Dynamic Interactions of Post Cleaved NS2B Cofactor and NS3 Protease Identified by Integrative Structural Approaches.

Diseases caused by flaviviruses such as dengue virus (DENV) and West Nile Virus (WNV), are a serious threat to public health. The flavivirus single-stranded RNA genome is translated into a polyprotein which is cleaved into three structural proteins and seven non-structural proteins by the viral and cellular proteases. Non-structural (NS) protein 3 is a multifunctional protein that has N-terminal protease and C-terminal helicase domains. The NS3 protease requires co-factor NS2B for enzymatic activity and folding. Due to its essential role in viral replication, NS2B-NS3 protease is an attractive target for antiviral drugs. Despite the availability of crystal structures, dynamic interactions of the N- and C-termini of NS2B co-factor have been elusive due to their flexible fold. In this study, we employ integrative structural approaches combined with biochemical assays to elucidate the dynamic interactions of the flexible DENV4 NS2B and NS3 N- and C-termini. We captured the crystal structure of self-cleaved DENV4 NS2B47NS3 protease in post cleavage state. The intermediate conformation adopted in the reported structure can be targeted by allosteric inhibitors. Comparison of our new findings from DENV4 against previously studied ZIKV NS2B-NS3 proteins reveals differences in NS2B-NS3 function between the two viruses. No inhibition of protease activity was observed for unlinked DENV NS2B-NS3 in presence of the cleavage site while ZIKV NS2B-NS3 cleavage inhibits protease activity. Another difference is that binding of the NS2B C-terminus to DENV4 eNS2B47NS3Pro active site is mediated via interactions with P4-P6 residues while for ZIKV, the binding of NS2B C-terminus to active site is mediated by P1-P3 residues. The mapping of NS2B N- and C-termini with NS3 indicates that these intermolecular interactions occur mainly on the beta-barrel 2 of the NS3 protease domain. Our integrative approach enables a comprehensive understanding of the folding and dynamic interactions of DENV NS3 protease and its cofactor NS2B.

Crystal structure of the Rubella virus protease reveals a unique papain-like protease fold.

Rubella, a viral disease characterized by a red skin rash, is well controlled because of an effective vaccine, but outbreaks are still occurring in the absence of available antiviral treatments. The Rubella virus (RUBV) papain-like protease (RubPro) is crucial for RUBV replication, cleaving the nonstructural polyprotein p200 into two multifunctional proteins, p150 and p90. This protease could represent a potential drug target, but structural and mechanistic details important for the inhibition of this enzyme are unclear. Here, we report a novel crystal structure of RubPro at a resolution of 1.64 Å. The RubPro adopts a unique papain-like protease fold, with a similar catalytic core to that of proteases from Severe acute respiratory syndrome coronavirus 2 and foot-and-mouth disease virus while having a distinctive N-terminal fingers domain. RubPro has well-conserved sequence motifs that are also found in its newly discovered Rubivirus relatives. In addition, we show that the RubPro construct has protease activity in trans against a construct of RUBV protease-helicase and fluorogenic peptides. A protease-helicase construct, exogenously expressed in Escherichia coli, was also cleaved at the p150-p90 cleavage junction, demonstrating protease activity of the protease-helicase protein. We also demonstrate that RubPro possesses deubiquitylation activity, suggesting a potential role of RubPro in modulating the host's innate immune responses. We anticipate that these structural and functional insights of RubPro will advance our current understanding of its function and help facilitate more structure-based research into the RUBV replication machinery, in hopes of developing antiviral therapeutics against RUBV.

Structure-Based Optimization and Characterization of Macrocyclic Zika Virus NS2B-NS3 Protease Inhibitors.

Zika virus (ZIKV) is a human pathogenic arbovirus. So far, neither a specific treatment nor a vaccination against ZIKV infections has been approved. Starting from our previously described lead structure, a series of 29 new macrocyclic inhibitors of the Zika virus protease containing different linker motifs have been synthesized. By selecting hydrophobic d-amino acids as part of the linker, numerous inhibitors with Ki values < 5 nM were obtained. For 12 inhibitors, crystal structures in complex with the ZIKV protease up to 1.30 Å resolution were determined, which contribute to the understanding of the observed structure-activity relationship (SAR). In immunofluorescence assays, an antiviral effect was observed for compound 26 containing a d-homocyclohexylalanine residue in its linker segment. Due to its excellent selectivity profile and low cytotoxicity, this inhibitor scaffold could be a suitable starting point for the development of peptidic drugs against the Zika virus and related flaviviruses.

Molecular Insights into the Flavivirus Replication Complex.

Flaviviruses are vector-borne RNA viruses, many of which are clinically relevant human viral pathogens, such as dengue, Zika, Japanese encephalitis, West Nile and yellow fever viruses. Millions of people are infected with these viruses around the world each year. Vaccines are only available for some members of this large virus family, and there are no effective antiviral drugs to treat flavivirus infections. The unmet need for vaccines and therapies against these flaviviral infections drives research towards a better understanding of the epidemiology, biology and immunology of flaviviruses. In this review, we discuss the basic biology of the flavivirus replication process and focus on the molecular aspects of viral genome replication. Within the virus-induced intracellular membranous compartments, flaviviral RNA genome replication takes place, starting from viral poly protein expression and processing to the assembly of the virus RNA replication complex, followed by the delivery of the progeny viral RNA to the viral particle assembly sites. We attempt to update the latest understanding of the key molecular events during this process and highlight knowledge gaps for future studies.

2-Cyanoisonicotinamide Conjugation: A Facile Approach to Generate Potent Peptide Inhibitors of the Zika Virus Protease.

The rapid generation and modification of macrocyclic peptides in medicinal chemistry is an ever-growing area that can present various synthetic challenges. The reaction between N-terminal cysteine and 2-cyanoisonicotinamide is a new biocompatible click reaction that allows rapid access to macrocyclic peptides. Importantly, 2-cyanoisonicotinamide can be attached to different linkers directly during solid-phase peptide synthesis. The synthesis involves only commercially available precursors, allowing for a fully automated process. We demonstrate the approach for four cyclic peptide ligands of the Zika virus protease NS2B-NS3. Although all peptides display the substrate recognition motif, the activity strongly depends on the linker length, with the shortest cyclization linker corresponding to highest activity (K i = 0.64 µM). The most active cyclic peptide displays affinity 78 times higher than that of its linear analogue. We solved a crystal structure of the proteolytically cleaved ligand and synthesized it by applying the presented chemistry to peptide ligation.

Identification and structural characterization of small molecule fragments targeting Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) NS2B-NS3 protease is a validated antiviral target as it is essential for maturation of viral proteins. However, its negatively charged active site hinders the development of orthosteric small-molecule inhibitors. Fragment-based drug discovery (FBDD) is a powerful tool to generate novel chemical starting points against difficult drug targets. In this study, we scre ened a fragment compound library against the Zika protease using a primary thermal shift assay and identified twenty-two fragments which (bind to and) stabilize the protease. We then determined the X-ray crystal structures of two hits from different classes, all of which bind to the S1 pocket of the protease. We confirmed that these two fragments bind to the protease without inducing significant conformational changes using solution NMR spectroscopy. These fragment scaffolds serve as the starting point for subsequent lead compound development.

Biocompatible Macrocyclization between Cysteine and 2-Cyanopyridine Generates Stable Peptide Inhibitors.

Peptides featuring an N-terminal cysteine residue and the unnatural amino acid 3-(2-cyano-4-pyridyl)alanine (Cpa) cyclize spontaneously in aqueous solution at neutral pH. Cpa is readily available and easily introduced into peptides using standard solid-phase peptide synthesis. The reaction is orthogonal to all proteinogenic amino acids, including cysteine residues that are not at the N-terminus. A substrate peptide of the Zika virus NS2B-NS3 protease cyclized in this way produced an inhibitor of high affinity and proteolytic stability.

Butelase-Mediated Macrocyclization of d-Amino-Acid-Containing Peptides.

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein-protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l-peptides; their ability to cyclize d-amino-acid-containing peptides has rarely been documented. Herein we show that macrocycles consisting of d-amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp-specific ligase. Furthermore, by using a peptide-library approach, we show that butelase 1 tolerates most of the d-amino acid residues at the P1'' and P2'' positions.