The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation.

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.

SARS-CoV-2 Mpro inhibitors and activity-based probes for patient-sample imaging.

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed. Currently, there is no effective antiviral treatment for COVID-19. To address this emerging problem, we focused on the SARS-CoV-2 main protease that constitutes one of the most attractive antiviral drug targets. We have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases. On the basis of these findings, we designed and synthesized a potent SARS-CoV-2 inhibitor (Ac-Abu-DTyr-Leu-Gln-VS, half-maximal effective concentration of 3.7 µM) and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. We visualized active SARS-CoV-2 Mpro in nasopharyngeal epithelial cells of patients suffering from COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents and/or diagnostic tests.

Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease.

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of Paxlovid, a drug approved by the U.S. Food and Drug Administration for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here, we investigated the H172Y Mpro's conformational dynamics, folding stability, catalytic efficiency, and inhibitory activity using all-atom constant pH and fixed-charge molecular dynamics simulations, alchemical and empirical free energy calculations, artificial neural networks, and biochemical experiments. Our data suggest that the mutation significantly weakens the S1 pocket interactions with the N-terminus and perturbs the conformation of the oxyanion loop, leading to a decrease in the thermal stability and catalytic efficiency. Importantly, the perturbed S1 pocket dynamics weaken the nirmatrelvir binding in the P1 position, which explains the decreased inhibitory activity of nirmatrelvir. Our work demonstrates the predictive power of the combined simulation and artificial intelligence approaches, and together with biochemical experiments, they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the optimization of antiviral drugs. The presented approach, in general, can be applied to characterize mutation effects on any protein drug targets.

Characterization of an Allosteric Pocket in Zika Virus NS2B-NS3 Protease.

The NS2B-NS3 protease from Zika virus (ZIKV NS2B-NS3pro) cleaves the viral polyprotein, being essential for its replication and a therapeutic target. Inhibitors that target the active site of ZIKV NS2B-NS3pro have been developed, but they tend to have unfavorable pharmacokinetic properties due to their highly positive charge. Thus, the characterization of allosteric sites in this protease provides new strategies for inhibitor development. Here, we characterized a new allosteric pocket in ZIKV NS2B-NS3pro, analogous to the one previously described for the dengue virus protease. Molecular dynamics simulations indicate the presence of cavities around the residue Ala125, sampling protein conformations in which they are connected to the active site. This link between the residue Ala125 and the active site residues was reinforced by correlation network analysis. To experimentally verify the existence of this allosteric mechanism, we expressed and purified the Ala125Cys mutant of ZIKV NS2B-NS3pro and demonstrated that this variant is inhibited by the thiol-containing chemical probes 5,5'-dithiobis-(2-nitrobenzoic acid) and aldrithiol, which do not affect the activity of the wild-type protein. Inhibition of the mutant protein is reversed by the addition of strong reducing agents, supporting the involvement of Cys125 in covalent bond formation and enzyme inhibition. Together, our results provide experimental evidence for an allosteric pocket in ZIKV NS2B-NS3pro, in the region around Ala125, and computational insights on the structural connection between this region and the enzyme active site.

From Repurposing to Redesign: Optimization of Boceprevir to Highly Potent Inhibitors of the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the betacoronavirus SARS-CoV-2 is an attractive target for the development of treatments for COVID-19. Structure-based design is a successful approach to discovering new inhibitors of the Mpro. Starting from crystal structures of the Mpro in complexes with the Hepatitis C virus NS3/4A protease inhibitors boceprevir and telaprevir, we optimized the potency of the alpha-ketoamide boceprevir against the Mpro by replacing its P1 cyclobutyl moiety by a γ-lactam as a glutamine surrogate. The resulting compound, MG-78, exhibited an IC50 of 13 nM versus the recombinant Mpro, and similar potency was observed for its P1' N-methyl derivative MG-131. Crystal structures confirmed the validity of our design concept. In addition to SARS-CoV-2 Mpro inhibition, we also explored the activity of MG-78 against the Mpro of the alphacoronavirus HCoV NL63 and against enterovirus 3C proteases. The activities were good (0.33 µM, HCoV-NL63 Mpro), moderate (1.45 µM, Coxsackievirus 3Cpro), and relatively poor (6.7 µM, enterovirus A71 3Cpro), respectively. The structural basis for the differences in activities was revealed by X-ray crystallo-graphy. We conclude that the modified boceprevir scaffold is suitable for obtaining high-potency inhibitors of the coronavirus Mpros but further optimization would be needed to target enterovirus 3Cpros efficiently.

Synthesis, Structure-Activity Relationships, and Antiviral Profiling of 1-Heteroaryl-2-Alkoxyphenyl Analogs as Inhibitors of SARS-CoV-2 Replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has led to a pandemic, that continues to be a huge public health burden. Despite the availability of vaccines, there is still a need for small-molecule antiviral drugs. In an effort to identify novel and drug-like hit matter that can be used for subsequent hit-to-lead optimization campaigns, we conducted a high-throughput screening of a 160 K compound library against SARS-CoV-2, yielding a 1-heteroaryl-2-alkoxyphenyl analog as a promising hit. Antiviral profiling revealed this compound was active against various beta-coronaviruses and preliminary mode-of-action experiments demonstrated that it interfered with viral entry. A systematic structure-activity relationship (SAR) study demonstrated that a 3- or 4-pyridyl moiety on the oxadiazole moiety is optimal, whereas the oxadiazole can be replaced by various other heteroaromatic cycles. In addition, the alkoxy group tolerates some structural diversity.

Processing of the SARS-CoV pp1a/ab nsp7-10 region.

Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1.

Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

The Structure of the Zika Virus Protease, NS2B/NS3pro.

In this chapter, we first briefly review the history of Zika virus (ZIKV) over the past 70 years since its discovery. We then focus on the ZIKV NS2B/NS3 protease, a major potential target for anti-ZIKV therapeutics. We describe the structure of the complex between Zika virus NS2B-NS3 protease and a peptide boronic-acid inhibitor that we determined in early 2016. We then review other structural studies on the Zika virus protease, which have been published in the past few months. Three different types of construct for the protease have been investigated by X-ray crystallography and NMR spectroscopy: the traditional "linked" construct comprising the NS2B cofactor, a Gly4SerGly4 linker, and the NS3pro chain; a construct where the linker has been replaced by Lys-Thr-Gly-Lys-Arg, which leads to autocleavage; and the bimolecular "unlinked" protease consisting of the NS2B cofactor segment and NS3pro. In complex with an inhibitor, the protease adopts a closed, "active" conformation with the NS2B chain wrapped around the NS3pro and contributing to the S2 pocket. In the ligand-free state, the Gly4SerGly4-linked enzyme adopts an open or relaxed conformation, with the C-terminal half of the NS2B cofactor highly flexible and disordered. Very surprisingly, however, the "unlinked", bimolecular protease has been reported to adopt the closed conformation in the crystal, even though, apparently, no peptide was bound to the substrate-binding site. The Gly4SerGly4-linked enzyme has been used successfully in drug discovery efforts.

Viral Entry and NS1 as Potential Antiviral Drug Targets.

A general discussion on viral entry and NS1 as potential drug targets was held at the Tofo Advanced Study Week (TASW) on Emerging Viral Diseases in September 2016. The opportunities and gaps for developing therapeutic countermeasures, to take advantage of the high-resolution cryo-electron microscopy structures of dengue and Zika viruses as well as the novel features of NS1 revealed by the 3D structures, were deliberated.

Effects of NS2B-NS3 protease and furin inhibition on West Nile and Dengue virus replication.

West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH2 inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11 µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.

Structural basis of the proteolytic and chaperone activity of Chlamydia trachomatis CT441.

Chlamydia trachomatis is the most prevalent cause of preventable blindness worldwide and a major reason for infectious infertility in females. Several bacterial factors have been implicated in the pathogenesis of C. trachomatis. Combining structural and mutational analysis, we have shown that the proteolytic function of CT441 depends on a conserved Ser/Lys/Gln catalytic triad and a functional substrate-binding site within a flexible PDZ (postsynaptic density of 95 kDa, discs large, and zonula occludens) domain. Previously, it has been suggested that CT441 is involved in modulating estrogen signaling responses of the host cell. Our results show that although in vitro CT441 exhibits proteolytic activity against SRAP1, a coactivator of estrogen receptor α, CT441-mediated SRAP1 degradation is not observed during the intracellular developmental cycle before host cells are lysed and infectious chlamydiae are released. Most compellingly, we have newly identified a chaperone activity of CT441, indicating a role of CT441 in prokaryotic protein quality control processes.

The enterovirus 3C protease inhibitor SG85 efficiently blocks rhinovirus replication and is not cross-resistant with rupintrivir.

The novel enterovirus protease inhibitor (PI) SG85 effectively inhibits the in vitro replication of 14 rhinoviruses representative of species A and B (median 50% effective concentration, 0.04 µM). A low-level SG85-resistant variant was selected that carried amino acid substitutions S127G and T143A in the 3C protease. Both substitutions are required for low-level resistance to SG85, as demonstrated by reverse genetics. Interestingly, there is no cross-resistance to SG85 and rupintrivir (another PI); a structural explanation is provided for this observation.

Antiviral Activity of Broad-Spectrum and Enterovirus-Specific Inhibitors against Clinical Isolates of Enterovirus D68.

We investigated the susceptibility of 10 enterovirus D68 (EV-D68) isolates (belonging to clusters A, B, and C) to (entero)virus inhibitors with different mechanisms of action. The 3C-protease inhibitors proved to be more efficient than enviroxime and pleconaril, which in turn were more effective than vapendavir and pirodavir. Favipiravir proved to be a weak inhibitor. Resistance to pleconaril maps to V69A in the VP1 protein, and resistance to rupintrivir maps to V104I in the 3C protease. A structural explanation of why both substitutions may cause resistance is provided.

Acquisition of new protein domains by coronaviruses: analysis of overlapping genes coding for proteins N and 9b in SARS coronavirus.

Acquisition of new proteins by viruses usually occurs through horizontal gene transfer or through gene duplication, but another, less common mechanism is the usage of completely or partially overlapping reading frames. A case of acquisition of a completely new protein through introduction of a start codon in an alternative reading frame is the protein encoded by open reading frame (orf) 9b of SARS coronavirus. This gene completely overlaps with the nucleocapsid (N) gene (orf9a). Our findings indicate that the orf9b gene features a discordant codon-usage pattern. We analyzed the evolution of orf9b in concert with orf9a using sequence data of betacoronavirus-lineage b and found that orf9b, which encodes the overprinting protein, evolved largely independent of the overprinted orf9a. We also examined the protein products of these genomic sequences for their structural flexibility and found that it is not necessary for a newly acquired, overlapping protein product to be intrinsically disordered, in contrast to earlier suggestions. Our findings contribute to characterizing sequence properties of newly acquired genes making use of overlapping reading frames.

Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis.

WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX(5)GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA.

Helix unfolding/refolding characterizes the functional dynamics of Staphylococcus aureus Clp protease.

The ATP-dependent Clp protease (ClpP) plays an essential role not only in the control of protein quality but also in the regulation of bacterial pathogen virulence, making it an attractive target for antibacterial treatment. We have previously determined the crystal structures of Staphylococcus aureus ClpP (SaClpP) in two different states, extended and compressed. To investigate the dynamic switching of ClpP between these states, we performed a series of molecular dynamics simulations. During the structural transition, the long and straight helix E in the extended SaClpP monomer underwent an unfolding/refolding process, resulting in a kinked helix very similar to that in the compressed monomer. As a stable intermediate in the molecular dynamics simulation, the compact state was suggested and subsequently identified in x-ray crystallographic experiment. Our combined studies also determined that Ala(140) acted as a "hinge" during the transition between the extended and compressed states, and Glu(137) was essential for stabilizing the compressed state. Overall, this study provides molecular insights into the dynamics and mechanism of the functional conformation changes of SaClpP. Given the highly conserved sequences of ClpP proteins among different species, these findings potentially reflect a switching mechanism for the dynamic process shared in the whole ClpP family in general and thus aid in better understand the principles of Clp protease assembly and function.

The capsid binder Vapendavir and the novel protease inhibitor SG85 inhibit enterovirus 71 replication.

Antivirals against enterovirus 71 (EV71) are urgently needed. We demonstrate that the novel enteroviral protease inhibitor (PI) SG85 and capsid binder (CB) vapendavir efficiently inhibit the in vitro replication of 21 EV71 strains/isolates that are representative of the different genogroups A, B, and C. The PI rupintrivir, the CB pirodavir, and the host-targeting compound enviroxime, which were included as reference compounds, also inhibited the replication of all isolates. Remarkably, the CB compound pleconaril was devoid of any anti-EV71 activity. An in silico docking study revealed that pleconaril-unlike vapendavir and pirodavir-lacks essential binding interactions with the viral capsid. Vapendavir and SG85 (or analogues) should be further explored for the treatment of EV71 infections. The data presented here may serve as a reference when developing yet-novel inhibitors.