Project description:The coronavirus replicase gene encodes one or two papain-like proteases (termed PL1pro and PL2pro) implicated in the N-terminal processing of the replicase polyprotein and thus contributing to the formation of the viral replicase complex that mediates genome replication. Using consensus fold recognition with the 3D-JURY meta-predictor followed by model building and refinement, we developed a structural model for the single PLpro present in the severe acute respiratory syndrome coronavirus (SCoV) genome, based on significant structural relationships to the catalytic core domain of HAUSP, a ubiquitin-specific protease (USP). By combining the SCoV PLpro model with comparative sequence analyses we show that all currently known coronaviral PLpros can be classified into two groups according to their binding site architectures. One group includes all PL2pros and some of the PL1pros, which are characterized by a restricted USP-like binding site. This group is designated the R-group. The remaining PL1pros from some of the coronaviruses form the other group, featuring a more open papain-like binding site, and is referred to as the O-group. This two-group, binding site-based classification is consistent with experimental data accumulated to date for the specificity of PLpro-mediated polyprotein processing and PLpro inhibition. It also provides an independent evaluation of the similarity-based annotation of PLpro-mediated cleavage sites, as well as a basis for comparison with previous groupings based on phylogenetic analyses.
Project description:Coronaviruses are a large group of enveloped, single-stranded positive-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a critical role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential first step in establishing infection, the proteolytic activation step is often critical for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large number of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity.
Project description:The main protease (M pro) of coronaviruses plays a key role in viral replication, thus serving as a hot target for drug design. PF-00835231 is a promising inhibitor of SARS-CoV-2 M pro. Here, we report the inhibitory potency of PF-00835231 against SARS-CoV-2 M pro and seven M pro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) from SARS-CoV-2 variants. The results confirm that PF-00835231 has broad-spectrum inhibition against various coronaviral M pros. In addition, the crystal structures of SARS-CoV-2 M pro, SARS-CoV M pro, MERS-CoV M pro, and seven SARS-CoV-2 M pro mutants (G15S, M49I, Y54C, K90R, P132H, S46F, and V186F) in complex with PF-00835231 are solved. A detailed analysis of these structures reveals key determinants essential for inhibition and elucidates the binding modes of different coronaviral M pros. Given the importance of the main protease for the treatment of coronaviral infection, structural insights into M pro inhibition by PF-00835231 can accelerate the design of novel antivirals with broad-spectrum efficacy against different human coronaviruses.
Project description:Papain-like proteases (PLpro) of coronaviruses (CoVs) support viral reproduction and suppress the immune response of the host, which makes CoV PLpro perspective pharmaceutical targets. Their inhibition could both prevent viral replication and boost the immune system of the host, leading to the speedy recovery of the patient. Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third CoV outbreak in the last 20 years. Frequent mutations of the viral genome likely lead to the emergence of more CoVs. Inhibitors for CoV PLpro can be broad-spectrum and can diminish present and prevent future CoV outbreaks as PLpro from different CoVs have conservative structures. Several inhibitors have been developed to withstand SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV). This review summarizes the structural features of CoV PLpro, the inhibitors that have been identified over the last 20 years, and the compounds that have the potential to become novel effective therapeutics against CoVs in the near future.
Project description:This review focuses on the important contributions that macromolecular crystallography has made over the past 12 years to elucidating structures and mechanisms of the essential proteases of coronaviruses, the main protease (M(pro) ) and the papain-like protease (PL(pro) ). The role of X-ray crystallography in structure-assisted drug discovery against these targets is discussed. Aspects dealt with in this review include the emergence of the SARS coronavirus in 2002-2003 and of the MERS coronavirus 10 years later and the origins of these viruses. The crystal structure of the free SARS coronavirus M(pro) and its dependence on pH is discussed, as are efforts to design inhibitors on the basis of these structures. The mechanism of maturation of the enzyme from the viral polyprotein is still a matter of debate. The crystal structure of the SARS coronavirus PL(pro) and its complex with ubiquitin is also discussed, as is its orthologue from MERS coronavirus. Efforts at predictive structure-based inhibitor development for bat coronavirus M(pro) s to increase the preparedness against zoonotic transmission to man are described as well. The paper closes with a brief discussion of structure-based discovery of antivirals in an academic setting.
Project description:The ongoing spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused hundreds of millions of cases and millions of victims worldwide with serious consequences to global health and economies. Although many vaccines protecting against SARS-CoV-2 are currently available, constantly emerging new variants necessitate the development of alternative strategies for prevention and treatment of COVID-19. Inhibitors that target the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation, represent a key class of antivirals. Here, we showed that a peptidomimetic compound with benzothiazolyl ketone as warhead, YH-53, is an effective inhibitor of SARS-CoV-2, SARS-CoV, and MERS-CoV Mpros. Crystal structures of Mpros from SARS-CoV-2, SARS-CoV, and MERS-CoV bound to the inhibitor YH-53 revealed a unique ligand-binding site, which provides new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures defined the key molecular determinants required for inhibition and illustrate the binding mode of Mpros from other coronaviruses. In consideration of the important role of Mpro in developing antivirals against coronaviruses, insights derived from this study should add to the design of pan-coronaviral Mpro inhibitors that are safer and more effective.
Project description:Similar to bacteria, eukaryotic pathogens may utilize common strategies of pathogenic secretion, because effector proteins from the oomycete Phytophthora infestans and virulence determinants from the human malaria parasite Plasmodium falciparum share a functionally equivalent host-cell-targeting motif (RxLR-dEER in P. infestans and RxLxE/D/Q in P. falciparum). Here we summarize recent studies that reveal that the malarial motif may function differently than previously envisioned. Binding of the lipid phosphatidylinositol 3-phosphate [PI(3)P] is a critical step in accessing the host for both pathogens, but occurs in different locations. Nanomolar affinity for PI(3)P by these short amino acid motifs suggests that a newly identified mechanism of phosphoinositide binding that unexpectedly occurs in secretory locations has been exploited for virulence by diverse eukaryotic pathogens.
Project description:Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.
Project description:Alphaviruses are important pathogens that continue to cause outbreaks of disease in humans and animals worldwide. Diseases caused by alphavirus infections include acute symptoms of fever, rash, and nausea as well as chronic arthritis and severe-to-fatal conditions including myocarditis and encephalitis. Despite their prevalence and the significant public health threat they pose, there are currently no effective antiviral treatments or vaccines against alphaviruses. Various genetic determinants of alphavirus virulence, including genomic RNA elements and specific protein residues and domains, have been described by researchers to play key roles in the development of disease, the immune response to infection, and virus transmissibility. Here, we focus on the determinants that are currently described in the literature. Understanding how these molecular determinants shape viral infections can lead to new strategies for the development of therapies and vaccines to combat these viruses.