JP2017074046A - Means and method for mediating protein interference - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inhibiting the function of a target protein.SOLUTION: The present invention provides a non-naturally occurring molecule that is able to down-regulate a biological function of a protein. Here, the non-naturally occurring molecule comprises at least one β-aggregation region exposed in the environment, which is derived from the protein that can be down-regulated. The β-aggregation region consists of five neighboring amino acids present in the protein. The non-naturally occurring molecule is fused to a moiety that prevents aggregation of the β-aggregation region. The non-naturally occurring molecule comprises part A and part B. Here, part A comprises a soluble region selected from a peptide, a protein domain, or a protein, which prevents the aggregation of part B; and part B comprises at least one β-aggregation region. Here, the region is derived from the protein that can be down-regulated.SELECTED DRAWING: None

Description

  The present invention belongs to the field of functional proteomics, in particular to the field of protein aggregation. The present invention discloses a method for inhibiting the function of a target protein and uses a non-natural, user-designed molecule called an interferor, which is specific for the target protein. And causes aggregation when contacted with the target protein. The present invention also discloses the interferor molecule and its use in therapeutic applications.

Background Information The information on the genome scale has increased, and biology has entered a vibrant era. As genome sequencing and high-throughput functional genomics approaches are being created every day, researchers need new ways to extract biological information. Functional genomes are making rapid progress, particularly with respect to assigning biological significance to genomic data. The information encoded in the genome consists of genes whose protein products mediate most of the functions of the organism and control elements. Recently, non-coding RNAs have also been identified to play an important role in the regulatory process, but proteins have been considered the most important effector in the cell.

  Several important biological questions are at the heart of ongoing genome projects and relate to all cellular organisms, from bacteria to humans. One challenge is to understand how the genes encoded in the genome function and interact to produce a complex biological system. A related challenge is determining the function of all sequence elements in the genome. A functional genomic toolbox will tell you when a gene is expressed, where its products are localized, what other gene products it interacts with, and what phenotype is when the gene is mutated Several systematic approaches are possible that can answer some basic questions about the majority of genes in the genome, such as what happens. Mutant phenotypic analysis is a powerful approach to determine gene function. Gene function can be altered through gene deletion, insertional mutation and RNA interference (RNAi). RNAi is a relatively recent development to reduce gene expression. It follows the report of gene silencing in plants and other model organisms, and it has been reported that adding double-stranded RNA (dsRNA) to cells often inhibits gene function in a sequence-specific manner. Based on observations of C. elegans. In many cases, the level of loss of functionality cannot be reasonably controlled, is incomplete, the level of specificity cannot be fully predicted, and RNAi does not function in some organisms (eg, yeast Candida・ Candida albicans).

  Functional genomics has changed the way biology works, and the field details the complexities underlying biological systems such as gene regulatory complex networks, protein interactions, and the biochemical reactions that form cells. It is clear that we are still in the early stages of writing. There is a clear need for the development of innovative technologies, especially in the field of functional proteomics, to facilitate discovery and maximize the potential of functional genomics supplements. Instead of targeting the mRNA that translates the protein or manipulating the gene that encodes it, you have the flexibility to be able to directly target the biological function of a specific extracellular or intracellular protein Would be desirable.

The conversion of normally soluble proteins into conformationalally modified insoluble proteins is, for example, amyloid beta peptide in Alzheimer's disease and cerebral amyloid angiopathy, alpha synuclein deposition in Parkinson's disease Lewy bodies, Creutzfeldt-Jakob disease It is thought to be a process that causes various diseases, such as prion in urine, superoxide dismutase in amyotrophic lateral sclerosis, and tau in neurofibrillary tangles in frontotemporal dementia and Pick disease. To date, protein aggregation has been primarily studied as an undesired etiology, and it is widely accepted that crossbeta-mediated aggregation is the most frequently occurring biologically relevant aggregation mechanism ² . Cross-beta aggregation is a term used to indicate that aggregation is nucleated through the formation of an intramolecular beta sheet where each aggregated molecule gives the same chain that usually contains at least three adjacent amino acids. There is currently abundant data showing that the individual chains interact to form an intramolecular beta sheet and that this structure forms the skeleton of an aggregate ^3,4 . The self-association region of the target protein can be determined by a computer program such as TANGO ⁶ developed for predicting the aggregation tendency of peptides and proteins. One particular form of aggregation, highly aligned amyloid fibrils, is already under exploration in the technical field for potential use in materials science ⁵ . International Publication No. 03102187 (Scegen, Pty Ltd) promotes the activity of a molecule by fusing the molecule with a membrane translocation sequence, and the resulting chimeric molecule self-aggregates into a high molecular weight aggregate. The method of doing is disclosed. US Patent 20050026165 (Arete Associates) discloses the use of conformational peptides capable of interacting with the beta sheet conformation of insoluble proteins such as prions as a diagnostic tool for prion diseases.

SUMMARY OF THE INVENTION The present invention relates to techniques for controlled inducible protein aggregation of specific target proteins. The present invention also provides a de novo designed molecule, designated herein as an interferor molecule, comprising at least one aggregation region, said aggregation region being derived from a target protein . In a preferred embodiment, the interferor molecule comprises at least one self-association region that fuses with a moiety that prevents aggregation of the self-association region. When contact occurs between a selected target protein and a specifically designed interferor molecule, specific coaggregation occurs between the target and the interferor, resulting in biological Functional knockout or down-regulation of function occurs. This protein knockdown is conditioned on the presence of aggregates induced by the presence of interferor molecules. As a further advantage, the intensity of protein interference can be experimentally controlled by changing the number of aggregated regions in the interferor molecule. The present invention not only provides an efficient tool to down-regulate the biological function of specific intracellular and extracellular proteins, but also has important therapeutic, agricultural and diagnostic applications.

Protein interference in E. coli using recombinant expression of four interferor constructs targeting target specific enzymes involved in amino acid biosynthesis. The B part of each interferor molecule consists of three synthetic self-association sequences divided by two amino acid linkers and one specific self-association region from the enzyme. The self-association region (synthetic and specific) couples with the protein NusA which acts as a moiety that prevents aggregation of the self-association region (ie, the A portion of the interferor molecule). S. cerevisiae cells harboring an endogenous wild type copy of URA3 were transformed with a free plasmid, a plasmid with only an aggregator sequence or a plasmid with an aggregator-Ura3 fusion construct. Cells were cultured overnight in glucose-containing medium, washed, and then plated on medium containing either glucose (left) or galactose (right). Dilute 10-fold, of which 5 μl was plated (maximum concentration OD ₆₀₀ = 1). C. albicans cells with two endogenous wild-type copies of TUP1 were transformed with the empty plasmid and the plasmid with the interferer-Tup1 fusion construct. Cells were cultured overnight in glucose-containing medium, washed, and then 20 colonies were plated in medium containing either glucose (left) or casamino acid (right). The upper panel shows the empty plasmid and the lower panel shows the interferor construct ("+ agreg.-TUP1" means the interferer-Tup1 construct). Photos were taken 4 days after incubation.

Objects and Detailed Description of the Invention In the present invention, we have developed a process to down-regulate the biological function of a protein using an interferor molecule specific for the target protein. Upon contact with the target protein, coaggregation occurs between the interferor molecule and the target. Aggregation pulls the target out of its soluble environment, resulting in functional knockdown of the target protein.

  Thus, in one embodiment, the invention provides a method of down-regulating a biological function of a protein comprising contacting the protein with a non-natural molecule comprising at least one self-association region isolated from the protein. To do.

  In another embodiment, the present invention provides a method for down-regulating a biological function of a protein comprising contacting the protein with a non-natural molecule comprising at least one self-association region isolated from the protein. To do.

In yet another embodiment, the present invention provides a method of down-regulating a biological function of a protein comprising contacting the protein with a non-natural molecule comprising at least one self-association region isolated from the protein. Wherein the self-association domain is fused to a portion that prevents aggregation of the self-association region.
In yet another embodiment, the present invention provides a method of down-regulating a biological function of a protein comprising contacting the protein with a non-natural molecule comprising at least one self-association region isolated from the protein. Wherein the self-association domain is fused to a portion that prevents aggregation of the self-association region.

  In yet another embodiment, the present invention provides a method of down-regulating a biological function of a protein comprising contacting said protein with a non-natural molecule comprising part A and part B, wherein I) part A is a peptide, or a protein domain or agarose bead that prevents aggregation of part B, and ii) part B comprises at least one self-association region consisting of at least three adjacent amino acids, and said region Is isolated from the protein that down-regulates function, and a linker is optionally present between part A and part B.

  In yet another embodiment, the present invention provides a method of down-regulating protein function comprising contacting said protein with a non-natural molecule comprising part A and part B, wherein i) Part A is a protein domain or agarose bead that prevents aggregation of peptide or part B, whereby part B is in direct contact with the solvent in which the molecule and the protein are present, and ii) part B is at least three adjacent At least one self-association region consisting of amino acids that are isolated from the protein that down-regulates function, and a linker is optionally present between the A and B portions.

  In another embodiment, part B of the non-natural molecule comprises at least two self-association regions, and at least one of said regions is derived from said protein whose function is inhibited.

  The term “non-natural molecule” means that the interferor molecule is artificial. For example, if the interferor molecule is a polypeptide (ie, both A and B peptides), the polypeptide will isolate B from the target protein (ie, self-association region) and (i) from another protein, Or (ii) Design by coupling the B part to the A part that can be derived from the same target protein when the A part is not directly adjacent to the B part. In other words, the target-derived self-association region fused to a moiety that prevents aggregation of the self-association region (if the interferor is a polypeptide, the part is also a polypeptide) is naturally produced by at least one natural amino acid. The fusion between the A part and the B part that occurs is different. Usually, the interferor molecule will not be present as a contiguous polypeptide within a gene-encoded protein in the non-recombinant genome.

  It should be clear that the interferor molecule can be designed in a modular fashion by inducing iteration and changing the order of part A and part B. The following is a non-limiting list of combinations: an interferor having an AB structure, an interferor having a BA structure, an interferor having an ABA structure, and a BAB structure. Interferer, interferor with A′-BA ”structure and interferor with B′-AB” structure: where the linker (spacer) is optionally A, A ′, A ″ part and B, It exists between B ′ and B ″. A, A ′, and A ″ are different or similar moieties (eg, different peptide sequences). B, B ′, and B ″ are different or similar self-association sequences (eg, B is self derived from the target protein) Association sequence, B ′ is a synthetic self-association sequence).

  In other words, the present invention provides a method for down-regulating the biological function of a protein comprising contacting the protein with a molecule comprising at least one self-association region isolated from the protein, Here, the self-association region is fused to a portion that prevents aggregation of the self-association region, so that the self-association region is in direct contact with the solvent in which the molecule and protein are present. From the above, it should be clear that the “part” is equivalent to the term A part and the B part is equivalent to the expression “at least one self-association region”.

  The expression “down-regulate protein function” reduces the normal biological activity of a protein (inhibition, down-regulation, reduction and interference are synonymous herein) or normalization of a protein to normal biology (For example, a protein resident in the endoplasmic reticulum is deleted when its function is down-regulated). Thus, by applying the method of the present invention, the function of the protein interferes with the protein by aggregating the protein by contacting the protein with the non-natural molecule of the present invention. Such non-natural molecules are referred to herein as “interferers” or “interferer molecules”. Aggregation means that a normally soluble protein is converted to an insoluble or aggregated protein through direct contact or binding with an interferor in a normal biological environment. The expression “down-regulate protein function” can also be replaced by the expression “knock down protein function” or “interfere negatively with protein function”. Down-regulation of protein function can also mean that the protein no longer exists in soluble form in the cell, or that the protein no longer exists in soluble form in its normal biological environment (eg, cells (internal ) Or extracellular localization). It can also mean that the aggregated protein is degraded via the cell's natural clearance mechanism and is no longer detectable in soluble or insoluble form. It may also mean that the transmembrane receptor protein can no longer bind to its normal ligand via aggregation of the transmembrane protein induced by the interferor. Thus, down-regulation of protein function can also mean, for example, that proteins resident in mitochondria are no longer present by means of protein interference. In certain embodiments, “down-regulation of protein function” or “negative inhibition of protein function” or “knock-down of protein function” is at least 20% compared to normal (100%) protein function, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even 100% loss of function.

  It can be conveniently determined by methods well known in the art that the protein is functioning or that the protein is not present in a normal biological environment (localization). For example, according to the target protein of interest, function can be determined by measuring a decrease in enzyme activity. Protein reduction in normal biological localization can be caused by, for example, insufficient complex formation, insufficient production of target protein in intracellular compartments, presence of soluble target protein, aggregation state (insoluble herein) ) And the presence of the target protein. Alternatively, the effect of down-regulation of the target protein can be measured by a cellular assay (eg, increase / decrease in proliferation, increase / decrease in invasion, increase / decrease in proteolytic activity).

  In one particular embodiment, the normal biological activity (or normal function or normal localization) of the protein can be inhibited intracellularly or extracellularly. “Intracellular” means within an organism or host cell (eg, cytoplasm, mitochondria, lysosome, vacuole, nucleus, chloroplast, endoplasmic reticulum (ER), cell membrane, mitochondrial membrane, chloroplast membrane ...). Means the localization of proteins. “Extracellular” means not only the localization of proteins in the extracellular medium of cells, but also proteins in contact with the extracellular medium, such as membrane-anchored proteins, transmembrane proteins and the like. Non-limiting examples of extracellular proteins include secreted proteins (eg proteases, antibodies and cytokines present in blood or plasma) or proteins present in the extracellular matrix (eg matrix metalloproteins and transmembrane proteins such as growth factor receptors) )).

  Cells or hosts that can be targeted by the methods of the invention include prokaryotic and eukaryotic cells. Non-limiting examples include viruses, bacteria, yeasts, fungi, protozoa, plants and mammals including humans.

  It should be apparent that 1, 2, 3, 4, 5 or even more proteins can be simultaneously inhibited using methods that down-regulate the biological function of the protein. In particular, since part B includes at least one self-association region, for example, part B includes different self-association regions that are each specific for a different protein. Interferers used to inhibit the biological function of at least one target protein are not naturally occurring and can be made by chemical synthesis or recombinant protein expression or a combination of the latter.

  Thus, the interferor molecule contains at least one self-association region (thus part B contains at least one self-association region). As used herein, a “self-association region” is defined as a contiguous sequence of amino acids that has a high tendency to closely assemble molecules with the same or very closely related sequences. The expression “high tendency to condense molecules” can also be interpreted as “high affinity”. Affinity is usually converted to a dissociation value (Kd value). The Kd value between the interferor and the target protein is usually in the micromolar to nanomolar range, but can also be below nanomolar or above micromolar. Examples of self-association regions include intermolecular beta sheet regions, alpha helix elements, hairpin loops, transmembrane sequences, and signal sequences. In one particular embodiment, at least one self-association region is present in part B. In another specific embodiment, at least two self-association regions are present in part B. In another specific embodiment, 3, 4, 5, 6 or more self-association regions are present in part B. The self-association regions can be interconnected by linker regions (eg, about 2 to about 4 amino acids). One (or at least one) self-association region present in part B is derived from the target protein. In certain embodiments, 2, 3, 4, 5, 6 or more self-association regions of part B are derived from the target protein. In another specific embodiment, 2, 3, 4, 5, 6, or more self-association regions of part B are derived from more than one target protein. In another specific embodiment, at least two self-association regions of part B are derived from the same target protein. As used herein, a target protein is defined as a protein whose function is to be inhibited. Accordingly, in order to create a B part specific for at least one protein, it is preferred that at least one self-association region of B part is “derived from” the target protein, or at least one self-association region is present in the target protein. Preferably it is present. “Derived from” means that at least one adjacent self-associating region is preferably identical to or adjacent to the adjacent region of the target protein. In a preferred embodiment, the at least one self-associating region is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% with a self-associating region present in the target protein region, Match at least 90%, at least 95% or at least 100%.

  The self-association region is preferably of a length consisting of at least 3 adjacent amino acids. In a preferred embodiment, the region consists of about 3 to about 30 amino acids. In another preferred embodiment, the region consists of about 3 to about 25 amino acids. In a particularly preferred embodiment, the region consists of about 5 to about 20 amino acids.

  The self-association region present in the B part of the interferor molecule can be identified and isolated from a protein other than the target protein, and the self-association region includes at least one self-association region derived from the target protein, To the spacer (or linker) between the self-association regions. For example, usable self-association regions can be derived from protein self-association regions that do not normally occur in hosts where the biological function of the target protein is down-regulated (thus not related to part B self-association regions). Some are obtained from living organisms). The nature of the self-association region determines the level of inhibition (ie, the strength of inhibition) of the target protein through induced aggregation. Two or more self-association regions can be used from the target protein in the interferor molecule, and a synthetic self-association region or a self-association region from a different target protein can be combined with one or more self-association regions obtained from the target protein. It is possible to use together.

  In certain embodiments, the self-association region consists of a synthetic sequence that is not naturally occurring because it is not derived from an existing protein. Examples of such synthetic self-association regions are described in Lopez de la Paz M et al. (2002) PNAS 99, 25, p. 16053, Table 1, which is incorporated herein by reference.

  If at least one self-association region (ie part B of the interferor molecule) is hydrophobic (due to aggregation-inducing properties), it prevents aggregation of the self-association region and is in direct contact with the solvent in which the interferor is present. The self-association region is preferably fused (or synonymous terms, linked or coupled) to the exposed portion (ie, part A of the interferor molecule). As such, in certain embodiments, part A has a solubilizing function that maintains part B in solution. In this embodiment, said part A is for example a peptide, a protein domain, a protein (preferably different from the target protein, see Example 2), a glycosylated structure, a (hydrophilic) chemical group or a cyclodextrin or these Is a derivative of In another specific embodiment, said part A is agarose beads, latex beads, cellulose beads, magnetic beads, silica beads, polyacrylamide beads, microspheres, glass beads or any solid support (eg polystyrene, plastic, nitrocellulose) Film, glass).

  In the interferor molecule, the B part and the A part may optionally be linked (or coupled) by a linker region (synonymous with spacer). The linker region can be, for example, a non-natural linker made by chemical synthesis (for example, a flexible linker such as a hydroxy-substituted alkane chain, dextran, polyethylene glycol, or the linker can be composed of amino acid homologs) ) Or the linker may consist of natural amino acids such as poly (threonine) or poly (serine). Preferably, when the linker comprises amino acids, the length of the linker region is about 3 to about 15 amino acids, more preferably about 5 to about 10 amino acids. While flexible linkers are frequently selected, it is envisioned that rigid linkers will also work. Flexible linker sequences can be obtained from nature, and usually the region connects domains in natural proteins such as the linker between the SH2 and SH3 domain src tyrosine kinases or the BRCT interdomain linker of BRCA1.

  The term “contacting” refers to the process by which an interferor and a target protein interact. In one form, an interferor is added to a sample containing the target protein (eg, the interferor is present at a specific concentration in the solution). In another form, the interferor molecule is injected into an organism with the target protein. For example, the contacting can also be carried out via a transformation process of one or more cells in a cell containing the target protein, such as an isolated cell of cell culture, a unicellular organism or a multicellular organism. Transformation can be accomplished using transfection or transformation methods known in the art as interferor molecules (eg calcium phosphate, DEAE-dextran, electroporation, microinjection, viral methods, use of cationic liposomes (eg Feigner, PL et al. (1987) , Proc. Natl. Acad. Sci USA 84, 7413), commercially available cationic lipid preparations such as Tfx 50 (Promega) or Lipofectamin 2000 (Life Technologies), by gene transfer techniques including particle guns, etc.) It means being introduced into (for example, a cell). The interferor molecule may be encoded by a recombinant vector (eg, plasmid, cosmid, viral vector) and can be synthesized in the host. In an alternative embodiment, the interferor molecule can be introduced into the cell via, for example, a liposome carrier or a carrier mediated by nanoparticles or injection. In yet another alternative embodiment, the interferor molecule can enter the cell via a sequence that mediates cell penetration (or translocation). In the latter case, the interferor molecule is further modified through recombination or synthetic attachment of cell penetrating sequences. Thus, interferor molecules (such as polypeptides) can be further fused or chemically coupled to sequences that facilitate transduction of the fusion protein or chemically coupled protein into prokaryotic or eukaryotic cells. Also good. Sequences that facilitate protein transduction are well known to those of skill in the art and include, but are not limited to, protein transduction domains. Preferably said sequence is selected from the group consisting of HIV TAT protein, polyarginine sequence, penetratin and pep-1. Still other common cell penetrating peptides (natural and artificial peptides) are disclosed in Joliot A. and Prochiantz A. (2004) Nature Cell Biol. 6 (3) 189-193.

  In one particular embodiment, the interferor consists essentially of amino acids. In some embodiments, the A and B sequences obtained from the interferor molecule are derived from the same target protein. In another embodiment, the interferor is a chimeric molecule, meaning that the sequences obtained from part A and part B are derived from different proteins, for example part A is derived from one protein, At least one aggregation region of part B is derived from the target protein. “Polypeptide” means a polymer in which the monomers are amino acids and are joined together through amide bonds, also referred to as a peptide. When the amino acid is an alpha-amino acid, either the L-optical isomer or the D-optical isomer can be used. Also included are unnatural amino acids such as beta-alanine, phenylglycine and homoarginine. In general, commonly found amino acids that are not encoded by genes may also be used in the present invention. All or some of the amino acids used in the interferor may be either the D- or L-isomer. Other peptidomimetics are also useful in the present invention. The inventors specifically investigated the development and use of peptidomimetics as antagonists of protein-protein interactions from Silerud LO and Larson RS (2005) Curr Protein Pept Sci. 6 (2): 151-69. Reference is made to this specification. In addition, D-amino acids are added to the peptide sequence to stabilize the turn properties (especially in the case of glycine). Another approach uses alpha, beta, gamma or delta turn mimics (alpha, beta, gamma or delta dipeptides are used to mimic peptide structural motifs and turn properties while at the same time providing stability against proteolysis, eg, conformation Other properties such as stability and solubility can be improved.

Isolation of self-association region from target protein :
Self-association sequences are often hydrophobic, but usually not always. For example, the self-associating region of yeast prions is rather polar. In fact, cross-beta aggregation of amino acid regions derived from polypeptides or proteins is (1) highly hydrophobic, (2) good in β-sheet propensity, (3) low effective charge, (4) solvent exposed Can be started. Thus, the self-associating protein region (“segment” is synonymous with “region”) is almost always embedded in the folded state and is not exposed to solvent. The latter is the case for many globular proteins during refolding, or when denaturation or partially folded is significantly dense, i.e. at high concentrations or under conditions of destabilization or mutation. It is confirmed by experimentally finding that aggregation occurs therein.

Based on these findings, we developed a computer algorithm that can predict self-association regions in proteins (synonymous with “β-associative range or segment”). One such algorithm, TANGO, is based on a statistical mechanics algorithm, which considers the three physicochemical parameters described above, and between different structural conformations: beta-turn, alpha-helix, beta-sheet aggregated structure and folded structure Competition is also considered (Fernandez-Escamilla, AM et al. (2004) Nat. Biotechnol. 22, 1302-1306, in particular the method section on pages 1305 and 1306 is hereby incorporated by reference. Supplementary notes 1 and 2 in the same section further detailed and dataset used for calibration and testing of the TANGO algorithm). Therefore, the self-association region in the target protein can be obtained by a computer algorithm such as TANGO. The self-association region is often buried within the core of the target protein ¹⁰ , which effectively protects the peptide from intramolecular association by an energy barrier corresponding to the stability of the target protein ¹¹ . In its normal environment (eg cytoplasm, extracellular matrix), the target protein is assisted by molecular chaperones that help the protein in maintaining a functional monomeric form ¹² . Model ^6, used by the TANGO algorithm, is designed to predict beta aggregation in peptides and proteins, with random coils and original conformations as well as other major conformational states: beta turns, alpha helices and It consists of a phase space containing a beta-aggregated structure. All segments of the peptide can assemble each of these states according to the Boltzmann distribution. Therefore, TANGO simply calculates the partition function of the phase space to predict the self-association region of the peptide. In order to estimate the aggregation tendency of a specific amino acid sequence, the following hypothesis is made: (i) In an aligned beta sheet aggregate structure, the main secondary structure is the beta chain. (Ii) The regions involved in the aggregation process are completely filled, so the full solvation budget, complete entropy can be used effectively to optimize the H-bond potential (ie the aggregate structure). The number of H-bonds occurring within is related to the number of donor groups supplemented by the acceptor, even if the donor or acceptor is in excess, it is unsatisfactory). (Iii) Complementary changes in the selected window establish favorable electrostatic interactions, and the overall net charge of peptides inside and outside the window hate aggregation. TANGO is accessible on the World Wide Web, http://tango.embl.de/. The zyggregator algorithm is another example (Pawar AP et al. (2005) J. Mol. Biol. 350, 379-392). These algorithms identify aggregation-prone sequences by comparing the aggregation propensity score for a given amino acid sequence with an average propensity calculated from a set of sequences of similar length.

In the present invention, we estimate that the TANGO score of the self-association region identified in the target protein is 5%, which represents 95% of the risk of aggregation in vitro ⁶ . We calculated that 85% of the protein from the human proteome unrelated to the disease has at least one region where the TANGO score is above the experimentally determined threshold of 5%. ing. This shows that over 85% of human proteins have at least one single self-association region but are prevented from aggregating with assistance from the normal stability of the protein and the chaperone mechanism. The present invention isolates these self-association regions from target proteins in order to prepare interferor molecules that are used for the specific induction of protein aggregation. Part B of the interferor molecule contains at least one aggregation region, which is derived from the target protein. By incorporating multiple aggregation regions of the target protein in the B part of the interferor molecule, the intensity of protein interference (for example, when the protein or a cell containing the protein comes into contact with a specific interferor molecule Loss of biological function of the target protein in%) is controllable. In fact, aggregation regions from target proteins with low TANGO scores (usually 5% to about 20%) can be repeated in the B portion of the interferor for 2, 3, 4 or more aggregation regions. As an alternative embodiment, 1, 2, 3 or 4 or more different aggregation regions with low TANGO scores from the same protein can be incorporated into the B part of the interferor. In another alternative embodiment, 1, 2, 3, 4 or more synthetic aggregation regions (and thus not from the target protein) are 1, 2, 3, 4 or more aggregation regions from the target protein and It is possible to bind within part B and promote the down-regulation of target proteins with low TANGO scores.

  Thus, in another embodiment, the present invention provides a non-natural molecule capable of aggregating a target protein. In one particular embodiment, the non-natural molecule is inherently proteinaceous. Proteinaceous means that the molecule comprises L-amino acids or D-amino acids or a mixture of L- and D-amino acids or a combination of natural amino acids and peptidomimetics.

  In yet another embodiment, the invention provides a non-natural molecule comprising at least one self-association region isolated from a water soluble protein domain, wherein the self-association region is fused with a moiety that prevents aggregation of the self-association region doing.

  In yet another embodiment, the present invention provides a non-natural molecule comprising at least one self-association region isolated from a water soluble protein domain, said self-association region comprising a moiety that prevents aggregation of said self-association region; Fused so that the self-association region is in direct contact with the solvent in which it is present.

  In yet another embodiment, the invention provides a non-natural molecule consisting of at least one self-association region isolated from a water soluble protein domain, wherein the self-association region is fused with a moiety that prevents aggregation of the self-association region doing.

  In yet another embodiment, the present invention provides a non-natural molecule consisting of at least one self-association region isolated from a water-soluble protein domain, said self-association region comprising a moiety that prevents aggregation of said self-association region; Fused so that the self-association region is in direct contact with the solvent in which it is present.

  In certain embodiments, the moiety is, for example, a peptide, agarose bead, protein domain or protein. In another specific embodiment, said non-natural molecule comprises at least two self-association regions whose at least one self-association region is derived from a target protein.

  In other words, the present invention provides a non-natural molecule comprising part A and part B, where i) part A comprises a region such as a peptide, protein domain, protein or agarose bead that prevents aggregation of part B. Ii) part B comprises at least one self-association region, wherein said region consists of at least three adjacent amino acids and is isolated from said protein whose function is inhibited, and the linker is optionally part A And B part.

  In other words, the present invention provides a non-natural molecule comprising part A and part B, wherein i) part A comprises a region such as a peptide, protein domain or agarose bead that prevents aggregation of part B; ii) part B comprises at least one self-association region consisting of at least three adjacent amino acids, the at least one self-association region being isolated from a protein whose function is inhibited, said region being a water-soluble protein The linker is optionally present between part A and part B, which is in direct contact with the environment in which the molecule and the protein are present.

  In other words, the present invention provides a non-natural molecule comprising part A and part B, wherein i) part A comprises a region such as a peptide, protein domain or agarose bead that prevents aggregation of part B; ii) Part B consists of at least one self-association region consisting of at least three adjacent amino acids, said at least one self-association region being isolated from a protein whose function is inhibited, said region being water-soluble It is derived from a domain obtained from a protein, and a linker is optionally present between part A and part B, which is in direct contact with the environment in which the molecule and the protein are present.

  The expression “isolated from (or derived from) a domain obtained from said water-soluble protein” means that the self-association region is a contiguous amino acid sequence isolated from the soluble domain of the protein. The latter also means that self-association regions from transmembrane regions or signal sequences are explicitly excluded in the claims for these interferor molecule products in this embodiment. I mean.

  In the present invention, at least one self-association region of an interferor molecule (ie, part B of the interferor molecule) is “in direct contact” with the environment (eg, solvent, cytosol) in which the interferor molecule is present. To further clarify this importance. In globular proteins, self-association sequences (also designated as “aggregated nucleation regions”) are generally embedded in the hydrophobic core of the globular protein, and as such, cooperative interactions that stabilize the original state. It is protected from solvents by a close network of action. Thus, under normal circumstances, there is no “direct contact” between the self-association region and the environment (eg, solvent). Only when the protein is unfolded, for example, it is synthesized on the ribosome or is destabilized by mutations, temperature changes, pH or loss of specific chaperones, thus allowing the unfolded state In some cases, the self-association area is exposed to the environment. The self-association region is usually embedded inside the protein (to prevent aggregation), and within the non-natural interferor molecule, the self-association region is isolated and the portion that prevents aggregation (ie, the A part of the interferor molecule) ) Is exposed to the environment by connecting the above-mentioned areas. Furthermore, in other words, the non-natural interferor molecule is not folded into a spherical structure, and therefore at least one self-association region (ie, part B) in the non-natural interferor molecule is a solvent in which the interferor molecule is present. In direct contact. Thus, “in direct contact” means a synonym for “implanted and protected from”.

  In one specific embodiment, the interferor molecule comprising at least one self-association region from a soluble protein domain is a polypeptide.

  In another specific embodiment, the present invention provides a recombinant vector comprising a polynucleotide encoding the interferor molecule.

  In another specific embodiment, the interferor molecule of the invention is used as a medicament.

Application of Interferor Molecules to Therapy Proteins are involved in biological activities ranging from numerous enzyme reactions, through signal transduction, to the provision of structures. Changes in protein structure, abundance or activity are the root cause of many diseases. Many drugs act through the specific interference of one or a limited number of proteins. The present invention provides a method for developing a new class of compounds capable of specifically inhibiting selected target proteins. These new types of compounds are called interferers.

  Thus, in yet another embodiment, the present invention provides the use of a non-natural molecule comprising at least one self-association region derived from a water soluble protein domain as a medicament, said self-association region being said self-association region It is fused to the part that prevents aggregation.

  In yet another embodiment, the present invention provides the use of a non-natural molecule comprising at least one self-association region derived from a water-soluble protein domain as a medicament, said self-association region comprising It is fused with a part that prevents aggregation, so that the self-association region is in direct contact with the solvent in which the molecule is present.

  In other words, the present invention provides the use of a non-natural interferor molecule comprising part A and part B as a drug, i) part A is a region such as a peptide or protein domain that prevents aggregation of part B. And ii) part B comprises at least one self-association region, said region comprising at least three adjacent amino acids from the target protein, and a linker is optionally present between part A and part B.

  In other words, the present invention provides the use of a non-natural interferor molecule comprising part A and part B as a drug, i) part A is a region such as a peptide or protein domain that prevents aggregation of part B. And ii) B part comprises at least one self-association region, said region comprising at least three adjacent amino acids from the target protein, and a linker is optionally present between part A and part B; Part B is in direct contact with the solvent in which the interferor molecule is present.

  The interferor molecule can be used in the treatment of a disease and / or in the manufacture of a medicament for treating a disease such as cancer associated with abnormal expression of at least one target protein such as an oncogenic protein. The term “abnormal expression” means, for example, (over) expression of an oncogenic protein in the case of cancer, which also includes expression of a dominant negative protein, undesired localization of a specific protein or splice mutation of a specific protein Body, undesired expression of a particular splice variant of a particular protein, high activity of a mutein or high activity of a particular protein.

  In certain embodiments, “abnormal expression” refers to an unwanted presence of a post-translationally modified protein or an undesirable presence of a non-translationally modified protein. Post-translational modifications alter the physicochemical properties of the modified amino acids, so that they alter the aggregation tendency of a given polypeptide segment that can be exploited to specifically target the form with the greatest aggregation tendency There is a possibility. Thus, if post-translational modification significantly reduces the tendency of the self-association region to aggregate, interference is most efficient with unmodified proteins. In contrast, interference is most efficient with modified proteins when post-translational modifications increase the tendency of the self-association region to aggregate. Based on hydrophobicity alone, it is speculated that modifications such as phosphorylation and glycosylation reduce the tendency to aggregate while lipid attachment increases the tendency to aggregate.

  The target protein to which the interferor molecule of the present invention is directed may be associated with a disease state. For example, the protein can be a pathogenic protein, such as a viral protein, a tumor-related protein or an autoimmune disease-related protein. In one aspect, the invention features a method of treating a subject at risk of or suffering from unwanted cell proliferation, eg, malignant or non-malignant cell proliferation. The method includes: providing an interferor molecule, eg, an interferor having a structure as described herein, wherein the interferor molecule is a function of a protein that promotes unwanted cell growth and / or Alternatively, it is possible to prevent (inhibit) the presence and administer the interferor to a subject, preferably a human specimen, thereby treating the subject.

  In a preferred embodiment, the protein is a growth factor or growth factor receptor, a kinase (eg, protein tyrosine, serine or threonine kinase), an adapter protein, a protein or transcription factor obtained from a receptor superfamily coupled to a G protein. . In a preferred embodiment, the interferor molecule inhibits the biological function of PDGF-beta protein, and thus has or is at risk for a disease characterized by unwanted PDGF-beta expression, such as testis and lung cancer. Can be used to treat the body. In another preferred embodiment, the interferor inhibits (knocks down) the function and / or presence of the Erb-B protein, and thus has or is at risk for a disease characterized by unwanted Erb-B expression. Can be used to treat a subject having In another preferred embodiment, the interferor is a disease characterized by unwanted Src expression, eg, colon cancer, that inhibits (or prevents the presence of) Src protein function (or synonymous with “prevents function”). Or can be used to treat a subject at risk. In another preferred embodiment, the interferor inhibits the function and / or presence of CRK protein, and thus subjects with or at risk for diseases characterized by unwanted CRK expression, such as colon cancer and lung cancer. Can be used to treat. In another preferred embodiment, the interferor inhibits the function and / or presence of GRB2 protein and thus treats a subject having or at risk for a disease characterized by unwanted GRB2 expression, eg, squamous cell carcinoma Can be used to do. In another preferred embodiment, the interferor molecule has a disease characterized by unwanted RAS expression, such as pancreatic cancer, colon cancer and lung cancer and chronic leukemia, or inhibits the function and / or presence of the RAS gene, or It can be used to treat a subject at risk. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of MEKK protein and thus has or is at risk for a disease characterized by unwanted MEKK expression, such as squamous cell carcinoma, melanoma or leukemia. It can be used to treat a subject with it. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of JNK protein, and thus is or is at risk for a disease characterized by unwanted JNK expression, such as visceral cancer or breast cancer. Can be used to treat. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of the RAF protein, and thus a subject having or at risk of having a disease characterized by unwanted RAP expression, such as lung cancer or leukemia. Can be used to treat. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of Erk1 / 2 protein, and thus has or is at risk for a disease characterized by unwanted Erk1 / 2 expression, such as lung cancer. Can be used to treat the body. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of PCNA (p21) protein, and thus has or is at risk for a disease characterized by unwanted PCNA expression, such as lung cancer. Can be used to treat. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of the MYB protein, and thus has or is at risk for a disease characterized by unwanted MYB expression, such as rectal cancer or chronic myeloid leukemia. It can be used to treat a subject with it. In preferred embodiments, the interferor molecule inhibits the function and / or presence of c-MYC protein and thus has a disease characterized by unwanted c-MYC expression, such as Burkitt lymphoma or neuroblastoma, or It can be used to treat a subject at risk. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of JUN protein and thus has or is at risk for a disease characterized by unwanted JUN expression, such as ovarian cancer, testicular cancer or breast cancer. It can be used to treat a subject with it. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of FOS protein and thus has or is at risk for a disease characterized by unwanted FOS expression, such as skin cancer or testicular cancer. Can be used to treat the body. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of BCL-2 protein and thus has a disease characterized by unwanted BCL-2 expression, such as lung cancer or testicular cancer or non-Hodgkin's lymphoma Or can be used to treat a subject at risk. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of cyclin D protein, and thus has or is at risk for diseases characterized by unwanted cyclin D expression, such as esophageal cancer and rectal cancer. It can be used to treat a subject with it. In another preferred embodiment, the interferor molecule has a disease characterized by unwanted VEGF expression, such as esophageal cancer, rectal cancer or pathological angiogenesis, which inhibits the function and / or presence of VEGF protein, or It can be used to treat a subject at risk. In a preferred embodiment, the interferor molecule inhibits the function and / or presence of EGFR protein, thus treating a subject having or at risk of having a disease characterized by unwanted EGFR expression, such as breast cancer. Can be used. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of cyclin A protein, and thus has or is at risk for diseases characterized by unwanted cyclin A expression, such as lung cancer and cervical cancer. It can be used to treat a subject with it. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of cyclin E protein, and thus has or is at risk for a disease characterized by unwanted cyclin E expression, such as lung cancer and breast cancer. Can be used to treat the body. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of WNT-1 protein and thus has or is at risk for a disease characterized by unwanted WNT-1 expression, such as basal cell carcinoma. It can be used to treat a subject with it. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of beta-catenin protein and thus has a disease characterized by unwanted beta-catenin expression, such as adenocarcinoma or hepatocellular carcinoma, or It can be used to treat a subject at risk. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of c-MET protein and thus has or is at risk for a disease characterized by unwanted c-MET expression, such as hepatocellular carcinoma. It can be used to treat a subject with it. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of protein kinase C (PKC) protein, and thus has or is at risk for a disease characterized by unwanted PKC expression. It can be used to treat a subject. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of the NF kappa-B protein and thus has or is at risk for a disease characterized by unwanted NF kappa-B expression. It can be used to treat a subject with it.

  In another preferred embodiment, the interferor molecule inhibits the function and / or presence of the STAT3 protein, thereby treating a subject having or at risk of having a disease characterized by unwanted STAT3 expression, such as testicular cancer. Can be used to do. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of survivin protein and thus has or is at risk for a disease characterized by unwanted survivin expression, such as cervical cancer or pancreatic cancer It can be used to treat a subject. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of the Her2 / Neu protein and thus has or is at risk for a disease characterized by unwanted Her2 / Neu expression. Can be used to treat the body. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of topoisomerase I protein, and thus has or is at risk for diseases characterized by unwanted topoisomerase I expression, such as ovarian cancer and rectal cancer. It can be used to treat a subject with it. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of topoisomerase II alpha protein, and thus has or is at risk for diseases characterized by unwanted topoisomerase II expression, such as breast cancer and rectal cancer. It can be used to treat a subject with it.

  In another aspect, the invention provides a method of treating a subject, eg, a human, at risk for or suffering from a disease or disorder that may be ameliorated by inhibition of angiogenesis, eg, cancer. The method includes: providing an interferor molecule, eg, an interferor molecule having a structure described herein, wherein the interferor molecule inhibits (or prevents function) a protein that mediates angiogenesis. Is possible, administering an interferor molecule to a subject, thereby treating the subject. In a preferred embodiment, the interferor molecule inhibits the function and / or presence of alpha v-integrin protein and thus has or is at risk for a disease characterized by unwanted alpha v-integrin, such as a brain tumor or epithelial derived tumor. Can be used to treat a subject having In another preferred embodiment, the interferor molecule inhibits the function and / or presence of Flt-1 receptor protein, and thus has a disease characterized by unwanted Flt-1 receptor, such as cancer and rheumatoid arthritis, Or it can be used to treat a subject at risk. In another preferred embodiment, the interferor molecule inhibits the function and / or presence of a tubulin protein and thus has or is at risk for diseases characterized by unwanted tubulin, such as cancer and retinal neovascularization. It can be used to treat a subject.

    In another aspect, the invention provides a method of treating a subject who is infected with, or at risk of, or suffering from a disease or illness associated with a viral infection. The method includes: providing an interferor molecule, eg, an interferor molecule having the structure described herein, wherein the interferor molecule is a viral protein or a viral function, eg, a cellular protein that mediates entry or growth. Homologous and capable of silencing; administering the interferor molecule to a subject, preferably a human specimen, thereby treating the subject. As such, the present invention relates to human papillomavirus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis A virus (HAV), hepatitis C virus (HCV), respiratory multinuclear virus (RSV). Patients infected with viruses such as herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein Barr virus (EBV), rhinovirus, West Nile virus, tick-borne encephalitis virus, measles virus (MV) or poliovirus Providing a method of using an interferor for the manufacture of a medicament to treat.

  In another aspect, the invention features a method of treating a subject infected with a pathogen, such as a bacterial, amoeba, parasite, or fungal pathogen. The method includes: providing an interferor molecule, eg, an interferor molecule having a structure described herein, wherein the interferor molecule inhibits the function of a pathogenic protein from the pathogen and is tested. It is possible to administer an interferor molecule to the body, preferably a human specimen, thereby treating the subject. Target proteins obtained from pathogens can be those involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism (eg Krebs cycle) or toxin production. Accordingly, the present invention includes, for example, Plasmodium falciparum, Mycobacterium ulcerans, Mycobacterium tuberculosis, Mycobacterium leprae, Staphylococcus aureus, Streptococcus pneumoniae A method of treating a patient infected with (Streptococcus pneumoniae), Streptococcus pyogenes, Chlamydia pneumoniae or Mycoplasma pneumoniae is provided.

  In another aspect, the invention relates to a subject at risk of or suffering from a disease or disorder characterized by an unwanted immune response, such as an inflammatory disease or disorder or an autoimmune disease or disorder, For example, a method of treating a human is provided. The method includes: providing an interferor molecule, eg, an interferor molecule having a structure described herein, wherein the interferor molecule inhibits the function and / or presence of a protein that mediates an unwanted immune response. (Or down-regulate) and administering the interferor molecule to the subject, thereby treating the subject. In preferred embodiments, the disease or disorder is ischemia or reperfusion injury such as acute myocardial infarction, unstable angina, cardiopulmonary bypass, surgical intervention (eg angioplasty such as percutaneous coronary angioplasty), transplantation Ischemic reperfusion or disease associated with response to an organ or tissue (eg transplanted heart or vascular tissue) or thrombolysis. In another preferred embodiment, the disease or disorder is restenosis, eg, restenosis associated with surgical intervention (eg, angioplasty such as percutaneous coronary angioplasty). In another preferred embodiment, the disease or disorder is an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. In another preferred embodiment, the disease or disorder is inflammation associated with infection or injury. In another preferred embodiment, the disease or disorder is asthma, lupus, multiple sclerosis, diabetes such as type 2 diabetes, arthritis such as rheumatic or psoriatic rheumatism. In another preferred embodiment, the interferor molecule inhibits the function of an integrin or a coligand thereof, such as VLA4, VCAM, ICAM. In another preferred embodiment, the interferor molecule inhibits the function of selectin or its co-ligand, such as P-selectin, E-selectin (ELAM), L-selectin or P-selectin glycoprotein (PSGL1). In another preferred embodiment, the interferor molecule inhibits the function of complementary components such as C3, C5, C3aR, C5aR, C3 convertase, C5 convertase. In another preferred embodiment, the interferor molecule is a chemokine or its receptor, such as TNF-α, IL-1α, IL-1, IL-2, IL-2R, IL-4, IL-4R, IL-5, Inhibits the function of IL-6, IL-8, TNFR I, TNFR II, IgE, SCYA11 or CCR3.

  In another aspect, the invention provides a method of treating a subject, eg, a human, at risk for or suffering from acute pain or chronic pain. The method includes providing an interferor molecule, e.g., an interferor molecule having a structure described herein, wherein the interferor molecule inhibits a protein that mediates a pain process and causes the subject to interact with the interferor molecule. It is possible to administer a Ferrer molecule, thereby treating the subject. In another preferred embodiment, the interferor molecule inhibits the function of a component of the ion channel. In another particular preferred embodiment, the interferor molecule inhibits neurotransmitter receptor or ligand function.

  In another aspect, the invention provides a method of treating a subject, eg, a human, at risk for or suffering from a nervous system disease or disorder. The method includes providing an interferor molecule, e.g., an interferor molecule having a structure described herein, wherein the interferor molecule inhibits a protein that mediates a nervous system disease or disorder and is directed to a subject. The interferor molecule can be administered, thereby treating the subject. In certain embodiments, the disease (or disorder) that can be treated includes Alzheimer's disease (in which case the interferor molecule is an APP, such as a gamma-secretase complex (eg, presenilin protein 1 or 2, Aph1 protein, Inhibits the function of secretase, which will process proteins involved in nicastrin, BACE1 or BACE2) Interferers inhibit the processing of APP and prevent the formation of insoluble amyloid beta. Other neurodegenerative diseases such as Huntington's disease, spinocerebellar ataxia (eg, SCA1, SCA2, SCA3 (Mashad Joseph disease), SCA7 or SCA8) can be prevented and / or treated.

  Accordingly, in one aspect, the invention provides a method of producing or manufacturing a medicament or pharmaceutical composition comprising at least one interferor molecule and mixing the interferor molecule with a pharmaceutically acceptable carrier. In a preferred embodiment, the interferor molecule is a polypeptide and can be made synthetically or as a recombinant protein. Recombinant proteins may be produced using recombinant expression systems such as bacterial cells, yeast cells, animal cells, insect cells, plant cells or transgenic animals or plants. The recombinant protein may be purified and / or mixed with additives by conventional, substantially homogeneous protein purification means.

  Administration of the pharmaceutical composition comprising the interferor molecule may be oral, inhalation, transdermal or parenteral (such as intravenous, intratumoral, intraperitoneal, intramuscular, intracavity and subcutaneous). The active compound may be administered alone or preferably formulated as a pharmaceutical composition. One unit is usually said to contain 0.01 to 500 mg, for example 0.01 to 50 mg, or 0.01 to 10 mg, or 0.05 to 2 mg of a compound or a pharmaceutically acceptable salt thereof. The unit dose is usually given once or more a day, for example 2, 3 or 4 times a day, more usually 1 to 3 times a day, so the total daily dose is usually A suitable total daily dose for a 70 kg adult is 0.01 to 700 mg, such as 0.01 to 100 mg, or 0.01 to 10 mg, or more generally, in the range of 0.0001 to 10 mg / kg. 0.05 to 10 mg.

  Preferably, the compound or pharmaceutically acceptable salt thereof is administered in the form of a unit dose composition such as a unit dose oral, parenteral, transdermal or inhalation composition. The composition is prepared by mixing and is preferably suitable for oral, inhalation, transdermal or parenteral administration, such as tablets, capsules, oral liquid preparations, powders, granules, troches, reconstituted powders, It may be in the form of injectable and injectable solutions or suspensions or suppositories or aerosols.

  Tablets and capsules for oral administration are usually provided in unit doses and contain conventional excipients such as binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavorings and wetting agents. contains. The tablets may be coated according to methods well known in the art. Suitable fillers for use include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include starch derivatives such as starch, polyvinylpyrrolidone and sodium starch glycolate. Suitable disintegrants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lariul sulfate. These solid oral compositions may be prepared by conventional methods such as mixing, filling and tableting. The mixing operation may be repeated to distribute the active agent throughout these compositions using large amounts of filler. The work is naturally practiced in the art.

  Oral liquid formulations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or provided as a dry product for reconstitution with water or other suitable vehicle prior to use May be. The liquid formulation comprises a suspension such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fat, emulsifier such as lecithin, sorbitan monooleate or acacia; non-aqueous vehicle ( Edible oils), eg oily esters such as peanut oil, coconut oil, esters of glycerin, propylene glycol or ethyl alcohol; preservatives, eg conventional additives such as methyl or propyl or sorbic acid p-hydroxybenzoate, In addition, a normal fragrance or coloring agent may be included as necessary. Oral formulations also include conventional sustained release formulations such as enteric coated tablets or granules.

  Preferably, the inhalation composition is provided for administration to the respiratory tract as an olfactory or nebulizer aerosol or solution, or as an insufflated ultrafine powder, alone or in combination with an inert carrier such as lactose. In this case, the diameter of the active compound particles is less than 50 microns, preferably less than 10 microns, for example 1-5 microns such as 2-5 microns. Alternatively, coated nanoparticles having a particle size of 30-500 nm can be used. Preferred inhalation doses are said to be in the range of 0.05-2 mg, such as 0.05-0.5 mg, 0.1-1 mg or 0.5-2 mg.

  For parenteral administration, fluid unit dosage forms are prepared containing a compound of the invention and a sterile vehicle. Depending on the vehicle and concentration, the active compound can be either suspended or dissolved. Parenteral solutions are usually prepared by dissolving the compound in a vehicle and filter sterilizing before filling into a suitable vial or ampoule and sealing. Conveniently, adjuvants such as local anesthetics, preservatives and buffering agents are also dissolved in the vehicle. To promote stability, the composition was frozen after filling into the vial and the moisture removed under vacuum. Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and it is sterilized by exposure to ethylene oxide before suspending in the sterile vehicle. is there. Conveniently, a surfactant or wetting agent is included in the composition that facilitates uniform distribution of the active compound. Optionally, small amounts of bronchodilators such as sympathomimetic amines such as isoprenaline, isoetarine, salbutamol, phenylephrine and ephedrine; xanthine derivatives such as theophylline and aminophylline and corticosteroids such as prednisolone and adrenal stimulants such as ACTH May be included.

  By convention, compositions are usually accompanied by written or printed instructions for use in relevant medical procedures.

  In a preferred embodiment, the interferor molecule further comprises a protein transduction domain. A series of small protein domains, called protein transduction domains (PTDs), can efficiently cross biological membranes independent of transporters or specific receptors and facilitate the delivery of peptides and proteins into cells. I know. For example, a TAT protein obtained from human immunodeficiency virus (HIV-1) can deliver a biologically active protein in vivo. Similarly, the 3rd alpha helix of the antennapedia homeodomain and the VP22 protein from herpes simplex virus facilitate the delivery of covalent peptides or proteins to cells (Ford KG et al. (2001) Gene Ther. 8, 1-4). reference). Protein delivery based on the short amphiphilic peptide carrier Prp-1 is a fully biologically active form, efficient for delivery of diverse peptides and proteins to several cell lines, Covalent coupling is not required (Morris MC et al. (2001) Nat. Biotechnol. 19, 1173-1176). The ability of the VP22 chimeric protein to spread from the first transduced cells to surrounding cells can improve the gene therapy approach (Zender L et al. (2002) Cancer Gene Ther. 9, 489-496). Sequences that facilitate protein transduction are well known to those of skill in the art and include, but are not limited to, protein transduction domains. Preferably, said sequence is selected from the group consisting of HIV TAT protein, polyarginine sequence, penetratin and pep-1. Yet other commonly used cell penetrating peptides (natural and artificial peptides) are disclosed in Joliot A. and Prochiantz A. (2004) Nature Cell Biol. 6 (3) 189-193.

  A second aspect of the pharmaceutical composition is the use of a nucleotide sequence encoding an interferor molecule. When using a nucleic acid sequence encoding an interferor molecule, the agent is preferably intended to deliver the nucleic acid into a cell in a gene therapy procedure. Numerous delivery methods are well known to those skilled in the art. Preferably, the nucleic acid is administered for use in in vivo or ex vivo gene therapy. Non-viral vector delivery systems include nucleic acids complexed with delivery vehicles such as DNA plasmids, naked nucleic acids and liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either an episome or an integrated genome after being delivered to cells. Non-viral delivery methods for nucleic acids include lipofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced DNA uptake. . Lipofection is described, for example, in US Pat. No. 5,049,386, US Pat. No. 4,946,787; and US Pat. No. 4,897,355, and lipofection reagents are commercially available (eg, Transfectam ™ and Lipofectin ™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Flegner, WO 91/17424, WO 91/16024. Delivery is possible to cells (ex vivo administration) or target tissues (in vivo administration). The preparation of lipid: nucleic acid complexes, including target liposomes such as immunolipid complexes, is well known to those skilled in the art (eg Crystal, 1995; Blaese et al., 1995; Behr, 1994; Remy et al., 1994; Gao and Huang, 1995; U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028 and 4,946,787).

  The use of RNA or DNA virus-based systems for nucleic acid delivery takes advantage of highly evolved processes to direct the virus to specific cells within the body and transport the viral load to the nucleus. Viral vectors can be administered directly to the patient (in vivo) or used to treat cells in vitro, and the modified cells are administered to the patient (ex vivo). Conventional virus-based systems for delivery of nucleic acids include retrovirus, lentivirus, adenovirus, adeno-associated and herpes simplex virus vectors, among others, for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer into target cells and tissues. Integration into the host genome is possible by retrovirus, lentivirus and adeno-associated virus gene transfer methods, and expression of the inserted transgene is long-lasting. Also, transduction efficiency is high in many different cell types and target tissues. If transient expression of the nucleic acid is preferred, an adenovirus-based system containing a replication defective adenoviral vector may be used. Vectors based on adenovirus allow very high efficiency transduction in many cell types and do not require cell division. High titers and expression levels are obtained with the vector. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors, including recombinant adeno-associated virus vectors, are also used to transduce cells with target nucleic acids, eg, in vitro generation of nucleic acids and peptides, and in vivo and ex vivo gene therapy tools (Eg, US Pat. No. 4,797,368; WO 93/24641; Kotin, 1994; The construction of recombinant AVV vectors is described in numerous publications such as US Pat. No. 5,173,414; Hermonat & Muzyczka, 1984; Samulski et al., 1989. Have been).

  Gene therapy vectors can be delivered in vivo by administration to individual patients, usually systemic administration (eg, intravenous, intraperitoneal, intramuscular, intratracheal, subcutaneous or intracranial injection) or local administration. In certain embodiments, the present invention also contemplates the use of hydrodynamic gene therapy. Hydrodynamic gene therapy is disclosed in US6627616 (Mirus Corporation, Madison) and is involved in the intravascular delivery of non-viral nucleic acids encoding interferors, whereby the permeability of the blood vessels is determined, for example, by the internal pressure of the blood vessels. Elevated by the application of elevation or by co-administration of a vascular permeability increasing compound such as papaverine.

  Alternatively, vectors can be delivered ex vivo to cells transplanted from individual patients (eg, lymphocytes, bone marrow aspirates, tissue biopsies) or cells such as universal supplier hematopoietic stem cells, and then cells that have incorporated the vector were selected. Later, the cells are usually reimplanted into the patient. Ex vivo cell transfection for diagnosis, investigation or gene therapy (eg, via reinfusion of transfected cells into a host organism) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from a test organism, transfected with a nucleic acid (gene or cDNA), and reinjected back into the test organism (eg, a patient). A variety of cell types suitable for ex vivo transfection are well known to those skilled in the art (for a discussion of how to isolate and culture cells from a patient, see, for example, Freshney et al., 1994, as well as the references cited herein. See literature).

  In yet another embodiment, the protein interference methods of the invention may be used to determine the function of a protein in a cell or organism capable of mediating protein interference. The cells can be prokaryotic or eukaryotic cells, or cell lines such as plant cells or mammalian cells such as fetal cells, pluripotent stem cells, tumor cells such as teratocarcinoma cells or virus infected cells It is. The organism is preferably a eukaryote, for example a plant or mammal, in particular an animal such as a human.

  The target protein to which the interferor molecule of the present invention is directed may be associated with a disease state. For example, the protein can be a pathogenic protein, such as a viral protein, a tumor-related protein or an autoimmune disease-related protein. The target protein may be a heterologous gene expressed in a recombinant cell or genetically modified organism. By inhibiting the function of the protein, various information and therapeutic effects in the agricultural field or pharmaceutical or veterinary field may be obtained. In a particularly preferred embodiment, the method of the invention uses a eukaryotic cell or eukaryotic non-human organism that exhibits a target protein-specific knockout phenotype comprising at least partially defective expression of at least one endogenous target protein. Wherein the cell or organism is at least one interferor molecule capable of inhibiting the function of at least one endogenous target protein, or the function of at least one endogenous protein and / or In contact with a vector encoding at least one interferor molecule capable of inhibiting its presence. It should be noted that the present invention also allows for target-specific knockout of several different endogenous proteins due to the specificity of the interferor molecule.

  The protein-specific knockout phenotype of a cell or non-human organism, in particular a human cell or non-human mammal, is used as an analytical tool, for example for functional and / or phenotypic analysis of complex physiological processes such as proteome analysis. It's okay. For example, a knockout phenotype of a human protein in cultured cells that is expected to be an alternative splicing process regulator may be prepared. Of these, the protein is in particular a member of the SR splicing factor family, such as ASF / SF2, SC35, SRp20, SRp40 or SRp55. Furthermore, the effect of SR protein on the mRNA profile of a predetermined or spliced gene such as CD44 may be analyzed.

  Using the protein-based knockout techniques described herein, the expression of the endogenous target protein can be inhibited in the target cell or target organism. An endogenous protein can be complemented by an exogenous nucleic acid that encodes the target protein or a variant or mutant form of the target protein, such as a gene or cDNA, which can optionally be a detectable peptide or It may be fused to an additional nucleic acid sequence encoding a polypeptide, for example an affinity tag, in particular a multiple affinity tag. A variant or mutant form of a target protein differs from an endogenous target protein in that it differs from an endogenous protein by amino acid substitution, insertion and / or deletion of one or more amino acids. A variant or mutant form may have the same biological activity as the endogenous target protein. On the other hand, the mutant or mutant target protein may have a biological activity different from that of the endogenous target protein, such as a partial deletion activity, a complete deletion activity, an enhanced activity, and the like. Complementarity is achieved by co-expressing a polypeptide encoded by an exogenous nucleic acid, such as a fusion protein comprising a target protein and an affinity tag, and an interferor molecule that knocks out the exogenous protein in the target cell. May be. This co-expression is achieved using a suitable expression vector that expresses a polypeptide encoded by the exogenous nucleic acid, for example, both the tag-modified target protein and the interferor molecule, or a combination of expression vectors. Alternatively, the interferor molecule may contact the target cell from outside the cell. Proteins and protein complexes that are synthesized de novo in target cells will include exogenous proteins, such as modified fusion proteins. In order to avoid suppression of exogenous protein function by the interferor molecule, the exogenous protein needs to have sufficiently different amino acids in the aggregation region selected to design the interferor molecule. Alternatively, an exogenous target protein can be complemented by a corresponding protein from another species, or an exogenous target protein can be complemented by a splice form of the target protein. The combination of exogenous protein knockout and rescue with a mutated, eg, partially deleted, exogenous target has advantages when compared to the use of knockout cells. Furthermore, this method is particularly suitable for identifying functional domains of target proteins.

  In a further preferred embodiment, a comparison is made of at least two cells or organisms, such as gene expression profiles and / or proteome and / or phenotypic characteristics. These organisms include (i) a control cell or control organism without target protein inhibition, (ii) a cell or organism with target protein inhibition, and (iii) exogenous encoding the target protein in addition to target protein inhibition. A cell or organism having target protein complementarity by the target nucleic acid.

The methods of the present invention are also suitable as a means to identify and / or characterize drugs, for example to identify new drugs from a collection of test substances and / or to characterize mechanisms of known drug action and / or side effects. Thus, the present invention also relates to a system for identifying and / or characterizing agents that act on at least one target protein including: (a) expressing at least one exogenous target gene encoding said target protein Eukaryotic cells or eukaryotic non-human organisms capable of being (b) at least one interferor molecule capable of inhibiting the expression of said at least one endogenous target gene, and (c) pharmaceutical properties A test substance or collection of test substances to be identified and / or characterized. Furthermore, the system described above preferably comprises: (d) at least one exogenous target nucleic acid encoding the target protein or a variant or mutant form or splice form of the target protein, wherein the exogenous target protein comprises: It differs from the endogenous target protein at the amino acid level of the aggregation region so that its function is not substantially inhibited by the interferor molecule rather than the expression of the endogenous protein.

  The invention also includes cells and organisms containing interferor molecules. The organism can be, for example, a transgenic plant carrying genetic information encoding an interferor. The transgenic plant is in a preferred embodiment a silenced plant (i.e. due to the presence of a specific interferor in which a particular target protein is within a cell or a subset of an organism or is present in all cells and organs of said plant. Plants that are down-regulated). A cell containing an interferor can be generated by contacting the cell with a specific interferor molecule or by electroporating the cell with a specific interferor molecule. In certain embodiments, cells comprising an interferor are produced via transfection (or transformation) in which the interferor is encoded by a recombinant expression vector such as a plasmid or viral vector.

Isolation: Separation and detection In another embodiment, the present invention provides a method for isolating a protein from a sample, the method comprising a non-naturally occurring region comprising at least one self-association region present in the protein. Contacting the molecule, and isolating the resulting co-aggregated molecule-protein complex from the sample.
In yet another embodiment, the invention provides a method for isolating a protein from a sample, the method comprising contacting the sample with a non-natural molecule comprising at least one self-association region isolated from the protein. And wherein the self-association region is fused with a moiety that prevents aggregation of the self-association region, and the resulting co-aggregated molecule-protein complex is isolated from the sample.
In yet another embodiment, the invention provides a method for isolating a protein from a sample, the method comprising contacting the sample with a non-natural molecule comprising at least one self-association region isolated from the protein. (Wherein the self-association region is fused with a portion that prevents aggregation of the self-association region, whereby the self-association region is in direct contact with the solvent in which the self-association region fused with the portion and the protein are present) And isolating the resulting co-aggregated molecule-protein complex from the sample.

In other words, the present invention provides a method for isolating a protein from a sample, comprising the following steps:
Contacting said protein with a non-natural molecule comprising part A and part B, wherein i) part A comprises a peptide, protein domain or agarose bead that prevents aggregation of part B; ii) part B is at least 1 Containing two self-association regions, wherein the region consists of at least three adjacent amino acids and is isolated from the protein, a linker optionally present between part A and part B, and-obtained Isolating the co-aggregated molecule-protein complex from the sample.

In other words, the present invention provides a method for isolating a protein from a sample, comprising the following steps:
Contacting said protein with a non-natural molecule comprising part A and part B, wherein i) part A comprises a peptide, protein domain or agarose beads that prevent aggregation of part B; ii) part B is at least 1 Comprising two self-associating regions, wherein said region consists of at least three adjacent amino acids and is isolated from said protein, a linker optionally present between part A and part B, wherein part B is said molecule And in direct contact with the environment in which the protein is present, and-isolating the resulting co-aggregated molecule-protein complex from the sample.

In other words, the present invention provides a method for isolating a protein from a sample, comprising the following steps:
Contacting said protein with a non-natural molecule comprising part A and part B, wherein i) part A comprises a peptide, protein domain or agarose beads that prevent aggregation of part B; ii) part B is at least 1 Consisting of at least three contiguous amino acids and isolated from the protein, a linker optionally present between part A and part B, wherein part B is the molecule And in direct contact with the environment in which the protein is present, and-isolating the resulting co-aggregated molecule-protein complex from the sample.

In other words, the present invention provides a method for isolating a protein from a sample, comprising the following steps:
Contacting said protein with a non-natural molecule comprising part A and part B, wherein i) part A comprises a peptide, protein domain or agarose bead that prevents aggregation of part B; ii) part B is at least 1 Consisting of two self-associating regions, wherein the region consists of at least three adjacent amino acids and is isolated from the protein, a linker optionally present between part A and part B, and-obtained Isolating the co-aggregated molecule-protein complex from the sample.

Separation In a further embodiment, the method of isolating at least one protein further comprises isolating at least one protein from the sample.

  One application for separating at least one protein from a sample is the removal (or depletion) of large amounts of protein from the sample. In fact, the main effort in protein target discovery and confirmation is how to clearly analyze complex protein samples (eg, plasma, urine, cerebrospinal fluid) and measure trace targets. Abundant proteins are often enriched by 6 to 10 orders of magnitude from lower amounts of protein. Thus, in order to detect and measure medically important trace proteins, abundant proteins must be removed. Albumin, IgG, antitrypsin, IgA, transferrin, and haptoglobin make up about 90% of the total protein content of human serum, so these unwanted abundant proteins are immediately depleted and small amounts of low molecular weight There is an urgent need for diagnostic tools that expose protein biomarkers. Several methods are already used in the art: 1) immunoglobulin G (IgG) as an affinity reagent to capture and separate abundant protein targets, 2) immunoglobulin yoke (IgY) An IgG-like antibody isolated from egg yolk, 3) pre-fractionated, separating the protein mixture into different fractions, removing certain proteins in the original mixture, and 4) protein A and Protein G is a bacterial cell wall protein specific for IgG antibodies, so protein A and G affinity resins remove IgG, and 5) IgG- and IgY-microbeads are used for protein detection.

In another specific embodiment of detection, the method of isolating at least one protein further comprises detecting at least one protein in said molecule-protein complex.

  Detection can be performed by separating the interferor molecule-target protein complex by, for example, electrophoresis, column chromatography, filtration, electrostatic attraction, magnetic or paramagnetic attraction, mass spectrometry, or the like.

  Biological detection technology based on the use of antibodies is most widely used. An antibody recognizes and binds to other molecules based on its shape and physicochemical properties. Antibodies are very suitable for detecting small amounts of target protein in the presence of a complex mixture of proteins. According to the present invention, the use of interferor molecules (part B is specific for and recognizes at least one specific protein) can be an alternative to the use of antibodies (recognition elements) for specific capture of target proteins I understand. In fact, interferor molecules can be used in a vast number of applications that commonly use antibodies. To name a few, applications are envisaged in diagnosis, microanalysis, forensic medicine, and specific detection of pathogens.

  For detection and separation applications of the present invention, it is preferred that part B of the interferor molecule is bound to a carrier designated herein as part A. The carrier can be a flat or chromatographic column such as plastic or nitrocellulose, but beads such as microsphere beads are preferred. General considerations regarding the various types of beads and microspheres serve the purpose of part A of the interferor molecule and are described on pages 9 and 10 of US6682940 and are specifically incorporated herein by reference. Is done.

  In a particular embodiment, the A part of the interferor molecule is a carbohydrate type carrier, such as cellulose or agarose. Part B can be covalently bound to the carbohydrate carrier by a cross-linking agent such as glutaraldehyde.

  In another specific embodiment, part A is a support such as cellulose, glass or a synthetic polymer. A covalent bond can be made between part A and part B via an amino acid residue in part B and an azide, carbodiimide, isocyanate or other chemical derivative in part A.

  In yet another specific embodiment, part A is a porous silane-modified glass microbead. Part B was formed by periodate oxidation of a glycidoxypropylsilane group chemically bonded to an atom of silica via its peptide amino group (Schiff reaction followed by reduction with sodium borohydride). It is possible to covalently bond to the A moiety towards the aldehyde group (this coupling is described in Sportsman and Wilson (1980) Anal. Chem. 52, 2013-2018).

  In a specific embodiment, the carrier A part is coated with a proteinaceous membrane in which the A part is cross-linked (see claims 1-50 of US4478946 and the examples relating to the carrier).

  In another specific embodiment, part A is a fluorescent bead such as a fluorescent latex particle. Patent US4550017, in particular page 4, describes fluorescent compounds that can be used to produce fluorescent beads.

  In another specific embodiment, the size of the bead A portion is variable and may include or be impregnated with a fluorescent dye. The varying size and dye of the beads allows multiple proteins to be detected and quantified in a single reaction. Means for developing the beads are described in US6159748.

  In yet another specific embodiment, the coupling between part A (beads) and part B is via poly (threonine), poly (serine), dextran or poly (ethylene glycol). Examples 6, 7, 8 and 9 of US6399317 describe how this coupling is performed.

  In yet another specific embodiment, part A is a magnetic bead. Magnetic beads, coupling between magnetic beads and protein drugs and their use are described on page 8 of application US6489092.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

  As used herein, the articles “a” and “an” mean one or more (ie, at least one) for article grammar purposes. By way of example, “a target protein” means one or more target proteins.

  As used herein, the term “about” is a quantity that is variable by as much as 30%, preferably as much as 20%, more preferably as much as 10% relative to a reference quantity, level, value, dimension, size or quantity. Means level, value, dimension, size or quantity.

  "Bifunctional cross-linking reagent" means a reagent that contains two reactive groups, so that the reagent has the ability to covalently bind to two elements such as the A and B parts of the interferor molecule. The reactive groups in the cross-linking reagent usually belong to a class of functional groups including haloacetamides such as succinimidyl esters, maleimides and iodoacetamides. Throughout this specification, unless the context requires otherwise, the terms “comprising” and “including” naturally encompass a defined step or element or group of steps or elements. It does not imply that other steps or elements or groups of steps or elements are excluded.

  By “expression vector” or “recombinant vector” is meant any self-genetic element that can be targeted for the synthesis of interferor molecules encoded by the vector. Such expression vectors are well known to those skilled in the art.

  A “derivative” is an interferor derived from a base sequence by modification, eg, by conjugation or conjugation with other chemical moieties (eg, pegylation) or by post-translational modification techniques as understood in the art. Means a molecule.

  In the context of modulating activity or treating or preventing a symptom, an “effective amount” refers to the dose of an interferor molecule in an individual in need of the modulation, treatment or prevention, modulation of the effect or condition thereof. Means to be administered alone or as part of a continuous administration that is effective in treating or preventing. The effective amount will vary depending on the health status and condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. The amount is expected to be in a relatively broad range that can be determined through routine trials.

  “Isolated” means that the material is substantially or essentially free from components that normally accompany the material in its original state. For example, an “isolated polypeptide” as used herein refers to a polypeptide that is purified from a sequence that naturally flank the polypeptide, eg, a self-association sequence that has been removed from a sequence that normally flank the sequence. . Self-association sequences (optionally coupled to moieties that prevent aggregation) can be generated by amino acid chemical synthesis or can be generated by recombinant production.

  As used herein, the term “oligonucleotide” is a polymer composed of multiple nucleotide units (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogs) linked through phosphodiester bonds. (Or related structural variants or synthetic analogues thereof). Although the term “polynucleotide” or “nucleic acid” is usually used for large oligonucleotides, oligonucleotides are usually about 10-30 nucleotides in length, but the term is a molecule of any length. Can mean. The term “polynucleotide” or “nucleic acid” as used herein refers to mRNA, RNA, cRNA, cDNA or DNA. The term usually refers to oligonucleotides longer than 30 nucleotides.

  The term “recombinant polynucleotide” as used herein refers to a polynucleotide formed in vitro by manipulating a nucleic acid in a form not normally found in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. Generally, the expression vector comprises a transcriptional regulatory nucleic acid and a translational regulatory nucleic acid operably linked to the nucleotide sequence.

  “Operably linked” means that the transcriptional regulatory nucleic acid and the translational regulatory nucleic acid are located relative to the polypeptide-encoding polynucleotide in such a way that the polynucleotide is transcribed and the polypeptide is translated. To do.

  As used herein, the terms “subject” or “individual” or “patient” mean any subject in need of treatment or prevention, particularly a vertebrate specimen, and more particularly a mammalian specimen. To do. Suitable vertebrates within the scope of the present invention include primates, birds, fish, reptiles, livestock (eg sheep, cows, horses, donkeys, pigs), laboratory animals (eg rabbits, mice, rats, guinea pigs, hamsters). ), Pets (eg, cats, dogs) and captive wild animals (eg, foxes, deer, dingos). However, it will be appreciated that the above terms do not imply the presence of symptoms.

  By “pharmaceutically acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating material that can be safely used for local or systemic administration to a patient.

  “Polypeptide”, “peptide” and “protein” are used interchangeably herein and refer to a polymer of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic unnatural amino acids, such as chemical analogs of the corresponding natural amino acids, as well as to natural amino acid polymers.

  “Recombinant polypeptide” means a polypeptide made using recombinant techniques, ie, by expression of a recombinant or synthetic polynucleotide. Where the chimeric polypeptide or bioactive portion thereof is produced recombinantly, it is also preferably substantially media free, i.e., the media is less than about 20% of the protein preparation volume, more preferably about 10%. Less than, most preferably less than about 5%.

  As used herein, the term “sequence identity” means a range in which the sequence is identical at every nucleotide or amino acid throughout the frame of comparison. Thus, the “percent sequence identity” compares two optimally aligned sequences over the entire window of comparison, and the same nucleobase (eg, A, T, C, G, I) or the same amino acid residue ( For example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Is calculated by calculating the number of corresponding positions, dividing the number of corresponding positions by the total number of positions within the comparison frame (ie, window size), and multiplying the result by 100 to obtain the percentage of sequence identity. . For purposes of the present invention, “sequence identity” is attached to the software by the DNASIS computer program (Windows version 2.5; purchased from Hitachi Software Engineering Co., Ltd., South San Francisco, Calif., USA). Naturally, this means the “corresponding percentage” calculated using the standard default used in the reference manual. “Similarity” means the percentage value of amino acids that are identical or constitute conservative substitutions. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). In this method, sequences of similar or substantially different length to the sequences cited herein are compared, for example by inserting a gap into the alignment as determined by the comparison algorithm used by GAP. May be.

  The term “transformation” means the modification of the genotype of an organism, such as a bacterium, yeast or plant, by the introduction of foreign or endogenous nucleic acids. Transformation vectors include plasmids, retroviruses and other animal viruses, YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome) and the like. By “vector” is meant a polynucleotide molecule, preferably a DNA molecule from a plasmid, bacteriophage, yeast or virus into which a polynucleotide can be inserted or cloned, for example. Preferably, the vector contains one or more unique restriction sites and is capable of self-replication in a defined host cell containing the target cell or tissue or its progenitor cells or tissue, or the cloned sequence can be regenerated. So that it can be integrated with the genome of a defined host. Thus, a vector is a self-replicating vector, i.e. a vector that exists as an extrachromosomal component and whose replication is independent of chromosomal replication, e.g. a linear or closed plasmid, extrachromosomal element, minichromosome or artificial chromosome. It is possible. The vector can include any means to ensure self-replication. Alternatively, the vector can be a vector that, when introduced into a host cell, is integrated into the genome and replicated along with the chromosome (s) into which it is integrated. A vector system can include a single vector or plasmid, two or more vectors or plasmids, both of which contain total DNA introduced into the genome or transposon of the host cell. The selection of the vector is usually said to depend on the compatibility of the vector with the host cell into which the vector is introduced. In a preferred embodiment, the vector is preferably a virus or virus-derived vector that is operably functional in an animal, preferably a mammalian cell. The vector can also contain a selectable marker such as an antibiotic resistance gene that can be used to select suitable transformants. Examples of such resistance genes are well known to those skilled in the art and include the nptII gene conferring resistance to the antibiotics kanamycin and G418 (Geneticin®) and the hph gene conferring resistance to the antibiotic hygromycin B. .

  Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials described below are useful. The materials, methods, and examples are illustrative only and not limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

Example
1. Design of Synthetic Peptide Interferor Molecules We have determined that the B part from only three synthetic self-association regions with a short linker of two amino acids (STLIVL-QN-STVIFE-QN-STVIFE) interconnecting the self-association regions A structured interferor molecule was constructed. The three self-association regions are hexapeptides having a strong tendency to aggregate. See FIG. 1 for the design of the interferor molecule. Note: In the text of the present invention, the entire amino acid sequence is expressed as starting from the amino terminus and is read in the direction of the carboxy terminus-thus "STLIVL" is read as "NH2-STLIVL-COOH"). The B part of the synthetic interferor molecule was fused at the N-terminus to the part (part A) where aggregation was prevented and the self-association region was in direct contact with the environment (here, E. coli cytosol). Represents the structure of the design). The part is a NusA protein that is frequently used as a solubilization tag in recombinant protein production ¹³ . The resulting synthetic interferor molecule (AB structure) could be prepared and purified by recombinant methods in E. coli.

  We have shown that overexpression of synthetic interferor molecules (which have no specific self-association sequence specific for a particular E. coli protein) does not interfere with bacterial growth. Therefore, BL21 E. coli cells were transformed with the synthetic interferer construct present in the pETM60 plasmid (assigned from G. Stier, EMBL). In the latter plasmid, the interferor is under the control of the chimeric T7 promoter (see Materials and Methods section). Recombinants were grown to a density of 0.6 OD, interferors were expressed for 3 hours at 37 ° C. with 0.5 μM IPTG, and the bacterial suspension was plated on agar plates. After 12 hours of incubation at 37 ° C., the plate was examined and found to be growing heavily in bacteria.

  In the next step, a flexible linker sequence (“KPGAAKG” —represented as “linker” in FIG. 1) is coupled to the COOH end of the synthetic interferor construct to allow fusion of self-association sequences from the target protein. did.

2. Protein interference in prokaryotes In this example, E. coli proteins were selected and down-regulated with functional protein interference allowing trait selection. From the E. coli proteome, target proteins were selected for cytosol localization and the presence of a suitable high TANGO score aggregation region. Because the conditional auxotrophy requirement for a single amino acid can be conveniently tested using a growth medium with limited composition, we have identified isoleucine (UniProt ¹⁵ registry: ILVI_ECOLI). ), Four candidate enzymes involved in the synthesis of methionine (UniProt ¹⁵ registry: METE_ECOLI and METK_ECOLI) and leucine (UniProt ¹⁵ registry: LEU1_ECOLI) were selected. Self-association sequences based on TANGO predicted scores of the four target proteins are: ILVI_ECOLI: “GVVLVTSG”, TANGO score: 44, METE_ECOLI: “LLLTTYF”, TANGO score: 32, METK_ECOLI: “LTLLV”, TANGO score: 20, And LEU1_ECOLI: “LAFIG”, TANGO score: 15. The genetic information about the synthetic interferor molecule of Example 1 was fused to a DNA sequence encoding the individual self-association regions of the four biosynthetic enzymes resulting in four specific interferor molecules. In order to demonstrate protein interference in vivo (essentially a coaggregation between a specific interferor and its biosynthetic enzyme (for biosynthetic enzymes)), we proceeded as follows. E. coli was transformed with plasmids containing individual interferor constructs and cultured in rich medium until the exponential growth phase began. At this time, expression of interferor protein was induced with IPTG (isopropyl-beta-D-thiogalactopyranoside). Protein expression is allowed to proceed at 37 ° C., cells are harvested, washed with salt, excess IPTG and nutrient rich medium removed, and a minimum a M9 medium-containing agar plate complete with 20 natural amino acids (M9 complete medium and And plated on minimal M9 medium (referred to as M9 selection) containing all amino acids other than those tested for auxotrophy. It was found that three of the four target enzymes were able to knock out function completely, ie, the bacteria formed colonies on M9 complete medium but not on M9 selective agar plates. . The expression of the interferor construct in the insoluble phase of the cell lysate and its exclusive presence was confirmed by Western blot. For the four enzymes tested here, there is a clear relationship between sensitivity to the coaggregation approach and the predicted propensity to aggregate according to the TANGO algorithm, which further includes functional cell context as well as the quality of the TANGO prediction. Is backed up. The ILVI protein with the highest TANGO score is almost completely knocked out due to leaky expression from the T7 promoter in the absence of all IPTG, and is completely functionally knocked out after 1 hour of overexpression. METE and METK enzymes show moderate TANGO scores and are not affected by overexpression of interferers for 1 hour. However, 3 hours after IPTG induction, the function was completely inactivated and colony formation could not be detected. LEU1 enzyme had the lowest aggregation score, and in this case overexpression only moderately down-regulated activity. Notably, the functional knockout of the target enzyme is reversible. When cells loaded with high levels of overexpressed interferor material were plated on LB agar, colonies were shown to be growing normally. When these colonies were copied to the M9 selection, normal growth was seen again. This suggests that agglomeration does not occur while the colony is growing, and the cell network is recovering normally.

3. Target protein interference self -association regions, including self-association regions with low self-association scores, are frequently adjacent to or contain charged residues such as R, K, D and E, and P and G (so-called gates) (See Rousseau, Serrano & Schymkowiz (2006) How Evolutionary Pressure Against Protein Aggregation Shaped Chaperone Specificity, J Mol Biol, doi: 10. 1016 / j. Jmb. 2005. 11. 035). These gatekeeper residues reduce the self-association propensity of the sequences with which they are associated. In order to optimize the sensitivity of the B part of an interferor molecule that co-aggregates with a given target protein, the self-association region of the target protein contained in the B part of the interferor can be mutated and thus Are replaced with aggregation-promoting residues such as L, V, I, F, W, and Y, and other residues that can increase the self-association tendency of the self-association region are also included. The mutant self-association region (derived from the target protein) contained in part B of the interferor molecule is at least 60%, preferably at least 70%, more preferably at least 80% and most preferably with the self-association region of the target protein Have at least 90% sequence homology. In addition, there are amino acids that are neutral with respect to aggregation, and substitution of the amino acids with amino acids that favor aggregation also results in certain regions (eg, S, T, C and Q, N, H, M are L, V, I, F, W It is believed that the self-association tendency can be increased by substitution with aggregation-prone residues such as Y, Y. These optimized integrator molecules are targeted at protein interferences that are predicted to have low aggregation scores Example 2 shows that the protein interference of the LEU1 enzyme in E. coli is not very efficient, in this example an interferor molecule specific for the LEU1 enzyme has been optimized. The identified self-association sequence is “LAFIG.” The latter sequence contains a gatekeeper residue.
In order to facilitate mutation of the target sequence, therefore, a strategy based on degenerate pcr is taken as follows: complementary primers are placed at each site of the codon to be mutated It is designed so that 20-25 bp overlap and the primer anneals efficiently to the template. Degenerate codons are introduced by incorporating an equimolar ratio of four bases during primer synthesis (so-called NNS codon). Using this degenerate primer Quickchange PCR protocol, a library containing 20 point mutations at adjacent positions is obtained. This library is amplified in Top 10 cells (Invitrogen) and the plasmid DNA is purified with the miniPrep kit (Qiagen). In testing, if the knockout efficiency increases, a mutant interferor (designed as in Example 2) for the LEU1 target is transformed into BL21 cells (Invitrogen) and plated on LB agar plates. Individual colonies were picked and seeded into each well in a 96 well plate containing 0.2 mL LB + antibiotic per well. Incubate the plate at 37 ° C. until the OD is 0.6. At this time, 0.5 μM IPTG is added to induce the expression of the mutant interferor at 37 ° C. for 3 hours. All contents of each well except 1 μL are plated on selective minimal medium containing all amino acids except leucine. For clones with varying degrees of growth damage on selective plates, TempliPhi reagent (GE Health Science) is added for DNA amplification and the plates are transferred to a sequencer. The sequence information provides interferor molecules mutated by optimized LEU1 over the entire range.

4). Use of interferor molecule for immunoglobulin G deficiency from serum In this depletion experiment, agarose beads were used as the part where the self-association region derived from the target protein is fused via amino-reactive chemical crosslinking (part A). select. The agarose material is commercially available, and includes NHS-activated Sepharose ™ 4 Fast Flow from GE healthcare. Human immunoglobulin G has two strong tango regions that can be used as self-association regions.
Have Since peptide consumption is proportional to the length of the amino acid, the peptide is designed to contain a fraction of 10 amino acids from the initial target region. In front of the target sequence (self-association region) is the linker sequence ADPRGAAEGA, which is synthesized at the unprotected end and maintains the reactive N-terminal amino group. The designed array is
(A), b) and c) contain the strong tango region I derived decapeptide),
Includes tango region II). To investigate the deficiency, 10 ml serum is incubated with 1 mg immobilized peptide for 1 hour at 25, 30, 37 and 45 ° C. Agarose beads are collected by centrifugation, serum is removed, and the agarose beads are washed with PBS buffer to remove residual impurities. The beads are then transferred to SDS buffer, incubated at 95 ° C. for 10 minutes, and vortexed extensively. The presence of IgG is investigated using SDS-PAGE. Confirm the identity of the target by mass spectrometry.

5. Use of Interferer Molecules for Detection Three commercially available recombinant proteins (porcine heart citrate synthase (Roche), E. coli-derived beta-galactosidase (Sigma) and Leuconostoc mesenteroides-derived glucose-6-phosphorus An interferor molecule was designed for specific detection of acid dehydrogenase (Sigma), where the first step determined the self-association region from the following target proteins by the TANGO algorithm:
a) Citrate synthase (CISY_PIG): “ALFWLLVT” (TANGO score 60),
b) Beta-galactosidase (BGAL_ECOLI): “AVIIWSLGN” (TANGO score 30) and “ALAVVLQ” (TANGO score 42), and
c) Glucose-6-phosphate dehydrogenase (G6PD_LEUME): “AFVDAISAVYTA” (TANGO score 41).

In the next step, biotin was coupled at the amino terminus to four different self-association regions, resulting in the following four different interferor molecules: i) Biotin-ALFWLLVT specific for citrate synthase, ii) beta -Biotin-AVIWSLGN and biotin-ALAVVLQ specific for galactosidase, and iii) Biotin-AFVDAISAVYTA specific for glucose-6-phosphate dehydrogenase. Biotinylated peptide was obtained from Jerine Peptide Technologies. Interferer design: The biotin-self-association region matches the AB structure in which biotin (part A) prevents aggregation of the self-association region (part B), and the biotin-self-association region is present in the self-association region Note that it is in direct contact with the solvent (PBST).
Individual dot blots were prepared by spotting 0.3 mg of each target protein onto a nitrocellulose membrane, then air dried and incubated overnight in 1% BSA-PBST (PBS containing 0.1% Tween20) Non-specific binding sites were blocked. The membrane was immersed in a 10 mM solution of biotinylated detection peptide and incubated at room temperature for 3 hours with stirring. After repeated washing with the buffer, the binding of the peptide to the protein was confirmed by visualization of the biotin moiety using streptavidin-HRP (Horse Radish Peroxidase, Pierce) and chemiluminescence detection using a CCD camera system.

6). Use of interferor molecules on mouse VEGF for the treatment of pathological retinal neovascularization Retinal neovascularization is the leading cause of blindness worldwide, and pathological retinal neovascularization is premature infant retinopathy (ROP), diabetic retina It is the last common path leading to blindness in diseases such as dementia and age related macular degeneration. Vascular endothelial growth factor (VEGF) has been shown to play an important role in the pathogenesis of pathological angiogenesis. The effect of interferor molecules on VEGF is studied in two mouse-induced retinopathy models. In the first model, neonatal mice (with immature retinal vasculature) are hyperoxygenated to occlude angiogenesis that supplies oxygen to the retina. When the mouse then returns to normoxia, the retina distal to the occluded blood vessel becomes ischemic, and VEGF production is induced, eventually resulting in a reproducible and quantifiable proliferative retinal neovascularization ( The model is described in detail in Pierce EA et al. (1995) Proc. Natl. Acad. Sci. 92 (3) 905-9 (see experimental procedure for -mouse model on page 905). That is, pup mice that had been with a lactating female parent for 7 days (P7) are placed in a hyperoxic state (75% oxygen) without opening the cage for 5 days in a specially designed oxygen chamber. At P12, the animal is returned to room air and maintained until P17, at which the retina is evaluated for maximum neovascular response. At P12, half of the animals are given an interferor molecule for VEGF and half are left untreated. Half of the administered mice are administered VEGF interferor by intravitreal injection, while the other half of the administered group is administered VEGF interferor by periocular injection (periocular or intravitreal injection is described in Shen J et al., (2006 ) As described in Gene Therapy, September 29, prior online announcement). Three different interferor molecules for the mouse VEGF165 isoform were used in a concentration range of 1-100 μg / ml.

a)
This interferor molecule has the ABA ′ structure of the interferor molecule. The self-association region from mouse VEGF165 (underlined) is adjacent to the solubilized region A (REAG and GGEERAG), ie, regions A and A ′ prevent aggregation of the self-association region (B part of the interferor molecule). To do.

b)
This interferor molecule has the B-A structure of the interferor molecule. Solubilized part A (GERAG) is shown in italics. Part B has the following structure: STVIIE (= synthetic self-association region) -GGAG (= linker) -NHVTLS (= synthetic self-association region) -GGAGQ (= linker) -FLLSWVHWTLLALLLYLHHG (= mouse VEGF165-derived self-association region).

c)
This interferor molecule has the B-A structure of the interferor molecule. Solubilized part A (GERAG) is shown in italics. Part B has the following structure: STVIIE (= synthetic self-association region) -GGAG (= linker between self-association regions) -FLLSWVHWTALLLYLYHH (= mouse VEGF165-derived self-association region).

At P17, anesthetized mice are perfused through the left ventricle with 1 ml of phosphate buffered saline containing 50 mg of 2 × 10 ⁶ molecular weight fluorescein-dextran. Eyes are removed and fixed in 4% paraformaldehyde for 3 hours (right eye) or 24 hours (left eye). The lens is removed from the right eye, the peripheral retina is cut, and the sample is made flat with glycerol-gelatin. The flat retina is analyzed with a fluorescence microscope. The left eye is embedded in paraffin, and successive 6 μm sections are cut sagittally parallel to the optic nerve to the cornea and stained with hematoxylin-eosin. The proliferative neovascular response is quantified by counting the number of new blood vessels (= tufts) and endothelial cells extending from the inner retina to the vitreous on the stained cross section. Angiographic techniques with fluorescent dextran perfusion are used in conjunction with this counting method for rapid screening of the retina or as an alternative scoring system for quantitative evaluation. In the second model, retinal neovascularization is experimentally mimicked by laser-induced retinal vein thrombus. The model is Saito Y et al. (1997) Curr. Eye Res. 16 (1): 26-33. Chi-Chun Lai et al. (2005) Acta Ophtalmologica Scandinavica 83: 590-594 ~ Materials and Methods section on page 592. VEGF interferor molecules are applied as previously described herein.

7). The regulation (induction or suppression) of protein interference apoptosis in human cell lines can be easily monitored in cell lines. Staurosporine is known to induce apoptosis in a p53-dependent manner. Thus, down-regulating p53 or down-regulating a protein that promotes p53 function (eg, ASPP1) suppresses staurosporine-induced apoptosis in animal (eg, human) cell lines. The recombinant expression vector encoding the interferor molecule is constructed based on the design of the synthetic interferor molecule described in Example 1, with the exception that the A part, the NusA protein changes to green fluorescent protein (GFP), The promoter is a constitutive mammalian promoter such as an actin or CMV promoter. The self-association sequence for p53 is ILTIITLE (tango score is 72), and this sequence is added in the B part of the synthetic interferor to generate an interferor molecule specific for p53. The self-association sequence for ASPP1 is MILTVFLSN (tango score is 63), and this sequence is added in part B of the synthetic interferor to generate an interferor molecule specific for ASPP1. HeLa cells are cultured and transfected with the recombinant vector. GFP (part A) visualizes overexpressed interferor molecules. Addition of 1 μM staurosporine to transfected and untransfected control cells induces different apoptotic responses.

8). An interferor molecule has been developed that points to the zebrafish VEGF protein interference of endothelial growth factor (VEGF) in zebrafish. Following specific inactivation (via aggregation) of secreted VEGF, it can interfere with angiogenesis in zebrafish embryos.

In the first step, the self-association region present in the zebrafish VEGF protein was determined by the TANGO algorithm. Clustering regions TANGO score is the highest is _NH 2 -FLAALLHLSA-COOH. Based on this self-association sequence, the following four synthetic interferor molecules were developed:
Interferer E serves as a control sequence and is derived from sequences outside this high TANGO region.

  Note that interferers A, B, and D include the entire TANGO region, while interferer C includes only a portion of the TANGO region. The sequence derived from the TANGO region is underlined.

These interferor molecules were added at different concentrations to the culture medium of transgenic Tg (fli1: EGFP) ^y1 zebrafish embryos. Transgenic Tg (fli1: EGFP) ^y1 zebrafish expresses enhanced green fluorescent protein (GFP) in its endothelial cells, said fish being Lawson ND and Weinstein BM (2002) Dev. Biol. 248, 307-318. ), Provided by the Zebrafish International Resource Center (University of Oregon) and published in the Zebrafish Book (Univ. Oregon Press, Eugene, 1994), a guide for experimenting with zebrafish (Danio rerio). Maintain as described.

  The corion-free embryos 20 hours after fertilization were arranged in 24-well plates (10 embryos / well) and exposed to several concentrations of selected interferor molecules (starting at 50 μM) for 24 hours. Live embryos were analyzed at 28 and 48 hpf (time after fertilization) by confocal imaging using a Zeiss laser scanning microscope LSM510.

  Although we monitor the growth of various vascular structures, we have (i) dorsal aorta (DA), posterior main vein (PCV) structure, Particular attention was paid to vascular (ISV) sprouting and vascular network (PV) formation. A summary of dose-dependent experiments is shown in Table 1. It is clear that Interferers A and C induce clear vascular injury during the growth of zebrafish larvae. Surprisingly, the interferor molecule is taken up by the zebrafish larvae through the skin and no injection of the interferor is necessary. Interferers B and D need to be administered at lower concentrations in order to be able to monitor certain vascular disorders.

9. Inactivation of the target (via aggregation ) of protein-interfering Ura3 protein in Saccharomyces cerevisiae can be easily read out, so that the yeast Ura3 enzyme is knocked down to show that protein interference acts in eukaryotes It was used. The budding yeast Ura3 enzyme is a basic enzyme involved in the uracil biosynthetic pathway. Therefore, a budding yeast mutant lacking the URA3 gene cannot grow in a medium not containing uracil, but growth can be recovered by adding uracil to the medium. In the first step, the self-association region present in the Ura3 protein was determined by the TANGO algorithm. The self-association region (or aggregation region) having the highest TANGO score is NH ₂ -VIGFIAQ-COOH (TANGO score: 74). This peptide sequence was used to generate an interferor expression construct. In order to clone the self-association sequence encoding this peptide in frame with a synthetic interferor construct (see Example 1), the following two oligonucleotides were used:

XbaI and SalI restriction sites are underlined. The start codon of the NusA protein is highlighted in bold. The stop codon is shown in italics and the sequence encoding the seven Ura3 amino acids is shown in bold.

The pETM60 plasmid (assigned from G. Stier, EMBL) containing the NusA protein coupled to the synthetic interferor / linker construct is used as a template for PCR (see Example 1). This vector contains the T7 promoter, confers resistance to kanamycin, resulting in six N-terminal expression tags for histidine. The resulting PCR product was subcloned into the pBEVY / GT vector (Miller CA 3 ^rd , Martinat MA and Hyman LE (1988) Nucleic Acids Res. 26: 3577-3583) using XbaI and SalI restriction sites. . Within this vector, the “NusA-synthetic interferer-linker-Ura3 self-association sequence” expression cassette was under the control of the budding yeast GAL1 / 10 promoter. The selectable marker for this vector is the TRP1 gene.

A sequence verification construct containing and not containing DNA encoding 7 Ura3 amino acids (from the self-association region) was introduced into Saccharomyces cerevisiae strain PVD2 by transformation and the selection for transformation was based on trp1 complementarity. The PVD2 strain is derived from the W303-1A strain (Thomas BJ and Rothstein R. (1989) Cell 56, 619-630) but is transformed with both HIS3 and URA3 wild type alleles. Nevertheless, the PVD2 strain has auxotrophy for leucine (LEU2), tryptophan (TRP1) and adenine (ADE2). Transformants were selected on SDglu-Trp medium (minimal yeast medium containing 2% glucose but no tryptophan). Colonies were streaked again on a new SDglu-Trp plate for single colonies. Two independent colonies were cultured overnight in liquid SDglu-Trp medium. Thereafter, the culture was adjusted so that the OD ₆₀₀ was 1, and a 10-fold serial dilution was diluted with 5 microliters of SDglu-Ura-Trp (SD medium containing 2% glucose but not uracil and tryptophan). Or spotted on SDgal-Ura-Trp (SD medium containing 2% galactose but no uracil and tryptophan) plates. The results of this experiment are shown in FIG. This experiment was repeated three times with similar results. When expressing an empty pBEVY / GT vector or a vector that expresses only the NusA-synthetic interferor construct (no ura3-derived self-association sequence), no growth inhibition is seen in uracil-free medium. However, expression of the NusA-synthetic interferer-Ura3 association domain construct strongly inhibited growth in uracil-free medium (when uracil was added to growth medium without growth defects), and endogenous Ura3 protein was specifically inhibited by protein interference. It can be seen that it is inactivated.

10. Forty percent of protein-interfering human fungal infections in the yeast Candida albicans are attributed to Candida albicans. This symbiont has a number of virulence factors. Apart from the ability to adhere to all types of plastic (which is a major problem in intensive care units) or the production of lipases and proteinases, it has the ability to take a variety of forms, because it is one of the major virulence factors The most extensively studied. Many transcription factors can induce the transition from yeast-like cells to hyphae or pseudohyphae. Other transcription factors are required to maintain the cells in a yeast-like form. One example of the repressor of mycelia is Tup1. As an example of protein interference in Candida albicans, we have targeted the Tup1 protein. It is preferred that down-regulation of Tup1 biological function induces hyphal formation. In the first step, the self-association region present in the Tup1 protein was determined by the TANGO algorithm. TANGO score best: self-association region (or aggregation region) is (TANGO score 30) is _NH 2 -VISVAVSL-COOH. This peptide was used to generate an interferor expression construct. To clone the self-assembling sequence encoding this peptide in frame with the synthetic interferor construct of Example 1, the following two oligonucleotides were used:

BsrGI and NheI sites are underlined. The stop codon is shown in italics and the reverse sequence encoding the target peptide is shown in bold.

The pETM60 plasmid containing the Nus-synthetic interferor construct (of Example 1) was used as a template for PCR. Using BsrGI and NheI restriction sites, the resulting PCR product was placed into the pPCK1-GFP plasmid (Barelle CJ, Manson CL, MacCallum DM, Odds F, Gow NAR, Brown AJP. Yeast 21: 333-340, 2004). Subcloned. In this vector, the interferor construct was cloned in frame with the GFP gene present in the vector, and the resulting interferor expression cassette “GFP-synthetic interferer-linker-“ Tup1 self-association region ”) was controlled by the PCK1 promoter. Below. In the latter construct, green fluorescent protein (GFP) is replaced with NusA, and GFP serves as the A part of the interferor molecule. The PCK1 promoter is strongly induced in casamino acid-containing media and repressed in glucose-containing media (Leuker CE, Sonneborn A, Delbruck S, Ernst JF. Gene 192: 235-240, 1997). The sequence verification plasmid is then C.I. It was transformed into albicans strain CA14 (Fonzi WA, Irwin MY. Genetics 134: 717-728, 1993). Transformants were selected on SDglu-ura (minimal yeast medium containing 2% glucose but no uracil). Transformants were cultured overnight in minimal medium containing glucose, and the cells were diluted to approximately 20 cells / 100 microliters, and this amount was plated on SDglu-ura or SD casamino acid-ura agar plates. Colony morphology was scored after 4 and 6 days of culture (see Figure 3). As can be seen in FIG. 3, Tup1 is down-regulated in the medium having casamino acid, and hyphal formation is clearly seen at the edge of the colony. Mycelium formation is not seen in the control transformants ((pPCK1-GFP plasmid without the interferor expression cassette)) and not in the glucose-containing medium. From the examples it can be seen that endogenous Tup1 is specifically inactivated by protein interference.

11. Application of protein interference in plants We describe protein interference in tobacco BY2 cells using already transformed BY2 cells with several GFP fusion genes (the genes are listed in Table 2). The Arabidopsis thaliana gene and its corresponding identified self-association region and tango score are listed in Table 2.

  Specific interferor molecules for each target in Table 2 are designed based on the synthetic interferor molecules described in Example 1, with the exception that the NusA protein changes to red fluorescent protein (RFP) and The target specific self-association region in Table 2 is added.

  The construct encoding the specific interferor molecule is introduced into a vector suitable for overexpression (In Vitrogen Life Technologies) using Gateway ™ technology. To this end, a set of Gateway compatible binary vectors for plant transformation was developed. For expression, the pK7WGD2 vector is used in which the gene is placed under the control of the p35S promoter. For plant cell transformation, a ternary vector system is applied. Plasmid pBBR1MCS-5. virGN54D is used as a ternary vector. A binary plasmid is introduced into Agrobacterium tumefaciens strain LBA4404 which already has a ternary plasmid by electrotransformation. Prior to transformation with a specific construct, a new BY-2 culture is constructed. 5-day culture BY-2 is seeded at 1:10 and cultured for 3 days (28 ° C., 130 rpm, light-shielded). Two days before BY-2 transformation, such as pK7WGD2-GUS (control vector), pK7WGD2-interferer 1 (eg specific to Aurora 1, pK7WGD2-interferer 2 (eg specific to Aurora 2), etc. Build a liquid culture of transformed Agrobacterium tumefaciens, etc. Seed 1 loopful of bacteria from solid medium in liquid LB medium containing 5 ml antibiotics (rifampicin, gentamicin, streptomycin and spectinomycin) Incubate the culture for 2 days (28 ° C., 130 rpm) Transform BY-2 in an empty Petri dish (φ4, 6 cm) by co-cultivation. Plated by petting and either 50 or 200 μl bacterial suspension added. Mix gently and leave on a layered bench for 3 days protected from light.After co-culture, select products (50 μg / ml kanamycin, 500 μg / ml vancomycin and 500 μg / ml to kill excess bacteria) The cells are plated with solid BY-2 medium containing 2 mg of carbenicillin. (Here aggregation between GFP and RFP constructs) is visualized by examining GFP, RFP expression and GFP and RFP localization under a fluorescence microscope.

Materials and methods for Examples 1 and 2 Constructs, cells and media The interferor molecule was cloned into the vector pETM60 (assigned from G. Stier, EMBL). This vector is under the control of RNA polymerase T7 (T7 RNA polymerase is under the control of regulatory elements from the E. coli lac operon), confer resistance to kanamycin, resulting in six N-terminal expression tags of histidine associated with NusA. A “synthetic gene” was prepared by PCR using a series of overlapping oligos encoding the sequence of the interferor B part. This gene was ligated into pETM60 via Nco1 and BamH1 restriction sites. Using a long anti-coding oligo, an interferor gene was generated by PCR of the interferor B part, and included a sequence for a flexible linker and a protein-specific self-association region (bait) at the C-terminus. These were ligated to pETM60 via Nco1 and BamH1 restriction sites. All oligos were purchased from Sigma-Genosys, all restriction enzymes were purchased from Fermentas and ligated using the Roche Quick Ligation Kit.

  Chemically competent BL21 (DE3) cells were generated in house and transformed according to standard protocols. Standard LB agar plates were prepared and all agar plates contained 50 μg / mL kanamycin. Standard M9 medium was supplemented with all 20 amino acids (50 μg / mL) and nucleotides, adenine, guanine, uracil and xanthine (20 μg / mL) to prepare M9 complete plates. The M9 selection plate was identical to the M9 complete plate except for one amino acid. Plates were prepared one day prior to use to control the potential for amino acid degradation during storage. LB was prepared according to the standard protocol and kanamycin was added at 50 μg / mL. All amino acids were purchased from Sigma and kanamycin and IPTG were purchased from Duchefa.

Protocol BL21 (DE3) cells were transformed with the expression construct, plated on LB agar supplemented with kanamycin, and incubated overnight at 37 ° C. A single colony was used to inoculate 10 mL of LB supplemented with kanamycin, and this was cultured overnight at 37 ° C. with shaking. On the next day, this culture solution was used to inoculate 10 mL of LB to which kanamycin 1: 100 was added, and this was cultured until OD600 became 0.6. The culture was then divided into two; IPTG (50 μM) was added to one culture to induce interferor expression, and both cultures were further incubated at 37 ° C. for the desired expression time. Cells were then harvested by centrifugation and washed twice with saline (0.85% w / v NaCl) (by resuspension and centrifugation). The cells were then resuspended in saline and finally OD600 was 0.05, and 200 μL of this cell suspension was plated on an agar plate. The agar plate was incubated overnight at 37 ° C. and the next day was watched for colony growth. If necessary, colonies were picked on fresh agar plates with a sterile toothpick.

table

Claims (15)

  A method of down-regulating a biological function of a protein, comprising contacting the protein with a non-natural molecule comprising at least one self-association region present in the protein.   The method of claim 1, wherein the self-association region is fused to a moiety that prevents aggregation of the self-association region.   The method of claim 2, wherein the moiety is a peptide, protein domain or agarose bead.   The method according to claim 1, wherein the self-association region consists of at least three adjacent amino acids.   The method according to claim 1, wherein a linker is present between the self-association region and the moiety.   6. The method of claim 5, wherein the linker is a polypeptide or has non-polypeptide properties.   The molecule is a polypeptide, encodes a nucleotide sequence present in a recombinant vector, and transformation into a cell or organism produces the polypeptide in the cell or organism. 6. The method according to 6.   A non-natural molecule comprising at least one self-association region isolated from a protein domain that can be soluble in water, wherein the self-association region is fused to a moiety that prevents aggregation of the self-association region The molecule.   9. A molecule according to claim 8, wherein the moiety is a peptide, protein domain or agarose bead.   A recombinant vector comprising a polynucleotide encoding the polypeptide of claims 8 and 9.   Molecules according to claims 8 to 10 for use as a medicament.   A method for isolating a protein from a sample comprising contacting the sample with a molecule comprising at least one self-association region isolated from the protein, and the resulting coaggregated molecule-protein complex comprising Isolating from a sample.   13. The method of claim 12, wherein the self-association region is fused to a moiety that prevents aggregation of the self-association region.   14. The method of claim 13, wherein the moiety is a peptide, protein domain or agarose bead.   15. The method of claims 12-14, further comprising detecting the protein in the sample.