key: cord-0316766-dnwh3via authors: Puhl, Ana C.; Gomes, Giovanni F.; Damasceno, Samara; Godoy, Andre S.; Noske, Gabriela D.; Nakamura, Aline M.; Gawriljuk, Victor O.; Fernandes, Rafaela S.; Monakhova, Natalia; Riabova, Olga; Lane, Thomas R.; Makarov, Vadim; Veras, Flavio P.; Batah, Sabrina S.; Fabro, Alexandre T.; Oliva, Glaucius; Cunha, Fernando Q.; Alves-Filho, José C.; Cunha, Thiago M.; Ekins, Sean title: Pyronaridine Protects Against SARS-CoV-2 in Mouse date: 2021-09-30 journal: bioRxiv DOI: 10.1101/2021.09.30.462449 sha: fe2aacabb804947286e8004c46e1c3151ef1ebce doc_id: 316766 cord_uid: dnwh3via There are currently relatively few small-molecule antiviral drugs that are either approved or emergency approved for use against SARS-CoV-2. One of these is remdesivir, which was originally repurposed from its use against Ebola and functions by causing early RNA chain termination. We used this as justification to evaluate three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg. Out of these we previously identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. Herein, the in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS CoV-2 infected mice. Furthermore, the pyronaridine treated group reduced lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Notably, pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 µM) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. Interestingly, pyronaridine also selectively inhibits the host kinase CAMK1 (IC50 of 2.4 µM). We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19. One sentence summary There is currently intense interest in discovering small molecules with direct antiviral activity against the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Pyronaridine, an antiviral drug with in vitro activity against Ebola, Marburg and SARS-CoV-2 has now statistically significantly reduced the viral load in mice along with IL-6, TNF-α, and IFN-β ultimately demonstrating a protective effect against lung damage by infection to provide a new potential treatment for testing clinically. There are currently relatively few small-molecule antiviral drugs that are either approved or emergency approved for use against SARS-CoV-2. One of these is remdesivir, which was originally repurposed from its use against Ebola and functions by causing early RNA chain termination. We used this as justification to evaluate three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg. Out of these we previously identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. Herein, the in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of . Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS CoV-2 infected mice. Furthermore, the pyronaridine treated group reduced lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Notably, pyronaridine inhibited the viral PL pro activity in vitro (IC50 of 1.8 µM) without any effect on M pro , indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. Interestingly, pyronaridine also selectively inhibits the host kinase CAMK1 (IC50 of 2.4 µM). We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PL pro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19. At the time of writing, we are in the midst of a major a global health crisis caused by the virus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that was originally reported in Wuhan, China in late 2019 (1, 2). Infection with this virus leads to extensive morbidity, mortality and a very broad range of clinical symptoms such as cough, loss of smell and taste, respiratory distress, pneumonia and extrapulmonary events characterized by a sepsis-like disease collectively called 2019 coronavirus disease (COVID-19) (3) . In the USA, there are currently three vaccines available, one of which has recently obtained full approval from the FDA to protect against SARS-CoV-2 (4-6). There are however few small-molecule drugs approved for COVID-19 (7) including remdesivir (8) , which originally demonstrated activity in Vero cells (9, 10) , human epithelial cells and in Calu-3 cells (10) infected with SARS-CoV-2 prior to clinical testing. Remdesivir represents a repurposed drug which was originally developed for Hepatitis C virus but was then repurposed for treating Ebola and has since reached clinical trials (11) . We therefore hypothesized that other drugs that were effective against Ebola might also be prioritized for evaluation in vitro against SARS-CoV-2. Previously, we had used a machine-learning model to identify tilorone, quinacrine and pyronaridine tetraphosphate (12) for testing against Ebola virus (EBOV) and subsequently these three inhibited EBOV and Marburg in vitro as well as demonstrating significant efficacy in the mouse-adapted EBOV (ma-EBOV) model (13) (14) (15) ). All of these molecules were identified as lysosomotropic, a characteristic that suggests these could be possible entry inhibitors (16) . Pyronaridine tetraphosphate is used as an antimalarial in several countries as part of a combination therapy with artesunate (Pyramax). Pyronaridine alone also demonstrated significant activity in the guinea pig-adapted model of EBOV infection (17). We and others (18) (19) (20) have recently shown that these compounds possess in vitro activity against SARS-CoV-2 and tilorone and pyronaridine are in clinical trials, the latter in combination with artesunate. The Cmax data for pyronaridine in our previous mouse pharmacokinetics studies (i.p. dosing) suggests that plasma levels that are above the average IC50 observed for SARS-CoV-2 inhibition in vitro (13) can be reached with dosing well below the maximum tolerated dose. Pyronaridine also has excellent in vitro ADME properties with a long half-life that makes a single dose treatment possible (13, 18) . We now expand on our earlier in vitro characterization of pyronaridine (18) by assessing the in vivo efficacy in a mouse model of COVID-19. Finally, in an attempt to further explore molecular mechanisms, we tested the activity of pyronaridine in vitro against viral and host targets. In vivo efficacy was assessed in K-18-hACE2 mouse model of COVID-19 (21-23). Pyronaridine (75 mg/kg, i.p) (13) was administered 1 h prior to infection. Mice that were given pyronaridine, received a single treatment. On the third day post-infection, mice were euthanized and lung viral load, cytokine levels and histopathology were evaluated ( Figure 1A ). In all groups tested, mice lost weight compared to uninfected animals that received only vehicle formulation ( Figure 1B) . Lung viral load was evaluated by RT-qPCR and the pyronaridine treated group showed a statistically significant decrease in the lung viral load ( Figure 1C ). Moreover, reduced levels of INF-1β were observed in infected mice, and pyronaridine restored the levels of INF-1β close to that found in uninfected animals ( Figure 1D ). Interestingly, the increased levels of IL-6 found in the infected untreated group were not observed in animals treated with pyronaridine ( Figure 1E ). In addition, pyronaridine reduced the high levels of CXCL1 and CCL4 observed in infected animals ( Figure 1D ). Pyronaridine did not however reduce the high levels of IL-10, TNF-α, CCL2, and CCL3 ( Figure 1F , G, K, L, M) below the elevated levels found in the infected mice. To determine the severity of lung damage, histological examination of hematoxylin and eosin (H&E) stained lung tissue was performed. Infected, untreated mice showed severe pathological changes with inflammatory cell infiltrates. In contrast, pyronaridinetreated animals exhibited improved morphology and milder infiltration ( Figure 2A ). Histological observations were confirmed by quantitative morphometric analysis of the H&E-stained slides showing a statistically significant reduction in inflammation ( Figure 2B ). SARS-CoV-2 proteases M pro and PL pro are essential for viral replication and have been widely studied for the discovery of new direct acting antivirals (24-26). Pyronaridine was therefore tested against both SARS-CoV-2 recombinant PL pro and M pro through FRETbased in vitro assays. Pyronaridine inhibited PL pro with an IC50 of 1.86 0.58 µM ( Figure 3A ) but did not show any appreciable activity against M pro at 20 µM (data not shown). Additional analogs of pyronaridine were also synthesized and tested against PL pro . The analogs 12126038, 12126039 and 12126040 showed similar inhibitory activity when compared with pyronaridine (as well as that reported for GRL0617 (27)), indicating that the aminophenol moiety together with pyrrole or tertiary amine substitutions at the meta position are tolerated for PL pro inhibition (Table 1, Figure 3A ). The deletion of these groups in analogs 10326099, 12126035, 12126036, 12126037 and 12126072 caused complete abolishment of the inhibitory activity of the series (Table 1) . The PL pro active site contains four subsites for peptide recognition, with a strong preference for positively charged amino acids at P3 and P4 subsites (24-26). In our molecular docking studies ( Figure 3B ), the positions where the 2,6-bis[(pyrrolidin-1-yl)methyl]phenol moiety of pyronaridine were docked at the P4 subsite showed a far higher score than the second highest cluster group (Glide score of -5.052 and HADDOCK score of -65.6). These positively charged aminogroups would likely satisfy the negatively charged cavity that forms the P4 subsite, forming hydrogen bounds with Asp164 and π-stacking with Tyr26 ( Figure 3C ). Compounds 12126038 and 12126039 also showed very similar poses after docking, indicating binding to the P4 subsite ( Figure 3B ). The primary docking poses from Glide for all compounds are provided in Fig. S1 . These results indicate that the in vitro and in vivo efficacy of pyronaridine may be related to PL pro inhibition. Due to the possible effect of pyronaridine on cytokines ( Figure 1 ) we have also assessed the effect on host targets, as SARS-CoV-2 can cause an imbalance in the immune system that may result in a cytokine storm(28) as well as leading to acute respiratory distress syndrome (ARDS), coagulation disorders, and eventually multiple organ failure (28, 29). Hence targeting the cytokine storm to address hyperinflammation represents another approach to the treatment of COVID-19 patients (30-33). In this regard, we have explored the effect of pyronaridine on human kinases which are responsible for host cell signaling (34, 35) . Screening of pyronaridine (tested at 1 µM) against 485 kinases identified only 2 as having mean percent inhibition greater than 30% including CAMK1 (35%) and MELK (31%) (Table S1 ). Subsequently, the IC50 was determined for CAMK1 (2.4 µM) ( Figure 4 ). Even with vaccines becoming widely available in many countries COVID-19 continues to exact a very heavy toll on those that are unvaccinated. We are in a race against time before the virus mutates and vaccines lose their effectiveness. There is therefore an urgent need for new antivirals and in particular small-molecule treatments that are orally delivered and can be used outside of a hospital setting. Finding, developing, and progressing small molecules to the clinic is generally a slow and expensive process (36), hence drug repurposing has been attempted by many groups to speed this up (either experimentally or computationally (37)) by identifying already approved or clinical stage candidates used for other applications or quickly follow up the few molecules that are being used already. The traditional prioritization of compounds in vitro before animal models and then humans is still repeated and so far with few successes with many molecules not demonstrating efficacy in vivo (38, 39). Our understanding of the antiviral mechanism of pyronaridine previously shown to inhibit the Ebola virus in vitro and in vivo (13) via binding to the viral glycoprotein(16) as well as through its potent lysosomotropic activity (40) and now the in vitro activity against SARS-CoV-2 (18) is also further expanded. Pyronaridine was previously identified with in vitro activity against SARS-CoV-2 in A549-ACE2 cells that was on a par with remdesivir in this cell line (18). In the current study, we have demonstrated that pyronaridine also has antiviral activity against SARS-CoV-2 in vivo. A single prophylactic dose of pyronaridine (75 mg/kg i.p) reduced the viral load in the lung of infected mice 3 days postinfection. In vitro assays suggest that pyronaridine possesses a direct antiviral effect showing activity against PL pro (IC50 1.86 µM, Figure 3 ), but did not inhibit SARS-CoV-2 M pro . Kinase profiling, resulted in determination of IC50 for CAMK1 (IC50 2.4 µM, Figure 4 ). In summary, our present study provided additional data on the efficacy of pyronaridine against SARS-CoV-2 infection as well as highlighting reduced lung pathology and inflammation in a mouse model of COVID-19. Furthermore, we have shown that pyronaridine may target Pl pro as well as CAMK1. There are few inhibitors of CAMK1 that have been identified to date (such as Barettin (62) or pyridine amides (63)) which has a role in inflammation targeting IL-10 (62). Previous in vitro work has shown that inhibiting CAMK1 in cells reduces IL-10, the master anti-inflammatory interleukin (64). In the present study there is not a significant difference in the IL-10 levels between the untreated and pyronaridine-treated infected groups so it seems unlikely that CAMK1 inhibition would be involved in the mechanism of action of inhibition of SAR-CoV-2. In conclusion, we propose that pyronaridine could be used alone as a potential therapeutic candidate for COVID-19. Finally, with the emerging virulence of novel SARS-CoV-2 strains, identifying repurposed drugs with novel mechanisms of action and whose antiviral activity translates from in vitro to in vivo are rare (39), and may lead to new treatments as well as their further optimization. SE is CEO of Collaborations Pharmaceuticals, Inc. ACP and TRL are employees at Collaborations Pharmaceuticals, Inc. The authors would like to kindly acknowledge their many collaborators around the world who have assisted in our various COVID-19 projects. The authors gratefully acknowledge the technical assistance of Marcella Daruge Grando, Ieda Regina dos Santos, Juliana Trench Abumansur, and Felipe Souza. We kindly acknowledge NIH funding: SE kindly acknowledges NIH funding The molecules were synthesized according to Scheme 1 or Scheme 2 and the specific methods and analytical results are described in the Supplemental Methods. Dose formulation for pyronaridine was prepared as previously described (13) On the day 3 post-infection animals were humanely euthanized, and lungs were collected. Right lung was collected, harvested, and homogenized in PBS with steel glass beads. The homogenate was added to TRIzol® (Invitrogen, CA, EUA) reagent (1:1), for posterior viral titration via RT-qPCR, or to lysis buffer (1:1), for ELISA assay, and stored at -70 °C. The left lung was collected in paraformaldehyde (PFA 4%) for posterior histological assessment. Total RNA from the right lungs were obtained using the Trizol® (Invitrogen, CA, EUA) method and quantified using NanoDrop One/Onec (ThermoFisher Scientific, USA). A total of 800 ng of RNA was used to synthesize cDNAusing the High-Capacity cDNA Reverse Five micrometer lung slices were submitted to Hematoxylin and Eosin staining. A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to a HDI camera for images capture. The total septal area and total area were analyzed with the aid of the Pro Plus 7 software (Media Cybernetics, Inc., MD, USA). Morphometric analysis was performed in accordance with the protocol established by the American Thoracic Society and European Thoracic Society (ATS/ERS) (72). All reagents and solvents were purchased from commercial suppliers and used without further purification. 1 H and 13 Solid 2-aminophenol (0.39 g, 3.6 mmol) was added to a suspension of starting 6,9dichloro-2-methoxyacridine 1 (0. A mixture of pyrrolidine (0.52 ml, 6.0 mmol), 0.7 ml of acetic acid, and 37% aqueous formaldehyde solution (0.28 ml, 3.6 mmol) were added to a suspension of 4-[(6-chloro-2methoxyacridin-9-yl)amino]phenol 2 (0.6 mmol) in 10 ml of dimethylformamide. The solution was stirred at reflux for 1 hour. The reaction mixture was cooled, an aqueous solution of NaHCO3 (11 mmol) was added and extracted with ethyl acetate (2 x 30 ml). The combined organic layer was washed with water (2 x 100 ml), dried by sodium sulfate and solvent was evaporated in vacuo. The resulting oil was dissolved in chloroform and applied to a chromatography column (chloroform/methanol 9:1) and product 4 was isolated. Pure methanol was used as eluent for separation product 5. -1-ylmethyl) Morpholine (0.23 ml, 2.7 mmol) was added to a suspension of starting 6,9-dichloro-2- Dimethyl acetal dimethylacetamide (1 ml, 7.2 mmol) was added to a suspension of cyanoacetamide 1 (0.5 g, 6 mmol) in 15 ml of absolute alcohol. The suspension was boiled for 3 hours, cooled, obtained precipitate was filtered off, washed with alcohol and diethyl ether. Aim product 2 was obtained with a yield of 82%. Mp 188-192 o С (isopropanol). Aniline (0.45 ml, 5 mmol) was added to a suspension of (2E)-2-cyano-3-(dimethylamino)but-2-enamide 2 (0.3 g, 2 mmol) in acetic acid (4 ml). The suspension was boiled for 3 hours, cooled, acetic acid was removed under vacuum/ The residue was treated by water, formed precipitate was filtered off and washed with water, isopropyl alcohol and diethyl ether. Aim product 3 was obtained with a yield of 87%. Mp 179-182 o С (isopropanol). Dimethylformamide diethyl acetal (1.3 ml, 7.5 mmol) was added to a suspension of (2E)-3-anilino-2-cyanobut-2-enamide 3 (0.6 g, 3 mmol) in 5 ml of absolute ethanol. The dark red solution was boiled for 6 hours. Part of the solvent was removed under vacuum until a thick suspension was obtained. The precipitate was filtered off, washed with absolute alcohol and dry diethyl ether. A mixture of compounds 4 and 5 was obtained. -2-oxo-1,2-dihydropyridine-3-carbonitrile 6 . The 4-anilino-5-formyl-2-oxo-1,2-dihydropyridine-3-carbonitrile 6 (2.3 g) was refluxed in phosphorus oxychloride (22 ml) for 1 hour. The reaction mixture was poured onto ice, stirred for 30 min. Formed precipitate was filtered off, washed with water, ethyl alcohol, diethyl ether. Aim product 7 was obtained in 80% yield. Mp 300-304 o С (DMF). To a suspension of 3-chloro-2,3-dihydrobenzo[b]-1,6-naphthyridine-4-carbonitrile 7 (0.44 g) in 15 ml of acetone was added m-chloroperbenzoic acid 55% (1.45 g) in small portions and refluxed for 9 hours. The reaction mixture was cooled, obtained precipitate was filtered off and washed with acetone, toluene, acetone. The aim product 8 was obtained with a yield 50%. Mp 305 o C. Morpholine (0.14 ml, 1.6 mmol) was added to a solution of 3-chloro-10-oxo-5,10- The viral cDNA template (GenBank MT126808.1) was kindly provided by Dr. Edison Kinase profiling was performed for pyronaridine (1 M) in duplicate by ThermoFisher (Life Technologies Corporation, Chicago, IL 60693) using Z'Lyte (76), Adapta (77) and LanthaScreen (78) assays for 485 purified kinases. Pyronaridine inhibition of CAMK1 was performed by Selected Services (Thermo Fisher) using the the Adapta universal kinase assay, which is a homogenous, fluorescent-based immunoassay for the detection of ADP. (TIE2) Y1108F -2 Lanthascreen TESK1 -2 Lanthascreen TESK2 -3 Lanthascreen TGFBR1 (ALK5) -4 Lanthascreen TGFBR2 -4 Lanthascreen TLK1 12 Lanthascreen TLK2 -2 Lanthascreen TNIK -1 Lanthascreen TNK2 (ACK) 1 Lanthascreen TTK 4 Lanthascreen ULK1 -4 Lanthascreen ULK2 2 Lanthascreen ULK3 8 Lanthascreen VRK2 -4 Lanthascreen WEE1 10 Lanthascreen WNK1 4 Lanthascreen WNK2 4 Lanthascreen WNK3 7 Docking poses from pyronaridine and all tested analogs. PL pro surface is colored according to its electrostatic potential. 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