubio and colleagues, who concluded that cytokine release is tamed by Nicotine. (Gonzalez-Rubio et al. 2020) The latter findings could lead to the therapeutic use of Nicotine patches after confirming this role through clinical trials.

Colchicine is a drug that is used in the management of inflammatory complications of Gout, as well as auto-inflammatory syndromes such as familial Mediterranean fever. Colchicine acts by targeting several inflammatory pathways, and has been found to target specifically endothelial inflammation and vascular degeneration through Furin. (Xu 2016 

Furin is a cellular protein. There is clear evidence that Furin interacts with intracellular ions.

Magnesium, the second most occurring intracellular ion after Potassium, has been shown to activate Furin. (Izidoro et al. 2010) Molloy and colleagues demonstrated that Furin is a calcium dependent enzyme. The levels of intracellular calcium are critical for the activation of Furin. (Molloy et al. 1992 There is no clear relationship between intracellular potassium levels and Furin activity; however amiodarone had been shown to block viral entry of some flaviruses. This role has been confirmed against COVID-19 by Gehring et al. (Gehring et al. 2014) This antiviral role of ion channel blockers is speculated to be more effective with low viral loads, and at the start of infection, when virus entry into target cells is the dominant step. 

The acquisition of polybasic cleavage site by the new SARS-CoV-2 seems to be an important feature for endothelial penetrability and pathogenicity of COVID-19. This might occur through its canonical activation by Furin, and through endothelial affinity to basic amino acid residues.

Additionally, the interaction of SARS-CoV-2 with Furin offers an explanation to the augmented manifestations of COVID-19 in obese and diabetic patients, as well as in males and elderly people.

Screening serum levels of Furin early in the course of COVID-19 complications might serve as an important strategy to anticipate COVID-19 poor outcomes, and preventing them.

Finally, and importantly, this relationship opens up new therapeutic strategies in the treatment of COVID-19, such as the use of Chinese herbals, ion channel inhibitors, nicotine patches, and Colchicine, for prevention of endothelial involvement and multi-organ failure in affected patients.

In view of the above, we strongly recommend the screening of baseline serum Furin levels early in the course of COVID-19 infections, and correlating such levels with COVID-19 complications and mortality; we recommend the launching of clinical trials involving the use of the aforementioned Furin inhibitors in COVID-19 patients. 

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