Upper Respiratory Infection Drives Clinical Signs and Inflammatory Responses Following Heterologous Challenge of SARS-CoV-2 Variants of Concern in K18 Mice.

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of several variants of concern (VOC) with increased immune evasion and transmissibility. This has motivated studies to assess protection conferred by earlier strains following infection or vaccination to each new VOC. We hypothesized that while NAbs play a major role in protection against infection and disease, a heterologous reinfection or challenge may gain a foothold in the upper respiratory tract (URT) and result in a self-limited viral infection accompanied by an inflammatory response. To test this hypothesis, we infected K18-hACE2 mice with SARS-CoV-2 USA-WA1/2020 (WA1) and, after 24 days, challenged with WA1, Alpha, or Delta. While NAb titers against each virus were similar across all cohorts prior to challenge, the mice challenged with Alpha and Delta showed weight loss and upregulation of proinflammatory cytokines in the URT and lower RT (LRT). Mice challenged with WA1 showed complete protection. We noted increased levels of viral RNA transcripts only in the URT of mice challenged with Alpha and Delta. In conclusion, our results suggested self-limiting breakthrough infections of Alpha or Delta in the URT, which correlated with clinical signs and a significant inflammatory response in mice.

AI-Accelerated Design of Targeted Covalent Inhibitors for SARS-CoV-2.

Direct-acting antivirals for the treatment of the COVID-19 pandemic caused by the SARS-CoV-2 virus are needed to complement vaccination efforts. Given the ongoing emergence of new variants, automated experimentation, and active learning based fast workflows for antiviral lead discovery remain critical to our ability to address the pandemic's evolution in a timely manner. While several such pipelines have been introduced to discover candidates with noncovalent interactions with the main protease (Mpro), here we developed a closed-loop artificial intelligence pipeline to design electrophilic warhead-based covalent candidates. This work introduces a deep learning-assisted automated computational workflow to introduce linkers and an electrophilic "warhead" to design covalent candidates and incorporates cutting-edge experimental techniques for validation. Using this process, promising candidates in the library were screened, and several potential hits were identified and tested experimentally using native mass spectrometry and fluorescence resonance energy transfer (FRET)-based screening assays. We identified four chloroacetamide-based covalent inhibitors of Mpro with micromolar affinities (KI of 5.27 µM) using our pipeline. Experimentally resolved binding modes for each compound were determined using room-temperature X-ray crystallography, which is consistent with the predicted poses. The induced conformational changes based on molecular dynamics simulations further suggest that the dynamics may be an important factor to further improve selectivity, thereby effectively lowering KI and reducing toxicity. These results demonstrate the utility of our modular and data-driven approach for potent and selective covalent inhibitor discovery and provide a platform to apply it to other emerging targets.

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2.

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M-1 s-1, achieves sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Dissecting Phenotype from Genotype with Clinical Isolates of SARS-CoV-2 First Wave Variants.

The emergence and availability of closely related clinical isolates of SARS-CoV-2 offers a unique opportunity to identify novel nonsynonymous mutations that may impact phenotype. Global sequencing efforts show that SARS-CoV-2 variants have emerged and then been replaced since the beginning of the pandemic, yet we have limited information regarding the breadth of variant-specific host responses. Using primary cell cultures and the K18-hACE2 mouse, we investigated the replication, innate immune response, and pathology of closely related, clinical variants circulating during the first wave of the pandemic. Mathematical modeling of the lung viral replication of four clinical isolates showed a dichotomy between two B.1. isolates with significantly faster and slower infected cell clearance rates, respectively. While isolates induced several common immune host responses to infection, one B.1 isolate was unique in the promotion of eosinophil-associated proteins IL-5 and CCL11. Moreover, its mortality rate was significantly slower. Lung microscopic histopathology suggested further phenotypic divergence among the five isolates showing three distinct sets of phenotypes: (i) consolidation, alveolar hemorrhage, and inflammation, (ii) interstitial inflammation/septal thickening and peribronchiolar/perivascular lymphoid cells, and (iii) consolidation, alveolar involvement, and endothelial hypertrophy/margination. Together these findings show divergence in the phenotypic outcomes of these clinical isolates and reveal the potential importance of nonsynonymous mutations in nsp2 and ORF8.

Virtual and In Vitro Antiviral Screening Revive Therapeutic Drugs for COVID-19.

The urgent need for a cure for early phase COVID-19 infected patients critically underlines drug repositioning strategies able to efficiently identify new and reliable treatments by merging computational, experimental, and pharmacokinetic expertise. Here we report new potential therapeutics for COVID-19 identified with a combined virtual and experimental screening strategy and selected among already approved drugs. We used hydroxychloroquine (HCQ), one of the most studied drugs in current clinical trials, as a reference template to screen for structural similarity against a library of almost 4000 approved drugs. The top-ranked drugs, based on structural similarity to HCQ, were selected for in vitro antiviral assessment. Among the selected drugs, both zuclopenthixol and nebivolol efficiently block SARS-CoV-2 infection with EC50 values in the low micromolar range, as confirmed by independent experiments. The anti-SARS-CoV-2 potential of ambroxol, amodiaquine, and its active metabolite (N-monodesethyl amodiaquine) is also discussed. In trying to understand the "hydroxychloroquine" mechanism of action, both pK a and the HCQ aromatic core may play a role. Further, we show that the amodiaquine metabolite and, to a lesser extent, zuclopenthixol and nebivolol are active in a SARS-CoV-2 titer reduction assay. Given the need for improved efficacy and safety, we propose zuclopenthixol, nebivolol, and amodiaquine as potential candidates for clinical trials against the early phase of the SARS-CoV-2 infection and discuss their potential use as adjuvant to the current (i.e., remdesivir and favipiravir) COVID-19 therapeutics.

An ACE2-IgG4 Fc Fusion Protein Demonstrates Strong Binding to All Tested SARS-CoV-2 Variants and Reduced Lung Inflammation in Animal Models of SARS-CoV-2 and Influenza.

Background: The continued emergence of SARS-CoV-2 variants has caused concern that a constantly evolving virus will escape vaccines and antibody therapies. New approaches are needed. Methods: We created and manufactured an ACE2 extracellular domain (ECD) fragment Fc fusion drug candidate, G921, and engineered the compound for enhanced delivery of drug to peripheral tissues by minimizing the size of the ACE2 ECD and by incorporating an Fc domain to enhance transcytosis. G921 was assessed for binding, neutralization, in vivo anti-inflammatory effect, and pharmacokinetic profile. Results: G921 was expressed as an IgG4 Fc fusion protein presenting two ACE2 domains to ACE2 ligands while avoiding risk of infection via antibody-dependent enhancement. G921 strongly binds to the SARS-CoV-2 Wuhan-Hu-1 spike protein and demonstrates further diminished off rate to the spike protein from each of the currently identified variants of concern. G921 demonstrates ACE2 enzymatic activity comparable to positive control and binding to the neonatal Fc receptor (FcRn) without binding to low affinity Fc-gamma receptors (FcγRs). G921 is effective in a concentration-dependent manner in a focus reduction neutralization assay with EC50=16.3±4.2 µg/mL without cytotoxicity in Vero E6 cells when tested at 200 µg/mL in an MTS cell proliferation assay. G921 demonstrates statistically significant reduction of lung inflammation in relevant models of both SARS-CoV-2 and influenza. The pharmacokinetic profile demonstrated dose-dependent exposure with a multi-day half-life in monkeys and rats. Conclusion: G921 data are consistent with both antiviral and anti-inflammatory modes of action. G921 is a novel approach for the prevention and treatment of COVID-19 and possible other diseases characterized by deficiency of ACE2.

Optimization of the Prodrug Moiety of Remdesivir to Improve Lung Exposure/Selectivity and Enhance Anti-SARS-CoV-2 Activity.

COVID-19 patients with severe symptoms still lack antiviral treatment options. Although remdesivir is the only FDA-approved drug for those patients, its efficacy is limited by premature hydrolysis to nucleoside (NUC), low accumulation in the disease-targeted tissue (lungs), and low antiviral potency. In this study, we synthesized a new series of remdesivir analogues by modifying the ProTide moiety. In comparison with remdesivir, the lead compound MMT5-14 showed 2- to 7-fold higher antiviral activity in four variants of SARS-CoV-2. By reducing premature hydrolysis in hamsters, MMT5-14 increased the prodrug concentration by 200- to 300-fold in the plasma and lungs but also enhanced lung accumulation of the active metabolite triphosphate nucleosides (NTP) by 5-fold. Compared to remdesivir, MMT5-14 also increased the intracellular uptake and activation in lung epithelial cells by 4- to 25-fold. These data suggest that MMT5-14 could be a potential antiviral drug to treat COVID-19 patients with severe symptoms.

Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity.

The current coronavirus disease 2019 (COVID-19) pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III). These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique sequence motif (sense strand, 5'-C; antisense strand, 3'-GGG) that mediates end-to-end dimer self-assembly. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory small interfering RNAs (siRNAs), their activity is independent of Toll-like receptor (TLR) 7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with immunostimulant poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad-spectrum inhibition of infections by many respiratory viruses with pandemic potential, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-NL63, and influenza A virus in cell lines, human lung chips that mimic organ-level lung pathophysiology, and a mouse SARS-CoV-2 infection model. These short double-stranded RNAs (dsRNAs) can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics.

Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infections During SARS-CoV-2.

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate infection with the SARS-CoV-2 USA-WA1/2020 strain increased the risk of pneumococcal (type 2 strain D39) coinfection in a time-dependent, but sex-independent, manner in the transgenic K18-hACE2 mouse model of COVID-19. Bacterial coinfection increased lethality when the bacteria was initiated at 5 or 7 d post-virus infection (pvi) but not at 3 d pvi. Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

Cardiopulmonary Injury in the Syrian Hamster Model of COVID-19.

The Syrian hamster has proved useful in the evaluation of therapeutics and vaccines for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). To advance the model for preclinical studies, we conducted serial sacrifice of lungs, large pulmonary vessels, and hearts from male and female Syrian hamsters for days 1-4, and 8 post-infection (dpi) following infection with a high dose of SARS-CoV-2. Evaluation of microscopic lung histopathology scores suggests 4 and 8 dpi as prime indicators in the evaluation of moderate pathology with bronchial hyperplasia, alveolar involvement and bronchiolization being key assessments of lung disease and recovery, respectively. In addition, neutrophil levels, red blood cell count and hematocrit showed significant increases during early infection. We present histological evidence of severe damage to the pulmonary vasculature with extensive leukocyte transmigration and the loss of endothelial cells and tunica media. Our evidence of endothelial and inflammatory cell death in the pulmonary vessels suggests endothelialitis secondary to SARS-CoV-2 epithelial cell infection as a possible determinant of the pathological findings along with the host inflammatory response. Lastly, pathological examination of the heart revealed evidence for intracardiac platelet/fibrin aggregates in male and female hamsters on 8 dpi, which might be indicative of a hypercoagulative state in these animals.

Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease.

Emerging SARS-CoV-2 variants continue to threaten the effectiveness of COVID-19 vaccines, and small-molecule antivirals can provide an important therapeutic treatment option. The viral main protease (Mpro) is critical for virus replication and thus is considered an attractive drug target. We performed the design and characterization of three covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2 created by splicing components of hepatitis C protease inhibitors boceprevir and narlaprevir, and known SARS-CoV-1 protease inhibitors. A joint X-ray/neutron structure of the Mpro/BBH-1 complex demonstrates that a Cys145 thiolate reaction with the inhibitor's keto-warhead creates a negatively charged oxyanion. Protonation states of the ionizable residues in the Mpro active site adapt to the inhibitor, which appears to be an intrinsic property of Mpro. Structural comparisons of the hybrid inhibitors with PF-07321332 reveal unconventional F···O interactions of PF-07321332 with Mpro which may explain its more favorable enthalpy of binding. BBH-1, BBH-2 and NBH-2 exhibit comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.

Iota-carrageenan and xylitol inhibit SARS-CoV-2 in Vero cell culture.

Last year observed a global pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome-coronavirus 2) infection affecting millions of individuals worldwide. There is an urgent unmet need to provide an easily producible and affordable medicine to prevent transmission and provide early treatment for this disease. Since the nasal cavity and the rhinopharynx are the sites of initial replication of SARS-CoV-2, a nasal spray may be an effective option to target SARS-CoV-2 infection. In this study, we tested the antiviral action of three candidate nasal spray formulations against SARS-CoV-2 in vitro. We determined that iota-carrageenan in concentrations as low as 6 µg/mL inhibits SARS-CoV-2 in vitro. The concentrations of iota-carrageenan with activity against SARS-CoV-2 in vitro may be easily achieved through the application of nasal sprays as commonly used in several countries. Recently a double-blind, placebo-controlled study showed that iota-carrageenan in isotonic sodium chloride reduces ca. five times the risk of infection by SARS-CoV-2 in health care personnel. Further, xylitol at a concentration of 50 mg/mL (ca. 329 mM) was found to exert some antiviral action, though this preliminary finding needs further confirmation.

Structural, Electronic, and Electrostatic Determinants for Inhibitor Binding to Subsites S1 and S2 in SARS-CoV-2 Main Protease.

Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure-activity relationship (SAR) study of noncovalent compounds that bind in the enzyme's substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor's P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.

Drug Repurposing to Identify Nilotinib as a Potential SARS-CoV-2 Main Protease Inhibitor: Insights from a Computational and In Vitro Study.

COVID-19, an acute viral pneumonia, has emerged as a devastating pandemic. Drug repurposing allows researchers to find different indications of FDA-approved or investigational drugs. In this current study, a sequence of pharmacophore and molecular modeling-based screening against COVID-19 Mpro (PDB: 6LU7) suggested a subset of drugs, from the Drug Bank database, which may have antiviral activity. A total of 44 out of 8823 of the most promising virtual hits from the Drug Bank were subjected to molecular dynamics simulation experiments to explore the strength of their interactions with the SARS-CoV-2 Mpro active site. MD findings point toward three drugs (DB04020, DB12411, and DB11779) with very low relative free energies for SARS-CoV-2 Mpro with interactions at His41 and Met49. MD simulations identified an additional interaction with Glu166, which enhanced the binding affinity significantly. Therefore, Glu166 could be an interesting target for structure-based drug design. Quantitative structural-activity relationship analysis was performed on the 44 most promising hits from molecular docking-based virtual screening. Partial least square regression accurately predicted the values of independent drug candidates' binding energy with impressively high accuracy. Finally, the EC50 and CC50 of 10 drug candidates were measured against SARS-CoV-2 in cell culture. Nilotinib and bemcentinib had EC50 values of 2.6 and 1.1 µM, respectively. In summary, the results of our computer-aided drug design provide a roadmap for rational drug design of Mpro inhibitors and the discovery of certified medications as COVID-19 antiviral therapeutics.

Genetic Dissection of the Regulatory Mechanisms of Ace2 in the Infected Mouse Lung.

Acute lung injury (ALI) is an important cause of morbidity and mortality after viral infections, including influenza A virus H1N1, SARS-CoV, MERS-CoV, and SARS-CoV-2. The angiotensin I converting enzyme 2 (ACE2) is a key host membrane-bound protein that modulates ALI induced by viral infection, pulmonary acid aspiration, and sepsis. However, the contributions of ACE2 sequence variants to individual differences in disease risk and severity after viral infection are not understood. In this study, we quantified H1N1 influenza-infected lung transcriptomes across a family of 41 BXD recombinant inbred strains of mice and both parents-C57BL/6J and DBA/2J. In response to infection Ace2 mRNA levels decreased significantly for both parental strains and the expression levels was associated with disease severity (body weight loss) and viral load (expression levels of viral NA segment) across the BXD family members. Pulmonary RNA-seq for 43 lines was analyzed using weighted gene co-expression network analysis (WGCNA) and Bayesian network approaches. Ace2 not only participated in virus-induced ALI by interacting with TNF, MAPK, and NOTCH signaling pathways, but was also linked with high confidence to gene products that have important functions in the pulmonary epithelium, including Rnf128, Muc5b, and Tmprss2. Comparable sets of transcripts were also highlighted in parallel studies of human SARS-CoV-infected primary human airway epithelial cells. Using conventional mapping methods, we determined that weight loss at two and three days after viral infection maps to chromosome X-the location of Ace2. This finding motivated the hierarchical Bayesian network analysis, which defined molecular endophenotypes of lung infection linked to Ace2 expression and to a key disease outcome. Core members of this Bayesian network include Ace2, Atf4, Csf2, Cxcl2, Lif, Maml3, Muc5b, Reg3g, Ripk3, and Traf3. Collectively, these findings define a causally-rooted Ace2 modulatory network relevant to host response to viral infection and identify potential therapeutic targets for virus-induced respiratory diseases, including those caused by influenza and coronaviruses.

Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights.

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants that may alter viral fitness highlights the urgency of widespread next-generation sequencing (NGS) surveillance. To profile genetic variants of the entire SARS-CoV-2 genome, we developed and clinically validated a hybridization capture SARS-CoV-2 NGS assay, integrating novel methods for panel design using double-stranded DNA (dsDNA) biotin-labeled probes, and built accompanying software. This test is the first hybrid capture-based NGS assay given Food and Drug Administration (FDA) emergency use authorization for detection of the SARS-CoV-2 virus. The positive and negative percent agreement (PPA and NPA, respectively) were defined in comparison to the results for an orthogonal real-time reverse transcription polymerase chain reaction (RT-PCR) assay (PPA and NPA, 96.7 and 100%, respectively). The limit of detection was established to be 800 copies/ml with an average fold enrichment of 46,791. Furthermore, utilizing the research-use-only analysis to profile the variants, we identified 55 novel mutations, including 11 in the functionally important spike protein. Finally, we profiled the full nasopharyngeal microbiome using metagenomics and found overrepresentation of 7 taxa and evidence of macrolide resistance in SARS-CoV-2-positive patients. This hybrid capture NGS assay, coupled with optimized software, is a powerful approach to detect and comprehensively map SARS-CoV-2 genetic variants for tracking viral evolution and guiding vaccine updates. IMPORTANCE This is the first FDA emergency-use-authorized hybridization capture-based next-generation sequencing (NGS) assay to detect the SARS-CoV-2 genome. Viral metagenomics and the novel hybrid capture NGS-based assay, along with its research-use-only analysis, can provide important genetic insights into SARS-CoV-2 and other emerging pathogens and improve surveillance and early detection, potentially preventing or mitigating new outbreaks. Better understanding of the continuously evolving SARS-CoV-2 viral genome and the impact of genetic variants may provide individual risk stratification, precision therapeutic options, improved molecular diagnostics, and population-based therapeutic solutions.

Common Themes in Zoonotic Spillover and Disease Emergence: Lessons Learned from Bat- and Rodent-Borne RNA Viruses.

Rodents (order Rodentia), followed by bats (order Chiroptera), comprise the largest percentage of living mammals on earth. Thus, it is not surprising that these two orders account for many of the reservoirs of the zoonotic RNA viruses discovered to date. The spillover of these viruses from wildlife to human do not typically result in pandemics but rather geographically confined outbreaks of human infection and disease. While limited geographically, these viruses cause thousands of cases of human disease each year. In this review, we focus on three questions regarding zoonotic viruses that originate in bats and rodents. First, what biological strategies have evolved that allow RNA viruses to reside in bats and rodents? Second, what are the environmental and ecological causes that drive viral spillover? Third, how does virus spillover occur from bats and rodents to humans?

Time to 'Mind the Gap' in novel small molecule drug discovery for direct-acting antivirals for SARS-CoV-2.

A pipeline of effective direct-acting antivirals (DAAs) remains a critical gap in addressing the current pandemic given vaccination hesitancy, the emergence of viral variants of concern, susceptible populations for which vaccination is ineffective or unavailable, and the possibility that coronavirus disease 2019 (COVID-19) is here to stay. Since the start of the pandemic, global efforts in small molecule drug discovery have focused largely on testing of FDA-approved drugs to accelerate evaluation in clinical trials in hospitalized patients. With 80% of the population who test positive for SARS-CoV-2 having asymptomatic to mild COVID-19, early stage, DAAs would be of enormous benefit to reduce spread, duration of symptoms and quarantine length. We highlight a few of the most promising DAAs in clinical trials and discuss considerations in how to navigate the challenges and pitfalls of novel small molecule discovery and thereby accelerate the advancement of new, safe, and oral DAAs.