The invention provides methods for identifying an anti-poxviral agents. In many embodiments, the methods involve contacting a poxviral p28 polypeptide with a candidate agent, and determining an effect of the agent on a ubiquitin ligase activity of the p28 polypeptide. The effect of the agent may be determined using a variety of different cell based or biochemical assays, such as polyubiquitylation assays and cell viability assays. The invention also provides methods for modulating poxvirus pathogenicity in a cell, and methods of treating an individual infected with a poxvirus. The subject methods find use in a variety of drug discovery, research and military applications.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for identifying an anti-poxviral agents. In many embodiments, the methods involve contacting a poxviral p28 polypeptide with a candidate agent, and determining an effect of the agent on a ubiquitin ligase activity of the p28 polypeptide. The effect of the agent may be determined using a variety of different cell based or cell-free biochemical assays, such as polyubiquitylation assays and cell viability assays. The invention also provides methods for modulating poxvirus pathogenicity (e.g., replication) in a cell, and methods of treating an individual infected with a poxvirus. The subject methods find use in a variety of drug discovery, research and military applications.

Before the present invention is described in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a candidate agent" includes a plurality of such candidate agents and reference to "the cell" includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

In describing this invention, screening assays will be described first, followed by a description of methods of modulating poxvirus pathogenicity in a cell. Finally, methods of treating poxvirus infections and kits are described.

Screening Methods

In one aspect, the invention features screening methods for, for example, identification of agents that modulate ubiquitin ligase activity of poxvirus p28 polypeptide. Components for use in these screening methods are described below, and then various exemplary screening formats are provided.

Assay Components

As noted above, "ubiquitin agents' as used herein refers to a collection of proteins that facilitates attachment of a ubiquitin moiety to a target protein via a ubiquitin ligase. The following section describes the various ubiquitin agents that may be present in the screening assays of the invention. In most embodiments, the ubiquitin ligase is a poxvirus p28 protein. This poxvirus p28 protein, because it can ubiquitylate itself, may also be the target protein. A discussion of many of these assay components may also be found in Wong et al (Drug Discovery Today 8:746 754, 2003) and published US Patent Application U.S. 20020042083, which are expressly incorporated herein in their entirety for all purposes.

Accordingly, assays usually include a ubiquitin, a ubiquitin activating agent, a ubiquitin conjugating agent, and a poxvirus p28 protein. In particular embodiments, the ubiquitin activating agent is usually an E1 protein, and the ubiquitin conjugating agent is usually an E2 protein.

Ubiquitin Ligating Agents

Most embodiments of the subject methods use a ubiquitin ligating agent. As used herein "ubiquitin ligating agent" refers to a ubiquitin agent, usually a protein (e.g., a ubiquitin ligating enzyme), capable of facilitating transfer or attachment of a ubiquitin from a ubiquitin conjugating agent to a target substrate molecule. In most embodiments, the ubiquitin ligating agent used in the subject methods is an E3 protein, e.g., a poxvirus p28 protein. Since certain ubiquitin ligating agents may autoubiquitylate, the ubiquitin ligating agent may also be a substrate for ubiquitylation, e.g., poxvirus p28 protein can serve as both the ubiquitin ligating agent and the substrate that is ubiquitylated.

"Poxvirus p28 protein" is a ring-zinc protein of approximately 28 kDa that is found in the genome of viruses of the Poxviridae family ("poxvirus" family). Accordingly, poxvirus p28 protein may be encoded by a genomes of Entomopoxviridae and Chordopoxvirinae viruses, including orthopoxvirus (e.g., variola, vaccinia, monkeypox, cowpox, rabbitpox, raccoon pox, tatera pox, buffalopox, camelpox, mousepox, etc.), avipoxvirus (fowlpox, canary pox, etc.), capripoxvirus (goatpox, sheeppox, lumpy skin disease, etc.) leporipoxvirus (myxoma, hare fibroma, etc.), parapoxvirus (orf, pseudo-cowpox, etc.), suipoxvirus (swinepox, etc.), molluscipoxvirus (molluscum contagiosum, etc.) and yatapoxvirus (tanapox, yaba, etc.), and others. The ectromelia (mousepox) p28 protein is generally described in Senkevich et al, (Virology 198, 118 128, 1994). The sequence of exemplary p28 proteins for use in the subject methods is set forth in the following Genbank Accessions: NP.sub.--671530.1 (Ectromelia virus EVM012), CAA64092.1 (Cowpox virus C7R protein), NP.sub.--536435.1 (Monkeypox virus D5R), NP.sub.--619812.1 (Cowpox virus CPXV023), NP.sub.--042048.1 (Variola virus D4R), AAA69414.1 (Variola virus D6R) and NP.sub.--570403.1 (Camelpox virus M-96). Unless otherwise indicated, the term "poxvirus" refers to any virus of the Poxviridae family of viruses.

Also included by the term "poxvirus p28 protein" are poxvirus p28 variants that retain ubiquitin ligase activity. In other words, the invention contemplates use of variants of the above proteins which retain a characteristic of a native ubiquitin ligating agent in being capable of facilitating transfer or attachment of a ubiquitin moiety to a target substrate protein. Guidance for which amino acids to change to produce a p28 variant that retains ligase activity can be obtained, for example, by aligning the amino acid sequences any of the poxivirus proteins listed above, identifying amino acids that are at identical positions in the proteins but are different between the proteins, and transferring the amino acids from one protein to the other. As discussed in greater detail below, the ring-finger domain is essential for ubiquitin ligase activity. Accordingly, poxvirus p28 proteins generally have an overall amino acid sequence identity of preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90% of an amino acid sequence provided above. In some embodiments the sequence identity will be as high as about 93% to 95% or 98%. In particular embodiments, the ring finger domain of the p28 proteins that find use in the subject methods have a high degree of sequence identity, e.g., at least 90%, at least 95%, at least 98% or at least 99% sequence identity. p28 variants having ubiquitin ligase activity are readily identified using the assays described above and below. Variants of these ubiquitin ligating agents and other components of the assays of the invention are described below in more detail.

Ubiquitin

By "ubiquitin" is meant any polypeptide which is transferred or attached to another polypeptide by ubiquitin agents. Ubiquitin as used in the assays below can be from any species of organism, usually a eukaryotic species, or any modified form thereof. In certain assays, the ubiquitin used is a mammalian ubiquitin, usually a human ubiquitin. Examples of ubiquitins suitable for use in the claimed methods are generally well known in the art, and include the human ubiquitin set forth in GenBank database PO2248 (which database entry is incorporated herein in its entirety), and the ubiquitin like modifier proteins known as NEDD8, ISGI5, SUMO1, SUMO2, SUMO3, APG12 and APG8, and the like. In many embodiments, the ubiquitin used in the subject assays is a naturally occurring allele or man-made variants of such polypeptides. Many ubiquitin polypeptides have an overall amino acid sequence identity of greater than about 75%, greater than about 80%, greater than about 85%, greater than 90% or even 93% to 95% or 98% or more of the amino acid sequence set forth in GenBank database PO.sub.2248. Further examples of ubiquitin molecules suitable for use in the claimed invention are described below.

As used in the subject methods, the ubiquitin polypeptides that be shorter or longer than the amino acid sequence of human ubiquitin depicted above. Thus, included within the definition of ubiquitin are portions or fragments of human ubiquitin. In one embodiment herein, fragments of the human ubiquitin protein are considered ubiquitin if they can be attached to a target polypeptide by ubiquitin agents.

In addition, as is more fully outlined below, ubiquitins of the present invention may be fusion proteins. Such fusion proteins may contain a ubiquitin polypeptide operably linked to a fusion sequence, e.g., a tag. In some embodiments, the tag may be an affinity tag, such as an epitope tag (e.g., HA, c-myc, etc) or a tag for attachment to a specific substrate (e.g., poly-his, poly-his-gly, GST, MBP) etc., In other embodiments, the tag may be a reporter tag, such as a fluorescent peptide, e.g., luciferase or Green Fluorescent Peptide (GFP), or variant thereof.

In certain embodiments, the ubiquitin moiety is endogenous to the cell which is to be used in a screening assay. That is, where the assay involves the use of cells, the ubiquitin moiety is naturally expressed in the cell to be assayed. However, in an alternative embodiment, the ubiquitin moiety, as well as other proteins of the present invention, are exogenous, e.g., recombinant proteins. A "recombinant protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as described below. In an exemplary embodiment, the ubiquitin moiety of the invention is made through the expression of a nucleic acid sequence corresponding to GENBANK accession number M26880 or AB003730, or a fragment thereof, and encodes the human ubiquitin, as discussed above.

Ubiquitin Activating Agents

As used herein "ubiquitin activating agent" refers to a ubiquitin agent, usually a protein (e.g., a ubiquitin activating enzyme), that transfers or attaches a ubiquitin moiety to a ubiquitin conjugating agent. Generally, the ubiquitin activating agent forms a high energy thiolester bond with ubiquitin moiety, thereby "activating" the ubiquitin moiety, and transfers or attaches the ubiquitin moiety to a ubiquitin conjugating agent (e.g., E2).

In a many embodiment the ubiquitin activating agent is an E1 protein, which can transfer or attach ubiquitin to an E2, defined below. Accordingly, E1 forms a high energy thiolester bond with ubiquitin, thereby "activating" the ubiquitin.

In exemplary embodiments, E1 proteins useful in the invention include those having the amino acid sequence of the polypeptide having ATCC accession numbers AAA61246, P22314, and CAA40296, incorporated herein by reference. E1 may be human E1. E1 is commercially available from Affiniti Research Products (Exeter, U.R.).

In further exemplary embodiments, nucleic acids which may be used for producing E1 proteins for the invention include, but are not limited to, those set forth in GenBank accession numbers M58028 and X56976, incorporated herein by reference. Variants of the cited E1 proteins, also included in the term "E1", can be made as described herein.

Further exemplary ubiquitin activating agents include those having the amino acid sequences or encoded by the nucleic acid sequences of a Genbank data base accession number listed in Table 1 (see Original Patent).

The invention also contemplates use of variants of a ubiquitin activating agents which retain a characteristic of a native ubiquitin activating agent in being capable of facilitating activation of a ubiquitin conjugating agent. Such ubiquitin activating agent variants generally have an overall amino acid sequence identity of preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90% of the amino acid sequence of a ubiquitin provided above. In some embodiments the sequence identity will be as high as activating agent about 93 to 95 or 98%. Variants of ubiquitin activating agents and other components of the assays of the invention are described below in more detail.

Ubiquitin Conjugating Agents

As used herein "ubiquitin conjugating agent" refers to a ubiquitin agent, usually a protein (e.g., a ubiquitin conjugating enzyme), capable of facilitating transfer or attaching a ubiquitin moiety to a substrate protein through interaction with a ubiquitin ligating agent. In some cases, the ubiquitin conjugating agent is capable of directly transferring or attaching ubiquitin moiety to lysine residues in a target substrate protein. The ubiquitin conjugating agent can be one capable of facilitating transfer or attachment of a ubiquitin moiety to a mono- or poly-ubiquitin moiety, which in turn can be attached to a ubiquitin agent or target protein.

In many embodiments, the ubiquitin conjugating agent is an E2, where the ubiquitin moiety is transferred from E1 to E2, in which the transfer results in a thiolester bond formed between E2 and ubiquitin moiety. In certain embodiments, E2 facilitates transfer or attachment of a ubiquitin moiety to a substrate protein through interaction with an E3 ubiquitin ligating agent, which is defined below.

In the methods and compositions of the present invention, the ubiquitin activating agent can comprise an amino acid sequence or a nucleic acid sequence corresponding to a sequence of an Genbank data base accession number listed in Table 2 (see Original Patent) and incorporated herein by reference. Ubiquitin conjugating agents of human cells (indicated by "Hs") are of particular interest.

Variants of the above ubiquitin conjugating proteins are suitable for use in the methods and compositions of the present invention. The ubiquitin conjugating agents and variants suitable for use in the methods and compositions of the present invention may be made as described herein.

In exemplary embodiments, the E2 used in the methods and compositions of the present invention comprises an amino acid sequence or nucleic acid sequence of a sequence corresponding to an Genbank data base accession number in the following list: AC37534, P49427, CAA82525, AAA58466, AAC41750, P51669, AAA91460, AAA91461, CAA63538, AAC50633, P27924, AAB36017, Q16763, AAB86433, AAC26141, CAA04156, BAA11675, Q16781, NP.sub.--003333, BAB18652, AAH00468, CAC16955, CAB76865, CAB76864, NP.sub.--05536, O00762, XP.sub.--009804, XP.sub.--009488, XP.sub.--006823, XP.sub.--006343, XP.sub.--005934, XP.sub.--002869, XP.sub.--003400, XP.sub.--009365, XP.sub.--010361, XP.sub.--004699, XP.sub.--004019, O14933, P27924, P50550, P52485, P51668, P51669, P49459, P37286, P23567, P56554, and CAB45853, each of which is incorporated herein by reference. Exemplary sequences of interest are those corresponding to Genbank data base accession numbers NP003331, NP003330, NP003329, P49427, AAB53362, NP008950, XP009488 and AAC41750, also incorporated by reference.

In further exemplary embodiments, E2 is one of Ubc5 (Ubch5, e.g., Ubch5c), Ubc3 (Ubch3), Ubc4 (Ubch4) and UbcX (Ubc10, Ubch10). In an exemplary embodiment, E2 is Ubc5c. In an exemplary embodiment, nucleic acids which may be used to make E2 include, but are not limited to, those nucleic acids having sequences disclosed in ATCC accession numbers L2205,229328, M92670, L40146, U393 17, U393 18, X92962, U58522, S81003, AF031141, AF075599, AJ000519, XM009488, NM007019, U73379, L40146 and D83004, each of which is incorporated herein by reference.

The skilled artisan will appreciate that many different E2 proteins and isozymes are known in the filed and may be used in the present invention, provided that the E2 has ubiquitin conjugating activity. Further exemplary E2 proteins for use in the invention are disclosed in PCT Publication No. WO 01/75145. Also specifically included within the term "E2" are variants of E2, which can be made as described herein.

The invention contemplates use of variants of a ubiquitin conjugating agents which retain a characteristic of a native ubiquitin conjugating agent in being capable of being activated by a ubiquitin activating agent and/or facilitating ubiquitylation of a target substrate protein in connection with a ubiquitin ligating agent. Such ubiquitin conjugating agent variants generally have an overall amino acid sequence identity of preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90% of the amino acid sequence of a ubiquitin conjugating agent provided above. In some embodiments the sequence identity will be as high as about 93 to 95 or 98%. Variants of ubiquitin conjugating agents and other components of the assays of the invention are described below in more detail.

In some embodiments, E2 has a tag, as defined herein, with the complex being referred to herein as "tag-E2". Exemplary E2 tags include, but are not limited to, labels, partners of binding pairs and substrate binding elements. In one embodiment of particular interest, the tag is an affinity tag, e.g., a His-tag or GST-tag.

Variant Polypeptides Differing in Amino Acid Sequence and Fragments

As noted above, the assays of the invention described herein can be conducted with various protein variants including variants of ubiquitin, E1, E2, and poxvirus 28 protein. These variants generally fall into one or more of three classes: substitution, insertion or deletion variants. Variants are generally described as having a sequence similarity (e.g., sequence identity) relative to that of a "reference" sequence, e.g., the sequence of the naturally-occurring protein. It will also be readily appreciated that proteins that share amino acid sequence similarity are encoded by nucleic acids that share nucleotide sequence similarity.

As is known in the art, a number of different programs can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence. Sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387 395 (1984), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127 149 (1988), Alan R. Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151 153 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403 410, (1990) and Karlin et al., PNAS USA 90:5873 5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266: 460 480 (1996); http://blast.wustl/edu/blast/README.html]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschul et al. Nucleic Acids Res. 25:3389 3402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to .about.22 bits.

A percent amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "shorter" sequence in the aligned region. The "shorter" sequence is the one having the least actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored). For example, if one polypeptide is longer than another polypeptide and contains the entire sequence of the shorter sequence, the polypeptides are 100% identical.

The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the reference amino acid sequence, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.

In one embodiment, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0", which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.

Variants of interest can ordinarily be prepared by site specific mutagenesis of nucleotides in the DNA encoding a protein of the present compositions, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant protein fragments having up to about 100 150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.

While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed variants screened for the optimal desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Rapid production of many variants may be done using techniques such as the method of gene shuffling, whereby fragments of similar variants of a nucleotide sequence are allowed to recombine to produce new variant combinations. Examples of such techniques are found in U.S. Pat. Nos. 5,605,703; 5,811,238; 5,873,458; 5,830,696; 5,939,250; 5,763,239; 5,965,408; and 5,945,325, each of which is incorporated by reference herein in its entirety. Screening of the mutants is performed using the activity assays of the present invention.

Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the protein are desired, substitutions of an original residue are generally made in accordance with exemplary substitutions listed below (see Original Patent for Table 3).

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in the above list. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.

In one embodiment, the variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the proteins as needed. Alternatively, the variant may be designed such that the biological activity of the protein is altered. For example, glycosylation sites may be altered or removed.

It will be appreciated that the nucleotide sequences of protein variants can be readily determined, for example based upon the amino acid sequence of the variant and the knowledge of the genetic code. Due to the degeneracy of the genetic code, a nucleotide sequence encoding a protein variant may exhibit a lower sequence identity with the corresponding native nucleotide sequence than the amino acid sequence identity between the variant protein and the native protein. For example, nucleotide sequences share as little as about 66% (i.e., about 2/3) nucleotide sequence identity can encode the same amino acid sequence due to the degeneracy of the genetic code. Thus, nucleic acid encoding a protein variant can have at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% sequence identity with a reference nucleic acid, for example, the corresponding nucleic acid encoding the native protein (i.e., the protein prior to modification) from which a variant protein sequence is derived.

The invention also contemplates use of E1, E2 and E3 proteins which are shorter or longer than the corresponding naturally occurring amino acid sequence. That is, portions or fragments of the proteins described herein can be used in the assays of the invention. The fragments of use in the invention retain a biological activity of the protein from which it was derived or with which it share amino acid sequence identity. For example, a ubiquitin fragment useful in the invention is one that can be transferred (or removed from) a substrate protein by the corresponding ubiquitin agents. Similarly, a fragment of a ubiquitin activating agent (e.g., a fragment of E1) of interest is one that retains activity in being modified by a ubiquitin moiety and activating a ubiquitin conjugating agent. A fragment of a ubiquitin conjugating agent (e.g., a fragment of E2) of interest is one that retains activity in interacting with an E3 to facilitate transfer of a ubiquitin moiety to a substrate protein. A ubiquitin ligating agent fragment retains activity in interacting with a target protein and an activated E2 to facilitate transfer of a ubiquitin moiety to the target protein. A target protein fragment of interest is one that can be modified by attachment of and/or removal of ubiquitin moieties by the relevant components of the ubiquitin cascade.

Production of Polypeptides

The subject proteins can be produced according to methods known in the art. In addition, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related or variant ubiquitin moieties, ubiquitin agents, and target proteins from humans or other organisms.

In one embodiment, the nucleic acids of the invention are part of an expression vector. Using the nucleic acids of the present invention which encode a protein, a variety of expression vectors are made. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the protein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. As another example, operably linked refers to DNA sequences linked so as to be contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus can be used to express the protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In one embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.

In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

In addition, in one embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

An exemplary expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference. Constructs also are described in U.S. Pat. No. 6,153,380, which is expressly incorporated herein by reference.

Proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding the protein, under the appropriate conditions to induce or cause expression of the protein. The conditions appropriate for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melanogaster cells, Pichia pastoris and P. methanolica, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, SF21 cells, C129 cells, Saos-2 cells, Hi-5 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells. Of greatest interest are A549, HeLa, HUVEC, Jurkat, BJAB, CHMC, primary T cells and macrophage.

In a one embodiment, the proteins are expressed in mammalian cells, especially human cells. Mammalian expression systems are also known in the art, and include retroviral systems. A mammalian promoter (i.e., a promoter functional in a mammalian cell) is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for a protein into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25 30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter can also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, are well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Where the host cell is a bacterial cell, a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of a protein into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. In E. coli, the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3 9 nucleotides in length located 3 11 nucleotides upstream of the initiation codon.

The expression vector may also include a signal peptide sequence that provides for secretion of the protein in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).

The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.

The protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, the protein may be made fusion nucleic acid encoding the peptide or may be linked to other nucleic acid for expression purposes. Similarly, proteins of the invention can be linked to tags that are protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc. The fusions may include other constructs as well, including separation sites such as 2a site and internal ribosomal entry sites IRES, which are particularly useful in the construct as IRES-label to provide a method of tracking infected cells.

Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others. The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others. In one embodiment, proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art. In another embodiment, proteins are produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii P. methanolica and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Promoter sequences for expression in yeast include the inducible GAL 1,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene. Yeast selectable markers include ADE2, HIS4, LEU2, TW1, and ALG7, which confers resistance to tunicamycin; the neomycin phosphotransferase gene, which confers resistance to G4 18; and the CUP 1 gene, which allows yeast to grow in the presence of copper ions.

Proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the ubiquitin protein may be purified using a standard anti-ubiquitin antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the protein. In some instances no purification will be necessary.

Expression of p28 in bacterial cells is described by Senkevich et al, (J. Virology 69: 4103 4111, 1995).

Covalently Modified Proteins, Including Detectably Labeled Ubiquitin Agents

In one embodiment, covalent modifications of polypeptides are included within the scope of this invention. Such covalent modifications generally find use in in vitro assays as described in more detail in U.S. Ser. No. 09/800,770, filed Mar. 6, 2001, which is expressly incorporated herein by reference.

Tagged Polypeptides

The subject polypeptides can be modified so that they comprise a tag. By "tag" is meant an attached molecule or molecules useful for the identification or isolation of the attached molecule(s), which can be substrate binding molecules. For example, a tag can be an attachment tag or a label tag. Components having a tag are referred to as "tag-X", wherein X is the component. For example, a ubiquitin moiety comprising a tag is referred to herein as "tag-ubiquitin moiety". Preferably, the tag is covalently bound to the attached component.

When more than one component of a combination has a tag, the tags will be numbered for identification, for example "tag1-ubiquitin moiety". Components may comprise more than one tag, in which case each tag will be numbered, for example "tag 1,2-ubiquitin moiety". Exemplary tags include, but are not limited to, a label, a partner of a binding pair, and a surface substrate binding molecule (or attachment tag). As will be evident to the skilled artisan, many molecules may find use as more than one type of tag, depending upon how the tag is used. In one embodiment, the tag or label as described below is incorporated into the polypeptide as a fusion protein.

As will be appreciated by those in the art, tagcomponents of the invention can be made in various ways, depending largely upon the form of the tag. Components of the invention and tags are preferably attached by a covalent bond. Examples of tags are described below.

Exemplary Tags Useful in the Invention

As noted above, "tags" can be any of a variety of labels, which can be detected either directly or indirectly. Tagged ubiquitylation cascade proteins, tagged substrate proteins, and tagged retroviral ubiquitylation modulator protein find particular use in the screening assays of the invention, described below in more detail.

By "label" or "detectable label" is meant a molecule that can be directly (i.e., a primary label) or indirectly (i.e., a secondary label) detected; for example a label can be visualized and/or measured or otherwise identified so that its presence or absence can be known. As will be appreciated by those in the art, the manner in which this is performed will depend on the label. Exemplary labels include, but are not limited to, fluorescent labels (e.g. GFP) and label enzymes.

In one embodiment, the tag is a polypeptide which is provided as a portion of a chimeric molecules comprising a first polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of a first polypeptide (e.g., a ubiquitin moiety, ubiquitin agent, or target protein) with a tag polypeptide. The tag is generally placed at the amino-or carboxyl-terminus of the polypeptide. The tag polypeptide can be, for example, a polypeptide which provides an epitope to which an anti-tag antibody can selectively bind, a polypeptide which serves as a ligand for binding to a receptor (e.g., to facilitate immobilization of the chimeric molecule on a substrate); an enzyme label (e.g., as described further below); or a fluorescent label (e.g., as described further below). Tag polypeptides provide for, for example, detection using an antibody against the tag polypeptide, and/or a ready means of isolating or purifying the tagged polypeptide (e.g., by affinity purification using an anti-tag antibody or another type of receptor-ligand matrix that binds to the tag). In an alternative embodiment, the chimeric molecule may comprise a fusion of a polypeptide disclosed herein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule. Tags for components of the invention are defined and described in detail below.

The production of tag-polypeptides by recombinant means is within the knowledge and skill in the art. Production of FLAG-labeled proteins is well known in the art and kits for such production are commercially available (for example, from Kodak and Sigma). Methods for the production and use of FLAG-labeled proteins are found, for example, in Winston et al., Genes and Devel. 13:270 283 (1999), incorporated herein in its entirety, as well as product handbooks provided with the above-mentioned kits.

Production of proteins having His-tags by recombinant means is well known, and kits for producing such proteins are commercially available. Such a kit and its use is described in the QIAexpress Handbook from Qiagen by Joanne Crowe et al., hereby expressly incorporated by reference.

By "fluorescent label" is meant any molecule that may be detected via its inherent fluorescent properties, which include fluorescence detectable upon excitiation. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Bluer.TM., Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.

Suitable fluorescent labels include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148):802 805 (Feb. 11, 1994); and EGFP; Clontech--Genbank Accession Number U55762), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8.sup.th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques 24(3):462 471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178 182 (1996)), enhanced yellow fluorescent protein (EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol. 150(12):5408 5417 (1993)), $-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603 2607 (April 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat. Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; and 5,925,558), and Ptilosarcus green fluorescent proteins (pGFP) (see WO 99/49019). All of the above-cited references are expressly incorporated herein by reference.

In some instances, multiple fluorescent labels are employed. In one embodiment, at least two fluorescent labels are used which are members of a fluorescence resonance energy transfer (FRET) pair. FRET can be used to detect association/dissociation of, for example, a ubiquitin ligating agent (e.g., an E3) and a target substrate protein; a ubiquitin conjugating agent (e.g., an E2) and a target substrate protein; a ubiquitin ligating agent (e.g., an E3) and a ubiquitin conjugating agent (e.g., an E2); and the like.

FRET is phenomenon known in the art wherein excitation of one fluorescent dye is transferred to another without emission of a photon. A FRET pair consists of a donor fluorophore and an acceptor fluorophore. The fluorescence emission spectrum of the donor and the fluorescence absorption spectrum of the acceptor must overlap, and the two molecules must be in close proximity. The distance between donor and acceptor at which 50% of donors are deactivated (transfer energy to the acceptor) is defined by the Forster radius, which is typically 10 100 angstroms. Changes in the fluorescence emission spectrum comprising FRET pairs can be detected, indicating changes in the number of that are in close proximity (i.e., within 100 angstroms of each other). This will typically result from the binding or dissociation of two molecules, one of which is labeled with a FRET donor and the other of which is labeled with a FRET acceptor, wherein such binding brings the FRET pair in close proximity.

Binding of such molecules will result in an increased fluorescence emission of the acceptor and/or quenching of the fluorescence 15 emission of the donor. FRET pairs (donor/acceptor) useful in the invention include, but are not limited to, EDANS/fluorescien, IAEDANS/fluorescein, fluoresceidtetramethylrhodamhe, fluoresceidLC Red 640, fluoresceidcy 5, fluoresceidCy 5.5 and fluoresceidLC Red.

In another aspect of FRET, a fluorescent donor molecule and a nonfluorescent acceptor molecule ("quencher") may be employed. In this application, fluorescent emission of the donor will increase when quencher is displaced from close proximity to the donor and fluorescent emission will decrease when the quencher is brought into close proximity to the donor. Useful quenchers include, but are not limited to, DABCYL, QSY 7 and QSY 33. Useful fluorescent donodquencher pairs include, but are not limited to EDANS/DABCYL, Texas RedLDABCYL, BODIPYDABCYL, Lucifer yellowDABCYL, coumarin/DABCYL and fluoresceidQSY 7 dye.

The skilled artisan will appreciate that FRET and fluorescence quenching allow for monitoring of binding of labeled molecules over time, providing continuous information regarding the time course of binding reactions. It is important to remember that ubiquitin is ligated to substrate protein by its terminal carboxyl group to a lysine residue, including lysine residues on other ubiquitin. Therefore, attachment of labels or other tags should not interfere with either of these active groups on the ubiquitin Amino acids may be added to the sequence of protein, through means well known in the art and described herein, for the express purpose of providing a point of attachment for a label. In one embodiment, one or more amino acids are added to the sequence of a component for attaching a tag thereto, with a fluorescent label being of particular interest. In one embodiment, the amino acid to which a fluorescent label is attached is Cysteine.

By "label enzyme" is meant an enzyme which may be reacted in the presence of a label enzyme substrate which produces a detectable product. Suitable label enzymes for use in the present invention include but are not limited to, horseradish peroxidase, alkaline phosphatase and glucose oxidase. Methods for the use of such substrates are well known in the art. The presence of the label enzyme is generally revealed through the enzyme's catalysis of a reaction with a label enzyme substrate, producing an identifiable product. Such products may be opaque, such as the reaction of horseradish peroxidase with tetramethyl benzedine, and may have a variety of colors. Other label enzyme substrates, such as Luminol (available from Pierce Chemical Co.), have been developed that produce fluorescent reaction products. Methods for identifying label enzymes with label enzyme substrates are well known in the art and many commercial kits are available. Examples and methods for the use of various label enzymes are described in Savage et al., Previews 247:6 9 (1998), Young, J. Virol. Methods 24:227 236 (1989), which are each hereby incorporated by reference in their entirety.

By "radioisotope" is meant any radioactive molecule. Suitable radioisotopes for use in the invention include, but are not limited to 14C, 3H, 32P, 33P, 35S, 125I, and 131I. The use of radioisotopes as labels is well known in the art.

In addition, labels may be indirectly detected, that is, the tag is a partner of a binding pair. By "partner of a binding pair" is meant one of a first and a second moiety, wherein said first and said second moiety have a specific binding affinity for each other. Suitable binding pairs for use in the invention include, but are not limited to, antigendantibodies (for example, digoxigeninlanti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, Fluoresceidanti-fluorescein, Lucifer yellow/anti-lucifer yellow, and rhodamine anti-rhodamine), biotirdavid (or biotirdstreptavidin) and calmodulin binding protein (CBP)/calmodulin. Other suitable binding pairs include polypeptides such as the FLAG-peptide (Hopp et al., BioTechnol, 6:1204 1210 (1988)); the KT3 epitope peptide (Martin et al., Science, 255:192 194 (1992)); tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266: 15 163 15 166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyemuth et al., Proc. Natl. Acad. Sci. USA, a:6393 6397 (1990)) and the antibodies each thereto. Generally, in one embodiment, the smaller of the binding pair partners serves as the tag, as steric considerations in ubiquitin ligation may be important. As will be appreciated by those in the art, binding pair partners may be used in applications other than for labeling, such as immobilization of the protein on a substrate and other uses as described below.

As will be appreciated by those in the art, a partner of one binding pair may also be a partner of another binding pair. For example, an antigen (first moiety) may bind to a first antibody (second moiety) which may, in turn, be an antigen for a second antibody (third moiety). It will be further appreciated that such a circumstance allows indirect binding of a first moiety and a third moiety via an intermediary second moiety that is a binding pair partner to each. As will be appreciated by those in the art, a partner of a binding pair may comprise a label, as described above. It will further be appreciated that this allows for a tag to be indirectly labeled upon the binding of a binding partner comprising a label. Attaching a label to a tag which is a partner of a binding pair, as just described, is referred to herein as "indirect labeling".

In one embodiment, the tag is surface substrate binding molecule. By "surface substrate binding molecule" and grammatical equivalents thereof is meant a molecule have binding affinity for a specific surface substrate, which substrate is generally a member of a binding pair applied, incorporated or otherwise attached to a surface. Suitable surface substrate binding molecules and their surface substrates include, but are not limited to poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags and Nickel substrate; the Glutathione-S Transferase tag and its antibody substrate (available from Pierce Chemical); the flu HA tag polypeptide and its antibody 12CA5 substrate (Field et al., Mol. Cell. Biol., 8:2159 2165 (1988)); the c-myc tag and the 8F9,3C7,6E107 G4, B7 and 9E10 antibody substrates thereto (Evan et al., Molecular and Cellular Biol, 5:3610 3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody substrate (Paborsky et al., Protein Engineering, 3(6):547 553 (1990)). In general, surface binding substrate molecules useful in the present invention include, but are not limited to, polyhistidine structures (His-tags) that bind nickel substrates, antigens that bind to surface substrates comprising antibody, haptens that bind to avidin substrate (e.g., biotin) and CBP that binds to surface substrate comprising calmodulin.

Production of antibody-embedded substrates is well known; see Slinkin et al., Bioconj, Chem. 2:342 348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconi. Chem. 33323 327 (1992); King et al., Cancer Res. 54:6176 6185 (1994); and Wilbur et al., Bioconjugate Chem. 5:220 235 (1994) (all of which are hereby expressly incorporated by reference), and attachment of or production of proteins with antigens is described above. Calmodulin-embedded substrates are commercially available, and production of proteins with CBP is described in Simcox et al., Strategies 8:40 43 (1995), which is hereby incorporated by reference in its entirety.

Where appropriate, functionalization of labels with chemically reactive groups such as thiols, amines, carboxyls, etc. is generally known in the art. In one embodiment, the tag is functionalized to facilitate covalent attachment.

Biotinylation of target molecules and substrates is well known, for example, a large number of biotinylation agents are known, including amine-reactive and thiol-reactive agents, for the biotinylation of proteins, nucleic acids, carbohydrates, carboxylic acids; see, e.g., chapter 4, Molecular Probes Catalog, Haugland, 6.sup.th Ed. 1996, hereby incorporated by reference. A biotinylated substrate can be attached to a biotinylated component via avidin or streptavidin. Similarly, a large number of haptenylation reagents are also known. Methods for labeling of proteins with radioisotopes are known in the art. For example, such methods are found in Ohta et al., Molec. Cell 3:535 541 (1999), which is hereby incorporated by reference in its entirety.

The covalent attachment of the tag may be either direct or via a linker. In one embodiment, the linker is a relatively short coupling moiety, that is used to attach the molecules. A coupling moiety may be synthesized directly onto a component of the invention, ubiquitin for example, and contains at least one functional group to facilitate attachment of the tag. Alternatively, the coupling moiety may have at least two functional groups, which are used to attach a functionalized component to a functionalized tag, for example. In an additional embodiment, the linker is a polymer. In this embodiment, covalent attachment is accomplished either directly, or through the use of coupling moieties from the component or tag to the polymer.

In one embodiment, the covalent attachment is direct, that is, no linker is used. In this embodiment, the component can contain a functional group such as a carboxylic acid which is used for direct attachment to the functionalized tag. It should be understood that the component and tag may be attached in a variety of ways, including those listed above. What is important is that manner of attachment does not significantly alter the functionality of the component. For example, in tag-ubiquitin, the tag should be attached in such a manner as to allow the ubiquitin to be covalently bound to other ubiquitin to form polyubiquitin chains.

As will be appreciated by those in the art, the above description of covalent attachment of a label and ubiquitin applies equally to the attachment of virtually any two molecules of the present disclosure. In one embodiment, the tag is functionalized to facilitate covalent attachment, as is generally outlined above. Thus, a wide variety of tags are commercially available which contain functional groups, including, but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to covalently attach the tag to a second molecule, as is described herein. The choice of the functional group of the tag 32 will depend on the site of attachment to either a linker, as outlined above or a component of the invention. Thus, for example, for direct linkage to a carboxylic acid group of a ubiquitin, amino modified or hydrazine modified tags will be used for coupling via carbodiimide chemistry, for example using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as is known in the art (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; see also the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both of which are hereby incorporated by reference). In one embodiment, the carbodiimide is first attached to the tag, such as is commercially available for many of the tags described herein.

In one embodiment, ubiquitin moiety is in the form of tag-ubiquitin moiety, wherein, tag is a partner of a binding pair. In one example is the tag is FLAG and the binding partner is anti-FLAG. In this embodiment, a label is attached to the FLAG by indirect labeling. In another embodiment, the label is a label enzyme, which can be, for example, horseradish peroxidase, which is reacted with a fluorescent label enzyme substrate. In one embodiment, the label enzyme substrate is Luminol. Alternatively, the label is a fluorescent label.

In another embodiment, the ubiquitin moiety is in the form of tag-ubiquitin moiety, wherein the tag is a fluorescent label. In one embodiment of interest, the ubiquitin moiety is in the form of tag1-ubiquitin and tag2-ubiquitin, wherein tag1 and tag2 are the members of a FRET pair. In an alternate embodiment, the ubiquitin moiety is in the form of tag1-ubiquitin and tag2-ubiquitin, wherein tag1 is a fluorescent label and tag2 is a quencher of the fluorescent label. In a related embodiment, when the tags ubiquitin and tag2-ubiquitin moieties are bound through the activity of a ubiquitin ligase, the tag1 and tag2 are within about 100, 70, 50, 40, or 30 or less angstroms of each other.

In another embodiment, ubiquitin is in the form of tag1,2-ubiquitin and tag1,3-ubiquitin, wherein tag1 is a member of a binding pair, e.g., FLAG, tag2 is a fluorescent label and tag3 is either a fluorescent label such that tag2 and tag3 are members of a FRET pair or tag3 is a quencher of tag2. In one embodiment, one or more amino acids are added to the ubiquitin sequence, using recombinant techniques as described herein, to provide an attachment point for a tag, e.g., a fluorescent label or a quencher. In one embodiment, the one or more amino acids are Cys or Ala-Cys. Preferably, the one or more amino acids are attached to the N-terminal of the ubiquitin. In one exemplary embodiment, the one or more amino acids intervenes the sequence of a FLAG tag and the ubiquitin. In an exemplary embodiment, the tag, e.g., a fluorescent label or a quencher, is attached to the added Cysteine.

Glycosylation Variants and Other Variants

Another type of covalent modification of a polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence polypeptide.

Addition of glycosylation sites to polypeptides may be accomplished by altering the amino acid sequence thereof. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence polypeptide (for O-linked glycosylation sites). The amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the polypeptide at pre-selected bases such that codons are generated that will translate into the desired amino acids.

Alternatively, the variant may be designed such that the biological activity of the protein is altered. For example, glycosylation sites may be altered or removed. Covalent modifications of polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking a protein to a water-insoluble support matrix or surface for use in the method for screening assays, as is more fully described below. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, -hydroxy-succinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl-propionate), bifunctional maleimides such as bis-N-maleimido-1, % octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the "-amino groups of lysine, arginine, and histidine side chains (Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79 86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Further means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259 306 (1981). 25 Removal of carbohydrate moieties present on the polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 25952 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzynol., 138:350 (1987). Another type of covalent modification of a protein comprises linking the polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Candidate Agents

The assays of the invention are designed to identify candidate agents that modulate the ubiquitin ligase activity of a poxvirus p28 protein. By "modulate" is meant a compound which can facilitate an increase or decrease ubiquitylation, with agents that decrease ubiquitylation being of particular interest.

By "candidate", "candidate agent", "candidate modulator", "candidate ubiquitylation modulator" or grammatical equivalents herein, which terms are used interchangeable herein, is meant any molecule, e.g. proteins (which herein includes proteins, polypeptides, and peptides), small (i.e., 5 1000 Da, 100 750 Da, 200 500 Da, or less than 500 Da in size), or organic or inorganic molecules, polysaccharides, polynucleotides, etc. which are to be tested for ubiquitination modulator activity. Candidate agents encompass numerous chemical classes. In one embodiment, the candidate agents are organic molecules, particularly small organic molecules, comprising functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, usually at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and or aromatic or polyaromatic structures substituted with one or more chemical functional groups.

Candidate modulators are obtained from a wide variety of sources, as will be appreciated by those in the art, including libraries of synthetic or natural compounds. As will be appreciated by those in the art, the present invention provides a rapid and easy method for screening any library of candidate modulators, including the wide variety of known combinatorial chemistry-type libraries.

In one embodiment, candidate modulators are synthetic compounds. Any number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94/24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods. As described in WO 94/24314, one of the advantages of the present method is that it is not necessary to characterize the candidate modulator prior to the assay; only candidate modulators that affect ubiquitylation of a target substrate protein of interest need be identified.

In another embodiment, the candidate modulators are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.

In one embodiment, candidate modulators include proteins, nucleic acids, and chemical moieties. In one embodiment, the candidate modulators are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be tested, as is more fully described below. In this way libraries of procaryotic and eucaryotic proteins may be made for screening against any number of ubiquitin ligase compositions. Other embodiments include libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.

In one embodiment, the candidate modulators are peptides of from about 2 to about 50 amino acids, with from about 5 to about 30 amino acids being usual, and from about 8 to about 20 being particularly of interest. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position.

The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents. A library of all combinations of a peptide 7 to 20 amino acids in length has the potential to code for 20.sup.7 to 20.sup.20 different peptides. Thus, with libraries of 10.sup.7 to 10.sup.8 different molecules the present methods allow a "working" subset of a theoretically complete interaction library for 7 amino acids, and a subset of shapes for the 20.sup.20 peptide library. Thus, in one embodiment, at least 10.sup.6, 10.sup.7, or 10.sup.8. Maximizing library size and diversity is of interest.

In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In one embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

A number of molecules or protein domains are suitable as starting points for the generation of biased randomized candidate modulators. A large number of small molecule domains are known, that confer a common function, structure or affinity. In addition, as is appreciated in the art, areas of weak amino acid homology may have strong structural homology. A number of these molecules, domains, and/or corresponding consensus sequences, are known, including, but are not limited to, SH-2 domains, SH-3 domains, Pleckstrin, death domains, protease cleavage/recognition sites, enzyme inhibitors, enzyme substrates, Traf, etc.

As described above generally for proteins, nucleic acid candidate modulator may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of genomes may be used as is outlined above for proteins. Where the ultimate expression product is a nucleic acid, at least 10, at least 12, more usually at least 15, normally at least 21 nucleotide positions need to be randomized, with more preferable if the randomization is less than perfect. Similarly, at least 5, at least 6, more usually at least 7 amino acid positions need to be randomized; again, more are preferable if the randomization is less than perfect.

In one embodiment, the candidate modulators are organic moieties. In this embodiment, as is generally described in WO 94/24314, candidate agents are synthesized from a series of substrates that can be chemically modified. "Chemically modified" herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions may be done on the moieties to form new substrates or candidate agents which can then be tested using the present invention. Exemplary molecules for use in the subject assays may be found in: "Rhodanine Compositions for use as Antiviral Agents", Ser. No. 60/514,951 filed on Oct. 28, 2003; U.S. provisional patent application Ser. No. 60/509,780; and US provisional patent application entitled "Ubiquitin Ligase Inhibitors", Ser. No. 60/514,951 filed on 9.sup.th Oct. 2003, which applications are incorporated herein by reference in their entirety.

Assay Formats

The invention provides methods for assessing the effect of a candidate agent upon the ubiquitin ligase activity of a poxvirus p28 protein. In these assays, the influence of candidate agent on the ubiquitin ligase activity of a poxvirus p28 protein can be observed and assessed.

In general, the assays of the invention are carried out by bringing into contact various ubiquitylation agents, including a poxvirus p28 protein, and assessing the effect of the candidate agent upon substrate protein ubiquitylation.

Identification of Agents that Decrease Ubiquitylation

In one embodiment, the method involves combining (e.g., in a test sample) a candidate agent, ubiquitin, a ubiquitin activating agent, a ubiquitin conjugating agent, and a poxvirus p28 protein under conditions suitable for ubiquitylation of a substrate polypeptide, e.g., the poxvirus p28 protein. The level of ubiquitylated substrate polypeptide is assessed either qualitatively or quantitatively. A decrease in ubiquitylated substrate polypeptide in the presence of the candidate agent relative to a level in the absence of the candidate agent indicates the agent causes a decrease in ubiquitylation of the substrate protein by p28.

As would be apparent to one of skill in the art, these assays may be performed in conjunction with suitable controls, which controls may include an E3 protein that is not a poxvirus p28 protein, assays that do not contain a candidate agent, and the like, to determine whether an agent specifically acts on the ubiquitin ligase activity of poxvirus p28 protein, or some other aspect of ubiquitylation.

An agent that reduces ubiquitylation by reducing the ligase activity of poxvirus p28 protein finds use as a therapeutic agent for treatment of poxvirus infections. In most embodiments, an agent that reduces poxvirus p28 protein ligase activity will decrease activity (and thereby decrease the amount of ubiquitylation) by greater than about 20%, greater than about 40%, greater then about 60%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 98%, or more, as compared to controls that do not contain the agent. In many embodiments, agents that have an IC.sub.50 (the IC.sub.50 is the concentration of agent that reduces activity by 50%, as compared to controls in the absence of an agent) of about 500 nM or less, about 100 nM or less, about 50 nM or less, about 10 nM or less, about 5 nM or less, or about 1 nM or less, are of especial interest as therapeutic agents for poxvirus infection.

In related embodiments, the assay uses a tagged ubiquitin moiety (tag-Ub), which can be tagged as discussed above.

Cell-Free Screening Assays

In general, the subject method involves combining at least a minimum number of required ubiquitin agents, e.g., ubiquitin, an E1, an E2 and a poxvirus p28 protein, and assessing either qualitatively or quantitatively a level of ubiquitylation activity. Ubiquitylation can be assessed by detection of mono-ubiquitylation, poly-ubiquitylation, or both, and can be assessed by detection of auto-ubiquitylation of p28 polypeptide.

Assessing ubiquitylation activity can be accomplished in a variety of ways. In general, the assay methods involve combining ubiquitin agents and with other components, such as a candidate agent. By "combining" is meant the addition of the various components into a receptacle under conditions in which ubiquitylation of a substrate may take place.

In one embodiment, the receptacle is a well of a 96 well plate or other commercially available multiwell plate. In another embodiment, the receptacle is the reaction vessel of a FACS machine. Other receptacles useful in the present invention include, but are not limited to 384 well plates and 1536 well plates. Still other receptacles useful in the present invention will be apparent to the skilled artisan.

The addition of the components may be sequential or in a predetermined order or grouping, as long as the conditions amenable to ubiquitin ligase activity are obtained. Such conditions are well known in the art, and optimization of such conditions is routine in the art.

The components of the present compositions may be combined in varying amounts. In one embodiment, ubiquitin is combined at a final concentration of 5 ng to 200 ng per 100 .mu.l reaction solution, preferably at about 100 ng per 100 .mu.l reaction solution. For example, a ubiquitin activating agent (e.g, E1) can be combined at a final concentration of from 1 to 50 ng per 100 .mu.l reaction solution, more preferably from 1 ng to 20 ng per 100 .mu.l reaction solution, most preferably from 5 ng to 10 ng per 100 .mu.l reaction solution. In another example, a ubiquitin conjugating agent (e.g., E2) is combined at a final concentration of 10 to 100 ng per 100 .mu.l reaction solution, more preferably 10 50 ng per 100 .mu.l reaction solution. In another example, a poxvirus p28 protein is combined at a final concentration of from 1 ng to 500 ng per 100 .mu.l reaction solution, more preferably from 50 to 400 ng per 100 .mu.l reaction solution, still more preferably from 100 to 300 ng per 100 .mu.l reaction solution, most preferably about 100 ng per 100 .mu.l reaction solution.

The components of the invention are combined under reaction conditions that favor ubiquitylation activity (e.g., ubiquitin ligase activity of p28 polypeptide). Generally, this will be physiological conditions. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40.degree. C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.5 and 1.5 hours will be sufficient.

A variety of other reagents may be included in the compositions. These include reagents like salts, solvents, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal ubiquitylation enzyme activity and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The compositions can also include adenosine tri-phosphate (ATP).

The mixture of components may be added in any order that promotes ubiquitylation or de-ubiquitylation as appropriate, or optimizes identification of candidate modulator effects. In one embodiment, ubiquitin is provided in a reaction buffer solution, followed by addition of the ubiquitylation enzymes. In an alternate embodiment, ubiquitin is provided in a reaction buffer solution, a candidate modulator is then added, followed by addition of the ubiquitylation enzymes.

In one example, at least one of the components is immobilized on a substrate, e.g., the poxvirus p28 protein. Binding of assay components may be done directly or indirectly (e.g., via covalent or non-covalent binding to a component which is bound to the substrate). Binding of the component can be via a tag moiety, which may or may not provide a detectable signal. In another embodiment, ubiquitin conjugating agent (e.g., E2) is bound to a surface substrate. In general, any substrate binding molecule can be used.

As will be appreciated by those of skill in the art, the surface substrate binding element and substrate to which the element binds can be selected according to the design of the assay and the desired characteristics, e.g., an element-substrate combination that will be effective for facilitating the separation of bound and unbound ubiquitin. The substrate used in embodiments involving immobilization of an assay component can be any suitable substrate, e.g., a well of a multi-well plate, a bead, and the like.

In another embodiment, the ubiquitin agents and other assay components are free in solution. In this embodiment, ubiquitylation activity can be monitored using a system that produces a signal which varies with the extent of ubiquitylation, such as the fluorescence resonance energy transfer (FRET) system described in detail below. In one embodiment, the ubiquitin is labeled, either directly or indirectly, as further described below, and the amount of label is measured. This allows for easy and rapid detection and measurement of ligated ubiquitin, making the assay useful for high-throughput screening applications. In one embodiment, the signal of the label varies with the extent of ubiquitylation, such as in the FRET system described below. One of ordinary skill in the art will recognize the applicability of the present invention to screening for agents which modulate ubiquitylation.

In a related embodiment, the assay composition comprises tag1-ubiquitin, tag2-ubiquitin, E1, E2 and poxvirus p28. In one embodiment, tag1 and tag2 are labels, preferably fluorescent labels, most preferably tag1 and tag2 are a FRET pair. In this embodiment, ubiquitylation is measured by measuring the fluorescent emission spectrum. This measuring may be continuous or at one or more times following the combination of the components. Alteration in the fluorescent emission spectrum of the combination as compared with unligated ubiquitin indicates the amount of ubiquitylation. The skilled artisan will appreciate that in this embodiment, alteration in the fluorescent emission spectrum results from ubiquitin bearing different members of the FRET pair being brought into close proximity, either through the formation of polyubiquitin and/or by binding nearby locations on a protein, preferably a target protein

Detection of Ubiquitylation

Once combined, the level of ubiquitylation can be assessed in a variety of ways. For example, the level of ubiquitylated substrate protein and/or the degree of ubiquitylation of the substrate protein can be assessed; the level of free ubiquitin can be assessed; the association of substrate protein with a ubiquitin conjugating agent; association of a substrate protein, ubiquitin conjugating agent, and ubiquitin ligating agent; and other variations that will be readily appreciated by the ordinarily skilled artisan. As will also be apparent to the skilled artisan, the detection of ubiquitin bound will encompass not only the particular ubiquitin bound directly to the corresponding protein (e.g., ubiquitin activating agent, ubiquitin conjugating agent, ubiquitin ligating agent, and/or substrate protein), but also the ubiquitin proteins bound in a polyubiquitin chain. In one embodiment, the assay is conducting by assessing ubiquitin ligase activity as described in PCT Publication No. WO 01/75145, which application is incorporated by reference herein in its entirety.

In one embodiment, ubiquitylation is measured, which can be accomplished by, for example, detection of a tag attached to the ubiquitin moiety, e.g., a fluorescent label. In another embodiment, the tag attached to the ubiquitin moiety is an enzyme label or a binding pair member which is indirectly labeled with an enzyme label. In this latter embodiment, the enzyme label substrate produces a fluorescent reaction product. In either of these embodiments, the amount of ubiquitin bound is measured by luminescence. As used herein, "luminescence" or "fluorescent emission" means photon emission from a fluorescent label. In an embodiment where FRET pairs are used, fluorescence measurements may be taken continuously or at time-points during the ligation reaction. Equipment for such measurement is commercially available and easily used by one of ordinary skill in the art to make such a measurement.

Other modes of measuring bound ubiquitin are well known in the art and easily identified by the skilled artisan for each of the labels described herein. For instance, radioisotope labeling may be measured by scintillation counting, or by densitometry after exposure to a photographic emulsion, or by using a device such as a PhosphorImager. Likewise, densitometry may be used to measure bound ubiquitin following a reaction with an enzyme label substrate that produces an opaque product when an enzyme label is used.

In one embodiment, the assay is conducted to detect ubiquitin ligase activity. In this embodiment, the assay can be performed by adapting the assays described in PCT Publication No. WO 01/75145, which describes assay for detecting ubiquitin ligase activity, including such assays conducted in a cell-free environment.

As well as identifying agents that may be used as antiviral agents, the subject assays may be modified to identify targets for the treatment of poxvirus infection. In general, the methods involve contacting a poxvirus p28 protein with a candidate cellular polypeptide in the presence of ubiquitin, (and usually an E1 and an E2 protein), and determining if the candidate cellular polypeptide is ubiquitylated by the p28 polypeptide. In such assays, for example, a cDNA library may be used to produce a plurality of cellular proteins in a corresponding plurality of cells in which a ubiquitin, a poxvirus p28, an E1 protein and an E2 protein are also produced. The cells, or lysates thereof, may be assayed to determine if the protein encoded by the cDNA is ubiquitylated. If the protein encoded by the cDNA is ubiquitylated, the cDNA may be sequenced and the identify of the encoded protein, i.e., the cellular target for poxvirus p28, can become known.

Cell-Based Assays

In one embodiment, the assay is conducted in a cell, usually a mammalian cell. In some embodiments, the assays are carried out in cells that are susceptible to poxvirus infection and/or permissive to poxvirus replication. In another embodiment, the cell is a mammalian cell that constitutively or inducibly expresses a poxvirus p28 polypeptide from a recombinant construct which may be either extrachromosomal or chromosomally integrated.

In general, in this embodiment the ubiquitin agents, are provided in a host cell, e.g., by expression of an endogenous or exogenous nucleic acid encoding the polypeptides, or by introduction of the polypeptides by, e.g., viral delivery.

Where co-expression of assay components is desired, co-expression may be achieved by introducing into the cell a vector comprising nucleic acids encoding two or more of the assay components, or by introduction of separate vectors, each comprising a single component of the desired assay components. In one embodiment, the candidate agents are peptides, e.g., randomized peptides, which can also be expressed in the host cell.

In general, the host cells used in cell-based assays of the invention mammalian cells, particularly human cells. Where mammalian cells are used, essentially any mammalian cells can be used, with mouse, rat, primate and human cells being particularly preferred.

The ordinarily skilled artisan will appreciate that various assay designs with respect to the assay component and to the methods of detection of ubiquitylation activity described above can be readily adapted for implementation in a cell-based assay.

In one embodiment, the assay is conducted by assessing ubiquitin ligase activity as described in PCT Publication No. WO 01/75145, which application is incorporated by reference herein in its entirety. Further methods for assessing ubiquitylation activity (e.g., using functional assays) are described in U.S. application serial no. U.S. Ser. No. 10/232,951, filed Aug. 30, 2002, and in PCT application serial no. PCT/US03/026843, filed Aug. 29, 2003, each of which applications is incorporated herein by reference in its entirety.

In general, cell-based assays involve contacting a cell containing the assay components with a candidate agent, and culturing the cell for a suitable period and under suitable conditions to allow for ubiquitylation to occur with respect to the substrate protein. The ordinarily skilled artisan will appreciate that precise culture methods will vary according to, for example, the host cell used, and is susceptible to ready optimization. Methods and means for detecting ubiquitylation activity can be adapted from those described above for cell-free assays.

In one embodiment, the assay is designed so as to be readily amenable for use in high-throughput assays. Preferably, in this embodiment, ubiquitylation activity can be detected without the need for isolation of, for example, ubiquitylated substrate protein or lysis of the host cell. For example, the FRET embodiment can be employed so that a level of ubiquitylation activity can be readily associated with a detectable signal that can be extrapolated to a level of ubiquitylation activity. For example, the intensity of the detectable signal can be associated with a level of ubiquitylation activity in the cell.

The cells can be cultured in any suitable receptacle, preferably in a receptacle that is amenable for high throughput assays (e.g., a multi-well plate).

High-Throughput Assays

In one embodiment, multiple assays are performed simultaneously in a high throughput screening system. In this embodiment, multiple assays may be performed in multiple receptacles, such as the wells of a 96 well plate or other multi-well plate. As will be appreciated by one of skill in the art, such a system may be applied to the simultaneous assay of multiple candidate agents.

It is understood by the skilled artisan that the steps of the assays provided herein can vary in order. It is also understood, however, that while various options (of compounds, properties selected or order of steps) are provided herein, the options are also each provided individually, and can each be individually segregated from the other options provided herein. Moreover, steps which are obvious and known in the art that will increase the sensitivity of the assay are intended to be within the scope of this invention. For example, there may be additionally washing steps, blocking steps, etc. it is understood that the exemplary embodiments provided herein in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are expressly incorporated by reference in their entirety.

Cell and Animal Based Screening Assays for Poxvirus Pathogenesis

Once identified, modulators of poxvirus p28 ligase activity may be tested in cellular and/or non-human animal models for poxvirus pathogenesis.

Such cellular and non-human animal models are generally described in Brick et al, (J. General Virology 81: 1087 1097, 2000), Senkevich et al, (Virology 198: 118 128, 1994) and Senkevich et al, (J. Virology 69: 4103 4111, 1995). As is known in the art, the effect of a candidate agent on a cell or an animal infected with poxvirus may be assayed a number of different ways, including measuring virus titer, replication, infectivity, etc., as well as cellular phenotypes, e.g., proliferation or viability, etc. In particular embodiments, the Moscow strain of ectromelia virus, propagated using BSC-1 cells, may be used.

Any cell that is permissive to poxvirus replication is suitable cells for assaying poxvirus pathogenesis, including COS, HEK-293, BHK, CHO, TM4, CVI, VERO-76, HELA, MDCK, BRL 3A, NIH/3T3 cells, etc. Additional cell lines will become apparent to those of ordinary skill in the art, and a wide variety of suitable cell lines are available from the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110 2209. Cells of particular interest include immune system cells, including lymphocytes (B and T cells e.g., T helper cells) and leucocytes (e.g., granulocytes, lymphocytes, macrophage and monocytes), cells from lymph, spleen and bone marrow tissues, epithelial cells, and cells from or derived from internal organs.

In in vivo assays, any mammal that may be susceptible to poxvirus infection may be used, and in performing assays for poxvirus pathogenesis, any organ or tissue of the mammal may be examined. For example, tissues having immune system cells, e.g., lymph, spleen and bone marrow, tissues from internal organs such as liver, heart, kidney, brain, spleen, etc., and any other tissues, e.g., epithelial tissues from skin, mouth, lungs and internal passages, may be examined.

In one embodiment, p28 ligase activity modulators may be tested to determine if they have an effect on cell viability. In these embodiments, a susceptible cell is transfected with a vector or poxvirus encoding a p28 protein to make it become sensitive to a variety of apoptosis agents, including ultraviolet light (UV), Fas and TNF (Brick et al, supra). The agent is tested to determine if it can protect the transfected cell from those apoptosis agents. In general, apoptosis assays are well known in the art and may be done using standard techniques (e.g., DAPI analysis).

In other embodiments, a susceptible cell, e.g., a macrophage such as a resident peritoneal macrophage, is transfected with a vector or poxvirus encoding a p28 protein to stop dividing or become apoptotic (Senkevich et al, J. Virology 69: 4103 4111, 1995). The agent is then tested to determine if it can increase the viability of the cell or increase cell proliferation. Again, cell viability and cell proliferation assays are well known in the art and may be done using standard techniques.

In other embodiments, a susceptible mammal, e.g., a mouse, may be used for in vivo testing of p28 ligase modulators. If a mouse is used, it may be a pathogen-free mouse of 6 10 weeks of age, or a severe combined immunodeficiency (SCID) mouse (e.g., strain C.B 17). To infect the mice with virus, 5.times.10.sup.4 PFU of ectromelia virus may be injected subcutaneously into their footpads. After 6 10 days, the mice may be sacrificed and assayed for the presence of virus. In most embodiments, viral titer in organs of the mice may be assessed. In particular, viral titer in liver, and/or liver damage may be assessed.

Suitable controls for the above experiments include p28 plasmids or viruses, such as those with an altered ring-finger domain, that are known in the art (Senkevich et al, Virology 198: 118 128, 1994).

Candidate agents possessing poxvirus p28 polypeptide ubiquitin ligase-modulatory activity may be further screened to identify those agents that are specific to the p28 polypeptide by testing the agent in assays that contain other E3 ubiquitin ligases, e.g., cellular ubiquitin ligases such as those listed in literature incorporated by reference above, for example. A "poxvirus p28-specific inhibitory agent" is an agent that inhibits p28 ligase activity without significantly inhibiting the ligase activity of other cellular E3 proteins or other assay components (e.g., E1 or E2 proteins).

Kits

Also provided are reagents and kits thereof for practicing one or more of the above-described methods. The subject reagents and kits thereof may vary greatly. Typically, the kits at least include poxvirus p28 protein or a nucleic encoding such a protein, and other proteins for performing ubiquitylation assays. The subject kits may also include one or more additional reagents, e.g., reagents employed in detecting a label.

In addition to the above components, the subject kits can further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

Methods of Reducing Poxvirus Pathogenicity

In another aspect, the invention features methods of reducing poxvirus pathogenicity in a cell by inhibiting the ubiquitin ligase activity of poxvirus p28 protein.

In one embodiment, pathogenicity of a poxvirus in a host cell is reduced by contacting a mammalian cell infected with a poxvirus with an agent that inhibits ubiquitin ligase activity of poxvirus p28 protein in the infected cell, where the agent is provided in amount effective to reduce poxvirus pathogenicity in the cell. The poxvirus may be any poxvirus mentioned above, or a recombinant form thereof.

As discussed above, viral pathogenicity can be determined using a number of different assays, including measuring virus titer, replication, infectivity, transmission, etc., as well as cellular phenotypes, e.g., cell proliferation, viability, expression of markers, etc. Accordingly, the term "pathogenicity" is used herein to indicate any aspect of viral biology that may be measured, including those listed in the previous sentence.

Subjects to be Treated

Any subject having a retroviral infection may be treated according to the invention. Mammalian subjects, especially human subjects, are of particular interest. The terms "individual," "host," "subject," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.

As used herein, the terms "treatment", "treating", and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

The subjects to be treated thus include those having or at risk of poxvirus infection. The subjects may be symptomatic or asymptomatic. Diseases and symptoms associated with poxvirus infection include, but are not limited to fever, chills, headache, nausea, vomiting severe muscle aches macules, papules, vesicles, pustules and scabbing and other clinical pathologies and symptoms such as bleeding. The methods of the invention can be continued until a desired clinical endpoint is attained (e.g., symptoms diminish or are otherwise improved), viral clearance (e.g. as detected by a decrease in viral titer or undetectably viral titer, etc.).

In particular, the subject invention finds most use for treating, military personnel, healthcare workers, researchers of poxvirus biology, and other persons such as key government officials, since they are at immediate risk from poxvirus, e.g., smallpox, infection.

In particular embodiments, the subject agents may be used to reduce side effects of smallpox vaccine, and, as such, may be administered at the same time as, prior to, or after, administration of such a vaccine.

Formulations and Routes of Administration

Antiviral agents suitable for use in the invention in the methods of inhibiting poxvirus replication (referred to herein as "the agents" or "the active agents" for convenience) as described herein can be formulated in a variety of ways suitable for administration. In general, these compounds are provided in the same or separate formulations in combination with a pharmaceutically acceptable excipient(s). A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7.sup.th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3.sup.rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

In some embodiments, the agents are formulated separately or in combination, e.g., in an aqueous or non-aqueous formulation, which may further include a buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strength from 5 mM to 100 mM. In some embodiments, the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride, and sugars e.g., mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80.

Optionally the formulations may further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4.degree. C. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.

In the subject methods, the active agents may be administered to the host using any convenient means capable of resulting in the desired therapeutic effect. Thus, the agents can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. Agents can also be provided in sustained release or controlled release formulations, e.g., to provide for release of agent over time and in a desired amount (e.g., in an amount effective to provide for a desired therapeutic or otherwise beneficial effect).

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the agents calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms for use in the present invention depend on the particular compound employed and the effect to be achieved, the pharmacodynamics associated with each compound in the host, and the like.

Dosage forms of particular interest include those suitable to accomplish intravenous or oral administration, as well as dosage forms to provide for delivery by a nasal or pulmonary route (e.g., inhalation), e.g., through use of a metered dose inhaler and the like.

In general, agents for use in the invention is formulated in either parenteral or enteral forms, usually enteral formulations, more particularly oral formulations. Agents for use in the invention are formulated for parenteral administration, e.g., by subcutaneous, intradermal, intraperitoneal, intravenous, or intramuscular injection. Administration may also be accomplished by, for example, enteral, oral, buccal, rectal, transdermal, intratracheal, inhalation (see, e.g., U.S. Pat. No. 5,354,934), etc.

The invention also contemplates administration of additional agents with the antiviral agents according to the invention, such as other antiviral agents that work through the same of different mechanism.
 

Claim 1 of 32 Claims

1. A method of ubiquitylating a substrate, comprising: combining an E1 polypeptide, an E2 polypeptide, ubiquitin and a poxvirus P28 protein having an amino acid that is at least 80% identical to a p28 protein encoded by the genome of an orthopoxvirus under ubiquitylation reaction conditions; and detecting a ubiquitylated substrate.

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