The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2

SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner; a stronger antiviral effect for AVI-4206 is observed in human airway organoids. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results provide pharmacological proof of concept that Mac1 is a valid therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major threat to public health.Despite the approval of several biologic and small molecule therapeutics, there is an urgent need for new small molecule antivirals with distinct mechanisms of action to overcome potential resistance to existing agents (Li et al. 2023b;von Delft et al. 2023).While most antivirals target an essential aspect of viral entry or replication, a potential avenue for new antivirals with alternative mechanisms is to target viral proteins that act to blunt the host immune response (Minkoff and tenOever 2023).For example, SARS-CoV-2 has evolved multiple mechanisms to evade and counter interferon signaling (Kim and Shin 2021).The viral proteins involved in such evasion would be valuable drug targets if their inhibition renders the host immune response sufficient to control virus replication and reduce disease severity.The macrodomain (Mac1) of non-structural protein 3 (NSP3) in SARS-CoV-2 is one such target that plays an antagonistic role to the host interferon response (Schuller et al. 2023).Macrodomains are found across the tree of life and catalyze the hydrolysis of ADP-ribose covalent modifications on protein side chains (Dasovich and Leung 2023).Viral macrodomains are found in alphaviruses, hepatitis E virus, and many betacoronaviruses (Leung et al. 2022) and in some systems, like murine hepatitis virus (MHV), their activity can be essential for viral replication (Voth et al. 2021).While SARS-CoV-2 bearing either catalytically inactivating point mutations (Taha et al. 2023b) or deletion of the Mac1 domain (Alhammad et al. 2023) have minor phenotypes in cell culture, their replication is profoundly attenuated in animal models.This discordance likely reflects the inability of cellular models to recapitulate the complex intercellular and systemic signaling required for proper viral-host immune interactions.The underlying mechanism of action results from the enzymatic activity of Mac1, which counters the wave of ADP-ribosylation that is catalyzed by poly-adenosine diphosphate-ribose polymerase (PARP) proteins during the interferon response (Kerr et al. 2023;Kar et al. 2024;Parthasarathy et al. 2024).While the critical proteins and sites modified by interferon-induced PARPs are not fully characterized, the inhibition of Mac1 should allow ADP-ribosylation and the resulting downstream signaling to persist (Kar et al. 2024).Indeed, multiple interferon genes are down-regulated upon infection with wild-type SARS-CoV-2 relative to a Mac1 deficient mutant, consistent with the hypothesis that antiviral interferon signaling could be productively enhanced by Mac1 inhibition (Alhammad et al. 2023;Taha et al. 2023b).We (Schuller et al. 2021;Gahbauer et al. 2023), and others (O'Connor et al. 2023;Schuller et al. 2023;Wazir et al. 2024), have previously developed inhibitors of Mac1 with activity in vitro.However, the therapeutic hypothesis that pharmacological Mac1 inhibition would restore host immune responses and lead to a survival benefit after SARS-CoV-2 infection has not yet been tested.Here, we build on our experimental fragment (Schuller et al. 2021) and virtual screening approach (Gahbauer et al. 2023) with medicinal chemistry, to develop a potent lead compound, AVI-4206, that engages Mac1 in cellular models and has suitable pharmacological properties to test antiviral efficacy in vivo.In an animal model of SARS-CoV-2 infection, AVI-4206 reduces viral replication, restores an interferon response, and leads to a survival benefit.Therefore, our results validate Mac1 as a therapeutic target via a novel immune-restoring mechanism that could synergize with existing therapies targeting essential aspects of viral replication.The approach could be more widely used to target other macrodomains in viruses beyond SARS-CoV-2.

Results
Optimization of in vitro potency against the SARS-CoV-2 Macrodomain Previously, we described two novel Mac1 inhibitors, AVI-92 and AVI-219 (Gahbauer et al. 2023), which evolved from fragment screening and virtual screening hits, respectively.heirpotency was determined using an ADPr-conjugated peptide displacement-based homogeneous time resolved fluorescence (HTRF) assay (Figure 1).The superposition of the Mac1 crystal structures in complex with both leads inspired a parallel approach to optimization, which was supported by additional high resolution X-ray structures of the complexes (Figure 1A,E, Supplementary Table 1, Supplementary Figure 1).First, we generated a merged compound that used the urea function of AVI-92 in the more lead-like AVI-219 scaffold, thus avoiding the phenolic and carboxylate functionalities present in AVI-92.Indeed, the X-ray structure of the resulting complex between Mac1 and AVI-4051 shows that it preserves and favorably orients the two hydrogen bonding contacts with the carboxylate of Asp22 and exhibits a ~four-fold lower IC50 value as compared to AVI-219 in the HTRF assay (Figure 1B,E).Further structure activity relationship (SAR) studies revealed a strong preference for urea (e.g., AVI-1500, IC50 of ~120 nM) over acetamide (AVI-1501) or carbamate (AVI-3367) in productively engaging Asp22 (Figure 1B,E).Second, at the other end of the adenosine site, we observed that the pyrrolidinone carbonyl of AVI-219 could accept hydrogen bonds from the backbone amides of Phe156/Asp157 (Figure 1C,E).To improve contacts with non-polar residues in this sub-site, we next explored substitutions of the pyrrolidinone ring.While C-5 substituents as large as phenyl were tolerated (AVI-3762 and AVI-3763), these analogs showed reduced ligand efficiency compared to AVI-219.By contrast, a methyl group at C-5 in either stereochemical configuration (AVI-3764 and AVI-3765) improved potency and ligand efficiency.Introducing two methyl groups at C-5 afforded the achiral, gem-dimethyl pyrrolidinone AVI-4636 with an impressive five-fold improvement in potency (IC50 of ~200 nM) compared to AVI-219 (Figure 1C,E).Ultimately, combining the ethyl urea side chain of AVI-1500 with the gem-dimethyl pyrrolidinone of AVI-4636 produced AVI-4206, the most potent Mac1 inhibitor identified from this series, with an IC50 value of ~20 nM (Figure 1A,E).This potency approaches the floor of our HTRF assay, which uses 12 nM of enzyme, and indicates that AVI-4206 is at least ~25-100-fold more potent than the AVI-92 and AVI-219 starting points, respectively.The high resolution co-crystal structure of AVI-4206 confirmed that the desired interaction elements and conformations were maintained from the separate optimization paths (Figure 1e).To confirm that the high affinity binding of AVI-4206 was reflected in inhibition of Mac1 catalytic activity, we used auto ADP-ribosylated PARP10 and a coupled NudT5/AMP-Glo assay to measure ADP-ribose released by the enzymatic reaction (Kasson et al. 2021) (Supplementary Figure 2).This assay demonstrated AVI-4206 potently inhibits Mac1 with an IC50 of 64 nM (Figure 1D).

AVI-4206 engages Mac1 in cells with high specificity
Having discovered AVI-4206 as a potent inhibitor of Mac1, we next determined whether this compound could enter cells and bind to Mac1 in this context.Therefore, to assess cellular target engagement, we developed a nanoluciferase-based CEllular Thermal Shift Assay (CETSA-nLuc) assay (Martinez et al. 2018) to measure thermal stabilization of Mac1 upon compound binding.A549 cells transiently expressing a HiBiT-and FLAGtagged Mac1 protein were treated with compounds for 1 hour and then incubated across a gradient of temperatures.After heat exposure, cells were lysed and incubated with LgBiT protein which binds to soluble HiBiT-Mac1 protein reconstituting nanoluciferase and producing a luminescent signal.We observed that the Tagg (the temperature at which 50% of protein is soluble) shift for compounds at 10 µM mirrored the affinities measured by the HTRF assay, suggesting a dominant role for Mac1 affinity, rather than bioavailability, or another factor, in determining target engagement in cells (Figure 2A).Furthermore, we observed a dosedependent shift in Tagg by AVI-4206, with a marked ~10°C shift in cells treated with 10 µM of compound compared to DMSO-treated control cells (Figure 2B).The observations were also validated by western blotting with a FLAG-specific antibody (Supplementary Figure 3).After confirming Mac1 target engagement in cells, we next tested the selectivity of AVI-4206 for Mac1 over two human macrodomains, Targ1 and MacroD2.In an adapted HTRF assay, both human proteins bind to ADP-ribose in the same low-μM range as Mac1 (Figure 2C), AVI-4206 does not bind appreciably to either protein in this assay (Figure 2D).The selectivity of AVI-4206 for the active site of Mac1 can be rationalized by the presence of larger residues at key positions in the binding pocket in the human orthologs.In Targ1, Cys104 occupies the analogous position to Ala52 of Mac1, leading to a putative clash with the urea moiety (Figure 2E).Similarly, in MacroD2, Arg122 occupies the analogous position to Leu 126; both the larger arginine side chain and accompanying backbone shift are predicted to clash with the gem-dimethyl of AVI-4206 (Figure 2E).Due to the shared adenosine motif in the substrates for macrodomains and kinases, and the therapeutic importance of protein kinases, we assessed AVI-4206 at 10 μM against a panel of diverse kinases and found no inhibition >35% (Supplementary Table 2).Lastly, we used mass spectrometry-based thermal proteome profiling (TPP) (Savitski et al. 2014) to evaluate the selectivity of AVI-4206 against a complex proteome We added 50 nM recombinant Mac1 protein into cellular lysates from A549 cells that were treated either with DMSO or with 100 µM of AVI-4206.We find that Mac1, but no native protein from the A459 lysate, displays a statistically significant shift in melting temperature (3.02°C, adjusted P value = 0.045) (Supplementary Figure 4).Collectively, these results indicate that AVI-4206 can cross cellular membranes and engage with high specificity for Mac1.

AVI-4206 displays limited efficacy in cellular models
To determine whether AVI-4206 can inhibit viral replication in cellular models of SARS-CoV-2 infection, we treated IFN-deficient Vero cells stably expressing TMPRSS2 (Vero-TMPRSS2) and IFN-competent A549 cells stably expressing high levels of ACE2 (A549-ACE2 h ) with AVI-4206 and infected them with an mNeon reporter SARS-CoV-2 WA1 strain (Xie et al. 2020).We observed that treatment with AVI-4206 did not reduce viral replication in Vero-TMPRSS2 or A549-ACE2 h cells (Figure 3A,B), consistent with previous studies showing that Mac1 deficient SARS-CoV-2 can replicate efficiently in several cell lines ( (Alhammad et al. 2023;Taha et al. 2023b)).This result stands in contrast to a SARS-CoV-2 protease inhibitor, nirmaltrevir, which potently inhibited replication in both cell lines (EC50 275 nM and 9.4 nM, respectively) (Figure 3A,B).Nonetheless, this experiment, together with a viability assay, indicated no direct cytotoxicity of AVI-4206 at concentrations as high as 100 μM (Supplementary Figure 5A,B).Next, we explored whether interferon pretreatment could potentiate the response of AVI-4206 using SARS-CoV-2 replicons (Taha et al. 2023a) as we previously demonstrated for a Mac1 deficient SARS-CoV-2 replicon (WA1 N40D mutant) (Alhammad et al. 2023;Taha et al. 2023b) (Figure 3C).We did not observe a reduction in viral RNA replication of the Mac1 deficient replicon compared with the wild-type replicon in Vero cells stably expressing ACE2 and TMPRSS2 (VAT) or A549-ACE2 h cells treated with or without AVI-4206 and 1000 IU/ml of IFN-gamma (Supplementary Figure 5C,D).However, there was a modest dose-dependent decrease in replication of the wild-type, but not Mac1 deficient, replicon in A549-ACE2 h cells (Supplementary Figure 5D).When the IFNgamma dose was increased to 10000 IU/ml, we observed a small (~1.6-fold) effect for the Mac1 deficient replicon relative to the wild-type replicon (Figure 3D).Treatment with highest dose (100 μM) of AVI-4206 led to a statistically significant, but small (~1.7-fold), reduction in replication for the wild-type, but not Mac1deficient, replicon (Figure 3D).From these experiments, we conclude that cellular models of SARS-CoV-2 infection give, at best, only a narrow window for assessing the efficacy of Mac1 inhibition and that high concentrations of AVI-4206 can achieve a limited anti-viral response without cytotoxicity in an IFN-and Mac1 catalytic activity-dependent manner.To test AVI-4206 in a system that more closely replicates both the structural and functional characteristics of the human airway epithelium, we used human airway organoids (HAOs), which are derived from primary stem cells generated from human lungs and grow as complex three-dimensional structures (Sachs et al. 2019).These cells can be differentiated into the various cell types found in the airway epithelium, including ciliated, goblet, and basal cells (Li et al. 2023a;Simoneau et al. 2024).We (Simoneau et al. 2024) and others (Li et al. 2023a) have utilized differentiated HAOs as a more relevant infection model that encompasses more robust innate immune functions.We therefore sought to test the efficacy of AVI-4206 in HAOs infected with SARS-CoV-2 (Figure 3E).The Mac1 deficient virus (WA1 N40D mutant) showed no reduction, 10-fold reduction, and 1000-fold reduction in viral particle production at 24, 48, and 72 hours post-infection compared to the wild-type virus (Figure 3F).AVI-4206 treatment reduced viral particle production 10-and 100-fold at 48 and 72 hours post-infection, respectively, and 20 μM AVI-4206 reduced viral particle production by 10-fold at 72 hours post-infection (Figure 3F).As we have observed previously (Alhammad et al. 2023;Taha et al. 2023b), the faster clearance of infection in AVI-4206 treated HAOs, similar to that seen with the Mac1 deficient virus, is likely due to a potent innate immune response rather than a direct effect of Mac1 on viral replication.While these cellular and organoid experiments gave some indication of an effect of AVI-4206, testing in animal models was required to establish whether this compound had significant activity in reducing viral pathogenesis.

AVI-4206 has favorable pharmacological properties
Prior to testing the efficacy of AVI-4206 in animal models, we assessed the pharmacological properties of the compound to predict a dosing regime that would provide sufficient target coverage to test efficacy.In parallel with optimizing compounds for potent inhibition of Mac1, as described above, we employed data from standard in vitro assays of metabolism, permeability, and physicochemical properties to drive our medicinal chemistry campaign.Thus, the series leading to AVI-4206 was optimized for stability in mouse liver microsomes and human hepatocytes, low plasma protein binding (good free fraction), and high aqueous solubility (Figure 4A).However, the introduction of the urea functionality in this series negatively impacted permeability in Caco-2 monolayers when compared to the parent AVI-219, predicting low oral bioavailability.Indeed, in mouse pharmacokinetic (PK) studies, AVI-4206 showed poor oral bioavailability (<4%), while intrinsic clearance was moderate, about 60% of hepatic blood flow (Supplementary Table 3).Bioavailability via the intraperitoneal (IP) route however, was excellent and free drug concentrations ~100-fold above the biochemical IC50 were achieved for 8 hours following a single IP dose at 100 mg/kg (Figure 4B, Supplementary Table 3).In a separate PK experiment employing a 10 mg/kg IP dose, total exposure of AVI-4206 in lung was higher than in plasma at later time points, (Figure 4C), suggesting its suitability for an in vivo infection model to validate Mac1 as an antiviral target Moreover, AVI-4206 showed minimal inhibition of common cytochrome P450 (CYP) isoforms (Figure 4D) and a broader panel of potential off targets (Figure 4E, Supplementary Table 4), identified no significant liabilities among major channels, receptors, or enzymes.Overall, the biochemical potency and PK profile of AVI-4206 suggested the likelihood of sustained target engagement in mice with twice daily doses (BID) of 100 mg/kg by the IP route allowing us to test proofof-concept.

AVI-4206 is effective in a mouse model of SARS-CoV-2 infection
To assess the efficacy of AVI-4206 in vivo, we used the K18-hACE2 mouse model, which mimics severe SARS-CoV-2 infection (Zheng et al. 2021).The K18-hACE2 mouse is a stringent model to test efficacy, as previous studies using potent protease inhibitors did not lead to full survival, unless combined with molnupiravir (Papini et al. 2024).In our experiment, animals were divided into three groups (wild-type WA1 virus with compound or vehicle treatment, and a Mac1 deficient mutant-infected positive control) with treatment (AVI-4206 at 100 mg/kg intraperitoneally, nirmatrelvir at 300 mg/kg orally, or vehicle control for each drug) initiated one day prior to infection (Figure 5A).AVI-4206 or nirmatrelvir were administered twice daily until 5 days post-infection, during which the mice were closely monitored for disease parameters such as weight loss and hunched posture.Both vehicle-treated groups experienced weight loss starting at 3-4 days post-infection and continued losing weight until the end of the study at 7 days post-infection (Figure 5B,C).The AVI-4206 and nirmatrelvir treated groups experienced weight loss starting at 4 days post-infection, but the extent of weight loss was about 5-10% lower on average at days 5-7 post-infection compared with their respective vehicle-treated groups (Figure 5B,C).Consequently, ~70% of AVI-4206 treated group and 40% of the nirmatrelvir treated group survived, whereas all mice in the vehicle treated groups died by the end of the study based on the humane endpoints of hunched posture or >20% decrease in body weight (Figure 5D,E).Consistent with previous studies (Taha et al. 2023b), none of the mice in the mutant-infected positive control group experienced weight loss, and all survived the infection (Figure 5D).These results indicate that AVI-4206 can significantly reduce disease severity and prevent death in the K18-hACE2 model and is comparable to the FDA-approved protease inhibitor nirmatrelvir.
To understand the mechanism of AVI-4206 action during the course of infection, mice from each group were euthanized at either day 4 or 7. We observed that AVI-4206 treatment reduced viral load in the lungs by ~10fold and ~100-fold at 4 and 7 days post-infection, respectively, and reduced transmission to the brain (Figure 5F).Although it is possible that AVI-4206 crosses the blood-brain barrier, it is more likely that the reduction of viral load in the brain is as a consequence of a reduction in overall systemic viral load.The prevention of virus localization to the brain is especially important in this model because human ACE2 overexpression allows virus replication and spread to brain tissue which ultimately leads to encephalitis and the death of infected mice (Bao et al. 2020;Oladunni et al. 2020).The faster clearance of viral load in the lungs for AVI-4206 treated and Mac1 deficient virus infected mice compared with the vehicle-treated mice, rather than an early antiviral effect post-infection, is consistent with an immune response mediated mechanism rather than a direct antiviral mechanism.
To further investigate the antiviral mechanism of AVI-4206, we measured the abundance of the antiviral cytokines IP-10, IL-2, IL-6, and TNF-α in lung tissue at 4 and 7 days post-infection (Figure 5G).We found that levels of all of these cytokines were elevated at 4 days post-infection.At 7 days post-infection, the AVI-4206 treated and Mac1 deficient virus infected mice maintained significantly higher levels of IP-10, IL-2, and IL-6 (P < 0.05) compared to the vehicle treated group; TNF-α showed a similar trend but did not reach statistical significance (Figure 5G).The lower levels of cytokines in the vehicle-treated group at 7 days postinfection is likely mediated by the immune-suppressive capability of SARS-CoV-2 macrodomain.However, when the macrodomain is inactivated, either through AVI-4206 treatment or infection with Mac-1 defective variant, the antiviral response is enhanced, which blocks viral replication.The cumulative cytokine abundance (IP-10, IL-2, and IL-6) indicates an antiviral immune response, likely mediated through the activation of the NF-κB pathway (Neufeldt et al. 2022;Robertson et al. 2023).Finally, we tested the efficacy of AVI-4206 at a lower dosage of 30 mg/kg using the same experimental setup.Even at this lower dose, AVI-4206 enhanced survival and produced lower viral load at 7 days post-infection relative to vehicle (Supplementary Figure 6), but to a more modest degree than at the higher dose (Figure 5).Collectively, our observations of enhanced survival of mice, reduced viral load, and an increase in antiviral cytokines suggest that AVI-4206 is capable of potentiating the host immune response, thereby reducing disease severity.For mice that reached the humane endpoint before day 7 post-infection, the tissues were collected and analyzed with mice at the 7 day time point.

Discussion
Here we provide strong pharmacological evidence validating Mac1 and de-ADP-ribosylation as a therapeutic target for SARS-CoV-2.AVI-4206 is a competitive inhibitor that blocks the ADP-ribosylhydrolase activity of Mac1.This activity antagonizes the PARP-mediated ADP-ribosylation that is part of the antiviral interferon response.Although mechanistic links are still emerging between specific post-translational modifications and an effective antiviral response, our pharmacologic studies add to the genetic and biochemical evidence of the importance of this signaling axis for viral replication in vivo (Alhammad et al. 2023;Taha et al. 2023b).Our work also adds to the growing role of modulating ADP-ribosylation signaling in therapeutic development (Dasovich and Leung 2023).For example, inhibitors of PARP1, which catalyzes the addition of poly-ADPribose marks, have been developed for treating tumors with mutations in either BRCA1 or BRCA2 (Lord and Ashworth 2017) and inhibitors of PARG, which catalyzes the removal of α(1′′-2′) O-glycosidic linkages in PAR chains, are under investigation for a variety of cancers (Slade 2020).The presence of macrodomains and experimental evidence for them as interferon signaling antagonists in other diverse viruses, such as Chikungunya (McPherson et al. 2017), suggests that inhibiting this target class may be effective for treatment of other virally-induced diseases beyond COVID.
While AVI-4206 is protective in an animal model of infection, it was developed without many of the normal intermediate markers of improvement in cellular models.This discordance was expected based on the mechanism of action, as interferon-based antiviral activity likely requires intra-and inter-cellular and systemic communication between different cell types (Platanias 2005).The limited replication defect difference between wild-type and Mac1 deficient viruses in cellular models renders them largely ineffective as a model to test the effects of macrodomain inhibition, which has led others to question the validity of Mac1 as target (Lee et al. 2024).AVI-4206 did in fact demonstrate modest antiviral activity only in the presence of exogenous IFN in cells, which is consistent with most other studies that have examined Mac1 activity (Alhammad et al. 2023;Taha et al. 2023b;Kerr et al. 2024).While a larger replication defect is observed in HAOs likely due to their more relevant antiviral innate immune responses (Simoneau et al. 2024), the highest dose of AVI-4206 does not achieve the magnitude of the replication defect of the Mac1 deficient virus.This may reflect an unoptimized prophylactic dosing schedule or the need to better tune the pharmacological properties of the inhibitor.Taken together, the concordance of in vitro (HTRF) and cellular target engagement assays (CETSA) stands out as particularly important in the development path of macrodomain inhibitors.AVI-4206 blocks the viral enzymatic removal of post-translational modifications important for the immune response, which is an important mechanism for blocking virus replication and reducing disease severity.Notably, Mac1 represents a second pharmacologically validated enzymatic domain within Nsp3: a recently developed inhibitor of the papain-like protease (PLpro) domain also shows efficacy in animal models and acts by removing a distinct set of host post-translational modifications (ubiquitin and interferon-stimulated gene 15 (ISG15)) (Tan et al. 2024).In summary, AVI-4206 provides proof-of-concept for the validation of a novel antiviral target, Mac1.By restoring the antiviral immune response, the novel mechanism of action of AVI-4206 could be synergistic or additive with orthogonally acting direct antivirals, such as protease and polymerase inhibitors, in combination therapies for the treatment of SARS-CoV-2 infection and beyond.

X-ray Crystallography:
Mac1 crystals (P43 construct, residues 3-169) were grown by sitting-drop vapor diffusion in 28% w/v polyethylene glycol (PEG) 3000 and 100 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) pH 9.5 as described previously (Schuller et al. 2021;Gahbauer et al. 2023).Compounds prepared in DMSO (100 mM) were added to crystal drops using an Echo 650 acoustic dispenser (Collins et al. 2017) (final concentration of 10 mM).Crystals were incubated at room temperature for 2-4 hours prior to vitrification in liquid nitrogen without additional cryoprotection.X-ray diffraction data were collected at the Advanced Light Source (ALS beamline 8.3.1) or the Stanford Synchrotron Light Source (SSRL beamline 9-2).Data were indexed, integrated and scaled with XDS (Kabsch 2010) and merged with Aimless (Evans and Murshudov 2013).The P43 Mac1 crystals contain two copies of the protein in the asymmetric unit (chains A and B).The active site of chain A is open, however chain B is blocked by a crystal contact.We previously observed that potent Mac1 inhibitors dissolve crystals, likely through the displacement of the B chain crystal contact (Gahbauer et al. 2023).In addition, crystal packing in the chain A active site restricts movement of the Ala129-Gly134 loop, leading to decreased occupancy for compounds with substituents on the pyrrolidinone.To aid modeling the resulting conformational and compositional disorder, we used the PanDDA method (Pearce et al. 2017) to model ligands where the occupancy was low (<25%,  or where there was substantial disorder (AVI-4636).After modeling ligands, structures were refined using phenix.refine(Liebschner et al. 2019) as described previously (Gahbauer et al. 2023).Data collection settings and statistics are reported in Supplementary Table 1.To achieve higher ligand occupancy for AVI-4206, we co-crystallized an alternative Mac1 construct previously reported to crystallize in P1, P21 and C2 (residues 2-170) (Michalska et al. 2020;Correy et al. 2022).Crystals grew by sitting-drop vapor diffusion in 200 mM lithium acetate and 20% w/v PEG 3350 with 30 mg/ml Mac1 (1.6 mM) and 3.2 mM AVI-4206 (3.2% DMSO).Crystals were vitrified directly in liquid nitrogen and diffraction data to 0.8 Å were collected at the ALS (beamline 8.3.1).Data were reduced in P1 using the same pipeline as the P43 crystals.Solvent content analysis suggested that there were two chains in the asymmetric unit.Phases were obtained using Phaser (McCoy et al. 2007) and apo Mac1 coordinates (PDB code 7KQO, chain A).Structural refinementent was performed with phenix.refinefollowing the previously described procedures for ultra-high resolution data (Correy et al. 2022).After several rounds of refinement, positive difference density was clear for a second, relatively low occupancy, conformation of the entire chain A and B, each representing a ~3.1 Å translation relative to the major conformation.Modeling and inspection of the minor conformations suggested that they cannot be occupied simultaneously, therefore they were modeled with distinct alternative location identifiers (altlocs).The major conformation (protein, AVI-4206 and water) was modeled with altloc A and the minor conformations (protein, AVI-4206 and water) were modeled with altlocs C and D. In addition to the rigid body disorder, there was clear density for a third conformation of the residue 57-75 α-helix.In chain A, this was modeled with altloc B, while the density in chain B was too weak to allow modeling.The FO-FC difference electron density maps or PanDDA event maps used to model ligands are shown in Supplementary Figure 1.

Inhibition assay:
Inhibition of Mac1 ADP-ribosylhydrolase activity by AVI-219 and AVI-4206 was determined using the NUDT5/AMP-Glo assay (Dasovich et al. 2021;Taha et al. 2023b).The substrate for the ration was human PARP10 (catalytic domain, residues 819-1007), purified and auto-mono-ADP-ribosylated using NAD+ as described previously (Taha et al. 2023b).Briefly, AVI-219 and AVI-4206 were dispensed into 384-well white assay plates (Corning, 3824) using an Echo 650 acoustic dispenser to achieve a final concentration range from 1 mM to 0.4 nM (8 μl reaction volume, 1% DMSO).Purified Mac1 (P43 construct, 2 μl, 10 nM final concentration) and NUDT5 (2 μl, 100 nM final concentration) were added to wells and the plates were incubated for five minutes at room temperature.Mono-ADP-ribosylated PARP10 was added to wells (4 μl, 2 μM final concentration) and the plates were incubated at room temperature for an additional hour.The concentration of AMP was measured with an AMP-Glo assay kit (Promega, V5011) following the manufacturer's instructions using a BioTek Synergy HTX plate reader.Percentage inhibition was calculated relative to control wells containing no inhibitor (DMSO only, 0% inhibition) or no Mac1 (100% inhibition) and IC50 values were determined by fitting a four-parameter sigmoidal dose-response equation using GraphPad Prism (version 10.1.1),with the top and bottom constrained to 100 and 0% inhibition respectively.Data are presented as the mean ± SD of four technical replicates.A control reaction with increasing concentrations of Mac1 indicated that <50% of the mono-ADP-ribosylated PARP10 was hydrolyzed in the 0% inhibition control (Supplementary Figure 2).In addition, a counterscreen to test for NUDT5 inhibition or assay interference was performed with identical reactions, except Mac1 was omitted and ADP-ribose was added to a final concentration of 2 μM (Sigma, A0752).

HTRF:
Binding of the compounds to macrodomain proteins was assessed by the displacement of an ADPr conjugated biotin peptide from His6-tagged protein using an HTRF-technology based screening assay which was performed as previously described (Schuller et al. 2021).The protein sequences used for SARS-CoV-2 Mac1, and the human macrodomains TARG1 and MacroD2, are listed in Supplementary Table 5.All proteins were expressed and purified as described previously for SARS-CoV-2 Mac1 (Schuller et al. 2021).Compounds were dispensed into ProxiPlate-384 Plus (PerkinElmer) assay plates using an Echo 650 Liquid Handler (Beckman Coulter).Binding assays were conducted in a final volume of 16 μl with 12.5 nM NSP3 Mac1 protein, 200 nM peptide ARTK(Bio)QTARK(Aoa-RADP)S (Cambridge Peptides), 1:20000 Eu 3+ cryptate conjugated to a His6-specific antibody (HTRF donor, PerkinElmer AD0402) and 1:500 Streptavidin-XL665 (HTRF acceptor, PerkinElmer 610SAXLB) in assay buffer (25 mM 4-(2-hydroxyethyl)-1-piperazine-1ethanesulfonic acid (HEPES) pH 7.0, 20 mM NaCl, 0.05% bovine serum albumin and 0.05% Tween-20).TARG1 and MacroD2 binding were measured at 25nM and 12.5 nM, respectively.Assay reagents were dispensed manually into plates using an electronic multichannel pipette.Macrodomain protein and peptide were dispensed and preincubated for 30 min at room temperature before HTRF reagents were added.Fluorescence was measured after a 1 hour incubation at room temperature using a Perkin Elmer EnVision 2105-0010 Dual Detector Multimode microplate reader with dual emission protocol (A = excitation of 320 nm, emission of 665 nm, and B = excitation of 320 nm, emission of 620 nm).Compounds were tested in triplicate in a 14-point dose response.Raw data were processed to give an HTRF ratio (channel A/B × 10,000), which was used to generate IC50 curves.The IC50 values were determined by nonlinear regression using GraphPad Prism (version 10.1.1).Data are presented as mean ± SD of three technical replicates.
For CETSA-nLuc experiments, 30 μl of suspended cells were dispensed into a 96-well PCR plate (Biorad) and heated for 3.5 min using a preheated gradient thermal cycler (Eppendorf).A Nano-Glo® HiBiT Lytic Detection System (Promega) was used to quantify HiBiT-tagged proteins in cell lysates.30 μl of a mixture containing Lytic Buffer, LgBiT protein, HiBiT Lytic Substrate were added to the cell suspension, and luminescence intensity was measured using a Biotek Synergy H1.Luminescence values for each sample were normalized to the lowest temperature on the range and Tagg (T-aggregate) values were determined by fitting data with a four-parameter sigmoidal dose-response equation using non-linear regression in GraphPad Prism (version 10.1.1).Delta values were calculated by subtracting the Tagg DMSO value from the Tagg drug value.Data are presented as mean ± SD of two technical replicates.For CETSA-WB experiments, 45 μl of suspended cells were dispensed into PCR strip tubes and heated for 3.5 min using a pre-heated gradient thermal cycler (Eppendorf).Samples were then placed in an aluminum PCR block on a dry ice/ethanol bath for 3 min, followed by incubating at 37°C for 3 min, and vortexing for 3 seconds.This freeze-thaw cycle was repeated three more times.Insoluble proteins were transferred to a 1.5 ml microcentrifuge tube, separated by centrifugation (20,000g, 15 min, 4°C) and 40 μl of supernatant corresponding to soluble proteins was kept for WB.Samples were separated on an SDS-polyacrylamide gel and transferred to a PVDF membrane.The following antibodies were used for immunoblotting: anti-FLAG antibody (Sigma, F1804, 1:1000 overnight), anti-mouse HRP antibody (CST, 7076S, 1:3000 for 1 hour).Images were captured using the Azure c600 Western Blot Imaging System, quantified using ImageJ and plotted as above.

SARS-CoV-2 culture:
As described in our previous report (Taha et al. 2023b), the pBAC SARS-CoV-2 WT (WA1) and N40D mutant constructs on WA1 background were made by co-transfecting them with an N expression vector into BHK-21 cells.Following three days of transfection the cell supernatants were used to infect Vero cells stably expressing TMPRSS2, followed by passaging to achieve a high viral titer.All viruses generated or used in this study were verified by NGS using the ARTIC Network's protocol.A previously reported mNeon SARS-CoV-2 infectious clone (ic-SARS-CoV-2-mNG) (Xie et al. 2020) was passaged on Vero-TMPRSS2 and used for Incucyte-based antiviral assays.

SARS-CoV-2 replicon assay:
The SARS-CoV-2 replicon assay was conducted as described previously (Taha et al. 2023a(Taha et al. , 2023b)).Briefly, the pBAC SARS-CoV-2 ΔSpike WT or nsp3 Mac1 N40D modified plasmids (40 μg), were transfected into BHK-21 fibroblast cells along with N and S expression vectors (20 μg each) in a 15-cm 2 tissue culture dish.The culture media was replaced with fresh growth medium 12 hours post-transfection.The media containing single-round infectious particles was collected and 0.45 μm-filtered 72 hours post-transfection and stored at -80 C until use.
Vero-ACE2-TMPRSS2 (VAT) and A549 ACE2 h cells were plated 2.5x10 4 cells per well in 96-well plate in media containing 0, 1000, or 10000 IU/ml of IFN-γ.After 16 hours, the media was replaced with 50 μl media containing 5x the final desired concentration of IFN-γ and AVI-4206.After 2 hours, 200 μl of supernatant containing WA1 or WA1 nsp3 Mac1 N40D single-round infectious particles was added.After 8 hours, the cells were washed with 200 μl culture medium and 100 μl of culture medium was added.After 16 hours, 50 μl from each well was transferred to a white 96-well plate to measure nanoluciferase activity by adding 50 μl of Nano-Glo luciferase assay buffer and substrate and analyzed on an Infinite M Plex plate reader (Tecan).

SARS-CoV-2 in vitro antiviral assay:
Antiviral activity of compounds was assessed using the Incucyte Ⓡ live-cell analysis system.2x10 4 A549-ACE2h cells per well were seeded in Edge 2.0 96-well plates filled with 1.5 ml PBS in the outer moats and 100 µl in-between wells and incubated at 37°C and 5% CO2.The next day, cells were pre-treated with compounds for 2 hours, followed by the removal of the compounds and infection with 50 µl of icSARS-CoV-2-mNG at a MOI 0.1 for 2 hours.Subsequently, virus inoculum was removed and fresh compounds diluted in DMEM (10% FBS, 1% L-Glutamine, 1× P/S, 1× NEAA, Incucyte® Cytotox Dye) were added.Infected cells were placed in an Incucyte S3 (Sartorius) and infection and cell death were measured over 48 hours at 1hour intervals using a 10x objective, capturing 3 images per well at each time point under cell maintenance conditions (37°C, 5% CO2).Infection and cell death were quantified as Total Green Object Integrated Intensity (300 ms acquisition time) and Red Object Integrated Intensity (400 ms acquisition time), respectively.After in-built software analysis, raw data was exported and antiviral efficacy was determined as the percentage of viral replication normalized to the vehicle control.Nirmatrelvir (HY-138687, MedChemExpress) and uninfected cells were used as intra-assay positive and negative controls, respectively.Unless otherwise stated, experiments were conducted in triplicate with 3 technical replicates.EC50 values were calculated using GraphPad PRISM 10 (La Jolla, CA, USA) employing a dose-response inhibition equation with a non-linear fit regression model.

Antiviral efficacy in human airway organoids:
The differentiated HAOs were utilized to analyze the dose-dependent anti SARS-CoV-2 efficacy of AVI-4206.Briefly, 100,000 cells of differentiated HAOs were seeded in a V-bottom plate (Greiner Bio-One).The cells were pretreated for 2 hours prior to infection with various concentrations of AVI-4206 (0, 0.16 μM, 0.8 μM, 4 μM, 20 μM, 100 μM).After pretreatment, the HAOs were washed and infected with SARS-CoV-2 WA1 at a multiplicity of infection (MOI) of 1.A WA1-N40D mutant strain lacking the macrodomain activity was used as a positive control.Following 2 hours of infection, the HAOs were washed three times.Each washing step involved replacing the media with PBS and centrifuging the cells at 1000 rpm for 3 minutes.After three washes, the PBS was replaced with 100 μl of HAO differentiation medium, with or without varying concentrations of AVI-4206, and the plate was incubated for 72 hours at 37°C with 5% CO2.Supernatants collected at 24-hour intervals were used to analyze mature virus particle formation via plaque assay.

Drug cytotoxicity assay:
A549-ACE2h cells were seeded and incubated as for the in vitro antiviral assay.Cells were treated with compounds at the respective concentrations and vehicle control for 50 hours at 37°C and 5% CO2.Subsequently, Cell Titer-Glo Ⓡ reagent was added in a 1:1 ratio to the cells and incubated at room temperature for 5 minutes before transferring 100 µl of the mixture to a white 96-well plate.Luciferase activity was measured using an Infinite M Plex plate reader (Tecan).Cell viability was determined as the percentage of viability normalized to the vehicle control.Compound cytotoxicity was assessed in parallel with infection experiments using cells of the same passage.

Thermal proteome profiling (TPP) assay:
Pelleted A549 cells were resuspended in extraction buffer (1× PBS + phosphatase and protease inhibitors (phosSTOP (Roche) and cOmplete Mini Protease Inhibitor Cocktail (Roche)) with gentle pipetting followed by rotation at 4°C for 30 minutes.Lysates were centrifuged at 1000g for 10 minutes at 4°C and supernatant was transferred to new tubes.Recombinant Mac1 was spiked into lysate to a final concentration of 0.05 μM.Lysates + Mac1 were incubated with compound at a final concentration of 100 µM AVI-4206 or DMSO for 30 minutes at 25°C.Lysates (2 replicates per condition) were distributed into 10 aliquots (20 μl each) in PCR tubes.Samples were heated from 37°C to 64°C in 3°C increments on a BioRad C1000 Touch Thermal cycler, and held for four minutes at the specified temperature.Samples were held at room temperature for three minutes.Samples were then subjected to 2 cycles of flash freezing and thawing at 35°C.Aggregated proteins were removed by centrifugation at 20,000g for 60 minutes.20 μl of lysis buffer (8 M urea, 100 mM Tris, pH ~7.5) was added to each well and samples were incubated for 30 minutes at room temperature.Samples were reduced and alkylated by the addition of TCEP (100mM final) and 2-chloroacetamide (44mM final) followed by incubation at room temperature for 30 minutes.Urea concentration was diluted to 1 M by the addition of 100 mM tris (pH ~7.5).Samples were digested overnight with LysC (Wako, 1:100 enzyme: protein ratio) and trypsin (Promega, 1:50 enzyme:protein ratio).Samples were desalted with a 96-well mini 20MG PROTO 300 C18 plate (HNS S18V, The Nest Group) according to manufacturer's directions.Peptide concentration was determined by NanoDrop (Thermo).
Following digestion, peptides were injected onto a timsTOF SCP (Bruker) connected to either an EASY-nLC 1200 system (Thermo) or VanquishNeo (Thermo).Peptides were separated on a PepSep reverse-phase C18 column (1.9 μm particles, 15 cm, 150 mm ID) (Bruker) with a gradient of 5-28% buffer B (0.1% formic acid in acetonitrile) over buffer A (0.1% formic acid in water) over 20 minutes, an increase to 32% B in 3 minutes, and held at 95% B for 7 minutes.DIA-PASEF analyses were acquired from 100 to 1700 m/z over a 1/Kø of 0.70 to 1.30 Vs/cm 2 , with a ramp and accumulation time set to 75 ms.Library DDA PASEF runs were collected over the same m/z and 1/Kø range and a cycle time of 1.9 s.
All data was searched against the Uniprot Human database (downloaded 05/25/23) appended with the SARS-CoV-2 database (downloaded 02/20/2024) using a combined DDA and DIA library in Spectronaut (Biognosys, version 19.0).Default settings, including trypsin digestion, variable modifications of methionine oxidation and N-termini acetylation, and fixed modification of cysteine carbamidomethylation, were used.Missing values were imputed for each run using background intensity.Data was filtered to obtain a false discovery rate of 1% at the peptide spectrum match and protein level.Lysate experiments were normalized to the lowest temperature (37°C) and melting points were determined in R using the Inflect package (McCracken et al. 2021).

ADMET target and kinase studies:
The kinase assessment was performed using contract services by Eurofins using their scanEDGE KINOMEscan Assay Platform (Study Code: US073-0032699).Assessment of ADMET targets (cardiac channel profiling, CYP induction, peptidase selectivity panel and secondary pharmacology profiling) was performed via NIAID's suite of preclinical services for in vitro assessment (Contract No. HHSN272201800007I/75N93022F00001).

Pharmacokinetic Studies:
The pharmacokinetic study of AVI-4206 with IV (10 mg/kg), PO (50 mg/kg), and IP (100 mg/kg) dosing (Fig. 4B and Supplementary Table 2) was performed in male CD1 mice (n = 3 per group) using a formulation of 10% DMSO: 50% PEG 400: 40% of a 20% HP-β-CD in water.Microsampling (40 ml) via facial vein was performed at 0, 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h into K2EDTA tubes.The blood samples were collected and centrifuged to obtain plasma (8000 rpm, 5 min) within 15 minutes post sampling.Nine blood samples were collected from each mouse; three samples were collected for each time point.Data was processed by Phoenix WinNonlin (version 8.3); samples below the limit of quantitation were excluded in the PK parameters and mean concentration calculation.

Animal experiments:
All the mice experiments were approved (AN169239-01) by the Institutional Animal Care and Use committees at the University of California, San Francisco and Gladstone Institutes and performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animal.For screening of lead Macrodomain inhibitors we employed a transgenic mice model capable of expressing human ACE2.Female mice were divided into three groups: test, positive control, and negative control.The positive control groups were infected (5×10 2 PFUs) with the N40D mutant of SARS-CoV-2, while the other mice were infected with the WA1 strain.Intraperitoneal treatments were administered twice daily which began at a day prior infection and continued until 5 days post-infection, with close monitoring for disease parameters such as weight loss, hypothermia, and hunched posture.At 4 and 7 days post-infection, a subset of mice from each group was euthanized, and their lungs and brain tissues were harvested for virus titration by plaque assay and cytokine expression.

Plaque assay:
The mature virus particles in the lung homogenates were analyzed using plaque assay.Briefly, VAT cells were seeded in a 12-well plate and incubated overnight.The cells were inoculated with 10 to 10 6 dilutions of the respective lung homogenates.After 1h incubation, the lung homogenates in the wells were overlaid with 2.5% Avicel (RC-591, Dupont).And the plates were incubated at 370C and 5% CO2 for 48h .After incubation the overlay media was removed and the cells were fixed in 10% formalin.The plaques were visualized by staining the cells with crystal violet.Data analysis was performed by using GraphPad Prism version 10.

Figure 1 :
Figure 1: Iterative structure-based design and optimization of AVI-4206 activity against Mac1.(A) Evolution of the early lead AVI-219 to AVI-4206 by introducing and optimizing urea functionality as found in AVI-92 to contact Asp22 and introducing geminal dimethyl substitution of the pyrrolidinone ring.HTRF-based IC50 values from (B) and (C), and PDB codes from (E) are indicated.(B and C) HTRF-based dose response curves showing peptide displacement of an ADPr-conjugated peptide from Mac1 by compounds from the urea (B) and the pyrrolidinone ring (C) optimization paths.Data is plotted as % competition mean ± SD of three technical replicates.Data were fitted with a sigmoidal dose-response equation using non-linear regression and the IC50 values are quoted with 95% confidence intervals.(D) Mac1 catalytic activity dose response curve for indicated compounds.Data is plotted as % inhibition mean ± SD of four technical replicates.IC50 values are quoted with 95% confidence intervals.(E) X-ray structures indicating conserved interactions during the optimization path from AVI-92 and AVI-219 (left) to AVI-4206 (right).Structures of compounds from the urea and the pyrrolidinone ring optimization paths are presented in the top and bottom middle panels, respectively.Multiple ligand conformations were observed for AVI-3367, AVI-3762 and AVI-4636 (labeled A and B).The FO-FC difference electron density map calculated prior to ligand modeling is shown for AVI-4206 (purple mesh contoured at 5 σ).Electron density maps used to model ligand other ligands are shown in Supplementary Figure 1.

Figure 2 :
Figure 2: AVI-4206 engages Mac1 with high potency and selectivity in cells.(A) CETSA-nLuc shows differential Mac1 stabilization after treatment of A549 cells with 10 μM of indicated compounds.Data are presented as mean ± SD of two technical replicates.Data were fitted with a sigmoidal dose-response equation using non-linear regression (gray line) and the Tagg values are quoted with 95% confidence intervals.(B) CETSA-nLuc shows a dose-dependent thermal stabilization of Mac1 after treatment of A549 cells with increasing concentrations of AVI-4206.Data are presented as mean ± SD of two technical replicates.(C and D) HTRF-based dose response curves showing displacement of an ADPr-conjugated peptide from indicated proteins by ADP-ribose (C) or AVI-4206 (D).ADP-ribose was used as a positive control.Data are presented as mean ± SD of three technical replicates.IC50 values are quoted with 95% confidence intervals.(E) Structural modeling of MacroD2 (top, PDB code 4IQY) and Targ1 (bottom, PDB code 4J5S) showing design elements that prevent AVI-4206 cross reactivity.The atoms of clashing residues (Cys140 in MacroD2, Arg122 in Targ1) are shown with a dot representation.The ADP-ribose present in both human macrodomain structures has been omitted for clarity.

Figure 3 :
Figure 3: AVI-4206 shows limited efficacy in cellular models of SARS-CoV-2 infection.(A and B) Vero-TMPRSS2 (A) or A549-ACE2 h (B) cells were pretreated with compounds and infected with mNeonGreen reporter SARS-CoV-2.mNeonGreen expression was measured by the Incucyte system.Graphs represent mean +/-SD of % replication normalized to the DMSO control 24 post-infection of three independent experiments performed in triplicate.Data were fitted with a sigmoidal dose-response equation using non-linear regression (gray line) and the EC50 values are quoted with 95% confidence intervals.(C) Schematic of the replicon assay to test the efficacy of AVI-4206 in A549 ACE2 h cells.(D) Luciferase readout of A549 ACE2 h cells infected with WA1 or WA1 Mac1 N40D replicons and treated with or without AVI-4206 and IFN-ɣ at indicated concentrations; *, P < 0.05 by two-tailed Student's t-test relative to the no AVI-4206 and no IFN-ɣ control.Results are plotted as normalized mean ± standard deviation luciferase values of a representative biological experiment containing two technical replicates.(E) Schematic of the HAO experiment.(F) Viral particle production was measured by plaque assay at indicated time points and AVI-4206 concentrations.Error bars indicate s.e.m. **, P < 0.01; *, P < 0.05 by two-tailed Student's t-test relative to the vehicle control.

Figure 4 :
Figure 4: AVI-4206 has a favorable pharmacological profile.(A) Pharmacokinetic properties of AVI-4206.(B) Unbound plasma exposure time course of AVI-4206, corrected for plasma protein binding, following administration by IV, PO, or IP routes in male CD-1 mice at the indicated doses.(C) Free plasma exposure of AVI-4206 and total exposure in lung homogenate following an IP dose of 10 mg/kg in female C57BL/6 mice.(D) Inhibition of CYP isoforms by AVI-4206 at a fixed concentration of 10 μM.Two experiments were performed with CYP3A4 using different positive controls.(E) Heatmap of AVI-4206 activity in an off-target safety panel including receptors, ion channels, and proteases, showing no antagonist response >15% at 10 μM.

Figure 5 :
Figure 5: AVI-4206 reduces viral replication and increases survival and cytokine abundance in vivo.(A) K18-hACE2 mice were intranasally infected and dosed as indicated with either AVI-4206 (n=15, intraperitoneally), nirmatrelvir (n=5, per os) or vehicle (n=10 for the AVI-4206 group or n=5 for the nirmatrelvir group).Mice infected with WA1 N40D mutant, which lacks Mac1 catalytic activity, served as a positive control (n=10).Lungs were harvested at indicated time points for virus titration by plaque assay.(B) The percent body weight loss for all animals treated with AVI-4206 (100 mg/kg IP) (C) or nirmatrelvir (300 mg/kg PO).The data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student's t-test relative to the vehicle control at each timepoint.(D) Survival curve plotted based on the percent weight loss humane endpoint (20%) for AVI-4206 and (E) nirmatrelvir.(F) Viral load in the lungs and brain of infected mice at the indicated time points.The data are shown as mean ± s.e.m. *, P < 0.05; **, P < 0.01 by Mann Whitney's test relative to the vehicle control.(G) Schematics and graphs demonstrating the abundance of indicated cytokines at 4 and 7 days post-infection in the lungs of infected mice.The data are presented as mean ± s.e.m. *, P < 0.05; **, P < 0.01 by two-tailed Student's t-test relative to the vehicle control at each timepoint.None of the mice reached the humane endpoint at day 4 post-infection.For mice that reached the humane endpoint before day 7 post-infection, the tissues were collected and analyzed with mice at the 7 day time point.