Classifications

• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • 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
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • 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
  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
  • C12N2760/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• Influenza virus is a major human health problem. It causes a highly contagious acute respiratory illness known as influenza.
• the 1918-1919 pandemic of the "Spanish influenza” was estimated to cause about 500 million cases resulting in 20 million deaths worldwide (Robbins, 1986) .
• the genetic determinants of the virulence of the 1918 virus have still not been identified, nor have the specific clinical preventatives or treatments that would be effective against such a re-emergence. See, Tumpey, et al . , PNAS USA 99(15) :13849-54 (2002).
• influenza virus infection causes some 20,000-30,000 deaths per year in the United States alone (Wright & Webster, (2001) Orthomyxoviruses. In “Fields Virology, 4th Edition” (D. M. Knipe, and P. M. Howley, Eds.) pp. 1533-1579. Lippincott Williams & Wilkins, Philadelphia, PA).
• influenza A virus undergoes continual antigenic change resulting in the isolation of ne , strains each year. Plainly, there is a continuing need for new classes of influenza antiviral agents.
• Influenza viruses are the only members of the orthomyxoviridae family, and are classified into three distinct types (A, B, and C) , based on antigenic differences between their nucleoprotein (NP) and matrix (M) protein (Pereira, (1969) Progr. Molec. Virol . 11:46) .
• the orthomyxoviruses are enveloped animal viruses of approximately 100 nm in diameter.
• the influenza virions consist of an internal ribonucleoprotein core (a helical nucleocapsid) containing a single-stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (M) .
• the segmented genome of influenza A virus consists of eight molecules (seven for influenza C virus) of linear, negative polarity, single-stranded RNAs which encode ten polypeptides, including: the RNA-directed RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the matrix proteins (Ml, M2) ; two surface glycoproteins which project from the lipoprotein envelope: hemagglutinin (HA) and neuraminidase (NA) ; and nonstructural proteins whose function is elucidated below (NS1 and NS2) . Transcription and replication of the genome takes place in the nucleus and assembly occurs via budding on the plasma membrane . The viruses can reassort genes during mixed infections.
• PB2, PB1 and PA RNA-directed RNA polymerase proteins
• NP nucleoprotein
• Ml, M2 matrix proteins
• HA hemagglutinin
• NA neuramini
• NP virus-encoded proteins
• PB1, PB2 virus-dependent RNA polymerase
• PA virus-encoded proteins
• the NP is the major structural component of the virion, which interacts with genomic RNA, and is required for anti-termination during RNA synthesis (Beaton & Krug, 1986, Proc. Natl. Acad. Sci. USA 83:6282-6286).
• NP is also required for elongation of RNA chains (Shapiro & Krug, 1988, J. Virol.
• Influenza virus adsorbs via HA to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome which facilitates membrane fusion, thus triggering uncoating.
• the nucleocapsid migrates to the nucleus where viral mRNA is transcribed as the essential initial event in infection.
• Viral mRNA is transcribed by a unique mechanism in which viral endonuclease cleaves the capped 5 '-terminus from cellular heterologous mRNAs which then serve as primers for transcription of viral RNA templates by the viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the template- independent addition of poly(A) tracts. Of the eight viral mRNA molecules so produced, six are monocistronic messages that are translated directly into the proteins representing HA, NA, NP and the viral polymerase proteins, PB2 , PB1 and PA.
• Influenza viruses have been isolated from humans, mammals and birds, and are classified according to their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) .
• the other two transcripts undergo splicing, each yielding two mRNAs, which are translated in different reading frames to produce Ml, M2, non-structural protein-1 (NSl) and and non-structural protein-2 (NS2) .
• Eukaryotic cells defend against viral infection by producing a battery of proteins, among them interferons .
• the NSl protein facilitates replication and infection of influenza virus by inhibiting interferon production in the host cell.
• the NSl protein of influenza A virus is variable in length (Parvin et al., (1983) Virology 128:512-517) and is able to tolerate large deletions in the carboxyl terminus without affecting its functional integrity (Norton et al . , (1987) 156 (2) .204-213) .
• the NSl protein contains two functional domains, namely a domain that binds double- stranded RNA (dsRNA) , and an effector domain.
• the effector domain is located in the C-terminal domain of the protein. Its functions are relatively well established. Specifically, the effector domain functions by interacting with host nuclear proteins to carry out the nuclear RNA export function. (Qian et al . , (1994) J. Virol. 68 (4) :2433-2441) .
• the dsRNA-binding domain of the NS1A protein is located at its amino terminal end (Qian et al . , 1994).
• An amino-terminal fragment which is comprised of the first 73 amino-terminal amino acids [NSlA(l-73) ] , possesses all the dsRNA-binding properties of the full-length protein (Qian et al, (1995) RNA 1:948-956).
• NMR solution and X-ray crystal structures of NSlA(l-73) have shown that in solution it forms a symmetric homodimer with a unique six- helical chain fold (Chien et al . , (1997) .Nature Struct. Biol .
• Each polypeptide chain of the NSlA(l-73) domain consists of three alpha-helices corresponding to the segments Asn 4 -Asp 24 (helix 1) , Pro 31 -Leu 50 (helix 2), and Ile 54 -Lys 70 (helix 3).
• the present invention exploits Applicants' discoveries regarding exactly how the NSl protein, and particularly the dsRNA binding domain in the N-terminal portion of the protein participate in the infectious process of influenza virus.
• Applicants have discovered that the RNA-binding domain of the NSIA protein is critical to the replication and pathogenicity of influenza A virus.
• Applicants have discovered that when the binding domain of NSIA binds dsRNA in the host cell, the cell is unable to activate portions of its anti-viral defense system that inhibit production of viral protein.
• dsRNA binding by NSIA causes the enzyme, double- stranded-RNA-activated protein kinase ("PKR") to remain inactivated such that it cannot catalyze the phosphorylation of translation initiation factor eIF2c, which would otherwise be able to inhibit viral protein synthesis and replication.
• PKA protein kinase
• NSIA arginine 38 (R 38 )
• NS1B arginine 50 (R 50 )
• arginine 53 (R 53 ) arginine 53
• Applicants have invented a set of assays for characterizing interactions between NSIA or NS1B, and dsRNA, which can be used in small scale and/or high-throughput screening for inhibitors of this interaction.
• an amino-terminal fragment which is comprised of the first 93 amino-terminal amino acids [NS1B (1-93) ] , possesses all the dsRNA-binding properties of the full-length NSl protein of influenza B virus.
• One aspect of the present invention is directed to a method of identifying compounds having inhibitory activity against an influenza virus, comprising: a) preparing a reaction system comprising an NSl protein of an influenza virus or a dsRNA binding domain thereof, a dsRNA that binds said protein or binding domain thereof, and a candidate compound; and b) detecting extent of binding between the NSl protein and the dsRNA, wherein reduced binding between the NSl protein and the dsRNA in the presence of the compound relative to a control is indicative of inhibitory activity of the compound against the influenza virus .
• the compounds identified as having inhibitory activity against influenza virus can then be further tested to determine whether they would be suitable as drugs. In this way, the most effective inhibitors of influenza virus replication can be identified for use in subsequent animal experiments, as well as for treatment (prophylactic or otherwise) of influenza virus infection in animals including humans.
• another aspect of the present invention is directed to a method of identifying compounds having inhibitory activity against an influenza virus, comprising: a) preparing a reaction system comprising an NSl protein of an influenza virus or a dsRNA binding domain thereof, a dsRNA that binds said protein or binding domain thereof, and a candidate compound; b) detecting extent of binding between the NSl protein and the dsRNA, wherein reduced binding between the NSl protein and the dsRNA in the presence of the compound relative to a control is indicative of inhibitory activity of the compound against the influenza virus; and c) determining extent of a compound identified in b) as having inhibitory activity to inhibit growth of influenza virus in vi tro .
• the method further entails d) determining extent of a compound identified in c) as inhibiting growth of influenza virus in vitro, to inhibit replication of influenza virus in a non-human animal .
• a further aspect of the present invention is directed to a method of preparing a composition for inhibiting replication of influenza virus in vitro or in vivo, comprising: a) preparing a reaction system comprising an NSl protein of an influenza virus or a dsRNA binding domain thereof, a dsRNA that binds said protein or binding domain thereof, and a candidate compound; b) detecting extent of binding between the NSl protein and the dsRNA, wherein reduced binding between the NSl protein and the dsRNA in the presence of the compound relative to a control is indicative of inhibitory activity of the compound against the influenza virus; c) determining extent of a compound identified in b) as having inhibitory activity to inhibit growth of influenza virus in vitro; d) determining extent of a compound identified in c) as inhibiting growth of influenza virus in vitro, to inhibit replication of influenza virus in a non-human animal; and e) preparing the composition by formulating a compound identified in d) as inhibiting replication of influenza virus in a non
• some embodiments entail labeling the NSl protein or the dsRNA with a fluorescent molecule, and then determining extent of binding via fluorescent resonance energy transfer or fluorescence polarization.
• the control is extent of binding between the dsRNA and the NSl protein or a dsRNA binding domain that lacks amino acid residues R 38 and/or K 41 .
• Other embodiments entail methods of assaying for influenza virus NSl protein/dsRNA complex formation.
• Yet still other embodiments entail methods of using a influenza virus NSl protein/dsRNA complex formation in screening for or optimizing inhibitors. These embodiments include NMR chemical shift perturbation of the NSl protein or RNA gel filtration sedimentation equilibrium and virtual screening using the structure of NSl protein and the model of the NS1-RNA complex
• a further aspect of the present invention is directed to a composition
• a composition comprising a reaction mixture comprising a complex of an NSl protein of influenza virus, or a dsRNA binding fragment thereof, and a dsRNA that binds said protein.
• the NSl protein is an NSIA protein, or the dsRNA binding fragment thereof, the 73 N-terminal amino acid residues of the protein.
• the NSl protein is an NS1B protein, or the dsRNA binding fragment thereof, the 93 N-terminal amino acid residues of the protein.
• the composition further contains a candidate or test compound being tested for inhibitory activity against influenza virus .
• a still further aspect of the present invention is directed to a method of identifying a compound that can be used to treat influenza virus infections comprising using the structure of a NSl protein or a dsRNA binding domain thereof, NSlA(l-73) or NS1B(1- 93) , and the three dimensional coordinates of a model of the NS1- RNA complex in a drug screening assay.
• FIG. 1 Gel shift assay for different duplexes on their ability to bind NSlA(l-73) . This experiment was performed under standard conditions using indicated 32 P-labeled double-stranded nucleic acids (1.0 nM) and either with (+) ; or without (-) 0.4 ⁇ M NSlA(l-73) .
• FIG. 2 Gel filtration chromatography profiles of different duplexes in the presence of NSlA(l-73) : (A) dsRNA; (B) RNA-DNA hybrid; (C) DNA-RNA hybrid; (D) dsDNA. The major peaks between 20 and 30 min correspond to the duplexes, except for the first peak in (A) which is from the NSlA(l-73) -dsRNA complex.
• FIG. 3 Gel filtration chromatograms of the purified NS1A(1- 73)-dsRNA complex.
• A 4 ⁇ M, 100 ⁇ l of the fresh complex sample;
• B 4 ⁇ M, 100 ⁇ l of the complex sample after one month.
• FIG. 4. (A) Determination of the stoichiometry based on sedimentation equilibrium at 16000 rpm on three samples with loading concentrations of 0.6 (D) , 0.3 ( ⁇ ) and 0.5 (not shown, to avoid the overlap of data points) absorbance unit.
• the solid line is the joint fit of the three sets of data assuming a 1:1 stoichiometry of the dsRNA:NSl complex; the insert shows the random residual plots of the fit.
• the dotted line is drawn assuming a 1:2 stoichiometry of the dsRNA:NSl complex.
• FIG. 6 (A) Ribbon diagram of NSlA(l-73) showing the results of chemical shift perturbation measurements. Residues of NS1A(1- 73) which give shift perturbations in NMR spectra of the NS1A(1- 73)-dsRNA complex are colored in cyan, residues that are not changed in the chemical shifts of their amide 15 N and X H are colored in pink, and white represents the chemical shift assignments of the residues that cannot be identified in 2D HSQC spectra due to the overlapped cross peaks. (B) Side chains shown in Figure 6B are also displayed here with all the basic residues labeled. Note that the binding epitope of NSIA (1-73) to dsRNA appears to be on the bottom of this structure.
• FIG. 7 CD spectra of the purified NSIA (1-73 ) -dsRNA complex (A), and the mixtures of duplexes and NSIA (1-73): RNA-DNA hybrid (B) , and DNA-RNA hybrid (C) .
• Orange experimental CD spectra of the mixtures (1:1 molar ratio of duplex and protein dimer) . Red: duplex alone. Blue: NSlA(l-73) alone. Green: calculated sum spectra of duplex and NSIA (1-73) .
• FIG. 8 A model of the dsRNA binding properties of NS1A(1-
• the present invention provides methods of designing specific inhibitors of dsRNA binding domains of NSl proteins from both influenza A and B viruses .
• the amino acid sequences of the dsRNA binding domains of NSl proteins of influenza A, particularly the R 38 and K 41 amino acid residues, are substantially conserved. Multiple sequence alignments for the NSl protein of various strains of influenza A virus is described in Table 1.
• amino acid sequence of the NSl protein of various strains of influenza A virus is set forth below.
• VAGSLCIRMD 121 QAIMDKNITL KANFSIIFDR LETLILLRAF TEEGAIVGEI SPVPSLPGHT
• VAGPLCIRMD 121 QAIMDKNIIL KANFSVIFDR LETLILLRAF TEAGAIVGEI SPLPSLPGHT
• amino acid sequence of the NSl protein of various strains of influenza B virus is set forth below.
• the amino acid sequence of the NSl protein of the influenza B virus (B/Lee/ 40) :
• the amino acid sequence of the NSl protein of the influenza B virus B/Memphis/296 1 MADNMTTTQI EVGPGATNAT INFEAGILEC YERLSWQRAL DYPGQDRLNR LKRKLESRIK 61 THNKSEPESK RMSLEERKAI GVKMMKVLLF MDPSAGIEGF EPYCMKSSSN
• the amino acid sequence of the NSl protein of the influenza B virus (B/Shangdong/7/97) :
• the amino acid sequence of the NSl protein of the influenza B virus (B/Nagoya/20/99) :
• the amino acid sequence of the NSl protein of the influenza B virus (B/Saga/S172/99) :
• the amino acid sequence of the NSl protein of the influenza B virus (B/Kouchi/193/99) :
• any one NSl protein or fragment thereof that binds dsRNA (and which has intact R 38 , K 41 residues for NSIA, and intact R 50 , R 53 residues for NS1B) will serve to identify compounds having inhibitory activity against strains of influenza A virus, as well as strains of influenza B virus, respectively.
• the present invention does not require that the proteins be naturally occurring.
• Analogs of the NSl protein that are functionally equivalent in terms of possessing the dsRNA binding specificity of the naturally occurring protein may also be used.
• Representative analogs include fragments of the protein, e.g., the dsRNA binding domain.
• analogs may differ from the naturally occurring protein in terms of one or more amino acid substitutions, deletions or additions. For example, functionally equivalent amino acid residues may be substituted for residues within the sequence resulting in a change of sequence.
• Such substitutes may be selected from other members of the class to which the amino acid belongs; e.g., the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; the positively charged (basic) amino acids include arginine, lysine, and histidine; the negatively charged (acidic) amino acids include aspartic and glutamic acid.
• the R 38 and K 41 residues for NSIA can be changed but there are limitations.
• dsRNA binding domain is intended to include analogs of the NSl protein that are functionally equivalent to the naturally occurring protein in terms of binding to dsRNA.
• the NSl proteins of the present invention may be prepared in accordance with established protocols .
• the NSl protein of influenza virus, or a dsRNA binding domain thereof may be derived from natural sources, e.g., purified from influenza virus infected cells and virus, respectively, using protein separation techniques well known in the art; produced by recombinant DNA technology using techniques known in the art (see e.g., Sambrook et al .
• peptides can be synthesized by solid phase techniques, cleaved from the resin and purified by preparative high performance liquid chromatography (see, e.g., Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman & Co., N.Y., pp. 50-60) .
• NSIA (1-73) dsRNA-binding domain of influenza virus nonstructural protein 1 differs from the predominant class of dsRNA-binding domains, referred to as dsRBMs, that are found in a large number of eukaryotic and prokaryotic proteins.
• the proteins which contain the dsRBM domain include eukaryotic protein kinase R (PKR) (Nanduri et al., 1998), a kinase that plays a key role in the cellular antiviral response, Drosophila melonogaster Staufen (Ramos et al . , 2000), and Escherichia coli Rnase III (Kharrat et al .
• the dsRBM domain comprises a monomeric ⁇ - ⁇ - ⁇ - ⁇ fold. Structural analysis has established that this domain spans two minor grooves and the intervening major groove of the dsRNA target (Ryter & Schultz, 1998) .
• Several amino acids of the dsRBM domain are involved in direct and water-mediated interactions with the phosphodiester backbone, ribose 2' -OH groups, and a small number of bases.
• the canonical A-form dsRNA duplex is distorted upon complex formation. This binding is relatively strong, with a K ⁇ j of approximately 1 nmolar.
• the methods of the present invention exploit a phenomenon that occurs exclusively between a viral protein and dsRNA present in the infected eucaryotic cell. Therefore, compounds identified by the methods of the present invention might not otherwise affect normal cellular function.
• Applicants also discovered that one of the intracellular functions of the RNA-binding domain of the NSIA protein is to prevent the activation of PKR by binding dsRNA.
• Applicants generated recombinant A/Udorn/72 viruses that encode NSIA proteins whose only defect is in RNA binding. Because the R at position 38 (R 38 ) and K at position 41 (K 41 ) are the only amino acids that are required solely for RNA binding, we substituted A for either one or both of these amino acids .
• the three mutant viruses are highly attenuated: the R 38 and K 41 mutant viruses form pin-point plaques, and the double mutant (R38/K41) does not form visible plaques.
• PKR PKR is activated, eIF2a is phosphorylated, and viral protein synthesis is inhibited. Surprisingly, after its activation, PKR is degraded. The R38/K41 double mutant is most effective in inducing PKR activation.
• NSlA(l-73) binds dsRNA, but not dsDNA or RNA/DNA hybrids.
• NSlA(l-73) and the full length NSIA protein have been shown to bind double-stranded RNAs (dsRNAs) with no sequence specificity (Lu et al., (1995) Virology 214 , 222-228, Qian et al., (1995) RNA 1, 948- 956, Wang et al . , 1999), but until the present invention, it had not been determined whether NSlA(l-73) or the NSIA protein bind RNA-DNA hybrids or dsDNA. Applicants incubated NSIA (1-73) with
• dsRNA is found to form a complex with NSlA(l-73) producing a 30% gel shift (lane 2), whereas all the other duplexes fail to bind to the protein (lanes 4, 6, and 8) .
• NSIA (1-73) specifically recognizes the conformational and/or structural features of dsRNA (A-form conformation) which are distinct from those of dsDNA (B- form conformation) or RNA/DNA hybrids (intermediate A/B conformations) under these conditions.
• dsRNA length and ribonucleotide sequence of the dsRNA are not critical. As described in some working examples herein, methods of the present invention may be conducted using a short synthetic 16- base pair (bp) dsRNA, which identifies key features of the mode of protein RNA interaction.
• This dsRNA molecule has a sequence derived from a commonly used 29-base pair dsRNA-binding substrate which can be generated in small quantities by annealing the sense and antisense transcripts of the polylinker of the pGEMl plasmid
• Circular dichroism (CD) spectra of the purified NSlA(l-73) -dsRNA complex are very similar to the sum of CD spectra of free dsRNA and NSlA(l-73) , demonstrating that little or no change in the conformations of either the protein or its A-form dsRNA target occur as a result of binding.
• NSIA (1-73) binds to neither the corresponding DNA-DNA duplex nor a DNA-RNA hybrids duplex
• NSIA (1-73) appears to recognize specific conformational features of canonical A-form RNA, thus highlighting yet another way in which the methods of the present invention extraordinarly mimics the interaction between the NSl protein of influenza and its host.
• Methods of the present invention are advantageously practiced in the context of a high throughput in vitro assay.
• the assay system could use either or both of the standard methods of fluorescence resonance energy transfer or fluorescence polarization with labeled dsRNA molecules, either NSIA or NSlA(l-73) , or NS1B or NSlB(l-93) molecules to monitor interactions between these protein targets and various dsRNA duplexes and to measure binding affinities.
• These assays would be used to screen compounds to identify molecules, which inhibit the interactions between the NSl targets and the RNA substrates, based on the above-disclosed structure of the NSl protein.
• a wide variety of compounds may be tested for inhibitory activity against influenza virus in accordance with the present invention, including random and biased compound libraries.
• Biased compound libraries may be designed using the particular structural features of the NSl target - RNA substrate interaction sites e.g., deduced on the basis of published results. See, e.g., Chien, et al . , Nature Struct. Biol. 4:891-95 (1997); Liu, et al . , Nature Struct. Biol. 4:896-899 (1997); and Wang, et al . , RNA 5:195-205 (1999) .
• binding partners The NSl protein of influenza virus, or a dsRNA binding domain thereof, and dsRNA which interact and bind are sometimes referred to herein as "binding partners". Any of a number of assay systems may be utilized to test compounds for their ability to interfere with the interaction of the binding partners. However, rapid high throughput assays for screening large numbers of compounds, including but not limited to ligands (natural or synthetic) , peptides, or small organic molecules, are preferred.
• the basic principle of the assay systems used to identify compounds that interfere with the interaction between the NSl protein of influenza virus, or a dsRNA binding domain thereof, and dsRNA involves preparing a reaction mixture containing the NSl protein of influenza virus, or a dsRNA binding domain thereof, and dsRNA under conditions and for a time sufficient to allow the two binding partners to interact and bind, thus forming a complex, in order to test a compound for inhibitory activity, the reaction is conducted in the presence and absence of the test compound, i.e., the test compound may be initially included in the reaction mixture, or added at a time subsequent to the addition of NSl protein of influenza virus, or a dsRNA binding domain thereof, and dsRNA; controls are incubated without the test compound or with a placebo.
• Still another aspect of the present invention comprises a method of virtual screening for a compound that can be used to treat influenza virus infections comprising using the structure of a NSl protein or a dsRNA binding domain thereof NSIA (1-73) or NSlB(l-93), and the three dimensional coordinates of a model of the NS1-RNA complex in a drug screening assay.
• Another aspect of the present invention comprises a method of using the three dimensional coordinates of the model of the complex for designing compound libraries for screening.
• the present invention provides methods of identifying a compound or drug that can be used to treat influenza virus infections.
• One such embodiment comprises a method of identifying a compound for use as an inhibitor of the NSl protein of influenza virus or a dsRNA binding domain thereof and a dataset comprising the three-dimensional coordinates obtained from the NSl protein of influenza A or B virus or a dsRNA binding domain thereof.
• the selection is performed in conjunction with computer modeling.
• the potential compound is selected by performing rational drug design with the three-dimensional coordinates determined for the NSl protein of influenza virus, or a dsRNA binding domain thereof.
• the selection is performed in conjunction with computer modeling.
• the potential compound is then contacted with and interferes with the binding of the NSl protein of influenza virus or a dsRNA binding domain thereof and dsRNA, and the inhibition of binding is determined (e.g., measured).
• a potential compound is identified as a compound that inhibits binding of the NSl protein of influenza virus or a dsRNA binding domain thereof and dsRNA when there is a decrease in binding.
• the potential compound is contacted with and/or added to influenza virus infected cell culture and the growth of the virus culture is determined.
• a potential compound is identified as a compound that inhibits viral growth when there is a decrease in the growth of the viral culture.
• the method further comprises molecular replacement analysis and design of a second-generation candidate drug, which is selected by performing rational drug design with the three-dimensional coordinates determined for the drug. Preferably the selection is performed in conjunction with computer modeling.
• the candidate drug can then be tested in a large number of drug screening assays using standard biochemical methodology exemplified herein.
• the three-dimensional coordinates of the NSIA protein and the model of NSlA-dsRNA complex or the model of NSlB-dsRNA complex provide methods for (a) designing inhibitor library for screening,
• ASSAY COMPONENTS One of the binding partners used in the assay system may be labeled, either directly or indirectly, to measure extent of binding between the NSl protein or dsRNA binding portion, and the dsRNA. Depending upon the assay format as described in detail below, extent of binding may be measured in terms of complexation between NSl protein of influenza virus, or a dsRNA binding domain thereof, and dsRNA, or extent of disassocation of a pre-formed complex, in the presence of the candidate compound. Any of a variety of suitable labeling systems may be used including but not limited to radioisotopes such as 125 I; enzyme labelling systems that generate a detectable colorimetric signal or light when exposed to substrate; and fluorescent labels.
• fusion proteins that can facilitate labeling, immobilization and/or detection.
• the coding sequence of the NSl protein of influenza virus, or a dsRNA binding domain thereof can be fused to that of a heterologous protein that has enzyme activity or serves as an enzyme substrate in order to facilitate labeling and detection.
• the fusion constructs should be designed so that the heterologous component of the fusion product does not interfere with binding of the NSl protein of influenza virus, or a dsRNA binding domain thereof, and dsRNA.
• Indirect labeling involves the use of a third protein, such as a labeled antibody, which specifically binds to NSl protein of influenza virus, or a dsRNA binding domain thereof.
• a third protein such as a labeled antibody, which specifically binds to NSl protein of influenza virus, or a dsRNA binding domain thereof.
• Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by an Fab expression library.
• various host animals may be immunized by injection with the NSl protein of influenza virus, or a dsRNA binding domain thereof.
• Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few.
• Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
• BCG Bacille Calmette-Guerin
• Monoclonal antibodies may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein, (Nature, 1975, 256:495-497), the human B-cell hybridoma technique (Kosbor et al . , 1983, Immunology Today, 4:72, Cote et al., 1983, Proc. Natl. Acad. Sci., 80:2026-2030) and the EBV- hybridoma technique (Cole et al . , 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In addition, techniques developed for the production of "chimeric antibodies"
• such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
• Fab expression libraries may be constructed (Huse et al . , 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
• ASSAY FORMATS The assay can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring one of the binding partners onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested.
• test compounds that interfere with the interaction between the binding partners can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the NSl protein of influenza virus, or a dsRNA binding domain thereof, and dsRNA.
• test compounds that disrupt preformed complexes e.g. compounds with higher binding constants that displace one of the binding partners from the complex, can be tested, by adding the test compound to the reaction mixture after complexes have been formed.
• the various formats are described briefly below.
• one binding partner e.g., either the NSl protein of influenza virus, or a dsRNA binding domain thereof, or dsRNA
• a solid surface and its binding partner, which is not anchored, is labeled, either directly or indirectly.
• the anchored species may be immobilized by non-covalent or covalent attachments.
• an immobilized antibody specific for the NSl protein of influenza virus, or a dsRNA binding domain thereof may be used to anchor the NSl protein of influenza virus, or a dsRNA binding domain thereof to the solid surface.
• the surfaces may be prepared in advance and stored.
• the binding partner of the immobilized species is added to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surf ce.
• the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the binding partner was pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
• an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the binding partner (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody) .
• test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.
• the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for the NSl protein of influenza virus or a dsRNA binding domain thereof to anchor any complexes formed in solution.
• test compounds which inhibit complex or which disrupt preformed complexes can be identified.
• a homogeneous assay can be used.
• a preformed complex of the influenza viral NSl protein or dsRNA binding domain thereof and dsRNA is prepared in which one of the binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein, which utilizes this approach for immunoassays) .
• the addition of a test substance that competes with and displaces one of the binding partners from the preformed complex will result in the generation of a signal above background. In this way, test substances, which disrupt the NSl protein of influenza virus, or a dsRNA binding domain thereof, and dsRNA interaction can be identified.
• the NSl protein of influenza virus, or a dsRNA binding domain thereof can be prepared for immobilization using recombinant DNA techniques described supra. Its coding region can be fused to the glutathione-S- transferase (GST) gene using the fusion vector pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein.
• GST glutathione-S- transferase
• NSl protein or a dsRNA binding domain thereof can be purified and used to raise a monoclonal antibody, specific for NSl or an NSl fragment, using methods routinely practiced in the art and described above.
• This antibody can be labeled with the radioactive isotope 125 I, for example, by methods routinely practiced in the art.
• the GST-NS1 fusion protein can be anchored to glutathione-agarose beads.
• dsRNA can then be added in the presence or absence of the test compound in a manner that allows dsRNA to interact with and bind to the NSl portion of the fusion protein.
• unbound material can be washed away, and the NSl-specific labeled monoclonal antibody can be added to the system and allowed to bind to the complexed binding partners.
• the interaction between NSl and dsRNA can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
• the GST-NS1 fusion protein and dsRNA can be mixed together in liquid in the absence of the solid glutathione- agarose beads . The test compound can be added either during or after the binding partners are allowed to interact . This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the binding partner interaction can be detected by measuring the radioactivity associated with the beads.
• a given compound found to inhibit one virus may be tested for general antiviral activity against a wide range of different influenza viruses.
• a compound which inhibits the interaction of influenza A virus NSl with dsRNA by binding to the NSl binding site can be tested, according to the assays described infra, against different strains of influenza A viruses as well as influenza B virus strains .
• the identified inhibitors of the interaction between NSl targets and RNA substrates may be further tested for their ability to inhibit replication of influenza virus, first in tissue culture and then in animal model experiments. The lowest concentrations of each inhibitor that effectively inhibits influenza virus replication will be determined using high and low multiplicities of infection.
• VIRAL GROWTH ASSAYS The ability of an inhibitor identified in the foregoing assay systems to prevent viral growth can be assayed by plaque formation or by other indices of viral growth, such as the TCID 50 or growth in the allantois of the chick embryo.
• an appropriate cell line or e bryonated eggs are infected with wild-type influenza virus, and the test compound is added to the tissue culture medium either at or after the time of infection.
• the effect of the test compound is scored by quantitation of viral particle formation as indicated by hemagglutinin (HA) titers measured in the supernatants of infected cells or in the allantoic fluids of infected embryonated eggs; by the presence of viral plaques; or, in cases where a plaque phenotype is not present, by an index such as the TCID 50 or growth in the allantois of the chick embryo, or with a hemagglutination assay.
• HA hemagglutinin
• An inhibitor can be scored by the ability of a test compound to depress the HA titer or plaque formation, or to reduce the cytopathic effect in virus- infected cells or the allantois of the chick embryo, or by its ability to reduce viral particle formation as measured in a hemagglutination assay.
• ANIMAL MODEL ASSAYS The most effective inhibitors of virus replication identified by the processes of the present invention can then be used for subsequent animal experiments.
• the ability of an inhibitor to prevent replication of influenza virus can be assayed in animal models that are natural or adapted hosts for influenza.
• Such animals may include mammals such as pigs, ferrets, mice, monkeys, horses, and primates, or birds.
• animal models can be used to determine the LD 50 and the ED 50 in animal subjects, and such data can be used to derive the therapeutic index for the inhibitor of the NSIA (1-73) or
• optimization of design of lead compounds may also be aided by characterizing binding sites on the surface of the NSl protein or dsRNA binding domain thereof by inhibitors identified by high throughput screening. Such characterization may be conducted using chemical shift perturbation NMR together with NMR resonance assignments. NMR can determine the binding sites of small molecule inhibitors for RNA. Determining the location of these binding sites will provide data for linking together multiple initial inhibitor leads and for optimizing lead design.
• PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION The identified compounds that inhibit viral replication can be administered to a patient at therapeutically effective doses to treat viral infection.
• a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of viral infection.
• Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) .
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 / ED S0 .
• Compounds, which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of infection in order to minimize damage to uninfected cells and reduce side effects.
• the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans .
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the therapeutically effective dose can be estimated initially from cell culture assays.
• a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal infection, or a half-maximal inhibition) as determined in cell culture.
• IC 50 i.e., the concentration of the test compound which achieves a half-maximal infection, or a half-maximal inhibition
• levels in plasma may be measured, for example, by high performance liquid chromatography.
• Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable
• the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
• the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• the dosage unit may be determined by providing a valve to deliver a metered amount .
• Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers
• pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers
• Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
• Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid) .
• suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
• emulsifying agents e.g., lecithin or acacia
• non-aqueous vehicles e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils
• preservatives e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid
• the preparations may also contain buffer
• Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides .
• the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may for example comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the invention is not limited to the embodiments described herein and may be modified or varied without departing from the scope of the invention
• Proteins were then purified from the supernatant by ion exchange and gel filtration chromatography using Pharmacia FPLC systems according to a procedure described elsewhere. (Qian et al . , (1995) RNA 1, 948- 956.) The overall yield of purified NSlA(l-73) was about 5 mg/1 of culture medium. Protein concentrations were determined by absorbance at 280 nm (A 280 ) using a molar extinction coefficient (e 28 o) for the monomer of 5750 M "1 cm "1 .
• RNA OLIGOMERS Two single-stranded (ss) 16-nucleotide (16-nt) RNAs, CCAUCCUCUACAGGCG (sense) and CGCCUGUAGAGGAUGG (antisense) , were chemically synthesized using standard phosphoramidite chemistry (Wincott et al., (1995) Nucleic Acids Res .
• RNA oligomers were then desalted over Bio-Rad Econo-Pac 10DG columns and purified by preparative gel electrophoresis on 20% (w/v) acrylamide, 7M urea denaturing gels. The appropriate product bands, visualized by UV shadowing, were cut out, crushed, and extracted into 90 mM Tris-borate, 2 mM EDTA, pH 8.0 buffer by gentle rocking overnight. The resulting solutions were concentrated by lyophilization and desalted again using Econo-Pac 10DG columns. Purified RNA oligomers are then lyophilized and stored at -20°.
• Analogous 16-nt sense and antisense DNA strands containing the same sequence can be purchased from Genosys Biotechnologies, Inc. Concentrations of nucleic acid samples were calculated on the basis of absorbance at 260 nm (A 260 ) using the following molar extinction coefficients (D 260 , M "1 cm “1 at 20 °C) : (+) RNA, 151 530; (-) RNA, 165 530; (+) DNA, 147 300; (-) DNA, 161 440; dsRNA, 262 580; RNA/DNA, 260 060; DNA/RNA, 273 330; dsDNA, 275 080.
• the extinction coefficients for the single strands were calculated from the extinction coefficients of monomers and dimers at 20 °C (Cantor et al . , (1965) J". Mol . Biol . 13 , 65-77) assuming that the molar absorptivity is a nearest-neighbor property and that the oligonucleotides are single-stranded at 20 °C (Hung et al . , (1994), Nucleic Acids Res . 22, 4326-4334) .
• Example 3 POLYACRYLAMIDE GEL SHIFT BINDING ASSAY: The single-stranded 16-nt synthetic RNA and DNA oligonucleotides were labeled at their 5 " ends with [ ⁇ 32 P]ATP using T4 polynucleotide kinase and purified by denaturing urea-PAGE. Approximate 1:1 molar ratios of single-stranded (ss) sense RNA (or DNA) and antisense RNA (or DNA) were mixed in 50 mM Tris, 100 mM NaCl, pH 8.0 buffer. Solutions were heated to 90 °C for two minutes and then slowly cooled down to room temperature to anneal the duplexes.
• ss single-stranded
• DNA antisense RNA
• binding buffer 50 mM Tris- glycine, 8% glycerol, 1 mM dithiothreitol, 50 ng/ ⁇ l tRNA, 40 units of RNasin, pH 8.8.
• the protein-nucleic acid complexes were resolved from free ds or ss oligomers by 15% nondenaturing PAGE at 150 V for 6 hours in 50 mM Tris-borate, 1 mM EDTA, pH 8.0 at 4 °C. The gel was then dried and analyzed by autoradiography.
• Example 4--ANALYTICAL GEL FILTRATION CHROMATOGRAPHY Micromolar solutions of the four 16-nt duplexes (RR, RD, DR, and DD) were prepared 10 mM potassium phosphate, 100 mM KCl, 50 ⁇ M EDTA, pH 7.0 buffer and annealed as described above. These duplexes are then purified from unannealed or excess ss species using a Superdex-75 HR 10/30 gel filtration column (Pharmacia) , and adjusted to a duplex concentration of 4 ⁇ M. Each ds nucleic acid was then combined with 1.5 mM NSlA(l-73) (monomer concentration) to give a 1:1 molar ratio of protein to duplex. Gel filtration chromatography can be performed on a Superdex 75 HR 10/30 column
• Example 5 PURIFICATION OF THE NSlA(l-73 ) -DSRNA COMPLEX The fraction corresponding to the first peak shown in the gel filtration chromatography of l.i molar ratio NSlA(l-73) dimer and dsRNA mixture was collected and concentrated to less than 1 ml using Centricon concentrators (Amicon, Inc.). This concentrated sample was then reloaded onto the same gel filtration column and the main fraction is collected again. The concentration of this purified NSIA (1-73 ) -dsRNA complex was determined by measuring the UV absorbance at 260 nm. The purity and stability of this complex was also examined using analytical gel filtration by loading 100 ⁇ l samples at 4 ⁇ M immediately following preparation and after 1 month.
• Example 6-SEDIMENTATION EQUILIBRIUM Sedimentation equilibrium experiments were carried out using a Beckman XL-I instrument at 25 °C. Short column runs using Beckman eight-channel 12 mm path charcoal- ⁇ pon cells at speeds 3OK to 48K rpm were conducted for NSlA(l-73) and dsRNA loading concentrations of 0.2 - 2 mg/ml and 0.2 - 0.6 mg/ml, respectively, in order to independently evaluate the behavior of these free components. Data were acquired using a Rayleigh interference optical system.
• the partial specific volume of NSIA (1-73 ), F NSI , and the solvent density, p are calculated to be 0.7356 and 1.01156, respectively, at 25 °C using the program Sednterp (Laue et al . , 1992).
• the specific volume of dsRNA, v i w is determined experimentally to be 0.5716 by sedimentation equilibrium of dsRNA samples (see Results for details).
• the specific volume of the NSIA (1-73 ) -dsRNA complex is calculated to be 0.672 assuming a 1:1 stoichiometry, using the method of Cohn and Edsall (Cohn & Edsall, 1943) .
• Example 7 CALCULATION OF THE DISSOCIATION CONSTANT: The calculation of the dissociation constant of a 1:1 NSIA (1-73 ) -dsRNA complex was based upon the assumption that there are equal molar amounts of free NSlA(l-73) protein and free dsRNA in the original solution. This assumption is valid if the gel-filtration purified samples of the complex used in these measurements is in fact a 1:1 stoichiometry. In this case, the amount of free dsRNA and free NSlA(l-73) correspond to that which has dissociated from the 1:1 complex. In addition, since the reduced molecular weight (defined below in Eq. 2) of NSlA(l-73) dimer and dsRNA differ only by 3%, the two free macromolecules are treated as the same hydrodynamic species during sedimentation. The concentration distribution of the ith species of an ideal system at sedimentation equilibrium can be expressed as ⁇
• the M ⁇ and V ⁇ in Eq. 2 are the molecular weight and the partial specific volume of the ith species, R is the gas constant, T is the absolute temperature and ⁇ is the angular velocity.
• the concentration is normally expressed in weight concentration scale
• the dsRNA can be expressed by
• m° refers to the concentration of the original solution
• m (r) refers to the concentration at radius r at sedimentation equilibrium.
• the subscripts ⁇ , RNA,t", “RNA, free” and w RNA,x” refer to the total amount of dsRNA, the free dsRNA and dsRNA in the NSIA (1-73) -dsRNA complex, respectively;
• r m and r b are radius values at the meniscus and base of the solution column, respectively.
• r' is set to be at the position of r m . Integration of equation 3 then yields:
• Eq.5 demonstrates that m (r , (r i ) NS i,free at the reference position, and thus, m (r) (r) NSlr f r ee at any radius r.
• E x (S RNA +S NSI ) ! , where ⁇ is the extinction coefficient and / is the optical path length.
• K a is the association constant in molar concentration scale, and is expressed as a function of m x and
• association system of NSlA(l-73) and dsRNA is reduced to a simple system of two components during sedimentation.
• Example 8 NMR SPECTROSCOPY: All NMR data were collected at 20°C on Varian INOVA 500 and 600 NMR spectrometer systems equipped with four channels. The programs VNMR (Varian Associates), NMRCompass (Molecular Simulations, Inc.), and AUTOASSIGN (Zimmerman et al., (1997) J “ . Mol . Biol . 269, 592-610) were used for data processing and analysis. Proton chemical shifts were referenced to internal 2, 2-dimethyl-2-silapentane-5-sulfonic acid; 13 C and 15 N chemical shifts were referenced indirectly using the respective gyromagnetic ratios, "C ⁇ H (0.251449530) and "N ⁇ H (0.101329118). (Wishart et al . , (1995) J. Biomol . NMR 6, 135-140.)
• Example 9 SEQUENCE SPECIFIC ASSIGNMENTS OF NSlA(l-73) : NMR samples of free 13 C, 15 N-NSlA(l-73) used for assignment were prepared at a dimer protein concentration of 1.0 to 1.25 mM in 270 ⁇ l of 95% H 2 0/5% D 2 0 solutions containing 50 mM ammonium acetate and 1 mM NaN 3 at pH 6.0 in Shigemi susceptibility-matched NMR tubes.
• Backbone X E, 13 C, 15 N, and 13 C D resonance assignments were determined by automated analysis of triple-resonance NMR spectra of 13 C, 15 N-enriched proteins using the computer program AUTOASSIGN (Zimmerman et al . , (1997) J”.
• the input for AUTOASSIGN includes peak lists from 2D ⁇ -"N HSQC and 3D HNCO spectra along with peak lists from three intraresidue [HNCA, CBCANH, and HA(CA)NH] and three interresidue [CA(CO)NH, CBCA(CO)NH, and HA(CA) (CO)NH] experiments. Details of these pulse sequences and optimization parameters were reviewed elsewhere (Montelione et al . , (1999), Ralph, L. J. , and Krishna, N. R. , Eds, Vol. 17, pp 81- 130, Kluwer Academic/Plenum Publishers, New York) .
• Peak lists for AUTOASSIGN were generated by automated peak-picking using NMRCompass and then manually edited to remove obvious noise peaks and spectral artifacts.
• Side chain resonance assignments (except for the 13 C assignments of aromatic side chains) were then obtained by manual analysis of 3D HCC(CO)NH TOCSY (Montelione et al . , (1992) J. Am . Chem. Soc . 114 , 10974-10975), HCCH-COSY (Ikura et al . , (1991) J". Biomol . NMR 1, 299-304) and 15 N-edited TOCSY (Fesik et al., (1988) J". Magn . Reson .
• N- enriched NSlA(l-73) was purified and prepared as described above.
• RNA duplex concentration 10 mM.
• This highly concentrated dsRNA solution was then used to titrate the NMR sample of free 15 N-enriched NS1A(1- 73), making protein-dsRNA samples with the ratios of [dimeric protein] to [dsRNA] as 2:1, 1:1, 1:1.5, and 1:2.
• these samples were prepared by slowly adding the free protein solution to the concentrated dsRNA.
• the HSQC spectra of free 15 N-enriched NSIA (1-73) were acquired with 80 scans per increment and 200 x 2048 complex data points, and transformed into 1024 x 2048 points after zero- filling in the tl dimension.
• HSQC spectra for the dsRNA titration experiments were collected with the same digital resolution using 320 scans per increment.
• Example 11 CD MEASUREMENTS: CD spectra were recorded in the 200-350 nm region at 20 oC using an Aviv Model 62-DS spectropolarimeter equipped with a 1 cm path-length cell. CD spectra for the four nucleic acid duplexes (RR, RD, DR, DD) were obtained on 1.1 ml, 4 ⁇ M samples in the phosphate buffer described above. Each duplex is then combined with 1.5 mM NSIA (1-73) (monomer concentration) to form a 1:1 molar ratio of protein to duplex. The CD spectra of these protein-duplex mixtures were collected under the same conditions, assuming that the total duplex concentration remained 4 ⁇ M for each sample.
• the calculated CD spectra of protein-duplex mixtures were obtained using the sum of CD data from free NSIA (1-73) and from each double-stranded nucleic acid alone. CD spectra were reported as 8 -S R , in units of ⁇ cm "1 per mol nucleotide.
• Example 12 CHARACTERIZATION AND PURIFICATION OF NSlA(l-73)-
• DSRNA COMPLEX BY GEL FILTRATION CHROMATOGRAPHY The four NSlA(l-73) nucleic acid duplex mixtures described above were further analyzed for complex formation using analytical gel filtration chromatography.
• the NSIA (1-73 ) -dsRNA mixture showed two major peaks in the chromatographic profile monitored at 260 nm ( Figure 2A) , whereas the mixtures containing dsDNA and RNA/DNA eluted as a single peak ( Figures 2B, C, D) . Since the chromatographic eluates were detected by absorbance at 260 nm, these chromatograms reflect the state (s) of the nucleic acid in these samples. In the dsRNA case (Fig.
• the faster and slower eluting peaks corresponded to the NSlA(l-73) -dsRNA complex and the unbound dsRNA duplex, respectively.
• the elution time and corresponding molecular weight ( ⁇ 26 kDa) for the more rapidly eluting peak were consistent with a complex with a 1:1 stoichiometry (protein dimer to dsRNA) .
• About 70% of the RNA and protein spann in the complex fraction under the chromatographic conditions used. No peak(s) corresponding to complex formation was observed for the other samples .
• the total amount of aggregate formation may vary with each sample and is separated from the dimer species at high speeds. This is indicative of a slow sample-dependent aggregation process. Consequently, samples of protein in complex with dsRNA are purified by gel filtration immediately prior to conducting sedimentation equilibrium measurements (see Figure 3) .
• the purified dsRNA sample behave as an ideal solution with a single component during sedimentation.
• the estimated reduced molecular weight obtained by fitting the data to the single component model of NONLIN does not change with the loading concentration and/or speed. This enables the calculation of the specific volume of dsRNA based on the estimated reduced molecular weight using Eqn. 2 (see above) .
• the value obtained, V KNA 0.57 units, agrees well with the typical partial specific volume values of DNA (0.55-0.59 units) and RNA (0.47-0.55 units) (Ralston,
• Example 14 STOICHIOMETRY AND THERMODYNAMICS OF COMPLEX FORMATION BASED ON SEDIMENTATION EQUILIBRIUM: The association of NSIA (1-73) protein with dsRNA was studied using samples of purified NSIA (1-73 ) -dsRNA complex prepared as described above and validated as homogeneous by analytical gel filtration (Fig. 3A) . The stoichiometry of the complex was determined on the basis of data collected at 16000 rpm (Fig. 4A) . At this low speed the free dsRNA and NSlA(l-73) protein have a ⁇ value less than 0.5 (Eqn. 2).
• NSIA (1-73) Essentially complete NMR resonance assignments for the free NSIA (1-73) protein, required for the analysis of its complex with dsRNA by NMR, were determined. In all, a total of 65/71 (92%) assignable 15 N- 1 H N sites were assigned automatically using AUTOASSIGN (Zimmerman et al . (1997) J". Mol . Biol . 269, 592-610) . This automated analysis provided 71/78 H ⁇ , 68/73 C ⁇ , 64/71 C*, and 44/68 C ⁇ resonance assignments via intraresidue and/or sequential connectivities.
• Example 16 EPITOPE MAPPING BY CHEMICAL SHIFT PERTURBATION Monitoring of the titration of 15 N-enriched NSIA (1-73) was accomplished with the 16 bp dsRNA by collecting a series of 1 H N - 15 N HSQC spectra. The chemical shifts of both X H and 15 N nuclei were sensitive to their local electronic environment and therefore are used as probes for interactions between the labeled protein and unlabeled RNA. The strongest perturbation of the electronic environment are observed for the residues that either come into direct contact with RNA or that are involved in major conformational changes upon binding to RNA.
• Example 17 CIRCULAR DICHROISM (CD) SPECTROSCOPY: Circular dichroism provides a useful probe of the secondary structural elements and global conformational properties of nucleic acids and proteins.
• CD Circular DICHROISM
• the 180 to 240 nm region of the CD spectrum mainly reflects the class of backbone conformations (Johnson, W. C, Jr. (1990) Proteins 7:205-214). Changes in the CD spectrum observed above 250 nm upon forming protein-nucleic acid complexes arise primarily from changes in the nucleic acid secondary structure (Gray, D. M. (1996) Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 469-501) .
• the CD profiles of the four 16 bp duplexes are distinct and characteristic of their respective duplex types ( Figure 7, red traces) .
• the RR duplex featured a slight negative band at 295 nm, strong negative band at 210 nm, and a positive band near 260 nm, characteristic of the A- form dsRNA conformation (Figure 7A) (Hung et al . , (1994) Nucleic Acids Res .
• NS1A(1- 73)-dsRNA complex was used to avoid interference due to the presence of free dsRNA (see Figures 2 and 3) .
• the spectrum of free NSlA(l-73) was also shown (blue traces) .
• NS1A(1- 73) dominated the CD spectra in the 200-240 nm range (Qian et al . , (1995) RNA 1:948-956) , while structural information for the nucleic acid duplexes dominated the 250-320 nm region.
• the CD spectrum of the dsRNA-NSlA(l-73) (yellow) and a spectrum computed by simply adding the spectra of free NSIA (1-73) and free dsRNA (green) were also quite similar in the 200-240 nm region, indicating the NSIA (1-73) backbone structure was also not extensively altered by complex formation.
• NSIA (1-73) did not bind to the other duplexes
• the CD spectra for each RD, DR, and DD mixed with an equimolar amount of NSIA (1-73) were obtained as controls ( Figure 7B, C, D) .
• NSIA (1-73) binds to dsRNA, but not to dsDNA or the corresponding hetero duplexes
• NSIA (1-73) -dsRNA complex exhibits 1:1 stoichiometry and dissociation constant of ⁇ 1 ⁇ molar
• iii) symmetry-related antiparallel helices 2 and 2' play a central role in binding the dsRNA target
• the structures of the dsRNA and the NSIA (1-73) backbone structure are not significantly different in their complex form than they are in the corresponding unbound molecules .
• dsDNA is characterized by a B-type conformation with C2'-endo sugar puckering
• dsRNA adopts an A-form structure featuring C3'-endo sugars
• DNA/RNA hybrids exhibit an intermediate conformation between the A- and B-motifs
• NSlA(l-73) exhibits slow irreversible self-aggregation under the conditions used in these studies. This hypothesis was also supported by the observation of larger molecules in the sedimentation equilibrium experiments when using laser light scattering as the method of detection. In addition, in some of the gel filtration runs of free NSlA(l-73) samples, a leading peak was observed before the elution of NS1A(1- 73) dimer, indicating the possible aggregation. However, when purified NSIA (1-73) -dsRNA complex was reloaded to the gel filtration column, no excessive free dsRNA was observed. The sample behaves like a tight complex with Kd in ⁇ M range, consistent with the estimation from sedimentation equilibrium experiments.
• the 1:1 stoichiometry observed in Applicants invention precludes the possible protein-protein interactions and other cooperative effects, which can occur in a multiple-binding mode of a larger system.
• the apparent affinity is modulated by configurational entropy effects when there are many possible sites for non-specific binding (Wang et al., (1999) RNA 5 , 195-205.
• Wang et al (1999) have reported that NSlA(l-73) has a 10- fold higher affinity for a 140-bp dsRNA substrate than for a similar 55-bp dsRNA substrate.
• RNA-BINDING SITE OF NSIA (1-73) Recent alanine scanning mutagenesis studies on NSlA(l-73) (Wang et al . , 1999) revealed that binding to larger dsRNA fragments as well as U6 snRNA established that i) the protein must be a dimer in order to bind its target; and ii) only R 38 is absolutely required for RNA binding, though K 41 also plays a significant role.
• the RNA-binding epitope of NSlA(l-73) identified by chemical shift perturbation of 15N-1H HSQC resonances described above supports and extends these mutagenesis data.
• the binding site of NSlA(l-73) consists of antiparallel helices 2 and 2' with an Arg-rich surface.
• a hypothetical model that is consistent with our cumulative knowledge of the dsRNA binding properties of NSIA (1-73) features a symmetric structure with the binding surface of the protein spanning the minor groove of canonical A-form RNA ( Figure 8) .
• outward-directed arginine and lysine side chains of antiparallel helices 2 and 2' interact in a symmetric fashion with the antiparallel ⁇ phosphate backbones that form the edges of the major groove, while the surface ion pairs between helices 2 and 2' form hydrogen-bonded interactions with bases in the minor groove.
• the putative NSIA (1-73) :dsRNA model claimed by this application constitutes a novel mode of protein-dsRNA complex formation.
• Arginine-rich ⁇ -helical peptides such as that derived from the HIV- 1 Rev protein, are known to bind dsRNA through specific interactions in the major groove (Battiste et al . , (1996), Science 273:1547-1551.)
• the major groove in canonical A-form duplexes is too narrow and deep to accommodate even a single ⁇ - helix.
• Rev-protein-RNA complex binding of the Arg-rich helix results in severe distortions to the structure of the nucleic acid. Id.
• dsRNA binding domain (dsRBD) (Fierro-Monti & Matthews, 2000)
• the invention has applications in control of influenza virus growth, influenza virus chemistry, and antiviral therapy.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Virology (AREA)
• Medicinal Chemistry (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Priority Applications (5)

Applications Claiming Priority (4)

Related Child Applications (1)

Publications (2)

Family

ID=32314599

Family Applications (1)

Country Status (5)

Cited By (6)

Families Citing this family (7)

Family Cites Families (3)

• 2003
  • 2003-11-13 WO PCT/US2003/036292 patent/WO2004043404A2/en not_active Ceased
  • 2003-11-13 CA CA002505949A patent/CA2505949A1/en not_active Abandoned
  • 2003-11-13 AU AU2003290842A patent/AU2003290842A1/en not_active Abandoned
  • 2003-11-13 US US10/534,782 patent/US20080234175A1/en not_active Abandoned
  • 2003-11-13 JP JP2005507162A patent/JP2006506101A/ja active Pending
• 2009
  • 2009-09-11 US US12/557,927 patent/US20100081126A1/en not_active Abandoned

Also Published As

Similar Documents

Legal Events