Racionálny prístup k identifikácii účinných kombinovaných antikoronavírusových terapií proti mačaciemu koronavírusu

Feline peritonitis (FIP), caused by a genetic mutant of feline enteric coronavirus known as FIPV, is a deadly disease in cats for which no FDA-approved vaccine or treatment is currently available. The spread of FIPV in affected cats leads to a number of clinical symptoms, including cavitation effusions, anorexia, fever and lesions of pyogranulomatous vasculitis and perivasculitis with or without central nervous system and / or eye involvement. There has been a critical need for effective and approved antiviral therapies against coronaviruses, including FIPV and zoonotic coronaviruses such as SARS-CoV-2, caused by COVID-19. For SARS-CoV-2, preliminary evidence suggests that there may be potential clinical and pathological features common to feline coronavirus disease, including enteric and neurological impairment. We examined 89 selected antiviral compounds and identified 25 compounds with antiviral activity against FIPV, which represent different classes of drugs and mechanisms of antiviral action. Based on successful combination therapy strategies in human patients with HIV infection or hepatitis C virus, we have identified drug combinations targeting different phases of the FIPV life cycle that lead to a synergistic antiviral effect. Similarly, we suggest that combination anti-cancer therapy (cACT) with multiple mechanisms of action and penetration into all potential anatomical sites of viral infection should be applied to the treatment of other coronaviruses, such as SARS-CoV-2.

Author Summary

We tested in vitro antiviral activity against FIPV in 89 compounds. The antiviral activity of these compounds consisted either of a direct effect on viral proteins involved in viral replication or an indirect inhibitory effect on normal cellular processes usurped by FIPV to promote viral replication. Twenty-five of these compounds showed significant antiviral activity. We have also found that certain combinations of these compounds are more effective than monotherapy alone.

Dictionary

In short	English expression	Slovak translation
PI	protease inhibitor	protease inhibitor
NPI	nucleoside polymerase inhibitor	nucleoside polymerase inhibitor
NNPI	non-nucleoside polymerase inhibitor	a non-nucleoside polymerase inhibitor
CPE	cytopathic effect	cytopathic effect
cACT	combined anti-coronaviral therapy	combined anticoronavirus therapy
cART	combined anti-retroviral therapy	combination antiretroviral therapy
CRFK cells	Crandell-Rees Feline Kidney cells	Crandell-Rees feline kidney cells

Introduction

Feline infectious peritonitis (FIP) is a highly fatal disease without an effective FDA-approved vaccine or treatment. Although the pathogenesis is not fully understood, FIP is generally thought to be the result of specific mutations in the viral genome of the minimally pathogenic and ubiquitous feline enteric coronavirus (FECV) that result in the virulent FIP virus (FIPV) [1–3]. These FECV mutations lead to a change in the tropism of the virus-infected host cell from intestinal enterocytes to peritoneal-type macrophages. FIPV productive macrophage infection, targeted extensive anatomical dissemination, and immune-mediated perivasculitis lead to the highly fatal systemic inflammatory disease FIP [4]. As a result of viral dissemination, FIP may present with clinical signs reflecting inflammation at various anatomical sites, which may potentially include the abdomen and intestines, the thoracic cavity, the central nervous system, and / or the eyes [5-8]. Due to its high mortality, FIP remains a devastating viral disease in cats and a challenge in making an accurate etiological diagnosis with a current lack of available and effective treatment options [7, 9]. The development of an effective FIP vaccine has been complicated by the role of antibody-dependent amplification (ADE) in the pathogenesis of FIP, where the presence of non-neutralizing anti-coronavirus antibodies has been shown to exacerbate FIP [10–12].

In mammals, coronaviruses infect and generally cause disease of the intestinal tract or respiratory system of infected hosts [13]. However, FIP often manifests itself as a multisystem inflammatory disease syndrome due to the widespread spread of FIPV-infected macrophages. The recent pandemic occurrence of SARS-CoV-2 in infected human patients results in various disease syndromes, collectively referred to as COVID-19. Although SARS-CoV-2 has overt tropism for respiratory epithelium leading to interstitial pneumonia, recent evidence suggests that COVID-19 may also present as a digestive disease and clinically manifest as diarrhea [14, 15]. The tropism for these tissues reflects the membrane expression of the ACE2 protein, the cellular target of SARS-CoV-2 [16]. Furthermore, SARS-CoV-2 has been shown to be able to infect and cause inflammatory disease in tissues outside the intestinal tract and respiratory tract, including the brain, eyes, reproductive organs, and cardiac myocardium [17-21]. Brain stem neuroinvasion and subsequent encephalitis caused by SARS CoV-2 may contribute to respiratory failure in patients with COVID-19 [20,22]. Experimentally, SARS CoV-2 is able to create a productive infection in cats [23]. Therefore, feline FIPV infection and SARS CoV-2 infection in human patients are more similar than originally thought.

There is an immediate and critical need for available and effective antiviral therapies for the treatment of these coronavirus diseases. FIPV-infected cats could serve as a translational model and provide useful insights useful for SARS-CoV-2-infected patients with COVID-19. Recent antiviral clinical trials in both experimental and naturally infected FIPV cats have provided hope for the treatment and cure of FIP with GS-441524, the nucleoside analog and metabolite of the prodrug Remdesivir (Gilead Sciences) or GC-376, a 3C-like FIPV protease inhibitor (Anivive) [24– 26]. Remdesivir, a prodrug of GS-441524, has recently been shown to be promising in the treatment of human patients infected with SARS-CoV-2 [27,28]. Despite these recent clinical successes, these antiviral compounds have yet to be approved and are not currently available for clinical veterinary use in cats with FIP.

Identifying and developing effective antiviral therapies can be costly and time consuming. Targeted screening and reuse of drugs already approved by the FDA or approved for research use can play an effective role in drug discovery. Using putative antiviral compounds selected on the basis of their proven efficacy in the treatment of other RNA viruses, we identified a subset of compounds with potent anti-FIPV activity and characterized their in vitro safety and efficacy profiles. Based on the great success of combination antiretroviral therapy (cART) against HIV-1 and combination treatment of hepatitis C virus [29], we have developed methods to identify effective combination therapies against FIPV. Initial monotherapies against HIV-1, such as azidothymidine (AZT), often led to viral escape mutations. The concomitant use of multiple antiviral compounds appears to block this adaptive viral evolutionary mechanism, as the development of HIV-1 is effectively arrested by modern cARTs [30]. The success of cART is the result of a pharmacological focus on multiple stages of the virus life cycle simultaneously, while achieving a synergistic antiviral effect [31].

Given the impressive success of cART, it could appear that the current targeting of FIPV at different stages of the viral life cycle by combined anti-coronavirus therapy (cACT) may offer a higher level of lasting and more complete success, compared to the monotherapies themselves. The inclusion of an antiviral agent in cACT capable of penetrating the blood-brain (BBB) and blood-eye barriers, and reaching pharmacologically relevant tissue concentrations, may facilitate the eradication of FIPV throughout the system. We describe a set of in vitro assays that facilitate rapid screening and identification of effective anti-coronavirus compounds. Active antiviral agents with different mechanisms of action and presumed distribution in the body were combined into cACT and tested for compound synergy. We hypothesized that the combined use of two or more effective antiviral monotherapies with different mechanisms of action would facilitate the identification of synergistic combinations providing excellent anticoronavirus efficacy compared to their use alone. Identification of successful cACT may also provide guidelines for the treatment of other emerging viral diseases, such as SARS-CoV-2.

The results

Compound testing

To identify compounds with anti-FIPV activity, a group of 89 compounds were tested in vitro (Additional table 1) from different classes of drugs and with different presumed mechanisms of action. Test compounds included nucleoside polymerase (NPI) inhibitors, non-nucleoside polymerase inhibitors (NNPIs), protease inhibitors (PIs), NS5A inhibitors, a set of novel anti-helicase chemical "fragments", and a set of compounds with unspecified mechanisms of action. Of this group of 89 compounds, a total of 25 different compounds were shown to have antiviral activity against FIPV, including NPI, PI, NS5A inhibitors, and two compounds with unspecified mechanisms of action (referred to as "others", Figure 1). These successful antivirals included toremifene citrate, daclatasvir, elbasvir, lopinavir, ritonavir, nelfinavir mesilate, K777 / K11777, grazoprevir, amodiaquin, EIDD 1931, EIDD 2801 and GS-441524 from three different Chinese manufacturers (Table 1). We tested several nucleoside analog compounds provided by Gilead Sciences structurally related to nucleoside analogs GS-441524 and Remdesivir for their antiviral properties and found several with potential (contained in the 25 identified compounds above), but did not follow these substances further. Thirteen antiviral agents were selected for further analysis. This total includes the previously identified 3-C protease inhibitor, GC-376 (Anivive).

Name of the compound	Drug category	EC50 (µM)
GC376	PI	0.04
EIDD 1931	NPI	0.09
Elbasvir	NS5A Inhibitor	0.16
EIDD 2801	NPI	0.4
GS-441524⁺	NPI	0.66
K777 / K11777	PI	0.67
Toremifene citrate	Other*	5
Amodiaquine	Other**	6.5
Lopinavir	PI	8.08
Ritonavir	PI	8.7
Grazoprevir	PI	12.13
Nelfinavir mesylate	PI	13.47

Table 1.
EC50 of compounds with anti-FIPV activity.
PI = Protease inhibitor; NPI = Nucleoside polymerase inhibitor
+ MedChem Express, HY-103586
* Selective estrogen receptor modulator
** 4-Aminoquinoline

**Figure 1**
**Test compounds by mechanism of action**
(A) All test compounds
(B) Compounds found to have anti-FIPV activity in in vitro assays

**Figure 2**
**An example of a test matrix using crystal violet staining to identify anti-FIPV activity at 10μM.**
The upper left row are control wells with CRFK cells only and without drug or FIPV. The upper right row is a positive control using GS-441524 with the known complete protection of CRFK cells from FIPV-induced cell death. The entire bottom row of wells represents CRFK cells infected with FIPV and without treatment. The remaining rows are test wells, with the left half evaluating cytotoxicity at 10μM (no FIPV infection) and the right half evaluating anti-FIPV activity at 10μM for any given compound. Loss of staining indicates loss of cells. Daklatasvir and velpatasvir demonstrated anti-FIPV activity, as evidenced by increased crystal violet staining (relatively intact cell monolayers) compared to control wells containing only FIPV (bottom row of the plate). Drugs 32, 33 and 34 showed absent to minimal antiviral activity, while drug 34 (Ravidasvir) also showed cytotoxicity at 10 μM based on the dramatic well cleaning observed in the left half of the FIPV-free matrix.

Determination of antiviral activity

Antiviral activity (EC50) was determined for 10 antiviral compounds. For these compounds, the EC50 ranged from 0.04μM to 13.47μM (Table 1, Figure 3). One of the antiviral agents, Daclatasvir, showed unacceptable cytotoxicity at 20 μM and was excluded from further testing. GS-441524 originating in China (MedChemExpress, HY-103586) was shown to have a comparable EC50 compared to previously published values for GS-441524 originating from Gilead Sciences [25].

Figure 3
Representative examples of non-linear EC50 regression assays for compounds with anti-FIPV activity.
Serial dilutions of each compound with anti-FIPV activity were performed to identify half-maximal effective concentration (EC50). The GS-441524 results reported herein represent a compound derived from MedChemExpress.

Cytotoxicity safety profiles

Cytotoxicity safety profiles (CSPs) were determined for ten different antiviral compounds in CRFK cells. At 5 μM, the seven test compounds showed essentially no cytotoxicity, while two of the antivirals, amodiaquine and toremifene, had 11 and 12% cytotoxicity, respectively (Fig. 4; Table 2). The 50% cytotoxic concentration (CC50) for GC376 is reported as> 150μM [32]. Interestingly, based on the Promega CellTox-Green Cytotoxicity assay, the cytotoxicity of both EIDD compounds was essentially undetectable up to 100μM. However, visual inspection of the EIDD wells just prior to fluorescent dye application and matrix reading revealed differences in cell morphology (cytopathic effect) between untreated CRFK cells and treated cells. Untreated CRFK cells showed adherent spindle morphology in a single monolayer, while EIDD wells showed a marked decrease in confluence compared to variable cell morphology, including cell rounding (cytopathic effect). The discrepancy between the subjective visual evaluation of the EIDD wells and the fluorescence assay is a mystery. It is possible that an overall reduction in the number of cells in the EIDD wells led to the loss and degradation of the nucleic acid necessary for fluorescent binding and detection in the CellTox assay.

Compound	Drug category	5μM	10μM	25μM	50μM	100μM
Elbasvir	NS5A Inhibitor	0.67	0.46	1.4	2	4.9
K777 / K11777	PI	0.61	0.29	2.3	9	16
Toremifene citrate	Other *	12	22	23	35	39
Amodiaquine	Other **	11	12	19	23	26
Lopinavir	PI	0.67	15	13	16	18
Ritonavir	PI	0.49	0.84	2	33	26
Grazoprevir	PI	0.55	0	0.81	3.9	19
EIDD1931	NPI	1.2	0.94	0.6	0.78	2.8
EIDD2801	NPI	0	0.95	0.63	1.7	0.8
GS-441524⁺	NPI	0.21	0	0	0	0

Table 2
Percent cytotoxicity by compound and concentration.
PI = Protease inhibitor; NPI = Nucleoside polymerase inhibitor
* Selective estrogen receptor modulator
** 4-Aminoquinoline
+ NMPharmTech

**Figure 4**
**Representative cytotoxicity profiles.**
Bar graphs of percent cytotoxicity +/- standard deviation (SD) for four compounds with anti-FIPV activity. Percent cytotoxicity values were determined by normalizing cytotoxicity for control wells with positive toxicity (set to 100% cytotoxicity) and untreated CRFK cells (set to 0% baseline cytotoxicity).

Quantification of inhibition of viral RNA production in monotherapy

A real-time RT PCR assay was used to measure the ability of each antiviral agent to inhibit coronavirus replication in monotherapy (Viral RNA knock-down assay). The compounds demonstrating the greatest inhibition of FIPV RNA production were GC376, a 3C-like coronavirus protease inhibitor, GS-441524, EIDD-1931 and EIDD-2801, the last three being nucleoside analogs (Fig. 5, Table 3). Substances with the least inhibitory effect on viral RNA production include elbasvir, nelfinavir and ritonavir. Ritonavir, a protease inhibitor, is used in combination with lopinavir to treat HIV-1 infection (Kaletra, AbbVie). Lopinavir monotherapy has unsatisfactory oral bioavailability in humans, but when used in combination, ritonavir has been shown to significantly improve lopinavir plasma concentrations [33]. Therefore, despite the relatively minimal inhibition of FIPV identified with ritonavir as monotherapy, this compound has been further tested, including combined anti-cancer evaluation.

Compound	Virus titer reduction fold
GC376 (20μM)	25000
GC376	7300
GS-441524 (NMPharmTech)	5280
EIDD-1931	3700
GS-441524 (MedChemExpress)	3500
EIDD-2801	2110
Lopinavir	309
Toremifene	10
K777	7
Grazoprevir	5
Amodiaquine	4
Elbasvir	2
Nelfinavir mesylate	1
Ritonavir	1

Table 3
Multiple reduction in viral RNA copy number for anti-FIPV compounds on monotherapy
* Unless otherwise indicated, all compounds were used at 10 μM.

Figure 5
Multiple reduction in FIPV RNA copy number using antiviral compounds as monotherapy.
FIPV-infected CRFK cells were incubated for 24 hours with compounds with detected anti-FIPV activity. The viral copy number was then determined by RT-qPCR and normalized to feline GAPDH copy number to determine the fold reduction effect for each compound. All compounds were tested at 10μM unless otherwise noted. All experimental treatments were performed in triplicate wells, and the fold decrease was calculated by dividing the average experimental normalized FIPV copy number by the average normalized FIPV copy number determined for untreated FIPV-infected wells.
¹GS-441524 - NMPharmTech (China).
²GS-441524 - MedChemExpress (China).

Quantification of inhibition of viral RNA production in cACT

To identify drug combinations with synergistic antiviral activity versus monotherapy, combinations of two or more compounds were selected based on (i) established combinations used in other viral infections such as HIV-1 and HCV, (ii) drugs with different mechanisms of action, (iii) potential changes in the systemic distribution of the compound (eg ability to cross the blood-brain or blood-eye barrier according to chemical classification) and (iv) minimal cytotoxicity (based on CSP). For each cACT, any resulting reduction in FIPV copy number in excess of the calculated additive effect for each drug used in the monotherapy regimen was considered synergistic (Table 4). The combination of GC376 and amodiaquin achieved the greatest synergistic effect with the highest overall fold reduction in viral RNA with a 76-fold reduction in viral RNA compared to the additive effect (Fig. 6). This particular synergistic combination was one of the surprising results, given that amodiaquin alone showed only limited inhibition of FIPV viral RNA copies as determined by qRT-PCR.

Compound 1	Compound 2	Compound 3	Virus titer reduction	Add	cACT / add
GC376 (20 μM)	Amodiaquine	-	1897000	25004	76
GC376 (20 μM)	Amodiaquine	Toremifene	256000	25014	10
GC376 (20 μM)	K777	-	248000	25007	10
GC376 (20 μM)	Toremifene	-	128000	25010	5.1
GC376 (20 μM)	Nelfinavir mesylate	-	91100	25001	3.6
Elbasvir (5 μM)	Lopinavir	-	14000	311	45
Elbasvir (5 μM)	GC376 (20 μM)	-	12600	25002	0.50
GC376 (10 μM)	Amodiaquine	-	11700	7304	1.6
GC376 (10 μM)	Grazoprevir	-	8290	7305	1.1
GC376 (20 μM)	GS-441524 (NMPharmTech)		8260	30280	0.27
GC376 (10 μM)	Amodiaquine	Elbasvir (5 μM)	7570	7306	1.0
GC376 (10 μM)	GS-441524 (MedChem)	-	6910	10800	0.64
GC376 (20 μM)	Ritonavir	-	6560	25001	0.26
K777	Lopinavir	-	5530	316	18
GC376 (10 μM)	GS-441524 (NMPharmTech)	-	4340	12580	0.34
GC376 (20 μM)	Lopinavir	-	3400	25309	0.13
Lopinavir	Ritonavir	-	3130	310	10
Lopinavir	Ritonavir	Toremifene	1740	320	5.4
GC376 (10 μM)	EIDD-2801	-	255	9410	0.03
K777	Toremifene	-	25	17	1.5

Table 4
Multiple reduction in FIPV viral RNA copy number in combination therapy (cACT).
The expected additive effect reflects the sum of the fold reductions in viral RNA of each compound used in monotherapy (Table 3).
* Unless otherwise indicated, all compounds were used at 10μM.
cACT / add - ratio of FIPV titer reduction in combination therapy versus the sum of fold reduction titers in monotherapy
Add - the sum of fold reduction titers in monotherapy

**Figure 6**
**Selected examples of fold FIPV RNA copy reduction using combination therapy (cACT).**
The bars represent the mean fold decrease in three wells of treated CRFK cells compared to the mean fold decrease in three untreated wells infected with FIPV. All compounds were tested at 10μM unless otherwise noted.

Due to the strong anti-FIPV activity of GC-376, as well as its potential availability for advances in in vivo pharmacokinetic studies, clinical trials, and promising use in cACT, this compound was selected for a series of "viral RNA knock-down" assays in mono and combination therapy (Fig. 7). Overall, GC376 demonstrated excellent anti-FIPV activity at 20 μM in both monotherapy and in vitro combination therapy. The most significant reduction in FIPV RNA occurred in the combination of GC376 at 20μM with amodiaquine at 10μM. The experiment combining GC376 with amodiaquine was repeated and both results are shown for comparison in FIG. 7C.

Figure 7
GC376 antiviral activity in monotherapy and in combination therapy at 10 and 20 μM
(A) FIPV RNA reduction quantified by RT-qPCR using GC376 as monotherapy at 10 and 20 μM. There is a significant difference between the two concentrations, with 20μM being better than 10μM. (unpaired t-test; p <0.0001).
(B) Combination in vitro therapy using GC376 at 10 μM.
(C) Combination in vitro anti-FIPV therapy using GC376 at 20 μM.

Discussion

Because there is currently no effective vaccine against FIP, there is a strong clinical and worldwide need for effective antiviral treatment options for FIPV-infected cats. We tested 89 compounds, which resulted in the identification of 25 antiviral agents with antiviral activity and strong safety profiles against feline coronavirus, FIPV. We also identified combinations of antiviral agents (cACT) that resulted in greater efficacy or synergism over monotherapy alone. Of particular interest was the finding regarding the use of elbasvirus, which repeatedly demonstrated excellent protection of CRFK against CPE-induced FIPV at concentrations below 1 μM based on multiple assays (EC50 0.16 μM). In principle, however, no difference in viral RNA copy number was found between infected cells treated with or without elbasvirus. Further visual analysis of FIPV-infected CRFK cells treated with elbasvirus revealed an atypical cell morphology relative to uninfected cells, which was characterized by variable enlargement, cell rounding, and partial cell detachment (cytopathic effect). These "atypical cells" were rarely detached from the culture plate, and as a result, absorbance values were comparable to uninfected control wells. This dichotomous result between platelet analysis and viral RNA knock-down assay suggests that the antiviral effect of elbasvir occurs after viral replication and, as a result, elbasvir may not protect cells from viral RNA accumulation. Elbasvir is used to treat patients infected with hepatitis C virus (HCV) and is thought to target the HCV NS5A protein, which prevents replication and also to complete virions [34]. Although no NS5A homolog has been identified for FIPV, it is possible that elbasvir exhibits a similar antiviral effect by preventing the assembly of FIPV virions without blocking viral RNA synthesis in CRFK cells. Additional evaluation of treated FIPV-infected CRFK cells by transmission electron microscopy may shed light on the effect of elbasvirus on protecting CRFK from FIPV-related damage and death.

Co-administration of ritonavir with lopinavir has been shown to significantly increase lopinavir plasma concentrations in rats, dogs and humans [33]. Ritonavir is a potent inhibitor of CYP3A, which is the primary enzyme responsible for protease inhibitor metabolism, and therefore its co-administration with other protease inhibitors results in increased systemic concentrations of co-administered protease inhibitors such as lopinavir [38, 39]. The increase in the antiviral effect of lopinavir associated with ritonavir was relatively minimal in the viral RNA elimination assays with only a 10-fold inhibition of FIPV over the additive effect. This may be the result of an in vitro testing artifact on a feline kidney cell line (ie CRFK cells) that lacks the enzyme CYP3, an enzyme that typically occurs at sites of high protease inhibitor metabolism at the first pass effect (ie enterocytes). and hepatocytes) [39]. These results suggest that in vitro tests alone may not fully predict the effect of antiviral agents in FIPV-infected cats in vivo.

Grazoprevir, a serine protease inhibitor NS3 / 4, has been used in combination with elbasvirus, an NS5A inhibitor, to treat HCV-infected patients (Zepatier, Merck) [40]. Here, we demonstrated that grazoprevir has anti-FIPV activity when used as monotherapy. The cysteine protease inhibitor K777 / K11777 has been investigated for its ability to block coronavirus (MERS-CoV and SARS-CoV-1) and ebolavirus entry and has been found to completely inhibit coronavirus infection, but only in target cell lines without virus-activating serine proteases. [41]. For other cell lines, K777 inhibited coronavirus cell entry in combination with a serine protease inhibitor [41]. It is possible that limited inhibition of FIPV RNA K777 production could be increased if combined with a serine protease inhibitor.

Amodiaquine is an antimalarial drug and belongs to the class of 4-aminoquinoline drugs. Amodiaquine, along with related 4-aminoquinolines such as chloroquine and hydroxychloroquine, was originally developed to treat malaria [42], and like chloroquine and hydroxychloroquine, it has a wide range of anatomical distributions, including the eyes and brain [43-49]. Penetration of antiviral agents into the CNS and / or ocular compartments is particularly important in FIPV-infected cats with neurological and / or ocular disorders. While several studies have defined the antiviral properties of chloroquine and hydroxychloroquine [28, 50, 51], the antiviral activity of amodiaquin has also been investigated with the identification of antiviral activity against dengue virus, Ebola virus and severe fever with viral thrombocytopenia syndrome (SFTS) [52–55]. The mechanism of action of amodiaquine may involve an increase in cytoplasmic lysosomal and / or endosomal pH, which prevents the release of viable virions into the cytoplasm [56]. Due to its unique drug class status and presumed ability to cross the blood-brain barrier [57], amodiaquine is a promising candidate for the combined treatment of neurological and / or ocular forms of FIP.

Toremifene citrate, a selective estrogen receptor modulator (SERM), is used to treat metastatic breast cancer in human patients. Recently, toremifene has been evaluated for its antiviral properties and has demonstrated anticorrosive activity against zoonotic coronaviruses, Middle East Respiratory Syndrome (MERS-CoV) and SARS-CoV-1 coronaviruses [58]. Toremifene has also been shown to be active against Ebola virus (EBOV) [59, 60]. Although the exact mechanism of antiviral action is not defined, the antiviral effect of toremifene against EBOV appears to be to destabilize the EBOV glycoprotein [59].

Interestingly, GC376 demonstrated confusing differences between 10 μM and 20 μM in combination therapy. When used at 10 μM with other compounds, synergism and in some cases a decrease in antiviral effect compared to additive values ranging from 0.03 to 1.6 were absent (Table 4). When used at 20 μM, there were still cases where combination with another compound resulted in a reduced antiviral effect compared to GC376 used as monotherapy at 20 μM. However, many more variations in the 20 μM combinations showed a fold effect compared to additive values ranging from 0.13 to 76 (Table 4). A specific example is the contrast between GC376 at 20 μM compared to GC376 at 10 μM combined with amodiaquine at 10 μM. The first caused the greatest inhibition of viral RNA as well as the greatest multiple of the additive (synergistic) effect, while the second caused almost a loss of synergism with a value of the additive multiple of 1.6.

The identification of effective antiviral strategies for the treatment of FIPV-infected cats has translational implications for the ongoing SARS-CoV-2 pandemic. FIPV infection in cats resembles coronavirus infection in ferrets [61, 62] and is compared with the pathogenesis of other chronic macrophage-dependent diseases such as tuberculosis [63]. Because the clinical and pathogenic details of SARS-CoV-2 infection in humans are still emerging, there appears to be some overlap with FIPV in anatomical distribution, clinical manifestations, and likely response to certain antiviral therapies. In cats, the feline enteric coronavirus biotype (FECV) is restricted to the gastrointestinal tract due to enterocyte tropism. Clinical symptoms in FECV-infected cats range from mild gastrointestinal disease (diarrhea) to the absence of symptoms. The mutated FIPV coronavirus feline biotype acquires macrophage tropism and preferentially targets serous abdominal and thoracic surfaces with a subset of cats that demonstrate CNS or eye involvement [7]. Similarly, in patients with COVID-19, there are reports of diarrhea and a subset of patients with CNS involvement [14]. Although the cellular receptor for SARS-CoV-2 has been identified as ACE2 [64], the cellular receptor for FIPV serotype I has yet to be determined. The cellular receptor for less clinically relevant FIPV serotype II has been identified as feline aminopeptidase peptidase (fAPN) [4]. A study using RNAseq to evaluate gene expression profiles of ascites cells obtained from cats with FIP did not identify ACE2 expression, suggesting that ACE2 is unlikely to be a FIPV serotype I receptor [63]. A more detailed examination of the identity of the FIPV serotype I receptor is needed.

Clinical successes with GS-441524 or GC-376 in cats with experimental and naturally occurring FIPs indicate that FIP can be effectively treated, but treatment of dry (granulomatous), neurological and ocular FIPs remains a challenge. The protease inhibitor 3C-like protease, GC-376, appears to be relatively effective in the treatment of FIPV effusion infection limited to body cavities, but may be less effective in the treatment of neurological or ocular forms of the disease [24]. These different results may be the result of ineffective penetration of the blood-brain and blood-eye barriers, making GC-376 a promising candidate for combination therapy with a CNS-penetrating antiviral drug.

Materials and methods

FIPV inoculation for in vitro experiments

Crandell-Reese cat kidney cells (CRFK, ATCC) were cultured in T150 flasks (Corning), seeded with FIPV serotype II (WSU-79-1146, GenBank DQ010921) and propagated in 50 ml of Dulbecco's modified Eagle's medium (DMEM) with 4 , 5 g / l glucose (Corning) and 10% fetal bovine serum (Gemini Biotec). After 72 hours of incubation at 37 ° C, extensive cytopathic effect (CPE) and large cell clearing / separation areas were noted. The flasks were then flash frozen at -70 ° C for 8 minutes, thawed briefly at room temperature, and the cells and supernatant were then centrifuged at 1500 g for 5 minutes, followed by a second centrifugation step at 4000 g for 5 minutes to isolate cell - free viral volumes. The supernatant containing the virus base was divided into 0.5 and 1.0 ml aliquots in 1.5 ml cryotubes (Nalgene) and archived at -70 ° C. After freezing, one tube was allowed to thaw and the virus titer was determined using biological assays (TCID50) and real-time RT PCR methods (below).

The tissue culture dose-50 infectious dose (TCID50) was determined using a viral plaque assay. CRFK cells were grown in a 96-well tissue culture plate (Genesee Scientific) until CRFK cells reached approximately 75-85% confluence. Serial 10-fold dilutions were prepared from FIPV stock solution and 200 μl samples from each dilution were added to 10-well replicates. 72 hours after infection, cells were fixed with methanol and stained with crystal violet (Sigma-Aldrich). Individual wells were visually evaluated for virus-induced CPE, evaluated as CPE positive or negative, and TCID50 was determined based on the log equation.₁₀TCID50 = [total number of # wells CPE positive / # replicates] + 0.5 to reflect infectious virions per milliliter of supernatant [68].

Quantification of FIPV by qRT-PCR

Cell-free viral RNA was isolated from the parent virus using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. The isolated RNA was treated with DNase (Turbo DNase, Invitrogen) and subsequently reverse transcribed using a High-Capacity RNA-to-CDNA kit (Applied Biosystems) according to the manufacturers' protocols. The copy number of FIPV and feline GAPDH cDNA was determined using an Applied Biosystems' QuantStudio 3 Real-Time PCR System and a PowerUp SYBR Green Master Mix according to the manufacturer's protocol for a 10 μL reaction. Each PCR reaction was performed in triplicate with an aqueous template as a negative control and plasmid DNA as a positive control. A reverse transcriptase-secreting control reaction was included in each set of real-time PCR assays. The cDNA templates were amplified using the FIPV forward primer, 5'-GGAAGTTTAGATTTGATTTGGCAATGCTAG and the FIP reverse primer, 5'-AACAATCACTAGATCCAGACGTTAGCT (terminal part of the FIPV gene 7b) [25]. Real-time PCR for the feline domestic GAPDH gene was performed simultaneously using primers, 5 GAPDH, 5'-AAATTCCACGGCACAGTCAAG and 3 GAPDH, 5'-TGATGGGCTTTCCATTGATGA. The cycling conditions for both FIPV and GAPDH amplicons were as follows: 50 ° C for 2 minutes, 95 ° C for 2 minutes, followed by 40 cycles of 95 ° C for 15s, 58 ° C for 30s, 72 ° C for 1 minute. The last step contained a dissociation curve to evaluate the specificity of the primer binding. The copy number of FIPV and GAPDH was calculated on the basis of standard curves generated in our laboratory. Copies of FIPV cIPNA determined by real-time RT PCR were normalized to 106 copies of feline GAPDH cDNA.

Development of anti-helicase chemical fragments

The drugs studied and described in this study were already known antiviral agents. In contrast, the helicase enzyme FIPV was cloned, expressed and used as a target for coronavirus and enzyme-specific viral discovery. The AviTag-FIP Helicase-HisTag target DNA sequence was optimized and synthesized. The synthesized sequence was cloned (Adeyemi Adedeji) into the Avi-His tagged pET30a vector to express the protein in E. coli. E. coli strain BL21 (DE3) was transformed with a recombinant plasmid. One colony was inoculated into 1 liter of auto-induced medium containing the antibiotic and the culture was incubated at 37 ° C at 200 rpm.

When the OD600 reached about 3, the cell culture temperature was changed to 15 ° C for 16 hours. Cells were harvested by centrifugation. The cell pellets were resuspended in lysis buffer followed by sonication. The centrifuge precipitate was dissolved with a denaturing agent. The target protein was obtained by one-step purification on a Ni column. The target protein was sterilized with a 0.22 μm filter. The yield was 7.2 mg at 0.90 mg / ml and was stored in PBS, 10% glycerol, 0.5 mM L-arginine, pH 7.4. The concentration was determined by the Bradford protein assay with BSA as a standard. Protein purity and molecular weight were determined by SDS-PAGE with Western blot confirmation.

Surface plasmon resonance (SPR) fragments were screened on a ForteBio Pioneer FE SPR platform. A HisCap sensor chip containing an NTA surface matrix was used. Channels 1 and 3 were filled with 100 μM NiCl 2, followed by injection of 50 μg / ml FIP protein. Channel 2 was left protein-free as well as NiCl 2 as a reference. Channel 1 was immobilized to a density of 88000 RU, while channel 3 contained approximately 12000 RU. Channel 1 was used. The buffer used for immobilization was 10 mM HEPES, pH 7.4, 150 mM NaCl and 0.1% Tween-20. DMSO was added to a final concentration of 4% for this assay. The proprietary compound library was diluted in the same DMSO-free buffer to a final DMSO concentration of 4% DMSO. Compounds from the library were tested at a concentration of 100 μM using the OneStep gradient injection method. The findings were selected based on RU and kinetics and used for cell screening.

Viral plaque test

To screen for antiviral activity of compounds, infected CRFK cells were treated with compounds in six-well replicates and compared to positive control wells (infected cells), negative controls (uninfected cells) and treatment controls (infected cells treated with a known active antiviral compound) simultaneously on each tissue plate. culture. CRFK cells were grown in 96-well tissue culture plates (Genesee Scientific) containing 200 μl of culture medium. At 7575-85% cell confluence, the medium in uninfected control wells was aspirated and replaced with 200 μl of fresh medium. The medium in the infected wells was aspirated and replaced with FIPV inoculated medium at a multiplicity of infection (MOI) of 0.004 infectious virion per cell. The tissue culture plate was incubated for 1 hour with periodic gentle agitation ("number eight" manipulations) every 15 minutes to facilitate virus-cell interaction. One hour after infection, each putative antiviral compound was added to six wells infected with FIPV (to determine the antiviral activity of the compound) and six uninfected control wells (to screen for cytotoxicity of the compound in CRFK cells). All compounds were initially screened at 10 μM, except for the "chemical fragment" compounds supplied by M. Olsen (Midwestern University), which were evaluated at 50 μM. Tissue culture plates were incubated at 37 ° C for 72 hours and then fixed with methanol and stained with crystal violet. Plates were scanned for absorbance at 620 nm using an ELISA plate reader (FilterMax F3, Molecular Devices; Softmax Pro, Molecular Devices). For each treatment condition, individual well absorbance values were recorded along with the mean absorbance value and mean error of the mean for 6-well experimental replicates.

For substances that demonstrated antiviral efficacy at initial screening at 10 or 50μM (protected from CPE-associated virus), the EC50 was determined by performing a series of progressive 2-fold dilutions of the compounds in a viral plaque assay. To determine the EC50, CRFK cells were grown in 96-well tissue culture plates similar to the antiviral screening assay. Except for uninfected control wells, all remaining wells were infected with FIPV as described above. A two-fold dilution series ranged from 20μM to 0μM and each concentration was performed in six well replicates. The number of dilution steps ranged from 6 to 14 and was compound dependent. Six well replicates of uninfected CRFK cells served as a control for normal CRFK cells; six FIPV-infected CRFK cell replicates served as untreated FIPV-infected control wells; and six well replicates of FIPV-infected CRFK cells treated with GS-441524 served as control wells for protection against virus-induced cell death based on published data on the efficacy of using GS-441524 in vitro in CRFK cells [26].

Tissue culture plates were incubated for 72 hours and then fixed with methanol, stained with crystal violet, and the absorbance at 620 nm was scanned using an ELISA plate reader. Individual absorbance values along with the mean absorbance value and standard deviation for 6-well experimental replicates were recorded for each treatment condition. The EC50 was calculated by plotting a non-linear regression equation (dose-response curve) using Prism 8 software (GraphPad).

Viral RNA knock-down test

Real-time RT-PCR assays were used to quantify inhibition of viral RNA production by the compound. CRFK cells were cultured in a 6-well tissue culture plate (Genesee Biotek). At approximately 75-85% cell fusion, the culture medium was replaced with fresh medium and the cells were infected with FIPV serotype II at an MOI of 0.2 (MOI based on TCID50 bioassay / pfu). The plates were incubated for one hour with periodic gentle shaking every 15 minutes. Wells infected with FIPV were treated with one (monotherapy), two or three (combined anti-cancer therapy) antiviral compounds; each experimental treatment was performed three times. The compound dose was based on the EC50 of the compounds and ranged from 0.001 to 20 μM. For each experimental set, three culture wells with FIPV-infected and untreated CRFK cells served as virus-infected controls. Infected cell cultures were then incubated for 24 hours and total RNA associated with the cells was isolated using a PureLink-RNA mini kit (Invitrogen). RNA was treated with DNAse (TurboDNAse, Ambion), reverse transcribed into cDNA using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems), and FIPV cIPNA and feline GAPDH cDNA were measured by real-time qRT-PCR as described above. . The fold reduction in viral titer was determined by dividing the normalized mean FIPV RNA copy number for untreated FIPV-infected CRFK cells into the normalized mean FIPV RNA copy number for CRFK treated cells with the desired compound (s). The expected additive effect was determined by adding a fold reduction for each monotherapy used in combination. The composite additive effect was determined by dividing the predicted additive effect by the combined multiple reduction value for a particular combination therapy.

Determination of cytotoxicity safety profiles (CSP).

The cytotoxicity of the compound in feline cells was assessed using a commercially available kit (CellTox Green Cytotoxicity Assay, Promega) according to the manufacturer's instructions. Untreated CRFK cells were used as negative controls and the cells were treated with a cytotoxic solution provided by the manufacturer as positive toxicity controls. Briefly, in addition to control wells, CRFK cells were plated in 96-well tissue culture plates (Genesee Scientific) in four well replicates with 5, 10, 25, 50 or 100 μM concentrations of the desired compound and incubated for 72 hours. After 72 hours, all wells were stained with the DNA kit, incubated at 37 ° C protected from light for 15 minutes, and the fluorescence intensity at 485-500 nm Ex / 520-530 nm EM was subsequently determined using a plate reader (FilterMax F3, Molecular Devices; Softmax Pro, Molecular Devices). The cytotoxicity of a compound at a particular concentration was thought to be proportional to the fluorescence intensity based on the selective penetration and binding of the dye to the DNA of degenerated, apoptotic or necrotic cells. The extent of cytotoxicity was determined by adjusting the fluorescence value for cells treated with the positive control reagent to 100% and untreated feline cells as 0% cytotoxicity. The average fluorescence value for the four wells containing each compound concentration was then interpolated as a percentage (percent cytotoxicity) ranging from 0 to 100%.

Conflict of interests

The authors declare that there has been no conflict of interest.

We appreciate funding provided by the Winn Feline Foundation (MTW 17-020; MTW 19-026) and the University of California, Davis, Center for Companion Animal Health (CCAH; 2018-92-F; 2018-94-FE) through multi-FIP research donations individual donors and organizations (SOCK FIP, Davis, CA) and foundations (Philip Raskin Fund, Kansas City, KS).

Additional information

Complete list of compounds tested in vitro for FIPV activity

Nucleoside polymerase inhibitors

12x GS Nuc Analogs	Nucleoside analog	NPI
GS-441524 (China-sourced)	Adenosine analog nucleoside	NPI
3-Deazaneplanocin A Hydrochloride	Adenosine analog nucleoside	NPI
Adefovir	Adenosine analog nucleoside	NPI
Galidesivir	Adenosine analog nucleoside	NPI
GS-441524 (Manufactured in China)	Adenosine analog nucleoside	NPI
MK-0608	Adenosine analog nucleoside	NPI
NITD008	Adenosine analog nucleoside	NPI
Didanosine	Adenosine analog nucleoside	NPI
Tenofovir alafenamide	Adenosine analog nucleoside	NPI
Tenofovir disoproxil fumarate	Adenosine analog nucleoside	NPI
EIDD 1931	Nucleoside analog Cytidine
EIDD 2801	Nucleoside analog Cytidine
2′-C-methylcytidine	Nucleoside analog Cytidine	NPI
Gemcitabine Hydrochloride	Nucleoside analog Cytidine	NPI
2-C-methylguanosine	Nucleoside analogue Guanosine	NPI
7-methylguanosine	Nucleoside analogue Guanosine	NPI
Entecavir	Nucleoside analogue Guanosine	NPI
Mizoribine	Nucleoside analogue Guanosine	NPI
Ribavirin	Nucleoside analogue Guanosine	NPI
PSI-6206	Nucleoside analog Uridine	NPI
6-Azauridine	Nucleoside analog Uridine	NPI
Balapiravir	Nucleoside analog Cytidine	NPI
Sofosbuvir	Nucleoside analog Uridine	NPI
Favipiravir	Nucleoside analog Purine	NPI

Total 36

Protease inhibitors

Grazoprevir	NS3 / 4A protease inhibitor	PI
Rupin trivir	Rhinoviral 3CP inhib	PI
Lopinavir	Antiretroviral PI	PI
Ritonavir	Antiretroviral PI	PI
Nelfinavir	Antiretroviral PI	PI
Disulfiram (tetraethyliuram disulfide)	Papain-like protease inhib	PI
K777 / K11777	Cysteine protease inhibitor	PI
Telaprevir	NS3 / 4A protease inhibitor	PI
Camostat mesylate	Serine protease inhibitor	PI
Paritaprevir	Serine protease inhibitor	PI
GC376	Coronavirus protease inhibitor	PI

Total 11

NS5A inhibitors

Velpatasvir	NS5A Inhibitor	NS5A Inhibitor
Ravidasvir / PPI-668	NS5A Inhibitor	NS5A Inhibitor
Ledipasvir	NS5A Inhibitor	NS5A Inhibitor
Ombitasvir	NS5A Inhibitor	NS5A Inhibitor
Pibrentasvir	NS5A Inhibitor	NS5A Inhibitor
Daclatasvir	NS5A Inhibitor	NS5A Inhibitor
Elbasvir	NS5A Inhibitor	NS5A Inhibitor

Total 7

NNPI

Dasabuvir

Non-nucleoside polymerase inhibitor

NNPI

Total 1

other

Monensin	Ionophore	other
Phenazopyridine hydrochloride	Crystalline solid	other
Pyrvinium pamoate hydrate	Androgen receptor inhibitor	other
Toremifene citrate	Selective estrogen receptor modulator	other
AM580	Retinobenzoic derivative	other
Homoharringtonine	Translation elongation inhib	other
Amodiaquine	4-aminoquinolone	other