pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Mutation Effects on Structure and Dynamics: Adaptive Evolution of the SARS-CoV-2 Main Protease.

The main protease of SARS-CoV-2 (Mpro) plays a critical role in viral replication; although it is relatively conserved, Mpro has nevertheless evolved over the course of the COVID-19 pandemic. Here, we examine phenotypic changes in clinically observed variants of Mpro, relative to the originally reported wild-type enzyme. Using atomistic molecular dynamics simulations, we examine effects of mutation on protein structure and dynamics. In addition to basic structural properties such as variation in surface area and torsion angles, we use protein structure networks and active site networks to evaluate functionally relevant characters related to global cohesion and active site constraint. Substitution analysis shows a continuing trend toward more hydrophobic residues that are dependent on the location of the residue in primary, secondary, tertiary, and quaternary structures. Phylogenetic analysis provides additional evidence for the impact of selective pressure on mutation of Mpro. Overall, these analyses suggest evolutionary adaptation of Mpro toward more hydrophobicity and a less-constrained active site in response to the selective pressures of a novel host environment.

Adaptive Mutation in the Main Protease Cleavage Site of Feline Coronavirus Renders the Virus More Resistant to Main Protease Inhibitors.

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.

Bad news for Paxlovid? Resistance may be coming.

In lab studies, SARS-CoV-2 finds ways to evade key drug. Some of the viral mutations are already found in people.

Pro108Ser mutation of SARS-CoV-2 3CLpro reduces the enzyme activity and ameliorates the clinical severity of COVID-19.

Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CLpro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CLpro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CLpro revealed that the Kcat/Km of the 3CLpro enzyme containing Ser108 was 58% lower than that of Pro108 3CLpro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CLpro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CLpro inhibitor.

Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells.

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.

Expression, purification, and biophysical characterization of recombinant MERS-CoV main (Mpro) protease.

MERS-CoV main protease (Mpro) is essential for the maturation of the coronavirus; therefore, considered a potential drug target. Detailed conformational information is essential to developing antiviral therapeutics. However, the conformation of MERS-CoV Mpro under different conditions is poorly characterized. In this study, MERS-CoV Mpro was recombinantly produced in E.coli and characterized its structural stability with respect to changes in pH and temperatures. The intrinsic and extrinsic fluorescence measurements revealed that MERS-CoV Mpro tertiary structure was exposed to the polar environment due to the unfolding of the tertiary structure. However, the secondary structure of MERS-CoV Mpro was gained at low pH because of charge-charge repulsion. Furthermore, differential scanning fluorometry studies of Mpro showed a single thermal transition at all pHs except at pH 2.0; no transitions were observed. The data from the spectroscopic studies suggest that the MERS-CoV Mpro forms a molten globule-like state at pH 2.0. Insilico studies showed that the covid-19 Mpro shows 96.08% and 50.65% similarity to that of SARS-CoV Mpro and MERS-CoV Mpro, respectively. This study provides a basic understanding of the thermodynamic and structural properties of MERS-CoV Mpro.

High-throughput design of symmetrical dimeric SARS-CoV-2 main protease: structural and physical insights into hotspots for adaptation and therapeutics.

Dimerization of SARS-CoV-2 main protease (Mpro) is a prerequisite for its processing activity. With >2000 mutations already reported in Mpro, SARS-CoV-2 may accumulate mutations in the Mpro dimeric interface to stabilize it further. We employed high-throughput protein design strategies to design the symmetrical dimeric interface of Mpro (300 000 designs) to identify mutational hotspots that render the Mpro more stable. We found that ∼22% of designed mutations that yield stable Mpro dimers already exist in SARS-CoV-2 genomes and are currently circulating. Our multi-parametric analyses highlight potential Mpro mutations that SARS-CoV-2 may develop, providing a foundation for assessing viral adaptation and mutational surveillance.

Non-covalent SARS-CoV-2 Mpro inhibitors developed from in silico screen hits.

Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 µM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.

Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor.

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.

Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography.

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).

Apigenin analogues as SARS-CoV-2 main protease inhibitors: In-silico screening approach.

The COVID-19 new variants spread rapidly all over the world, and until now scientists strive to find virus-specific antivirals for its treatment. The main protease of SARS-CoV-2 (Mpro) exhibits high structural and sequence homology to main protease of SARS-CoV (93.23% sequence identity), and their sequence alignment indicated 12 mutated/variant residues. The sequence alignment of SARS-CoV-2 main protease led to identification of only one mutated/variant residue with no significant role in its enzymatic process. Therefore, Mpro was considered as a high-profile drug target in anti-SARS-CoV-2 drug discovery. Apigenin analogues to COVID-19 main protease binding were evaluated. The detailed interactions between the analogues of Apigenin and SARS-CoV-2 Mpro inhibitors were determined as hydrogen bonds, electronic bonds and hydrophobic interactions. The binding energies obtained from the molecular docking of Mpro with Boceprevir, Apigenin, Apigenin 7-glucoside-4'-p-coumarate, Apigenin 7-glucoside-4'-trans-caffeate and Apigenin 7-O-beta-d-glucoside (Cosmosiin) were found to be -6.6, -7.2, -8.8, -8.7 and -8.0 kcal/mol, respectively. Pharmacokinetic parameters and toxicological characteristics obtained by computational techniques and Virtual ADME studies of the Apigenin analogues confirmed that the Apigenin 7-glucoside-4'-p-coumarate is the best candidate for SARS-CoV-2 Mpro inhibition.

Is amyloid fibrillation related to 3D domain swapping for the C-terminal domain of SARS-CoV main protease?

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.

Structural and functional significance of the amino acid differences Val35Thr, Ser46Ala, Asn65Ser, and Ala94Ser in 3C-like proteinases from SARS-CoV-2 and SARS-CoV.

Three dimensional structures of (chymo)trypsin-like proteinase (3CLpro) from SARS-CoV-2 and SARS-CoV differ at 8 positions. We previously found that the Val86Leu, Lys88Arg, Phe134His, and Asn180Lys mutations in these enzymes can change the orientation of the N- and C-terminal domains of 3CLpro relative to each other, which leads to a change in catalytic activity. This conclusion was derived from the comparison of the structural catalytic core in 169 (chymo)trypsin-like proteinases with the serine/cysteine fold. Val35Thr, Ser46Ala, Asn65Ser, Ala94Ser mutations were not included in that analysis, since they are located far from the catalytic tetrad. In the present work, the structural and functional roles of these variable amino acids at positions 35, 46, 65, and 94 in the 3CLpro sequences of SARS-CoV-2 and SARS-CoV have been established using a comparison of the same set of proteinases leading to the identification of new conservative elements. Comparative analysis showed that, in addition to interdomain mobility, which could modulate catalytic activity, the 3CLpro(s) can use for functional regulation an autolytic loop and the unique Asp33-Asn95 region (the Asp33-Asn95 Zone) in the N-terminal domain. Therefore, all 4 analyzed mutation sites are associated with the unique structure-functional features of the 3CLpro from SARS-CoV-2 and SARS-CoV. Strictly speaking, the presented structural results are hypothetical, since at present there is not a single experimental work on the identification and characterization of autolysis sites in these proteases.

Identification of genotypic variants and its proteomic mutations of Brazilian SARS-CoV-2 isolates.

The second wave of COVID-19 caused by severe acute respiratory syndrome virus (SARS-CoV-2) is rapidly spreading over the world. Mechanisms behind the flee from current antivirals are still unclear due to the continuous occurrence of SARS-CoV-2 genetic variants. Brazil is the world's second-most COVID-19 affected country. In the present study, we identified the genomic and proteomic variants of Brazilian SARS-CoV-2 isolates. We identified 16 different genotypic variants were found among the 27 isolates. The genotypes of three isolates such as Bra/1236/2021 (G15), Bra/MASP2C844R2/2020 (G11), and Bra/RJ-DCVN5/2020 (G9) have a unique mutant in NSP4 (S184N), 2'O-Mutase (R216N), membrane protein (A2V) and Envelope protein (V5A). A mutation in RdRp of SARS-CoV-2, particularly the change of Pro-to Leu-at 323 resulted in the stabilization of the structure in BRA/CD1739-P4/2020. NSP4, NSP5 protein mutants are more virulent in genotype 15 and 16. A fast protein folding rate changes the structural stability and leads to escape for current antivirals. Thus, our findings help researchers to develop the best potent antivirals based on the new mutant of Brazilian isolates.

The SARS-CoV-2 main protease Mpro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

Allosteric Regulation of 3CL Protease of SARS-CoV-2 and SARS-CoV Observed in the Crystal Structure Ensemble.

The 3C-like protease (3CLpro) of SARS-CoV-2 is a potential therapeutic target for COVID-19. Importantly, it has an abundance of structural information solved as a complex with various drug candidate compounds. Collecting these crystal structures (83 Protein Data Bank (PDB) entries) together with those of the highly homologous 3CLpro of SARS-CoV (101 PDB entries), we constructed the crystal structure ensemble of 3CLpro to analyze the dynamic regulation of its catalytic function. The structural dynamics of the 3CLpro dimer observed in the ensemble were characterized by the motions of four separate loops (the C-loop, E-loop, H-loop, and Linker) and the C-terminal domain III on the rigid core of the chymotrypsin fold. Among the four moving loops, the C-loop (also known as the oxyanion binding loop) causes the order (active)-disorder (collapsed) transition, which is regulated cooperatively by five hydrogen bonds made with the surrounding residues. The C-loop, E-loop, and Linker constitute the major ligand binding sites, which consist of a limited variety of binding residues including the substrate binding subsites. Ligand binding causes a ligand size dependent conformational change to the E-loop and Linker, which further stabilize the C-loop via the hydrogen bond between the C-loop and E-loop. The T285A mutation from SARS-CoV 3CLpro to SARS-CoV-2 3CLpro significantly closes the interface of the domain III dimer and allosterically stabilizes the active conformation of the C-loop via hydrogen bonds with Ser1 and Gly2; thus, SARS-CoV-2 3CLpro seems to have increased activity relative to that of SARS-CoV 3CLpro.

SARS-CoV-2 Nsp5 Demonstrates Two Distinct Mechanisms Targeting RIG-I and MAVS To Evade the Innate Immune Response.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.

Virtual Alanine Scan of the Main Protease Active Site in Severe Acute Respiratory Syndrome Coronavirus 2.

Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.

DNA-encoded chemistry technology yields expedient access to SARS-CoV-2 Mpro inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.