Publications by authors named "Rolf Hilgenfeld"

120 Publications

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

EMBO J 2021 Apr 20:e102277. Epub 2021 Apr 20.

Max-von-Pettenkofer Institute, Ludwig-Maximilians-University Munich, Munich, Germany.

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.
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http://dx.doi.org/10.15252/embj.2019102277DOI Listing
April 2021

Design, Synthesis, and Biological Evaluation of Peptidomimetic Aldehydes as Broad-Spectrum Inhibitors against Enterovirus and SARS-CoV-2.

J Med Chem 2021 Apr 19. Epub 2021 Apr 19.

State Key Laboratory of Drug Research, CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China.

A novel series of peptidomimetic aldehydes was designed and synthesized to target 3C protease (3C) of enterovirus 71 (EV71). Most of the compounds exhibited high antiviral activity, and among them, compound demonstrated potent enzyme inhibitory activity and broad-spectrum antiviral activity on a panel of enteroviruses and rhinoviruses. The crystal structure of EV71 3C in complex with determined at a resolution of 1.2 Å revealed that covalently linked to the catalytic Cys147 with an aldehyde group. In addition, these compounds also exhibited good inhibitory activity against the 3CL and the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially compound (IC = 0.034 μM, EC = 0.29 μM). According to our previous work, these compounds have no reasons for concern regarding acute toxicity. Compared with , compound also exhibited good pharmacokinetic properties and more potent anticoronavirus activity, making it an excellent lead for further development.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8084273PMC
April 2021

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease.

Science 2021 May 2;372(6542):642-646. Epub 2021 Apr 2.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, 22607 Hamburg, Germany.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.
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http://dx.doi.org/10.1126/science.abf7945DOI Listing
May 2021

Identification of non-covalent SARS-CoV-2 main protease inhibitors by a virtual screen of commercially available drug-like compounds.

Bioorg Med Chem Lett 2021 Mar 26;41:127990. Epub 2021 Mar 26.

Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA 93407, USA. Electronic address:

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http://dx.doi.org/10.1016/j.bmcl.2021.127990DOI Listing
March 2021

Structural biology in the fight against COVID-19.

Nat Struct Mol Biol 2021 01;28(1):2-7

Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China.

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http://dx.doi.org/10.1038/s41594-020-00544-8DOI Listing
January 2021

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

Nat Chem Biol 2021 02 22;17(2):222-228. Epub 2020 Oct 22.

Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, Wroclaw, Poland.

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 M. We visualized active SARS-CoV-2 M 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.
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http://dx.doi.org/10.1038/s41589-020-00689-zDOI Listing
February 2021

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors.

Science 2020 04 20;368(6489):409-412. Epub 2020 Mar 20.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germany.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (M, also called 3CL) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 M and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. On the basis of the unliganded structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 M The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.
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http://dx.doi.org/10.1126/science.abb3405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164518PMC
April 2020

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

Biochem J 2020 03;477(5):1009-1019

Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistraße 52, 20251 Hamburg, Germany.

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.
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http://dx.doi.org/10.1042/BCJ20200029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078746PMC
March 2020

α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment.

J Med Chem 2020 05 24;63(9):4562-4578. Epub 2020 Feb 24.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germany.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, (P2 = cyclopentylmethyl) and (P2 = cyclohexylmethyl), display low-micromolar EC values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098070PMC
May 2020

Profiling of flaviviral NS2B-NS3 protease specificity provides a structural basis for the development of selective chemical tools that differentiate Dengue from Zika and West Nile viruses.

Antiviral Res 2020 03 31;175:104731. Epub 2020 Jan 31.

Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370, Wroclaw, Poland. Electronic address:

West Nile virus (WNV) and Dengue virus (DENV) are mosquito-borne pathogenic flaviviruses. The NS2B-NS3 proteases found in these viruses are responsible for polyprotein processing and are therefore considered promising medical targets. Another ortholog of these proteases is found in Zika virus (ZIKV). In this work, we applied a combinatorial chemistry approach - Hybrid Combinatorial Substrate Library (HyCoSuL), to compare the substrate specificity profile at the P4-P1 positions of the NS2B-NS3 proteases found in all three viruses. The obtained data demonstrate that Zika and West Nile virus NS2B-NS3 proteases display highly overlapping substrate specificity in all binding pockets, while the Dengue ortholog has slightly different preferences toward natural and unnatural amino acids at the P2 and P4 positions. We used this information to extract specific peptide sequences recognized by the Dengue NS2B-NS3 protease. Next, we applied this knowledge to design a selective substrate and activity-based probe for the Dengue NS2B-NS3 protease. Our work provides a structural framework for the design of inhibitors, which could be used as a lead structure for drug development efforts.
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http://dx.doi.org/10.1016/j.antiviral.2020.104731DOI Listing
March 2020

Third Tofo Advanced Study Week on Emerging and Re-emerging Viruses, 2018.

Antiviral Res 2019 02 28;162:142-150. Epub 2018 Dec 28.

Institute of Biochemistry, University of Lübeck, Lübeck, Germany; German Center for Infection Research (DZIF), Hamburg - Lübeck - Borstel - Riems Site, Lübeck, Germany. Electronic address:

The Third Tofo Advanced Study Week on Emerging and Re-Emerging Viruses (3rd TASW) was held in Praia do Tofo, Mozambique, from September 02 to 06, 2018. It brought together 55 participants from 10 African countries as well as from Belgium, China, Germany, Singapore, and the USA. Meeting sessions covered aspects of the epidemiology, diagnosis, molecular and structural biology, vaccine development, and antiviral drug discovery for emerging RNA viruses that are current threats in Africa and included flaviviruses (dengue and Zika), alphaviruses (chikungunya), coronaviruses, filoviruses (Ebola), influenza viruses, Crimean Congo hemorrhagic fever virus, Rift Valley fever Virus, Lassa virus, and others. Data were presented on recent flavivirus and/or chikungunyavirus outbreaks in Angola, Burkina Faso, and Mozambique. In addition, these viruses are endemic in many sub-Saharan countries. The TASW series on emerging viruses is unique in Africa and successful in promoting collaborations between researchers in Africa and other parts of the world, as well as among African scientists. This report summarizes the lectures held at the meeting and highlights advances in the field.
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http://dx.doi.org/10.1016/j.antiviral.2018.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132404PMC
February 2019

The Structure of the Zika Virus Protease, NS2B/NS3.

Adv Exp Med Biol 2018;1062:131-145

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.

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 GlySerGly linker, and the NS3 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 NS3. In complex with an inhibitor, the protease adopts a closed, "active" conformation with the NS2B chain wrapped around the NS3 and contributing to the S2 pocket. In the ligand-free state, the GlySerGly-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 GlySerGly-linked enzyme has been used successfully in drug discovery efforts.
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http://dx.doi.org/10.1007/978-981-10-8727-1_10DOI Listing
November 2018

Viral Entry and NS1 as Potential Antiviral Drug Targets.

Adv Exp Med Biol 2018;1062:107-113

Emerging Infectious Diseases Program, Duke-NUS Medical School Singapore, Singapore, Singapore.

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.
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http://dx.doi.org/10.1007/978-981-10-8727-1_8DOI Listing
November 2018

Crystal structure of the C-terminal fragment of NS1 protein from yellow fever virus.

Sci China Life Sci 2017 12 29;60(12):1403-1406. Epub 2017 Nov 29.

Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, 100101, China.

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http://dx.doi.org/10.1007/s11427-017-9238-8DOI Listing
December 2017

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein.

Antiviral Res 2018 01 8;149:58-74. Epub 2017 Nov 8.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany; German Center for Infection Research (DZIF), Hamburg - Lübeck - Borstel - Riems Site, University of Lübeck, Germany. Electronic address:

The multi-domain non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome, with an average molecular mass of about 200 kD. Nsp3 is an essential component of the replication/transcription complex. It comprises various domains, the organization of which differs between CoV genera, due to duplication or absence of some domains. However, eight domains of Nsp3 exist in all known CoVs: the ubiquitin-like domain 1 (Ubl1), the Glu-rich acidic domain (also called "hypervariable region"), a macrodomain (also named "X domain"), the ubiquitin-like domain 2 (Ubl2), the papain-like protease 2 (PL2), the Nsp3 ectodomain (3Ecto, also called "zinc-finger domain"), as well as the domains Y1 and CoV-Y of unknown functions. In addition, the two transmembrane regions, TM1 and TM2, exist in all CoVs. The three-dimensional structures of domains in the N-terminal two thirds of Nsp3 have been investigated by X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy since the outbreaks of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2003 as well as Middle-East Respiratory Syndrome coronavirus (MERS-CoV) in 2012. In this review, the structures and functions of these domains of Nsp3 are discussed in depth.
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http://dx.doi.org/10.1016/j.antiviral.2017.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113668PMC
January 2018

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

Antiviral Res 2017 Oct 18;146:174-183. Epub 2017 Sep 18.

Center for Structural and Cell Biology in Medicine, Institute of Chemistry, University of Luebeck, Ratzeburger Allee 160, 23562 Luebeck, Germany. Electronic address:

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3 is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3 inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3, we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3. Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3. To identify the matching protein binding site, chemical shift perturbation studies employing H,N-TROSY-HSQC experiments with uniformly H,N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3 inhibitors.
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http://dx.doi.org/10.1016/j.antiviral.2017.09.008DOI Listing
October 2017

RNA-virus proteases counteracting host innate immunity.

FEBS Lett 2017 10 15;591(20):3190-3210. Epub 2017 Sep 15.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Germany.

Virus invasion triggers host immune responses, in particular, innate immune responses. Pathogen-associated molecular patterns of viruses (such as dsRNA, ssRNA, or viral proteins) released during virus replication are detected by the corresponding pattern-recognition receptors of the host, and innate immune responses are induced. Through production of type-I and type-III interferons as well as various other cytokines, the host innate immune system forms the frontline to protect host cells and inhibit virus infection. Not surprisingly, viruses have evolved diverse strategies to counter this antiviral system. In this review, we discuss the multiple strategies used by proteases of positive-sense single-stranded RNA viruses of the families Picornaviridae, Coronaviridae, and Flaviviridae, when counteracting host innate immune responses.
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http://dx.doi.org/10.1002/1873-3468.12827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163997PMC
October 2017

Lybatides from Lycium barbarum Contain An Unusual Cystine-stapled Helical Peptide Scaffold.

Sci Rep 2017 07 12;7(1):5194. Epub 2017 Jul 12.

School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

Cysteine-rich peptides (CRPs) of 2-6 kDa are generally thermally and proteolytically stable because of their multiple cross-bracing disulfide bonds. Here, we report the discovery and characterization of two novel cystine-stapled CRPs, designated lybatide 1 and 2 (lyba1 and lyba2), from the cortex of Lycium barbarum root. Lybatides, 32 to 33 amino acids in length, are hyperstable and display a novel disulfide connectivity with a cysteine motif of C-C-C-C-CC-CC which contains two pairs of adjacent cysteines (-CC-CC). X-ray structure analysis revealed the presence of a single cystine-stabilized (α + π)-helix in lyba2, a rare feature of CRPs. Together, our results suggest that lybatides, one of the smallest four-disulfide-constrained plant CRPs, is a new family of CRPs. Additionally, this study provides new insights into the molecular diversity of plant cysteine-rich peptides and the unusual lybatide scaffold could be developed as a useful template for peptide engineering and therapeutic development.
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http://dx.doi.org/10.1038/s41598-017-05037-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507927PMC
July 2017

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

J Enzyme Inhib Med Chem 2017 Dec;32(1):712-721

a Department of Pharmacy, Institute of Pharmaceutical Chemistry , Philipps University , Marburg , Germany.

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-NH 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.
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http://dx.doi.org/10.1080/14756366.2017.1306521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445162PMC
December 2017

Computer-Aided Structure Based Drug Design Approaches for the Discovery of New Anti-CHIKV Agents.

Curr Comput Aided Drug Des 2017 Nov;13(4):346-361

Department of Pharmaceutical Sciences & Technology, Birla Insitute of Technology, Mesra, Ranchi, Jharkhand, India.

Background: Chikungunya is a viral infection caused by Chikungunya virus (CHIKV), an arbovirus transmitted through mosquito (Aedes aegypti and Aedes albopictus) bite. The virus from sylvatic cycle in Africa mutated to new vector adaptation and became one of the major emerging and re-emerging viral infections in the past decade, affecting more than 40 countries. Efforts are being made by many researches to develop means to prevent and control the infection through vaccines and vector control strategy. On the other hand, search for novel chemotherapeutic agents for the treatment of infected patients is on. Approach of repurposed drug is one way of identifying an existing drug for the treatment of CHIKV infection.

Objective: Review the history of CHIKV nsp2 protease inhibitors derived through structure-based computer-aided drug design along with phytochemicals identified as anti-CHIKV agents.

Methods: A survey on CHIKV inhibitors reported till date has been carriedout. The data obtained were organized and discussed under natural substances and synthetic derivatives obtained as result of rational design.

Results: The review provides a well organized content in chronological order that has highly significant information for medicinal chemist who wish to explore the area of Anti-CHIKV drug design and development. Natural compounds with different scaffolds provides an opportunity to explore Ligand based drug design (LBDD), while rational drug design approaches provides opportunity to explore the Structure based drug design.

Conclusion: From the presented mini-review, readers can understand that this area is less explored and has lots of potential in anti-CHIKVviral drug design & development. of reported literature inferred that, unlike other viral proteases, the nsP2 protease can be targeted for CHIKV viral inhibition. The HTVS process for the identification of anti-CHIK agents provided a few successive validated lead compounds against CHIKV infections.
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http://dx.doi.org/10.2174/1573409913666170309145308DOI Listing
November 2017

Extended substrate specificity and first potent irreversible inhibitor/activity-based probe design for Zika virus NS2B-NS3 protease.

Antiviral Res 2017 Mar 26;139:88-94. Epub 2016 Dec 26.

Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland. Electronic address:

Zika virus is spread by Aedes mosquitoes and is linked to acute neurological disorders, especially to microcephaly in newborn children and Guillan-Barré Syndrome. The NS2B-NS3 protease of this virus is responsible for polyprotein processing and therefore considered an attractive drug target. In this study, we have used the Hybrid Combinatorial Substrate Library (HyCoSuL) approach to determine the substrate specificity of ZIKV NS2B-NS3 protease in the P4-P1 positions using natural and a large spectrum of unnatural amino acids. Obtained data demonstrate a high level of specificity of the S3-S1 subsites, especially for basic amino acids. However, the S4 site exhibits a very broad preference toward natural and unnatural amino acids with selected D-amino acids being favored over L enantiomers. This information was used for the design of a very potent phosphonate inhibitor/activity-based probe of ZIKV NS2B-NS3 protease.
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http://dx.doi.org/10.1016/j.antiviral.2016.12.018DOI Listing
March 2017

Peptide-Boronic Acid Inhibitors of Flaviviral Proteases: Medicinal Chemistry and Structural Biology.

J Med Chem 2017 01 14;60(1):511-516. Epub 2016 Dec 14.

Medicinal Chemistry, IPMB, Heidelberg University , INF-364, 69120 Heidelberg, Germany.

A thousand-fold affinity gain is achieved by introduction of a C-terminal boronic acid moiety into dipeptidic inhibitors of the Zika, West Nile, and dengue virus proteases. The resulting compounds have K values in the two-digit nanomolar range, are not cytotoxic, and inhibit virus replication. Structure-activity relationships and a high resolution X-ray cocrystal structure with West Nile virus protease provide a basis for the design of optimized covalent-reversible inhibitors aimed at emerging flaviviral pathogens.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01021DOI Listing
January 2017

Coxsackievirus B3 protease 3C: expression, purification, crystallization and preliminary structural insights.

Acta Crystallogr F Struct Biol Commun 2016 12 25;72(Pt 12):877-884. Epub 2016 Nov 25.

Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras, University Campus, 26500 Patras, Greece.

Viral proteases are proteolytic enzymes that orchestrate the assembly of viral components during the viral life cycle and proliferation. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis are presented of protease 3C, the main protease of an emerging enterovirus, coxsackievirus B3, that is responsible for many cases of viral myocarditis. Polycrystalline protein precipitates suitable for X-ray powder diffraction (XRPD) measurements were produced in the presence of 22-28%(w/v) PEG 4000, 0.1 M Tris-HCl, 0.2 M MgCl in a pH range from 7.0 to 8.5. A polymorph of monoclinic symmetry (space group C2, unit-cell parameters a = 77.9, b = 65.7, c = 40.6 Å, β = 115.9°) was identified via XRPD. These results are the first step towards the complete structural determination of the molecule via XRPD and a parallel demonstration of the accuracy of the method.
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http://dx.doi.org/10.1107/S2053230X16018513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137464PMC
December 2016

Zika virus NS1, a pathogenicity factor with many faces.

Authors:
Rolf Hilgenfeld

EMBO J 2016 12 27;35(24):2631-2633. Epub 2016 Oct 27.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Lübeck, Germany.

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http://dx.doi.org/10.15252/embj.201695871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167348PMC
December 2016

Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments.

Nat Commun 2016 Sep 28;7:12761. Epub 2016 Sep 28.

Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.

Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme-inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases.
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http://dx.doi.org/10.1038/ncomms12761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052702PMC
September 2016

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

Proc Natl Acad Sci U S A 2016 08 12;113(35):E5192-201. Epub 2016 Aug 12.

Max-von-Pettenkofer Institute, Ludwig-Maximilians-University Munich and German Center for Infection Research (DZIF), partner site Munich, 80336 Munich, Germany;

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.
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http://dx.doi.org/10.1073/pnas.1603435113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024628PMC
August 2016

Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

Science 2016 Jul 7;353(6298):503-5. Epub 2016 Jul 7.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany. German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems Site, University of Lübeck, Germany.

The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing.
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http://dx.doi.org/10.1126/science.aag2419DOI Listing
July 2016

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin.

Virol Sin 2016 Aug 30;31(4):288-99. Epub 2016 May 30.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562, Lübeck, Germany.

The papain-like protease (PL(pro)) of Middle-East respiratory syndrome coronavirus (MERS-CoV) has proteolytic, deubiquitinating, and deISGylating activities. The latter two are involved in the suppression of the antiviral innate immune response of the host cell. To contribute to an understanding of this process, we present here the X-ray crystal structure of a complex between MERS-CoV PL(pro) and human ubiquitin (Ub) that is devoid of any covalent linkage between the two proteins. Five regions of the PL(pro) bind to two areas of the Ub. The C-terminal five residues of Ub, RLRGG, are similar to the P5-P1 residues of the polyprotein substrates of the PL(pro) and are responsible for the major part of the interaction between the two macromolecules. Through sitedirected mutagenesis, we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PL(pro). The enzyme appears not to be optimized for catalytic efficiency; thus, replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities. Ubiquitin binding by MERS-CoV PL(pro) involves remarkable differences compared to the corresponding complex with SARS-CoV PL(pro). The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PL(pro).
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http://dx.doi.org/10.1007/s12250-016-3742-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090527PMC
August 2016

Production, crystallization and X-ray diffraction analysis of the protease CT441 from Chlamydia trachomatis.

Acta Crystallogr F Struct Biol Commun 2015 Dec 18;71(Pt 12):1454-8. Epub 2015 Nov 18.

Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.

The prokaryotic obligate intracellular pathogen Chlamydia trachomatis is the most prevalent cause of preventable blindness, affecting approximately six million people worldwide. In addition, C. trachomatis is the most commonly reported sexually transmitted pathogen in Europe and the US, causing pelvic inflammation, ectopic pregnancy and infertility. As in other intracellular pathogens, proteases play crucial roles during most stages of the complex life cycle of Chlamydia. CT441 is a chlamydial protease that has been reported to interfere with oestrogen signalling of the host cell. Here, the recombinant production, purification and crystallization of an inactive variant of CT441, designated CT441° (active-site Ser455 replaced by Ala), are described. CT441° was crystallized in space group P22121, with unit-cell parameters a = 86.7, b = 184.0, c = 209.6 Å. A complete diffraction data set was collected to a resolution of 2.95 Å.
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http://dx.doi.org/10.1107/S2053230X15020518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666471PMC
December 2015

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

Antimicrob Agents Chemother 2015 Dec 14;59(12):7782-5. Epub 2015 Sep 14.

KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium.

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.
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http://dx.doi.org/10.1128/AAC.01375-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4649165PMC
December 2015