WO2012159187A2 - Peptides dérivés du virus de la dengue denv et méthodes pour inhiber la réplication du flavivirus - Google Patents

Peptides dérivés du virus de la dengue denv et méthodes pour inhiber la réplication du flavivirus Download PDF

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WO2012159187A2
WO2012159187A2 PCT/BR2012/000162 BR2012000162W WO2012159187A2 WO 2012159187 A2 WO2012159187 A2 WO 2012159187A2 BR 2012000162 W BR2012000162 W BR 2012000162W WO 2012159187 A2 WO2012159187 A2 WO 2012159187A2
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protein
lds
denv
interaction
residues
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PCT/BR2012/000162
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WO2012159187A3 (fr
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Fabio Ceneviva Lacerda DE ALMEIDA
Ivo Cristiano DA ROCHA MARTINS
Andrea Thompson DA POIAN
Nuno Fernando Duarte Cordeiro Correia DOS SANTOS
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Universidade Federal Do Rio De Janeiro - Ufrj
Universidade De Lisboa
Instituto De Medicina Molecular
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the field of the viral replication processes, more particularly to the replication of the Flavivirus spp., in which the interaction of the viral C protein with biological membranes and/or RNA is necessary to the formation of new infective viral particles and to the spread of the virus in the host tissues and organs.
  • the invention provides methods for diminishing and/or avoiding viral particle formation via the use of peptide-based therapeutics.
  • Dengue virus (DENV) infection is one of the major causes of viral hemorrhagic fever in the world, with over 20,000 deaths, 500,000 hospitalizations and 500 million people newly infected every year (Beatty et al . , 2010) .
  • DHF dengue hemorrhagic fever
  • DSS dengue shock syndrome
  • DENV belongs to the Flavivirus genus, of the Flaviviridae family, together with other important human pathogens, such as yellow fever, West Nile, tick-borne encephalitis and Japanese encephalitis viruses (Kuhn et al .
  • Flaviviruses are icosahedral enveloped virus composed of a lipid bilayer surrounding a nucleocapsid, formed by a positive sense single stranded genomic RNA complexed with multiple copies of the C protein (Kuhn et al., 2002; Zhang et al . , 2003; Mukhopadhyay et al., 2005).
  • DENV C protein structure and surface charge distribution suggests that it plays a role in viral assembly and encapsidation via an hydrophobic ⁇ 2- ⁇ 2' interface, which may interact with lipid membranes, and a positively charged ⁇ 4- 4' C-terminal region, which is likely to strongly interact with the viral RNA (Ma et al . , 2004) .
  • LDs intracellular lipid droplets
  • this invention describes both an original identification of the role of the N-terminal in the interaction as well as the invention of the above-mentioned peptides, and the ability of these peptides to inhibit DENV and similar flaviviruses ability to interact with lipid droplets and other host lipid systems. It is also protected in the present invention the modification of the above mentioned peptides, whereby the introduction of cell penetrating peptide sequences to their N- and C-terminal renders them more efficient in dengue and other flaviviruses prophylactic or therapeutic approaches.
  • nucleic acid sequences codifying the peptides described above their insertion into expression vectors and transformation into a host cells; consequently, this application is also protected under the present claims.
  • Any pharmaceutical composition comprising the peptides as defined above, their codifying nucleic acid sequences, their expression vector containing the codifying nucleic acids, or other pharmaceutically acceptable carrier for the manufacture of a medicine for the prophylactic or therapeutic treatment of DENV or other Flavivirus infection is also protected here.
  • the present invention describes a Flaviviruses C protein- derived molecule :
  • a preferred embodiment of the present invention provides a peptide comprising N- and/or C-terminal segment.
  • the above mentioned peptide incorporates a Tat sequence and its variations with a disulfide bond between the cysteine residues at the N- or C-terminal segment.
  • An alternative embodiment of the present invention provides the above mentioned peptide incorporating a polyarginine sequence and its variations with a disulfide bond between cysteine residues at the N- or C-terminal segment.
  • Another embodiment of the present invention is a nucleic acid sequence that codifies the above mentioned peptide.
  • a preferred embodiment of the present invention is an expression vector comprising the nucleic acid sequence that codifies the peptide.
  • a host cell is transformed with the expression vector.
  • composition comprising a peptide, a nucleic acid sequence and/or an expression vector and a pharmaceutically acceptable carrier.
  • kits containing a peptide and/or nucleic acid sequence as described above for detection or diagnosis of DENV or other flaviviruses infection.
  • any peptidomimetic molecule including the strategy of employing peptides synthesized with D-amino acids for the prophylactic or therapeutic treatment of DENV or other Flavivirus infection.
  • Dengue virus a member of the Flavivirus genus, causes the most important human arbovirosis, for which no specific and effective treatment is currently available. This is partially due to the lack of knowledge on the basic molecular aspects of the viral life cycle. It has been however suggested that viral replication might depend on the ability of DENV C protein to interact with host intracellular lipid droplets (LDs) , in a somewhat similar fashion to what is observed for the Hepatitis C virus core protein (HCV) , a virus from the Hepacivirus spp. genus that also belongs to the Flaviviridade family.
  • LDs host intracellular lipid droplets
  • the HCV core protein shares no significant sequence or structural homology to the C protein from Flavivirus spp. However, a number of residues from its C-terminal section have been implicated in the interaction with LDs and, for this reason, the DENV C protein has been studied relating its ability to interact with LDs. It was found that a hydrophobic middle section of the protein structure, the ⁇ 2- ⁇ 2' region, is likely involved in this possible interaction. Nevertheless, this hypothesized interaction was never directly probed and the specific amino acids were never identified, which thus become the original focus of the present invention.
  • NMR analysis also showed that the LDs-C protein interaction occurs via specific residues located in the ⁇ 2- 2' region of the protein. Moreover, NMR also demonstrates that the protein N-terminal (residues 1-26) is intrinsically disordered. Surprisingly, residues within this intrinsically disordered section (residues 5-26) were shown not only to interact with lipid droplets, but also to take a major role in the interaction. Consistent with this finding, the BLAST analysis of DENV C protein against non- redundant proteins reveals an N-terminal conserved motif among flaviviruses .
  • the present invention contemplates the design of two innovative peptides based on these two regions of the C protein (pepl4-23 and pep5-26) .
  • pepl4-23 was able to substantially decrease the C protein interaction with lipid droplets .
  • the present invention also includes the characteristics that enable the in vitro viral load in hepatic models of dengue decrease, as a result of the cell incubation with pepl4-23.
  • the referred peptide and improved versions regarding the LDs affinity, stability and/or biological effect
  • Table 1 a list of different candidate peptide sequence alternatives able to interfere with the LDs-C protein interaction are hereby described.
  • Table 1 List of peptide sequences derived from pep!4
  • Sequences Pep_01 to Pep_20 are based on Flavivirus spp. conserved sequences homologous to the pepl4-23 (which corresponds to Pep_01)
  • Sequences Pep_21 to Pep_30 are based on the inclusion of a domain to facilitate the entrance into the cell to the N- and C-terminal of DENY and W V pepl4-23 homologous sequences.
  • Sequences Pep_31 to Pep_34 are based on the inclusion of domain to facilitate the entrance into the cell to the N- and C-terminal of shorter versions of the pepl4-23.
  • Sequences Pep_35 to Pep_52 are based on pepl4-23 shorter versions including arginines in the C and N terminus of the peptide to facilitate the entrance into the cell to the N- and C-terminal.
  • Sequences Pep_53 to Pep_56 are based on pepl4-23 and incorporate a Tat sequence with several variations where "-S-S-" or "S- blind-” represent a disulfide bond between the cystein residues (without a peptide bond) .
  • FIG. 1 The C protein N- terminal and 2- 2' residues are affected by the LDs-C protein interaction.
  • Panel A Superimposed HSQC spectra of 15 N- labelled DENV C protein with and without LDs .
  • Top graph full spectrum of the protein with and without LDs; 2 nd graph from the top: highlight on Gly40, located in an intermediate loop, which suffered a significant CSP; 3 rd graph from the top: highlight on amino acids 53 and 54, located in a-helix 2, which show a significant change in CSP; 4 th graph from the top: highlight on residues 5, 18 and 22, belonging to the unstructured N- terminal, which showed a significant CSP; 4 th graph from the top: highlight on residues 79, 80 and 98, that are part of a-helix 4, on residue 68, that is part of a-helix 3, and on residue 28, that is part of a-helix 1, all of which are a-helical sections of the protein that do not suffer any significant CSP.
  • Panel B NMR shows that specific residues are affected by the interaction with LDs.
  • Left graph CSP changes as a result of an interaction of the C protein with LDs; right graph: peak intensities ratio of bond/free C protein.
  • Residues for which the CSP changes were lower than 0.089 ppm i.e., the background level of chemical change cut-off (which is equal to the average CSP of 0.050 plus one half of the standard deviation of 0.078) are considered to have suffered a significant change in CSP and to be involved in the interaction.
  • Panel C Residues that show a significant change in CSP are highlighted (by showing their side chains) within the DENV C structure in two alternate views (DENV C protein strain 2, NMR ribbon structure) . It is clear that the N- terminal section of the protein is altered upon the DENV C protein interaction with LDs.
  • Figure 2 C protein residues involved in the LDs-C protein interaction are conserved among Flavivirus spp. Study of conservation of the C protein sequence and structure, when compared to similar flaviviruses in the context of the NMR findings .
  • Panel A Alignment of the DENV C protein sequence with similar flaviviruses , showing a consensus sequence.
  • Panel B Comparison of WNV (dark grey) and DENV (light grey) C protein structures, showing a highly conserved region (a-helix 2, 3 and 4) and a more flexible section (a- helix 1) . It is immediately clear that there are two regions of the protein, one being a core hydrophobic conserved fold (from residues 44 to the C- terminal) and the other a particularly flexible region, with alternative folds, and composed of the unstructured section (from residues 1 to 26) and the a-helix 1.
  • Sections involved in the interaction according to the NMR analysis i.e., residues for which a significant CSP was seen
  • residues for which a significant CSP was seen are highlighted by showing their side chains in the DENV C protein and contain amino acids belonging to the N-terminal unstructured region, the flexible loop and the a2 region of the DENV C protein.
  • the residues affected by the DENV C protein-LDs interaction are located within both regions, primarily in the a-helix 2 section of the structurally conserved core hydrophobic fold region and in the unstructured section of the flexible alternative folds region.
  • Figure 3 pepl4-23 peptide interacts with LDs as strongly as the C protein.
  • pepl4-23 interacts with LDs via specific conserved residues, (a) Sections of the 13 C- 1 H HSQC spectra of pepl4-23, in the presence (grey) and absence (black) of LDs, showing the Ha/Ca paired resonances (left) and side- chain ⁇ / ⁇ paired resonances (right) . (b) CSP changes for each pepl4-23 residue as a result of the interaction with LDs .
  • FIG. 5 DENV C protein binding to LDs is inhibited by pepl4-23.
  • Each histogram was constructed with the results of approximately 5000 single rupture-force measurements, done with an applied force of 0.2 nN, pulling speed of 2 ⁇ /s and loading rate of 4 nN/s.
  • FIG. 6 DENV C protein residues involved in the interaction with LDs are in the disordered N- terminal region and in a-helix 2.
  • Figure 7 Analysis of the backbone dynamics of DENV C protein.
  • FIG. 8 C protein residues involved in the LD interaction are conserved among Flavivirus spp.
  • Figure 9 Zeta potential analysis of DENV C protein and pepl4-23 interaction with LDs. Zeta potential measurements of the titration of LDs samples (filled symbols) , or LDs samples after limited proteolysis (empty symbol) were performed for DENV C protein (squares) , pepl4-23 (circles) , or pep5-26 (triangles) .
  • Figure 10 ( 15 N, 1 H) -HSQC spectra of DENV C protein in the presence and absence of LDs .
  • the data indicates that the overall protein structure is maintained in the presence of LDs and similar to the overall structure in the presence of LDs .
  • Figure 11 ( 13 C, 1 H) -HSQC spectra of pep5-26 in the presence and absence of lipid droplets. Ca region (top) , C region (middle) and methyl region (bottom) of the spectra. pep5-26 does not interact with LDs. Sections of the 13 C- 1 H HSQC spectra of pep5-26, in the presence and absence of LDs. Ha/Ca paired resonances in three different regions of the ( 13 C, 1 H) -HSQC spectra do not show significant chemical shift changes, reinforcing the previous conclusion of the absence of pep5-26-LD binding.
  • NLKRARNRV NLKRARNRV
  • RKKTGRPSFNMLKRARNRVSTV RKKTGRPSFNMLKRARNRVSTV
  • 15 N labelled ammonium chloride, 13 C-glucose and deuterated water were ordered from Cambridge Isotopes (USA) .
  • Protease cocktail- inhibitor was obtained from Roche Diagnostics GmBH (Germany) . All other chemicals were obtained commercially from Sigma-Aldrich.
  • C protein of DENV serotype 2 New Guinea strain was expressed and purified following established procedures (Jones et al . , 2003; Ma et al . , 2004). Briefly, DENV recombinant C protein was expressed in Escherichia coli BL21-DE2 Codon Plus cells transformed with C protein gene (encoding residues 1-100) cloned into pET21a plasmid. Cells were grown for 6 h in LB medium at 37 °C and shaking at 200 rpm in the presence of antibiotics (100 mg/mL ampicilin and 34 mg/mL chloramphenicol) .
  • One fifth of the culture was transferred to freshly prepared LB medium with the same antibiotics at 18 °C, with overnight recombinant protein expression being induced by the addition of 1 mM isopropyl beta-D-1-thiogagactopyranoside (IPTG) when the cell culture reached 0.8-1.0 OD 6 oonm- Cells were then centrifuged at 7000 x g for 20 min at 4°C and the supernatant rejected.
  • IPTG isopropyl beta-D-1-thiogagactopyranoside
  • the cell pellet was resuspended in minimum volume of buffer A (25 mM HEPES pH 7.4, 200 mM NaCl , 1 mM EDTA, 5% glycerol) and 10 mM protease inhibitor mix (PMSF, pepstatin, leupeptin, E64 and bestatin) .
  • buffer A 25 mM HEPES pH 7.4, 200 mM NaCl , 1 mM EDTA, 5% glycerol
  • 10 mM protease inhibitor mix PMSF, pepstatin, leupeptin, E64 and bestatin
  • the pellets were resuspended with buffer A, combined and injected into the column MonoS 10 x 100 GL (GE Healthcare) coupled to an Akta Purifier System (GE healthcare) at 0.5 mL/min flow.
  • the DENV C protein was eluted with increasing NaCl concentration (0.1 - 2M) gradient at 2 mL/min flow.
  • the 3-mL fractions containing the DENV C protein were confirmed by 18% SDS-PAGE.
  • the fractions were then pooled and dialyzed against 50 mM NaH 2 P0 4 , 1 M NaCl, pH 6.
  • the dialysis step was repeated 3 times with decreased NaCl concentration (0.5 M and 0.2 M) .
  • DENV C protein was concentrated with a centriprep (cut-off 3000 Da, Millipore) and stored at 80 °C or used immediately.
  • 15 N-uniformly- labelled protein was obtained in a similar manner, except that minimum medium was employed with 15 N labelled ammonium choride as the sole source of nitrogen. Following, the protein was concentrated using a centriprep device with 3000 kDa cut-off, spinning at 3000 x g, 4°C. The concentrated sample was then applied on an 18% SDS-PAGE and confirmed to be over 95% pure. Purification and storage of LPs from BHK cells
  • Baby Hamster Kidney cells (BHK-21) were maintained in Minimum Essential Medium alpha (a-MEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 g/ml streptomycin, at 37°C in a humidified 5% carbon dioxide incubator.
  • a-MEM Minimum Essential Medium alpha
  • BHK cells were treated with 10 mM oleic acid for 24 h. After this, cells were washed twice and resuspended in TEE buffer (20 mM Tris-HCl, 100 mM KCl, 1 mM EDTA and 1 mM EGTA, pH 7.4) in the presence of a protease cocktail- inhibitor .
  • NMR experiments were performed on a Bruker Avance III 800 (Rheinstetten, Germany) spectrometer operating at 800.13 MHz, equipped with triple-resonance ( ⁇ ⁇ , 13 C, 15 N) probes and shielded z-gradients. NMR experiments were acquired at 300 K. LDs were at an approximate concentration of 4 x 10 5 /mL in TEE buffer.
  • Sequential backbone resonance assignment was performed from a 1 H, 13 C HSQC experiment using intra-residual and sequential connectivity NOESY and TOCSY experiments acquired in 10% D20.
  • TOCSY spectrum using MLEV spin-lock time of 60 ms
  • NOESY spectra mixed time of 100 and 200 ms
  • Quadrature detection in Fl was done by States TTPI. Water suppression was achieved using the 3-9-19 WATERGATE sequence (Piotto et al., 1992).
  • Gradient selection 1 H, 13 C HSQC spectra were acquired at 800.13 MHz with 1024 complex points in F2 and 128 complex points in Fl .
  • Quadrature detection in Fl was done by echo-anti-echo.
  • DENV C protein sequence from strain New Guinea was aligned with other flaviviruses C proteins on the Basic Local Alignment Search Tool for proteins (BLASTp) from the National Center for Biotechnology Information (NCBI) (Altschul et al., 1990; Mount 2007). The search was performed against non-redundant protein sequences and excluding DENV group (TaxID: 11052). Only Flavivirus spp. gave a score high enough to be considered for further analysis. The best scored sequence of each different strain of virus was then selected.
  • BLASTp Basic Local Alignment Search Tool for proteins
  • NCBI National Center for Biotechnology Information
  • the DENV C protein determined by NMR spectroscopic analysis (PDB ID: 1R6R) (Ma et al . , 2004) and the West Nile Virus, strain Kunjin (WNV-K) C protein, determined via X-ray crystallography (PDB ID: 1SFK) (Dokland et al . , 2004).
  • DENV C protein model number 21 of the 1R6R structure file is the energy minimized averaged structure of the 53 NMR models obtained, and was employed in the superimposition plotted.
  • model number 2 was selected and shown in the images depicted, given that it presented the lowest Root Mean Squared Deviations (RMSD) of the a-carbons of the amino acids when superimposed with the selected DENV C protein NMR structure and analyzed via the Swiss- PdbViewer software package (Guex and Peitsch 1997) .
  • RMSD Root Mean Squared Deviations
  • the LDs interaction with the DENV C protein was analyzed by determining the zeta potential of LDs, at 25 °C, from the mean of 15 measurements (120 runs each) , in the absence and in the presence of different concentrations of DENV C protein, by phase analysis light scattering (PALS) , using disposable zeta cells with platinum gold-coated electrodes (Malvern) .
  • PALS phase analysis light scattering
  • a NanoWizard II atomic force microscope (JPK Instruments, Berlin, Germany) mounted on the top of an Axiovert 200 inverted optical microscope (Zeiss, Jena, Germany) was used for force spectroscopy experiments .
  • the AFM head is equipped with a 15-ym z-range linearized piezoelectric scanner and an infrared laser.
  • 50 ⁇ L of LDs suspension were placed on thin freshly cleaved muscovite mica and allowed to deposit for 30 min at room temperature.
  • Non-adherent LDs were removed by 5 sequential washes with TEE buffer.
  • the sample was loaded onto the AFM and allowed to equilibrate in TEE buffer for 10 min before measurements. Force spectroscopy measurements were performed using DENV C protein-functionalized in OMCL TR- 400-type silicon nitride tips (Olympus, Japan) .
  • tips were cleaned with an intense ultraviolet light source and silanized in a vacuum chamber with 3 -aminopropyl-triethoxysilane (APTES, 30 yL) and N, Ndiisopropylethylamine (10 L) for 1 h in an argon atmosphere, to be coated with a self-assembled monolayer of amines.
  • APTES 3 -aminopropyl-triethoxysilane
  • N, Ndiisopropylethylamine (10 L) for 1 h in an argon atmosphere, to be coated with a self-assembled monolayer of amines.
  • the probes were rinsed with fresh chloroform and dried in nitrogen gas .
  • the amine terminated AFM probes were then placed in glutaraldehyde solution 2.5% (v/v) for 20 rain and washed 3 times with TEE buffer pH 7.4.
  • the tips were placed during 30 min in a 167 ⁇ DENV C protein solution to covalently attach the protein
  • the softest triangular cantilevers with a tip radius of 15 nm and a resonant frequency of 11 kHz, were used.
  • the maximum values of the Gaussian peaks represent a single-molecule-based statistical measure of the strength of the molecular bond. For all measurements, either TEE buffer or pepl4-23 peptide in TEE buffer was then added to make the appropriate concentration of peptide.
  • CSP chemical shift perturbation index
  • This loop is located immediately before the a-helix 2 and may thus facilitate the interaction by providing flexibility to the a-helix 1 and the unstructured N-terminal region.
  • Table 2 summarizes the major changes in CSP and in peak intensity and identifies the residues of the DE V C protein likely involved in the interaction with LDs. C protein residues involved in the LDs interaction are located within conserved regions .
  • Phe53 is highly conserved as well, being part of the ⁇ 2- ⁇ 2' hydrophobic patch that interacts with LDs (Val51, Ala52, Phe53 and Leu54) , which, in turn, is part of a hAFL/F conserved sequence.
  • the residues affected by the DENV C protein-LDs interaction are located within both regions, primarily in the a- helix 2 section of the structurally conserved core hydrophobic fold region (residues Val51, Ala52, Phe53 and Leu54) and in the unstructured section of the flexible alternative folds region (residues Arg5, Lys7, Ala8 , Arg9, Phel3, Asnl4 , Metl5, Leul6, Argl8, Asn21, Arg22 and Thr25) .
  • pep5-26 corresponding to the sequence comprising residues 5 to 26, and pepl4-23, corresponding to residues 14 to 23.
  • residue Glul9 was modified to Ala, to make it more similar to the sequence of C protein of all other DENV strains as well as of all other flaviviruses , in which neutral amino acids are present in this position.
  • Pep5-26 includes all N-terminal amino acids of the protein affected by the interaction with LDs, starting in residue 5 and ending just before the start of a-helix 1, in residue 26.
  • pepl4-23 is a shorter version of pep5-26 that excludes the N-terminal residues located before the conserved motif NML+R and includes that motif plus all the following residues that are affected by the interaction with LDs before the start of a helix 1, i.e., up to residue 23.
  • pepl4-23 To map the interaction of pepl4-23 with LDs, we first assigned pepl4-23 using sequential assignment strategy (Wagner et al . , 1986) with a combination of TOCSY and NOESY spectra. We also used ⁇ , 13 C HSQC spectra to solve ambiguities and to assign the 13 C resonances. It was possible to unambiguously and fully assign Met2, Leu3 , Lys4, Ala6 and VallO . Probably due to line broadening, the amide resonance of Met2 and the N-terminal Asnl could not be found. Both Asnl and Asn8 were assigned, although they could not be unambiguously differentiated.
  • atomic force microscopy AFM -based force spectroscopy was used. LDs were lightly adsorbed to the mica surface, in buffer.
  • Tapping on LDs with DENV C protein-derivatized AFM tips allowed to measure the frequency of C-protein-LDs binding events (by the subsequent unbinding) , as well as to measure the force necessary to break the bond between a single C protein and a LD.
  • the frequency of (un) binding and the unbinding forces of DENV C protein-LDs interaction were determined in the absence of pepl4-23 and in the presence of different concentrations of this peptide ( Figure 5) . It was found that C protein-LDs interaction is blocked by the addition of the peptide, in a concentration-dependent manner.
  • the force necessary to break the binding decreases from 33 pN in the absence of the inhibitor peptide (Carvalho et al . , 2012) to 19 pN in the presence of 100 ⁇ pepl4-23.
  • Flavivirus spp C protein interacts with LDs via specific residues.
  • the unstructured flexible region may prompt an initial interaction with the negatively charged LDs, which may eventually facilitate further interactions between the hydrophobic core of the protein (residues 50-54 that are part of a hAFL/F conserved sequence) and hydrophobic sections of LDs.
  • the glycines of the flexible loop between a-helices 1 and 2 are affected by an interaction with LDs because, given their high conformational freedom to rotate, they may readjust in order to enable the access by the ligand (LDs) to the protein final interaction site (residues 50-54) .
  • This may facilitate the DENV C protein interaction with LDs and is certainly of special importance in the case of NV, where the a-helix 1 partially blocks the access to the a2- a2' C protein hydrophobic core.
  • zeta potential analysis allows to monitor the changes in the surface charge density of particles such as LDs as a result of an interaction with small ligands, such as the rationally designed peptides or the full C protein, without any further modification of the ligand being necessary (such as the addition of fluorophores , which could dramatically affect the ligand overall hydrophobicity and membrane affinity, especially in the case of short peptides as those studied) .
  • LDs are negatively charged, with zeta potential values in the vicinity of -14 mV. Testing the interaction of LDs with the rationally designed peptides showed that pep5-26 does not interact with LDs.
  • pepl4-23 (the shorter rationally designed peptide) interacts extensively with LDs, to the same extent as the full protein in which it is based on (reaching a zeta potential value of roughly +20 mV at higher concentrations, the same value that is observed for the full-length C protein) .
  • NML+R may become more tightly bound due to the contribution of hydrophobic interactions, which may also occur in the full length protein and, in this manner, anchor the C protein to the LDs and promote further interaction with the hydrophobic 2- ⁇ 2' core.
  • pepl4-23 inhibits the interaction of the C protein with LDs.
  • the AFM data clearly demonstrated the ability of pepl4-23 to significantly inhibit the interaction between DENV C protein and LDs.
  • AFM-based force spectroscopy was employed (Figure 5) . Based on a previously developed methodology (Carvalho et al .
  • the flexible unstructured N-terminal domain is crucial for the C protein of DENV (and possibly of other flaviviruses) key interaction with LDs.
  • the novel designed pepl4-23 is therefore an inhibitor peptide that can now be employed in new therapeutic strategies to treat DENV infection, by targeting a key step of the viral life cycle involving the C protein.
  • Other drug development strategies can be used to increase the efficiency of the present molecule against DENV, by small changes on its sequence.
  • an identical approach can be used to yield successful inhibitors to treat the infection by other flaviviruses , such as the West Nile virus.
  • NLKRARNRV NLKRARNRV
  • RKKTGRPSFNMLKRARNRVSTV RKKTGRPSFNMLKRARNRVSTV
  • the C protein of DENV serotype 2 New Guinea strain was expressed and purified following established procedures (Jones et al . , 2003; Carvalho et al . , 2012), except that M9 minimal medium supplemented with 15 N-NH 4 C1 was used for expression of 15 N- labeled C protein.
  • Baby Hamster Kidney cells (BHK-21) were maintained in Minimum Essential Medium alpha (a-MEM) supplemented with 10% fetal bovine serum, at 37°C, in 5% C0 2 . At 80% confluence, cells were treated with 10 ⁇ oleic acid for 24 h. LDs were isolated by nitrogen cavitation (Parr Instrument Company, model 4639, Moline, IL) and purified by sucrose gradient ultracentrifugation, as described elsewhere (Carvalho et al . , 2012) . To ensure LDs purity, only fractions negative for lactate dehydrogenase activity were used. LDs samples were kept at 4°C and used during two weeks .
  • a-MEM Minimum Essential Medium alpha
  • NMR experiments were performed at 300 K in Bruker Avance III 800 MHz and 600 MHz (Rheinstetten, Germany) , equipped with triple-resonance ( 1 H, 13 C, 15 N) probes.
  • C protein 200 L in 50 mM phosphate, 0.2 M NaCl buffer, pH 6.0, were added to 125 L of LDs ( ⁇ 4 x 105/mL) in TEE buffer (25 mM Tris HC1, 1 mM EDTA, 1 mM EGTA, pH 7.4), or to TEE buffer alone, to obtain a final concentration of uniformly- labeled 15 N-DENV C protein between 250 and 275 ⁇ .
  • Sequential backbone resonance assignment was performed for free peptides using 1 H, 13 C gradient selection HSQC (natural abundance of 13 C) , NOESYs (100 and 200 ms mixing time) and TOCSY (60 ms MLEV) in 10% D 2 0, acquired at 800.13 MHz, with 4096 x 512 complex points for NOESY and TOCSY, and 1024 x 128 for HSQC.
  • Quadrature detection in indirect dimension was done using States-TPPI (3 ⁇ 4) , for NOESY and TOCSY and echo-antiecho ( 13 C) for the HSQC. Water suppression was achieved using the 3-9-19 WATERGATE sequence for NOESY and TOCSY (Piotto et al . , 1992).
  • the C protein aligned sequence was in the first -110 amino acid residues.
  • the residues next to the NS2B-NS3 protease cleavage site were excluded from further analysis in all polyproteins , leaving the presumed C protein sequences, which were aligned by multiple alignments on the Clustal W2 tool of the European Bioinformatics Institute web server (Chenna et al . , 2003) against the DENV C protein sequence. Structural comparison of DENV and WNV-K C proteins
  • UCSF Chimera software package (Pettersen et al . , 2004) was used to superimpose DENV C protein model number 21 (PDB ID: 1R6R) (Ma et al . , 2004), which is the energy minimized averaged structure of the 53 NMR models obtained, with WNV- K C protein model number 2 (PDB ID: 1SFK) (Dokland et al . , 2004) , which presented the lowest Root Mean Squared Deviations (RMSD) of the a-carbons of the amino acids when superimposed with the selected DENV C protein structure and analyzed using Swiss-PdbViewer software package (Guex & Peitsch, 1997) .
  • RMSD Root Mean Squared Deviations
  • u is the electrophoretic mobility
  • ⁇ the viscosity of the solvent and ⁇ its dielectric constant.
  • LD samples were previously incubated with 10 ⁇ trypsin for 15 min at room temperature, as previously described Carvalho et al . , 2012). The reaction was stopped by the addition of 1 mM PMSF to the mixture. After 5 min of incubation with PMSF, pepl4-23 was added and the zeta potential measurements were carried out .
  • a NanoWizard II atomic force microscope JPK Instruments, Berlin, Germany mounted on an Axiovert 200 inverted optical microscope (Zeiss, Jena, Germany) , equipped with a 15- ⁇ z-range linearized piezoelectric scanner and an infrared laser, was used following standard procedures (Carvalho et al . , 2010), recently adapted to the probing of DENV C protein-LD interaction (Carvalho et al . , 2012). Briefly, 50 pL of LD suspension were allowed to deposit for 30 min at room temperature on a mica surface. Non-adherent LDs were removed by 5 washes with TEE buffer.
  • Force spectroscopy measurements were performed using OMCL TR-400- type silicon nitride tips (Olympus, Japan) functionalized with C protein. Molecular recognition was searched by pressing the DENV C protein- functionalized tips on different points of the LDs in the presence of different pepl4-23 concentrations. Force spectroscopy curves were analyzed using the JPK image processing v.3. Each experiment was performed at least three times, using different samples and different functionalized tips, with approximately 5000 force-distance curves collected, analyzed and fitted to the worm- like-chain model (WLC) (Carvalho et al . , 2012).
  • WLC worm- like-chain model
  • Histograms of the (un)binding forces of each data set were constructed choosing the ideal bin size to achieve the best fit (6 pN) . Force rupture values below 10 pN were considered to represent noise, artifacts or unspecific interactions. From each histogram, the most likely single DENV C protein-LD binding rupture force was determined fitting the distributions of the rupture forces with the Gaussian model. The maximum values of the Gaussian peaks represent a single-molecule-based statistical measure of the strength of the molecular bond.
  • the N-terminal region of the DENV C protein interacts with LDs
  • residues located elsewhere were almost not affected by the presence of LDs, as exemplified for residues within a-helix 1 (Gln28), a-helix 3 (Arg68), - helix 4 (Asn79 and Val80) or in the C-terminal (Arg98) .
  • Peak intensity change upon binding is also a good reporter for measuring protein recognition (De Paula et al . , 2011).
  • the intensities ratios between LD-bound and free (ILD/ I O ) C protein residues are shown in Figure 6f. For most of the residues, I LD /I 0 was slightly less than 1, as expected for non- interacting regions of a protein complex.
  • Residues in conformational exchange show low peak intensities due to the line broadening caused by the exchange contribution to transverse relaxation rate. Binding stabilizes one conformation, thus leading to an increase in peak intensity.
  • the results showed that a significant increase in ILD/ I O was observed for regions that are prone to be in conformational exchange, such as the N-terminal region and the ⁇ 2- ⁇ 2' dimer interface.
  • DENV C protein displays intrinsically disordered behavior for its N-terminal region
  • the central hydrophobic patch at a-helix 2 has previously been assigned to participate in C protein interaction with membranes (Ma et al . , 2004; Markoff et al . , 1997) and LDs (Samsa et al . , 2009) .
  • loop Ll-2 and the previously unstudied N-terminal region also play a role in C protein binding to LDs.
  • Talos prediction of secondary structure indicates the presence of one turn of a helix within these segments, comprising residues Gly36, Met37 and Leu38 (Figure 7c) .
  • Analysis of DENV C protein structure in solution (PDB ID:1R6R) corroborates the presence of the one-turn-helix involving these residues. Indeed, for this segment, a S2 value above 0.8 was found ( Figure 7b) . Additionally, reduced order parameters, but not as pronounced as for LI-2 loop, were observed for the L2-3 and L3-4 loops ( Figure 7b) . It is important to mention that the assignments of Leu35 and Arg55 were missing in the previously determined C protein structure (Ma et al . , 2004), possibly due to conformational exchange, which is interesting since Arg55 is within the central hydrophobic patch that is also involved in the binding to LDs .
  • a peptide corresponding to C protein conserved N-terminal motif binds to LDs
  • pep5-26 includes all N-terminal residues affected by the interaction with LDs, starting in residue 5 and ending just before the beginning of the a-helix 1, in residue 26.
  • pepl4-23 is a shorter version of pep5-26 that excludes the N- terminal residues and the hydrophobic segment that showed high order parameter.
  • pepl4-23 includes the conserved motif NML+R, beginning in the first residue of the conserved motif (Asnl4) and extending to the last residue affected by LD interaction, just before the beginning of a-helix 1.
  • NML+ is the LD-binding motif of pep!4-23
  • DENV C protein is known to interact with LDs and this association was shown to be mandatory for virus particle formation (Samsa et al . , 2009). Taking into account the biological relevance of that interaction, we investigated the determinant structural features of C protein for the binding to LDs . Local structural changes involving specific amino acids residues could be mapped in the disordered N- terminal region, in the central hydrophobic patch and in the LI -2 loop.
  • Intrinsically disordered proteins or regions undergo equilibrium among several conformational states (Dunker et al . , 2008; Turoverov et al . , 2010).
  • the pre-existent conformational states allow recognition of different targets. Binding to each of the targets, in turn, leads to conformational selection (James & Tawfik, 2003; Valente et al . , 2006; Henzler- Wildman & Kern, 2007) .
  • conformational selection as a recognition mechanism in C protein-LD interaction is supported by the increase in peak intensity ratios.
  • Conformational selection may offer an evolutionary advantage for multif nctional proteins, such as the C proteins of flaviviruses .
  • RNA binding Kromykh & Westaway, 1996; Majeau et al . , 2004
  • RNA chaperoning Ivanyi-Nagy et al . , 2008
  • RNA binding Kromykh & Westaway, 1996; Majeau et al . , 2004
  • RNA chaperoning Ivanyi-Nagy et al . , 2008
  • a nuclear localization signal has already been identified in the disordered N-terminal region, but mutations in this region had partial effects on nuclear migration (Sangiambut et al., 2008).
  • antibodies produced against DENV C protein presented specific reactivity to the disordered N-terminal region, suggesting that it corresponds to the predominant immunogenic segment (Puttikhunt et al . , 2009).
  • the high immunoreactivity of C protein N-terminal region may be related to the intrinsically disordered nature of this segment, enabling its interaction with multiple targets, including LDs, as shown here.
  • the NS5A from HCV also a member of Flaviviridae family
  • HCV also a member of Flaviviridae family
  • NS5A from HCV contains a relatively similar sequence (299YMLPKRR304 ) , further supporting the role of the NML+R sequence as a LD-binding motif.
  • the disordered region rich in positively-charged residues, containing the NML+ motif
  • the disordered region might prompt an initial interaction with the negatively charged LDs, after which a conformational rearrangement facilitated by the conformational freedom provided by the flexible Ll-2 loop enables the access of LDs to the later specific interactions with central ⁇ 2- ⁇ 2' hydrophobic dimer interface.
  • the increase in peak intensity observed for a2 -helix residues Ala52 and Phe53 corroborates the stabilization of binding through this region.
  • the data presented here show that the ⁇ 2- ⁇ 2' interface is in conformation exchange in the free state, possibly interconverting between an open and a close states.
  • residue Arg55 is missing in the previous assignment of DENV C protein structure (Ma et al . , 2004), and residues in l and a2 helices showed consistently lower peak intensities than those of a3 and a4 helices, indicating line broadening due to conformational exchange.
  • This facilitates the DENV C protein interaction with LDs and may have special importance in the case of NV-K, in which the a-helix 1 partially blocks the access to the ⁇ 2- ⁇ 2' hydrophobic core (Dokland et al . , 2004).
  • the disordered N-terminal residues are the key to initiate the interaction with LDs.
  • the NML+R residues may become more tightly bound, due to the contribution of hydrophobic interactions, which may also occur in the full length protein and, in this way, anchor the C protein to the LDs, allowing further interaction of the hydrophobic ⁇ 2- ⁇ 2' core.
  • both zeta potential and NMR data showed that pep5-26 does not interact with LDs. This result is similar to those found for other peptide- ligand interactions, in which shorter sequences were more effective in eliciting a response than longer versions (Maecker et al . , 2001). The longer size of this peptide may render the NML+R sequence inaccessible to LD binding.
  • pep5-26 The inaccessibility of the NML+R sequence in pep5-26 may result from the presence of the hydrophobic triad (Thrll-Prol2- Phel3) that displays high order parameter, which may impose some conformational constrain.
  • pepl4-23 comprises only the low order parameter segment. This binding behavior is another evidence of the intrinsically disordered nature of C protein N- terminal region.
  • Therapeutic approaches using peptides and peptide analogues were effective against other viruses, such as HIV (Lalezari et al . , 2003; Welch et al . , 2010) and HCV (Poordad et al . , 2011), a strategy that can also be followed for DENV and similar flaviviruses .
  • NMRPipe a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3): 277-93.
  • a TALOS+ a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44, 213- 223.
  • Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins .

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Abstract

Le virus de la dengue (DENV) est un flavivirus qui est à l'origine de l'arbovirose humaine la plus répandue, pour laquelle il n'existe pas de traitement spécifique. La protéine C, la protéine capside virale, est une protéine hélicoïdale homodimère symétrique qui interagit avec des gouttelettes lipidiques (LD) intracellulaires pendant la réplication virale, éventuellement par l'intermédiaire d'un patch central non polaire α2-α2' que l'on suppose interagir avec des lipides. Les sites de liaison de la protéine C aux LD ont été identifiés, ce qui a permis de révéler une nouvelle fonction pour un segment conservé dans la région désordonnée N-terminal, et d'indiquer qu'une sélection de conformation est impliquée dans la reconnaissance. Les résultats obtenus ont montré que la zone N-terminal chargée positivement de la protéine C sollicite l'interaction avec des LD chargées négativement, interaction à la suite de laquelle un réarrangement conformationnel permet au patch hydrophobe central d'accéder à la surface des LD. En somme, les résultats ont permis de concevoir un peptide ayant pour action d'inhiber la liaison protéine C - LD et de jeter les bases de nouvelles approches de développement de médicaments contre la dengue. La capacité de ce peptide à se lier aux LD de la même manière qu'à une protéine C a été démontrée au moyen de différentes techniques, comprenant entre autres la résonance magnétique nucléaire, l'analyse de potentiel zêta et la microscopie à force atomique. En outre, les peptides nouvellement conçus ont été testés concernant leur capacité à inhiber la liaison de la protéine C du virus de la dengue (DENV) aux LD, puisque l'inhibition effective de cette interaction a été mis en évidence, une découverte présentant une applicabilité directe pour la dengue et les pathologies liées à ce Flavivirus.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998031814A1 (fr) * 1997-01-15 1998-07-23 Centro De Ingenieria Genetica Y Biotecnologia (Cigb) Epitopes de la proteine pre-m/m du virus de la dengue, peptides synthetiques
US6017535A (en) * 1992-04-29 2000-01-25 Insititute Of Molecular And Cell Biology cDNA sequence of Dengue virus serotype 1 (Singapore strain)

Patent Citations (2)

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US6017535A (en) * 1992-04-29 2000-01-25 Insititute Of Molecular And Cell Biology cDNA sequence of Dengue virus serotype 1 (Singapore strain)
WO1998031814A1 (fr) * 1997-01-15 1998-07-23 Centro De Ingenieria Genetica Y Biotecnologia (Cigb) Epitopes de la proteine pre-m/m du virus de la dengue, peptides synthetiques

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MA, L. ET AL.: 'Solution structure of dengue virus capsid protein reveals another fold.' PNAS vol. 101, no. 10, 2004, pages 3414 - 3419 *
MARTINS, IC ET AL.: 'The disordered N-terminal region of dengue virus capsid protein contains a lipid-droplet-binding motif.' BIOCHEM. J. vol. 444, June 2012, pages 405 - 415 *
PUTTIKHUNT, C. ET AL.: 'Production and characterization of anti- dengue capsid antibodies suggesting the N terminus region covering the first 20 amino acids of dengue virus capsid protein is predominantly immunogenic in mice.' ARCH VIROL vol. 154, 2009, pages 1211 - 1221 *
SAMSA, MM. ET AL.: 'Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Formation.' PLOS PATHOGENS vol. 5, 2009, page E1000632. *
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