WO2002042451A2 - Aspergillus fumigatus cofilin - Google Patents
Aspergillus fumigatus cofilin Download PDFInfo
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- WO2002042451A2 WO2002042451A2 PCT/US2001/044622 US0144622W WO0242451A2 WO 2002042451 A2 WO2002042451 A2 WO 2002042451A2 US 0144622 W US0144622 W US 0144622W WO 0242451 A2 WO0242451 A2 WO 0242451A2
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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
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
Definitions
- the present invention relates to a gene involved in actin dynamics in the yeast Aspergillus fumigatus and more particularly to the identification, isolation and cloning of this gene. This invention also relates to a method of using this gene to screen for compounds with antifungal activity.
- the fungal actin cytoskeleton consists of actin and diverse associated and regulatory proteins. It guides polarized growth required for extension of buds, hyphae, pseudo- hyphae and mating projections, underlies cell division, and plays a critical role in maintaining cell integrity. It is therefore essential for viability and pathogenesis. While actin is not a suitable target for anti-fungal therapeutics because fungal and human actins are 90% identical in amino acid sequence, leading to the potential cross- reactivity of inhibitors, other candidates include the numerous proteins that function in concert with actin.
- cofilin stands out as a particularly promising target, being 22% identical between yeast and humans, respectively. It plays a crucial role in actin regulation. More specifically, in cells actin rapidly interconverts between monomers and polymers. Only the polymer form of actin is known to be functional in vivo. Actin filaments have an inherent polarity because all subunits within the filament have the same orientation. The so-called barbed end elongates ten times more rapidly than the so-called pointed end. Each actin monomer can bind to one molecule of ATP. Hydrolysis of this ATP is slow on monomers, but is promoted by actin assembly.
- Cofilin alone causes only a modest increase in actin treadmilling. This is because when bound to ADP-actin, cofilin inhibits nucleotide exchange.
- Profilin binds to actin monomers and accelerates treadmilling synergistically with cofilin because it dissociates the cofilin- ADP actin complex and promotes nucleotide exchange on actin. The resulting profilin ATP-actin complex assembles readily at filament barbed ends.
- Cofilin (15-19 kDa) is readily expressed in bacteria and purified.
- Well-established assays measure monomer and filament binding, inhibition of actin nucleotide exchange, and filament disassembly stimulation. It represents an important component of the eukaryotic cell division cycle as well as, by virtue of its interaction with the actin cytoskeleton, overall cellular integrity, vesicular transport, and cell polarity.
- the endemic mycoses also constitute a risk for patients.
- At particular risk for such infections are those with AIDS, those having undergone bone marrow or organ transplants, those receiving chemotherapy and those who have had debilitating illness, severe injury, prolonged hospitalization, or long- term treatment with antibacterial drugs (NIAID fact sheet, 1996).
- the present invention concerns an isolated nucleic acid molecule encoding A. fumigatus cofilin.
- the A. fumigatus cofilin has a sequence that has greater than 70%, 80%, or 90%) amino acid sequence identity to SEQ ID NO:2 (see Fig. 2) as measured using a sequence comparison algorithm.
- the invention provides an isolated nucleic acid sequence encoding A. fumigatus cofilin, wherein the cofilin has a sequence that has greater than 70%, 80%, or 90%) amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm.
- the protein further specifically binds to polyclonal antibodies raised against SEQ ID NO:2.
- the nucleic acid encodes A. fumigatus cofilin, or a fragment thereof. In another embodiment, the nucleic acid encodes SEQ ID NO:2. In another embodiment, the nucleic acid has a nucleotide sequence of SEQ ID NO:l (see Fig. 1). In one aspect, the nucleic acid comprises a sequence which encodes an amino acid sequence which has greater than 70%> sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%>, more preferably greater than 95%> or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
- the nucleic acid comprises a sequence which has greater than 55 or 60%) sequence identity with SEQ ID NO:l (see Fig. 1), preferably greater than 70%, more preferably greater than 80%>, more preferably greater than 90 or 95% or, in another embodiment, has 98 to 100%> sequence identity with SEQ ID NO:l.
- the nucleic acid hybridizes under stringent conditions to a nucleic acid having a sequence or complementary sequence of SEQ ID NO:l.
- the invention provides an expression vector comprising a nucleic acid encoding A. fumigatus cofilin, wherein the protein has a sequence that has greater than 70, 80, or 90%> amino acid sequence identity to SEQ ID NO:2 as measured using a sequence comparison algorithm.
- the invention further provides a host cell transfected with the vector.
- the protein comprises an amino acid sequence of SEQ ID NO:2.
- the protein provided herein comprises an amino acid sequence which has greater than 70%> sequence identity with SEQ ID NO:2, preferably greater than 80%», more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100% sequence identity with SEQ ID NO:2.
- the invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof.
- modulators of the target protein including agents for the treatment of fungal disorders.
- the agents and compositions provided herein can be used in variety of applications which include the formulation of sprays, powders, and other compositions. Also provided herein are methods of treating fungal disorders. BRIEF DESCRIPTION OF THE DRAWINGS
- Figure 1 shows an embodiment of a nucleic acid sequence encoding A. fumigatus cofilin (SEQ ID NO: 1).
- Figure 2 shows the amino acid sequence of A. fumigatus cofilin (SEQ ID NO:2).
- Figure 3 shows the results of representative pyrene actin assays for A. fumigatus cofilin.
- the effect of cofilin on preassembled pyrene-actin filaments is monitored.
- Two cofilin activities binding to actin polymer and enhancing depolymerization — combine to reduce the relative fluorescence intensity.
- Addition of stoichiometric amounts of cofilin results in a 3-5 fold reduction in fluorescence intensity.
- any inhibitor will result in an increase in relative fluorescence intensity.
- the x-axes show cycle number (1 cycle/30 sec) and the y-axes reflect fluorescence in arbitrary units..
- A. fumigatus cofilin refers to proteins or polypeptides present in A. fumigatus that are capable of binding actin and have the sequence described below.
- an "anti- A. fumigatus cofilin” antibody is an antibody or antibody fragment that specifically binds a polypeptide encoded by the A. fumigatus cofilin gene, cDNA, or a subsequence thereof.
- Bioly active target protein refers to a target protein that has one or more of the target protein's biological activities, including, but not limited to ability to bind actin.
- Bio sample as used herein is a sample of biological tissue or fluid that contains a target protein or a fragment thereof or nucleic acid encoding a target protein or a fragment thereof.
- a biological sample comprises at least one cell or cell extract.
- Control region refers to a nucleotide sequence that regulates expression of a nucleic acid or any subunit thereof, including but not limited to any promoter, silencer, enhancer, splice site, transcriptional initiation element, transcriptional termination signal, polyadenylation signal, translational control element, translational start site, translational termination site, and message stability element. Such control regions may reside 5' or 3' to the coding region or in introns interrupting the coding region.
- a “comparison window' includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 25 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
- Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
- This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
- T is referred to as the neighborhood word score threshold (Altschul et al, supra.).
- HSPs high scoring sequence pairs
- M return score for a pair of matching residues; always > 0
- N penalty score for mismatching residues; always ⁇ 0).
- a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the default parameters of the BLAST programs are suitable.
- the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix.
- the TBLATN program uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM 62 scoring matrix, (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
- the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
- One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)) . which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
- a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
- PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a dendrogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). As a general rule, PileUp can align up to 500 sequences, with any single sequence in the final alignment restricted to a maximum length of 7,000 characters.
- the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. A series of such pairwise alignments that includes increasingly dissimilar sequences and clusters of sequences at each iteration produces the final alignment and a consensus sequence of conserved positions.
- a “diagnostic” as used herein is a compound, method, system, or device that assists in the identification and characterization of a health or disease state.
- the diagnostic can be used in standard assays as is known in the art.
- an "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
- the expression vector can be part of a • plasmid, virus, or nucleic acid fragment.
- the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
- High stringency conditions may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1%. sodium dodecyl sulfate at 50-68°C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll/0.1%> polyvinylpyrrolidone / 50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%> sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42
- High throughput screening refers to an assay which provides for multiple candidate agents or samples to be screened simultaneously.
- examples of such assays may include the use of microtiter plates which are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
- host cell is meant a cell that contains an expression vector and supports the replication or expression of the gene contained within the expression vector.
- Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa and the like, or plant cells. Both primary cells and cultured cell lines are included in this definition.
- hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
- Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
- stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
- T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.
- stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.05 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
- Stringent conditions may also be achieved with the addition of DNA duplex destabilizing agents such as formamide.
- nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
- the percent identity exists over a region of the sequence that is at least about 25 amino acids in length, more preferably over a region that is 50 or 100 amino acids in length.
- This definition also refers to the complement of a test sequence, provided that the test sequence has a designated or substantial identity to a reference sequence.
- the percent identity exists over a region of the sequence that is at least about 25 nucleotides in length, more preferably over a region that is 50 or 100 nucleotides in length.
- sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g.,. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Alternatively, when one includes such conservative substitutions in the comparison, a percent "similarity" can be noted, as opposed to a percent “identity”. Means for making this adjustment are well known to those of skill in the art.
- the scoring of conservative substitutions can be calculated according to, e.g., the algorithm of Meyers & Millers, Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
- Isogenic refers to strains that have identical genomes but differ in a single gene, be it resident on the chromosome or a plasmid.
- isolated refers to material that is substantially or essentially free from components which normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In an isolated gene, the nucleic acid of interest is separated from open reading frames which flank the gene of interest and encode proteins other than the protein of interest.
- purified denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85%> pure, more preferably at least 95%> pure, and most preferably at least 99% pure.
- a “label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
- useful labels include fluorescent proteins such as green, yellow, red or blue fluorescent proteins, radioisotopes such as 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the polypeptide of SEQ ID NO:2 can be made detectable, e.g., by incorporating a radio-label into the peptide, and used to detect antibodies specifically reactive with the peptide).
- a "labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker, or through ionic, van der Waals, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
- Modely stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %>SDS) less stringent than those described above.
- moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20%> formamide, 5 x SSC (150 mMNaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
- the skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
- Modulators refer to modulatory molecules identified using in vitro and in vivo assays for target protein activity. Such assays include binding activity such as actin binding activity. Samples or assays that are treated with a candidate agent at a test and control concentration. The control concentration can be zero. If there is a change in target protein activity between the two concentrations, this change indicates the identification of a modulator.
- a change in activity which can be an increase or decrease, is preferably a change of at least 20%) to 50%, more preferably by at least 50% to 75%, more preferably at least 75% to 100%), and more preferably 150% to 200%), and most preferably is a change of at least 2 to 10 fold compared to a control. Additionally, a change can be indicated by a change in binding specificity or substrate.
- Multi-copy plasmid refers to a plasmid having 10 to 30 copies present in a cell.
- nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
- degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);
- nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
- Nucleic acid probe or oligonucleotide is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
- a probe may include natural (i.e., A, G, C, or T) or modified bases.
- the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
- probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
- probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
- the probes are preferably directly labeled with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
- polypeptide polypeptide
- peptide protein
- amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes, i.e., the one-letter symbols recommended by the IUPAC-IUB.
- a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
- a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA box element.
- a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
- a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
- An “inducible” promoter is a promoter that is under environmental or developmental regulation.
- operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
- a nucleic acid expression control sequence such as a promoter, or array of transcription factor binding sites
- the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
- Specific binding moieties typically have an affinity for one another of at least 10 6 M "1 .
- Preferred antibodies for use in diagnostics or therapeutics often have high affinities such as 10 7 , 10 8 , 10 9 or 10 10 M "1 .
- Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
- antibodies raised to A. fumigatus cofilin with the amino acid sequence encoded in SEQ ID NO:2 can be selected to obtain only those antibodies that are specifically immunoreactive with A. fumigatus cofilin and not with other proteins, except for polymorphic variants, orthologs, alleles, and closely related homologues of A. fumigatus cofilin. This selection may be achieved by subtracting out antibodies that cross react with molecules, for example, such as C. elegans unc-104 and human KiflA by affinity chromatography.
- a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
- solid-phase ELIS A immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immimoreactivity).
- a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
- Test composition refers to a molecule or composition whose effect on the interaction between one or more cytoskeletal components it is desired to assay.
- the “test composition” can be any molecule or mixture of molecules, optionally in a carrier.
- a “therapeutic” as used herein refers to a compound which is believed to be capable of modulating the target protein in vivo which can have application in both human and animal disease. Modulation of the cytoskeletal system would be desirable in a number of conditions including, but not limited to antifungal agents and anti- inflammatory agents.
- Variant applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCT all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
- nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
- each degenerate codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
- target proteins of the present invention are amino acid sequence variants of wild-type target proteins. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the target protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Variant target protein fragments having up to about 100-150 amino acid residues may be prepared by in vitro synthesis using established techniques.
- Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the target protein amino acid sequence.
- the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics.
- Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to about 20 amino acids, although considerably longer insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases, deletions may be much longer.
- substitutions, deletions, and insertions or any combinations thereof may be used to arrive at a final derivative. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger characteristics may be tolerated in certain circumstances.
- the present invention provides for the first time a nucleic acid encoding A. fumigatus cofilin.
- This protein is a member of the cofilin superfamily. More specifically, the A. fumigatus cofilin sequence of Fig. 2 shares approximately 30-40%> identity to S. pombe cofilin; Candida albicans cofilin; and S. cerevisiae cofilin over 126 amino acids.
- A. fumigatus cofilin can be defined by having at least one or preferably more than one of the following functional and structural characteristics. Functionally, A. fumigatus cofilin will be capable of binding actin.
- the novel nucleotides sequences provided herein encode A. fumigatus cofilin or fragments thereof.
- the nucleic acids provided herein are defined by the novel proteins provided herein.
- the protein provided herein comprises an amino acid sequence which has one or more of the following characteristics: greater than 70% sequence identity with SEQ ID NO:2, preferably greater than 80%, more preferably greater than 90%, more preferably greater than 95% or, in another embodiment, has 98 to 100%> sequence identity with SEQ ID NO:2.
- the sequence identity can be the same percentages or slightly lower due to the degeneracy in the genetic code.
- the invention also includes fragments of the nucleotide sequence shown in Fig. 1 (SEQ ID NO:l) having at least 10, 15, 20, 25, 50, 100, 200 or 300 contiguous nucleotides from SEQ ID NO:l or a degenerate form thereof. Some such fragments can be used as hybridization probes or primers.
- reference to nucleotide sequences shown in the Figures or sequences can refer to the sequence shown, its perfect complement or a duplex of the two strands.
- Also included within the definition of the target proteins are amino acid sequence variants of wild-type target proteins.
- Portions of the A. fumigatus cofilin nucleotide sequence may be used to identify polymorphic variants, orthologs, alleles, and homologues of A. fumigatus cofilin.
- This identification can be made in vitro, e.g., under stringent hybridization conditions and sequencing, or by using the sequence information in a computer system for comparison with other nucleotide sequences. Sequence comparison can be performed using any of the sequence comparison algorithms discussed below, with PILEUP as a preferred algorithm.
- the target proteins can be made in a variety of ways, including both synthesis de novo and by expressing a nucleic acid encoding the protein.
- Target proteins of the present invention may also be modified in a way to form chimeric molecules comprising a fusion of a target protein with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
- the epitope tag is generally placed at the amino or carboxyl terminus of the target protein. Provision of the epitope tag enables the target protein to be readily detected, as well as readily purified by affinity purification.
- Various tag epitopes are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his- gly) tags; the influenza HA tag polypeptide and its antibody 12CA5 (see, Field et al. (1988) Mol. Cell. Biol.
- Tag polypeptides include the Flag-peptide (see, Hopp et al. (1988) BioTechnology 6:1204); the KT3 epitope peptide (see, Martine et al. (1992) Science, 255:192); tubulin epitope peptide (see, Skinner (1991) J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag (see, Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.
- any of the peptides provided herein can be routinely confirmed by the assays known in the art.
- polymorphic variants, alleles, and orthologs, homologues of A. fumigatus cofilin are confirmed by using actin assays as known in the art.
- A. fumigatus cofilin The isolation of biologically active A. fumigatus cofilin for the first time provides a means for assaying for modulators of this protein.
- Biologically active A. fumigatus cofilin is useful for identifying modulators of A. fumigatus cofilin or fragments thereof. In vivo assays and uses are provided herein as well. Also provided herein are methods of identifying candidate agents which bind to A. fumigatus cofilin and portions thereof.
- A. fumigatus cofilin includes at least 7, 10, 15, 20, 35, 50, 75, 100, or 125 contiguous amino acids from the sequence shown in Fig. 2 (SEQ ID NO:2). Some fragments contain fewer than 125, 100, 75, 50, or 35 contiguous amino acids from SEQ ID NO:2. For example, exemplary fragments include fragments having 15-20 amino acids or 35-50 amino acids. Nucleic acids encoding such fragments are also included in the invention.
- nucleic acids sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel elecfrophoresis, from sequenced nucleic acids, or from published DNA sequences.
- kb kilobases
- bp base pairs
- proteins sizes are given in kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel elecfrophoresis, from mass spectroscopy, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
- Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al, Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel elecfrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 225:137-149 (1983).
- sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace et al, Gene 16:21-26 (1981).
- nucleic acid sequences encoding A. fumigatus cofilin and related nucleic acid sequence homologs are cloned from cDNA and genomic DNA libraries or isolated using amplification techniques with oligonucleotide primers.
- expression libraries can be used to clone A. fumigatus cofilin and A. fumigatus cofilin homologues by detected expressed homologues immunologically with antisera or purified antibodies made against A. fumigatus cofilin that also recognize and selectively bind to the A. fumigatus cofilin homologue.
- amplification techniques using primers can be used to amplify and isolate A. fumigatus cofilin from DNA or RNA.
- Amplification techniques using degenerate primers can also be used to amplify and isolate A. fumigatus cofilin homologues.
- Amplification techniques using primers can also be used to isolate a nucleic acid encoding A. fumigatus cofilin. These primers can be used, e.g., to amplify a probe of several hundred nucleotides, which is then used to screen a library for full-length A. fumigatus cofilin.
- Appropriate primers and probes for identifying the gene encoding homologues of A. fumigatus cofilin in other species are generated from comparisons of the sequences provided herein.
- antibodies can be used to identify A. fumigatus cofilin homologues.
- antibodies made to the conserved actin-binding domain of A. fumigatus cofilin or to the whole protein are useful for identifying A. fumigatus cofilin homologues.
- the genomic library is usually contained in, for example, a modified bacteriophage lambda or E. coli plasmid (Sambrook et al. supra).
- the cDNA library may be contained in such vectors as ⁇ gtlO, ⁇ gtl 1, or lambda ZAP.
- the mRNA is converted into cDNA using reverse transcriptase, made double stranded, ligated into a recombinant vector, and introduced into a recombinant host for propagation, screening and cloning. Methods for making and screening cDNA libraries are well known (see, e.g., Gubler & Hoffman, Gene 25: 263-269); Sambrook et al., supra; Ausubel et al., supra).
- An alternative method of isolating A. fumigatus cofilin nucleic acid and its homologues combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A guide to Methods and Applications (Innis et al, eds. 1990)).
- Methods such as polymerase chain reaction and ligase chain reaction can be used to amplify nucleic acid sequences of A. fumigatus cofilin directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
- Degenerate oligonucleotides can be designed to amplify A.
- fumigatus cofilin homologues using the sequences provided herein. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of A. fumigatus cofilin encoding mRNA in physiological samples, for nucleic sequencing or for other purposes. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
- Gene expression of A. fumigatus cofilin can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A + RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, quantitative PCR, and the like.
- Synthetic oligonucleotides can be used to construct recombinant A. fumigatus cofilin genes for use as probes or for expression of protein. This method is performed using a series of overlapping oligonucleotides usually 40-120 bp in length, representing both the sense and nonsense strands of the gene. These DNA fragments are then annealed, ligated and cloned. Alternatively, amplification techniques can be used with precise primers to amplify a specific subsequence of the A. fumigatus cofilin gene. The specific subsequence is then ligated into an expression vector.
- the gene for A. fumigatus cofilin is typically cloned into intermediate vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression.
- the intermediate vectors are typically prokaryote vectors or shuttle vectors.
- a cloned gene such as those cDNAs encoding ⁇ . fumigatus cofilin
- Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al.
- Bacterial expression systems for expressing the A. fumigatus cofilin protein are available in, e.g., E.
- Kits for such expression systems are commercially available.
- Eukaryotic expression systems are well known in the art and are also commercially available.
- the promoter used to direct expression of a heterologous nucleic acid depends on the particular application.
- the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
- the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the A. fumigatus cofilin encoding nucleic acid in host cells.
- a typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding A. fumigatus cofilin and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
- the nucleic acid sequence encoding A. fumigatus cofilin may typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell.
- Such signal peptides would include, among others, the signal peptides from tissue plasminogen activator, insulin, and neuron growth factor, and juvenile hormone esterase of Heliothis virescens. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
- the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
- the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
- the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23, and fusion expression systems such as GST and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc or histidine tags.
- Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, cytomegalo virus vectors, papilloma virus vectors, and vectors derived from Epstein Bar virus.
- exemplary eukaryotic vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, CMV promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
- Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
- markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
- high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a A. fumigatus cofilm encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
- the elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
- the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
- the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary. Standard transfection or transformation methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of A.
- fumigatus cofilin protein which are then purified using standard techniques (see, e.g., Colley et al, J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher ed., 1990)).
- Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact, 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology, 101:347-362 (Wu et al, eds, 1983). Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used.
- the transfected cells are cultured under conditions favoring expression of A. fumigatus cofilin, which is recovered from the culture using standard techniques identified below.
- A. fumigatus cofilin can be purified for use in functional assays.
- the target proteins are purified for use in the assays to provide substantially pure samples.
- the target proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
- Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, chromatofocussing, selective precipitation with such substances as ammonium sulfate; and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra).
- the target protein can be purified using a standard anti-target antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
- a preferred method of purification is use of Ni-NTA agarose (Qiagen) in combination with polyhistine-tagged proteins.
- the expressed protein can be purified by standard chromatographic procedures to yield a purified, biochemically active protein.
- the activity of any of the peptides provided herein can be routinely confirmed by the assays known in the art.
- Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is a preferred method of expression.
- Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein. Alternatively, it is possible to purify A. fumigatus cofilin from bacteria periplasm. After A . fumigatus cofilin is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art. To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet.
- the pellet is resuspended in a buffer containing 20% sucrose.
- the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO 4 and kept in an ice bath for approximately 10 minutes.
- the cell suspension is centrifuged and the supernatant decanted and saved.
- the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
- an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest.
- the preferred salt is ammonium sulfate.
- Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
- a typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins.
- the precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest.
- the precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration.
- Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.
- the molecular weight of A. fumigatus cofilin can be used to isolated it from proteins of greater and lesser size using ultrafilfration through membranes of different pore size (for example, Amicon or Millipore membranes).
- membranes of different pore size for example, Amicon or Millipore membranes.
- the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest.
- the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
- the recombinant protein will pass through the membrane into the filtrate.
- the filtrate can then be chromatographed as described below.
- immunoassays can be used to qualitatively or quantitatively analyze A. fumigatus cofilin. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).
- A. Antibodies to A. fumigatus cofilin Methods of producing polyclonal and monoclonal antibodies that react specifically with A. fumigatus cofilin are known to those of skill in the art (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler &
- Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al, Science 246:1275-1281 (1989); Ward et al, Nature 341 :544-546 (1989)).
- Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861 (incorporated by reference for all purposes). Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outersurfaces. Antibodies are usually displayed as Fv or Fab fragments.
- Phage displaying antibodies with a desired specificity are selected by affinity enrichment to A. fumigatus cofilin or fragments thereof.
- Human antibodies against A. fumigatus cofilin can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991) (each of which is incorporated by reference in its entirety for all purposes). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody.
- Human polyclonal antibodies can also be provided in the form of serum from human immunized with an immunogenic agent.
- such polyclonal antibodies can be concentrated by affinity purification using A. fumigatus cofilin as an affinity reagent.
- a number of A. fumigatus cofilin comprising immunogens may be used to produce antibodies specifically reactive with A. fumigatus cofilin.
- recombinant A. fumigatus cofilin or a antigenic fragment thereof is isolated as described herein.
- Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above.
- Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
- a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
- Naturally occurring protein may also be used either in pure or impure form.
- the product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.
- mice e.g., BALB/C mice
- rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
- the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to A. fumigatus cofilin.
- blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see Harlow & Lane, supra).
- Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al, Science 246:1275-1281 (1989).
- Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
- an immunoassay for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
- polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against m-A. fumigatus cofilin proteins or even other homologous proteins from other organisms
- Specific polyclonal antisera and monoclonal antibodies will usually bind with a K d of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
- A. fumigatus cofilin can be detected by a variety of immunoassay methods.
- immunoassay methods see Basic and Clinical Immunology (Stites & Terr eds., 7th ed. 1991).
- the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio ed., 1980); and Harlow & Lane, supra.
- Antibodies can be used for treatment or to identify the presence of A. fumigatus cofilin having the sequence identity characteristics as described herein. Additionally, antibodies can be used to identify modulators of the interaction between the antibody and A. fumigatus cofilin as further described below. While the following discussion is directed toward the use of antibodies in the use of binding assays, it is understood that the same general assay formats such as those described for "non-competitive" or “competitive” assays can be used with any compound which binds to A. fumigatus cofilin such as microtubules or the compounds described in Serial No. 60/070,772.
- A. fumigatus cofilin is detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
- immunological binding assays see also Methods in Cell Biology Volume 37: Antibodies in Cell Biology (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).
- Immunological binding assays typically use an antibody that specifically binds to a protein or antigen of choice (in this case the A.
- the antibody e.g., snti-A. fumigatus cofilin
- the antibody may be produced by any of a number of means well known to those of skill in the art and as described above.
- Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen.
- the labeling agent may itself be one of the moieties comprising the antibody/antigen complex.
- the labeling agent may be a labeled A. fumigatus cofilin polypeptide or a labeled anti- fumigatus cofilin antibody.
- the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the tibody/A. fumigatus cofilin complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived).
- Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent.
- the labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin.
- detectable moieties are well known to those skilled in the art.
- incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 4°C to 40°C.
- Immunoassays for detecting A. fumigatus cofilin in samples may be either competitive or noncompetitive.
- Noncompetitive immunoassays are assays in which the amount of antigen is directly measured.
- the anti- fumigatus cofilin antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture A. fumigatus cofilin present in the test sample. A. fumigatus cofilin is thus immobilized is then bound by a labeling agent, such as a second A. fumigatus cofilin antibody bearing a label.
- the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
- the second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety.
- the amount of A. fumigatus cofilin present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) A. fumigatus cofilin displaced (competed away) from an an ⁇ -A. fumigatus cofilin antibody by the unknown A. fumigatus cofilin present in a sample.
- a known amount of A. fumigatus cofilin is added to a sample and the sample is then contacted with an antibody that specifically binds to A. fumigatus cofilin.
- the amount of exogenous A. fumigatus cofilin bound to the antibody is inversely proportional to the concentration of A. fumigatus cofilin present in the sample.
- the antibody is immobilized on a solid substrate.
- the amount of A. fumigatus cofilin bound to the antibody may be determined either by measuring the amount of A. fumigatus cofilin present in a A. fumigatus cofilin antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein.
- the amount of A. fumigatus cofilin may be detected by providing a labeled A. fumigatus cofilin molecule.
- Immunoassays in the competitive binding format can also be used for crossreactivity determinations.
- a protein at least partially encoded by SEQ ID NO:2 can be immobilized to a solid support.
- Proteins e.g., other fungal or vertebrate cofilins
- the ability of the, added proteins to compete for binding of the antisera to the immobilized protein is compared to the ability of A. fumigatus cofilin encoded by SEQ ID NO:2 to compete with itself.
- the percent crossreactivity for the above proteins is calculated, using standard calculations.
- Those antisera with less than 10% crossreactivity with each of the added proteins listed above are selected and pooled.
- the cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e.g., distantly related homologues.
- the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps the protein of this invention, to the immunogen protein (i.e., A. fumigatus cofilin of SEQ ID NO:2).
- the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount of the protein encoded by SEQ ID NO:2 that is required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to a A. fumigatus cofilin immunogen.
- the technique generally comprises separating sample proteins by gel elecfrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind A. fumigatus cofilin.
- a suitable solid support such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter
- the ⁇ -A. fumigatus cofilin antibodies specifically bind to the A. fumigatus cofilin on the solid support.
- These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the ax ⁇ i-A. fumigatus cofilin antibodies.
- LOA liposome immunoassays
- the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
- the detectable group can be any material having a detectable physical or chemical property.
- Such detectable labels have been well- developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention.
- a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
- Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., H, I, S, C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.) or other labels that can be detected by mass spectroscopy, NMR spectroscopy, or other analytical means known in the art.
- magnetic beads e.g., DYNABEADSTM
- fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
- radiolabels e.g., H, I, S
- the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.
- Non-radioactive labels are often attached by indirect means.
- a ligand molecule e.g., biotin
- the ligand then binds to another molecules (e.g., streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
- the ligands and their targets can be used in any suitable combination with antibodies that recognize A. fumigatus cofilin, or secondary antibodies that recognize anti--4. fumigatus cofilin.
- the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
- Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidases, particularly peroxidases.
- Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
- Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
- Means of detecting labels are well known to those of skill in the art.
- the label is a radioactive label
- means for detection include a scintillation counter or photographic film as in autoradiography.
- the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence.
- the fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
- enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
- simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
- agglutination assays can be used to detect the presence of the target antibodies.
- antigen-coated particles are agglutinated by samples comprising the target antibodies.
- none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
- the present invention provides methods to identify candidate agents that bind to a target protein or act as a modulator of the binding characteristics or biological activity of a target protein.
- the method is performed in plurality simultaneously.
- the method can be performed at the same time on multiple assay mixtures in a multi-well screening plate.
- the invention provides a high throughput screening system.
- the methods of the present invention can be used to identify inhibitors of cofilin that have antifungal activity and that therefore provide the starting point for maturing a candidate compound for clinical development. These methods can be used to identify inhibitors that show biochemical inhibition of bacterially expressed Aspergillus fumigatus and Candida albicans cofilin in vitro and counterscreened against human cofilin.
- the method also comprises the further step of measuring the toxicity of the compounds of interest on mammalian cells.
- A. fumigatus cofilin was cloned by amplifying fragments of Aspergillus nidulans ESTs, then screening an Aspergillus fumigatus cDNA library at low stringency.
- the protein was expressed in E. coli, and was purified to homogeneity. Using this method, tens to hundreds of milligrams of purified protein per liter of culture, can be obtained. The protein exhibited the biochemical activities previously demonstrated for homologs isolated from other organisms.
- Candida albicans proteins were obtained by cloning the respective genes using publically available sequence information, designing appropriate primers, performing PCR, and expressing these cloned genes as described above.
- Candida albicans cofilin was purified to homogeneity as described for A. fumigatus cofilin. See, e.g., PCT Publication WO 97/31104, which is incorporated herein by reference for all purposes.
- cofilin Upon addition of cofilin to filamentous pyrene-actin, two well-characterized biochemical activities can be monitored. First, cofilin binds to the filaments and partially quenches the pyrene fluorescence. Second, it induces filament disassembly, further decreasing pyrene fluorescence. The decrease in fluorescence that results when cofilin is added to filamentous actin is monitored ( Figure 3). An inhibitor of the interaction of cofilin with pyrene-actin will result in an increase in fluorescence.
- Chicken skeletal actin was used because it can be purified in quantities needed for high throughput screening; it is 90% identical in amino acid sequence to fungal actin, and it can assemble into copolymers with fungal actin. Pyrene-labeled skeletal actin has been prepared and characterized as to its polymerization properties as well as for its interaction with fungal cofilin. Accordingly, the present invention provides for a high throughput method for the identification of small molecules that inhibit or modulate cofilin interactions with actin.
- Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons.
- Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
- the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
- Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
- Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.
- the candidate agents are organic chemical moieties, a wide variety of which are available in the literature.
- Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion. Such compounds include polypeptides, proteins, nucleic acids, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates.
- Compounds to be screened can also be obtained from governmental or private sources, including, e.g., the National Cancer Institute's (NCI) Natural Product Repository, Bethesda, MD, the NCI Open Synthetic Compound Collection, Bethesda, MD, NCI's Developmental Therapeutics Program, or the like.
- Compounds to be screened can also be obtained from governmental or private sources, including, e.g., the National Cancer Institute's (NCI) Natural Product Repository, Bethesda, MD, the NCI Open Synthetic Compound Collection, Bethesda, MD, NCI's Developmental Therapeutics Program, or the like.
- Fungal infections which can be inhibited or treated with compositions provided herein include but are not limited to: Candidiasis including but not limited to onchomycosis, chronic mucocutaneous candidiasis, oral candidiasis, epiglottistis, esophagitis, gastrointestinal infections, genitourinary infections, for example, caused by any Candida species, including but not limited to Candida albicans, Candida tropicalis, Candida (Torulopsis) glabrata, Candida parapsilosis, Candida lusitaneae, Candida rugosa and Candida pseudotropicalis; Aspergillosis including but not limited to granulocytopenia caused for example, by, Aspergillus spp. including but not limited to A.
- Zygomycosis including but not limited to pulmonary, sinus and rhinocerebral infections caused by, for example, zygomycetes such as Mucor.
- Rhizopus spp. Absidia, Rhizomucor, Cunningamella, Saksenaea, Basidobolus and Conidobolus
- Cryptococcosis including but not limited to infections of the central nervous system - meningitis and infections of the respiratory tract caused by, for example, Cryptococcus neoformans
- Trichosporonosis caused by, for example, Trichosporon beigelii
- Pseudallescheriasis caused by, for example, Pseudallescheria boydii
- Fusarium infection caused by, for example, Fusarium such as Fusarium solani, Fusarium moniliforme and Fusarium proliferatum
- other infections such as those caused by, for example, Penicillium spp.
- Treating "fungal infections” as used herein refers to the treatment of conditions resulting from fungal infections. Therefore, one may treat, for example, pneumonia, nasopharnygeal infections, disseminated infections and other conditions listed above and known in the art by using the compositions provided herein. In preferred embodiments, treatments and sanitization of areas with the compositions provided herein are provided to immunocompromised patients or areas where there are such patients. Wherein it is desired to identify the particular fungi resulting in the infection, techniques known in the art may be used, see, for example, Musial et al., Clin. Microbiol. Rev., American Society for Microbiology, 349-64 (1998), incorporated herein by reference.
- compositions of the invention are administered to cells.
- administered herein is meant administration of a therapeutically effective dose of the candidate agents of the invention to a cell either in cell culture or in a patient.
- therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
- cells herein is meant almost any cell in which mitosis or meiosis or in which cell morphology (e.g., filamentous hyphal growth, etc.) can be altered.
- a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications.
- the patient is a mammal, and in the most preferred embodiment the patient is human.
- Candidate agents having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%. The agents maybe administered alone or in combination with other treatments, i.e., radiation, or other chemotherapeutic agents.
- the pharmaceutical compositions are in a water soluble form, such as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
- compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
- compositions containing the therapeutically- active compounds can be used to make up compositions containing the therapeutically- active compounds.
- Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
- the pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.
- the administration of the candidate agents of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, intrathecally, vaginally, rectally, or infraocularly.
- the candidate agents may be directly applied as a solution or spray.
- a diagnostic as used herein is a compound or method that assists in the identification and characterization of a health or disease state in humans or other animals. More specifically, antibodies which specifically bind A.
- fumigatus cofilin may be used for the diagnosis of disorders characterized by expression of A. fumigatus cofilin or in assays to monitor patients being treated with A. fumigatus cofilin, or agonists, antagonists, or inhibitors of A. fumigatus cofilin. Diagnostic assays include methods which utilize the antibody and a label to detect A. fumigatus cofilin in human body fluids or in extracts of cells or tissues.
- the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecules. A wide variety of reporter molecules are known in the art and may be used.
- a variety of protocols for measuring A. fumigatus cofilin including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of A. fumigatus cofilin expression.
- Normal or standard values for A. fumigatus cofilin expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to A. fumigatus cofilin under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of A. fumigatus cofilin expressed in subject, control, and disease samples from biopsied tissues can be compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
- the polynucleotides encoding A. fumigatus cofilin may be used for diagnostic purposes.
- the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
- the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which A. fumigatus cofilin may be correlated with disease.
- the diagnostic assay may be used to determine absence, presence, and excess expression of A. fumigatus cofilin, and to monitor regulation of A. fumigatus cofilin levels during therapeutic intervention.
- hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences encoding A. fumigatus cofilin or closely related molecules may be used.
- the specificity of the probe whether it's made from a highly specific region or from a less specific region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding A. fumigatus cofilin, allelic variants, or related sequences.
- Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the A. fumigatus cofilin encoding sequences.
- the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:l or from genomic sequences including promoters, enhancers, and introns of ' the A. fumigatus cofilin gene.
- Means for producing specific hybridization probes for DNAs encoding A. fumigatus cofilin include the cloning of polynucleotide sequences encoding A. fumigatus cofilin or derivatives thereof into vectors for the production of mRNA probes.
- Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
- Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as P or S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems and the like.
- reporter groups for example, by radionuclides such as P or S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems and the like.
- the nucleotide sequences encoding A. fumigatus cofilin may be useful in assays that detect the presence of associated disorders.
- the nucleotide sequences encoding A. fumigatus cofilin may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding A. fumigatus cofilin in the sample indicates the presence of the associated disorder.
- Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
- Oligomers designed from the sequences encoding A. fumigatus cofilin may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro based on SEQ ID NO: 1. Oligomers will preferably contain a fragment of a polynucleotide encoding A. fumigatus cofilin, or a fragment of a polynucleotide complementary to the polynucleotide encoding A. fumigatus cofilin, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences. Quantitative PCR as practiced by those of skill in the art can be used to detect relative levels of A. fumigatus mRNA in a test sample.
- Methods which may be used to quantitate the expression of A. fumigatus cofilin include radiolabeling or biotinylating nucleotides, co amplification of a control nucleic acid, and interpolating results from standard curves.
- the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometer or colorimetric response gives rapid quantitation.
- a diagnostic as used herein is a compound or method that assists in the identification and characterization of a health or disease state in humans or other animals.
- kits for screening for modulators of the target protein can be prepared from readily available materials and reagents.
- such kits can comprise any one or more of the following materials: biologically active target protein, reaction tubes, and instructions for testing activity of the target protein.
- the kit contains biologically active target protein.
- kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. For example, the kit can be tailored for actin binding assays.
- Na 2 ATP is at 100 mM stock (pH 8.0) and MgCl 2 at 1M (pH 8.0)
- Solid KCL is at 0.6 M (45 g per L of buffer) and stir at 4°C for 30 mins.
- Add a small amount of cold buffer A (about 25 -30 ml) to each bottle to soften the pellets. Resuspend vigorously by shaking the capped bottles by hand.
- Buffers Lysis Buffer: lOmM Tris/HCl; 0.5mM EDTA; ImM DTT; ImM PMSF; pH 7.5
- Wash Buffer lOmM sodium phosphate; 2M ammonium phosphate; 0.5mM EDTA; ImM DTT; ImM PMSF; pH 7.5
- Elution Buffer lOmM sodium phosphate; IM ammonium phosphate; 0.5mM EDTA; ImM DTT;.
- GF Buffer lOmM Tris/HCl; 0.5mM EDTA; 50mM KCL; ImM DTT; ImM PMSF; pH 7.5
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2002227006A AU2002227006A1 (en) | 2000-11-27 | 2001-11-27 | Aspergillus fumigatus cofilin |
EP01995963A EP1343883A2 (en) | 2000-11-27 | 2001-11-27 | Aspergillus fumigatus cofilin |
CA002439184A CA2439184A1 (en) | 2000-11-27 | 2001-11-27 | Aspergillus fumigatus cofilin |
JP2002545156A JP2004522426A (en) | 2000-11-27 | 2001-11-27 | Cofilin of Aspergillus fumigatus |
US10/432,554 US20040138440A1 (en) | 2001-11-27 | 2001-11-27 | Aspergillus fumigatus cofilin |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US72283800A | 2000-11-27 | 2000-11-27 | |
US09/722,838 | 2000-11-27 |
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WO2002042451A2 true WO2002042451A2 (en) | 2002-05-30 |
WO2002042451A3 WO2002042451A3 (en) | 2003-02-27 |
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PCT/US2001/044622 WO2002042451A2 (en) | 2000-11-27 | 2001-11-27 | Aspergillus fumigatus cofilin |
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EP (1) | EP1343883A2 (en) |
JP (1) | JP2004522426A (en) |
AU (1) | AU2002227006A1 (en) |
CA (1) | CA2439184A1 (en) |
WO (1) | WO2002042451A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2004035092A2 (en) * | 2002-10-15 | 2004-04-29 | Oxford Glycosciences (Uk) Ltd | Use of an agent modulating cofilin expresion or activity for the treatment of inflammatory diseases |
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JP5774816B2 (en) * | 2007-05-28 | 2015-09-09 | 有限会社梅田事務所 | Method for producing phospholipid-containing functional material and method for producing plasmalogen-type glycerophospholipid |
-
2001
- 2001-11-27 JP JP2002545156A patent/JP2004522426A/en active Pending
- 2001-11-27 EP EP01995963A patent/EP1343883A2/en not_active Withdrawn
- 2001-11-27 CA CA002439184A patent/CA2439184A1/en not_active Abandoned
- 2001-11-27 AU AU2002227006A patent/AU2002227006A1/en not_active Abandoned
- 2001-11-27 WO PCT/US2001/044622 patent/WO2002042451A2/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
AIZAWA HIROYUKI ET AL.: "Identification, characterization, and intracellular distribution of cofilin in Dictyostelium discoideum " JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 18, 1995, pages 10923-10932, XP002215593 MD US * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004035092A2 (en) * | 2002-10-15 | 2004-04-29 | Oxford Glycosciences (Uk) Ltd | Use of an agent modulating cofilin expresion or activity for the treatment of inflammatory diseases |
WO2004035092A3 (en) * | 2002-10-15 | 2004-06-10 | Oxford Glycosciences Uk Ltd | Use of an agent modulating cofilin expresion or activity for the treatment of inflammatory diseases |
Also Published As
Publication number | Publication date |
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CA2439184A1 (en) | 2002-05-30 |
WO2002042451A3 (en) | 2003-02-27 |
AU2002227006A1 (en) | 2002-06-03 |
EP1343883A2 (en) | 2003-09-17 |
JP2004522426A (en) | 2004-07-29 |
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