Patent Grant
8871435
Hepatitis C virus (HCV) is an example of a Flaviviridae virus and is the principal etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 150 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected with this pathogen and many patients progress to a state of chronic liver disease, so-called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
HCV is an enveloped positive strand RNA virus. The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. In the case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one is a metalloprotease located in NS2 that cleaves the NS2-NS3 junction in cis; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, at the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been elucidated. In addition, chronic infection with the hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The incidence of HCC and the mortality rate associated with it are increasing dramatically. While chronic inflammation, fibrosis and liver cell proliferation are considered as a major pathogenic mechanism for the development of HCC, there is increasing data to suggest that direct viral effects may play a role as well.
Literature of interest includes: Hugle et al, 2001 Virology 284:70-81; Gorbalenya and Koonin 1989 Nucleic Acids Res 17:8413-8440; Bartenschlager and Lohmann 2000 Virology 81 Pt 7:1631-1648; Reed and Rice 2000 Current Topics in Microbiology and Immunology 242:55-84; Mirzayan and Wimmer 1992 Virology 189:547-555; Rodriguez and Carrasco 1993 J. Biol Chem 268:8105-8110; and Piccininni 2002 J. Biol Chem 277:45670-45679; Ray et al 1996 Virol 70:4438-43; Park et al, 2000 Biochemical and Biophysical Research Communications 267:581-587; Florese et al, 2002 Virus Res. December; 90(1-2):119-31; Chang et al. J Virol 72:3060-5; Gimenez-Barcons et al. J Interferon Cytokine Res 25:152-64; Ray et al. J Virol 70:4438-43; and published patent applications US20030087873, US20020147160, US20060199174 and WO99/01582.
The disclosure provides methods and compositions for identifying agents that modulate a neoplastic cellular phenotype mediated by the NS4B protein nucleotide binding motif (NBM) of hepatitis C virus (HCV). In general, the methods involve contacting a candidate agent with a mammalian cell expressing an NS4B NBM polypeptide of an HCV virus, wherein expression of the NS4B NBM polypeptide in the absence of candidate agent promotes a neoplastic cellular phenotype, and detecting the presence or absence of an effect of said candidate agent on NS4B-mediated promotion of a neoplastic cellular phenotype. The disclosed methods and compositions find use in a variety of therapeutic and screening applications.
Before the present invention is further described, 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 limit of that 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 in the smaller ranges, and are 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.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the 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”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an NS4B polypeptide” includes a plurality of such polypeptides and reference to “the NS4B nucleotide binding motif” includes reference to one or more NS4B nucleotide binding motifs 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.
A “nucleotide binding motif” or “NBM” as used herein refers to a region of a viral NS4B polypeptide that binds a nucleotide triphosphate (NTP), which NTP can be GTP, ATP, TTP, or CTP, and usually is at least GTP.
A “nucleotide binding NS4B protein” or “NS4B protein” is any viral NS4B protein, or functional equivalent thereof (e.g., proteins that are encoded by other viruses and are equivalent to NS4B, but not named NS4B), that contains a nucleotide binding motif, binds a nucleotide, and binds RNA in the presence of a nucleotide. NS4B proteins are typically characterized by an N-terminal amphipathic helix, at least two transmembrane domains, and an NBM, where the NBM facilitates nucleotide binding. NS4B proteins may be identified using a number of methods, e.g., by pairwise sequence alignment between the HCV NS4B protein and the proteins encoded by other viruses. The NS4B polypeptide may also itself be contained within a larger polypeptide such as one encoding a replication-competent form of a viral polyprotein.
A “variant” of a polypeptide (e.g., an NS4B polypeptide or an NS4B nucleotide binding motif of a polypeptide) is defined as a polypeptide that is altered by one or more amino acid residues. Such alterations include amino acid substitutions, deletions or insertions, or a combination thereof. Variants of NS4B, particularly those that have conservative amino acid substitutions, usually retain their basic structural features and biological activity. Variants of an NS4B nucleotide binding motif may retain a nucleotide binding activity, allow HCV virus replication and promote a neoplastic cellular phenotype. NS4B variants may alternatively have decreased nucleotide binding activity (e.g., a decreased binding affinity or avidity relative to a wildtype NS4B of, for example, the same viral origin), may have constitutive nucleotide binding activity, or may have enhanced nucleotide binding activity (e.g., an increased binding affinity or avidity relative to a wildtype NS4B of, for example, the same viral origin). A variant NS4B NBM may have decreased ability to promote a neoplastic cellular phenotype.
Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted (e.g., without abolishing activity) may be found by comparing the sequence of a polypeptide to the sequence of a polypeptide with a related structure and function e.g., between sequences from HCV strains or genotypes. Assays for HCV are readily available and straightforward, and can be readily applied to determine empirically which and how many amino acid residues may be substituted, inserted or deleted.
A “substitution” results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a polypeptide. If a substitution is conservative, the amino acid that is substituted into a polypeptide has similar structural or chemical properties (e.g., charge, polarity, hydrophobicity, and the like) to the amino acid that it is substituting. Conservative substitutions of naturally occurring amino acids usually result in a substitution of a first amino acid with second amino acid from the same group as the first amino acid, where exemplary amino acid groups are as follows: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. It is understood that NS4B NBM polypeptides may have conservative amino acid substitutions which have substantially no affect on HCV viral replication or promotion of a neoplastic cellular phenotype. In some embodiments, polypeptide variants may have “non-conservative” changes, where the substituted amino acid differs in structural and/or chemical properties.
A “deletion” is defined as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a naturally occurring polypeptide. In the context of a polypeptide or polynucleotide sequence, a deletion can involve deletion of about 2, about 5, about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. An NS4B NBM polypeptide may contain more than one deletion.
An “insertion” or “addition” is that change in an amino acid or nucleotide sequence which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a naturally occurring polypeptide. “Insertion” generally refers to addition of one or more amino acid residues within an amino acid sequence of a polypeptide, while “addition” can be an insertion or refer to amino acid residues added at the N- or C-termini. In the context of a polypeptide or polynucleotide sequence, an insertion or addition may be of up to about 10, up to about 20, up to about 30 or up to about 50 or more amino acids. A NS4B NBM polypeptide may contain more than one insertion.
A “biologically active” NS4B polypeptide refers to a polypeptide having structural and biochemical functions of a naturally occurring NS4B protein.
A “neoplastic cellular phenotype” refers to a cellular phenotype characterized by, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, continued cellular proliferation despite serum depletion, anchorage-independent growth potential, ability to promote tumor growth and/or development in an immuno-compromised non-human animal model, or any appropriate indicator of cellular transformation.
“Non-native”, “non-endogenous”, and “heterologous”, in the context of a polypeptide, are used interchangeably herein to refer to a polypeptide having an amino acid sequence or, in the context of an expression system or a viral particle, present in an environment different to that found in nature.
“Exogenous” in the context of a nucleic acid or polypeptide is used to refer to a nucleic acid or polypeptide that has been introduced into a host cell. “Exogenous” nucleic acids and polypeptides can be native or non-native to the host cell, where an exogenous, native nucleic acid or polypeptide provides for elevated levels of the encoded gene product or polypeptide in the recombinant host cell relative to that found in the host cell prior to introduction of the exogenous molecule.
The term “Ha-ras oncogene” refers to a Ha-ras gene which when expressed in a mammalian cell promotes a neoplastic cellular phenotype of the mammalian cell. The Ha-ras oncogene may be endogenous or exogenous to the mammalian cell and the ability to promote a neoplastic cellular phenotype of the mammalian cell may result from activating mutations in the DNA sequence encoding the Ha-ras gene or by virtue of overexpression of the Ha-ras gene and/or increased signaling by an upstream receptor.
As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
The terms “polypeptide” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and native leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, β-galactosidase, luciferase, etc.; and the like.
The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.
As used herein the term “isolated,” when used in the context of an isolated compound, refers to a compound of interest that is in an environment different from that in which the compound naturally occurs. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified.
As used herein, the term “substantially pure” refers to a compound that is removed from its natural environment and is at least 60% free, preferably 75% free, and most preferably 90% free from other components with which it is naturally associated.
A “coding sequence” or a sequence that “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in-vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are typically determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence. Other “control elements” may also be associated with a coding sequence. A DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence.
“Encoded by” refers to a nucleic acid sequence which codes for a gene product, such as a polypeptide. Where the gene product is a polypeptide, the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.
“Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given polypeptide that is operably linked to a HCV NS4B nucleotide binding motif binds nucleotides. In the case of a promoter, a promoter that is operably linked to a coding sequence will effect the expression of a coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
By “nucleic acid construct” it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like.
A “vector” is capable of transferring gene sequences to target cells. Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells, which can be accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.
An “expression cassette” comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Such cassettes can be constructed into a “vector,” “vector construct,” “expression vector,” or “gene transfer vector,” in order to transfer the expression cassette into target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
Techniques for determining nucleic acid and amino acid “sequence identity” are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986).
An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Ma dison, Wis.). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the internet address :www.ncbi.nlm.gov/cgi-bin/BLAST.
Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two DNA, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 80%-85%, preferably at least about 85%-90%, more preferably at least about 90%-95%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., infra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
Two nucleic acid fragments are considered to “selectively hybridize” as described herein. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit a completely identical sequence from hybridizing to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern blot, Northern blot, solution hybridization, or the like, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.
When utilizing a hybridization-based detection system, a nucleic acid probe is chosen that is complementary to a target nucleic acid sequence, and then by selection of appropriate conditions the probe and the target sequence “selectively hybridize,” or bind, to each other to form a hybrid molecule. A nucleic acid molecule that is capable of hybridizing selectively to a target sequence under “moderately stringent” typically hybridizes under conditions that allow detection of a target nucleic acid sequence of at least about 10-14 nucleotides in length having at least approximately 70% sequence identity with the sequence of the selected nucleic acid probe. Stringent hybridization conditions typically allow detection of target nucleic acid sequences of at least about 10-14 nucleotides in length having a sequence identity of greater than about 90-95% with the sequence of the selected nucleic acid probe. Hybridization conditions useful for probe/target hybridization where the probe and target have a specific degree of sequence identity, can be determined as is known in the art (see, for example, Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).
With respect to stringency conditions for hybridization, it is well known in the art that numerous equivalent conditions can be employed to establish a particular stringency by varying, for example, the following factors: the length and nature of probe and target sequences, base composition of the various sequences, concentrations of salts and other hybridization solution components, the presence or absence of blocking agents in the hybridization solutions (e.g., formamide, dextran sulfate, and polyethylene glycol), hybridization reaction temperature and time parameters, as well as, varying wash conditions. The selection of a particular set of hybridization conditions is selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). An example of stringent hybridization conditions is hybridization at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above representative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least about 90% as stringent as the above specific stringent conditions. Other stringent hybridization conditions are known in the art and may also be employed to identify nucleic acids of this particular embodiment of the invention.
A first polynucleotide is “derived from” a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above.
A first polypeptide is “derived from” a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above.
The terms “treatment”, “treating”, “treat”, 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 and/or relieving one or more disease symptoms. “Treatment” is also meant to encompass delivery of an agent in order to provide for a pharmacologic effect, even in the absence of a disease or condition. For example, “treatment” encompasses delivery of modified NS4B polypeptide-encoding nucleic acids that can provide for enhanced or desirable effects in the subject (e.g., reduction of pathogen load, increase in CD4 count, reduction of disease symptoms, etc.).
“Subject”, “host” and “patient” are used interchangeably herein, to refer to an animal, human or non-human, susceptible to or having an infection by an intracellular pathogen and amenable to therapy according to the methods of the invention. Generally, the subject is a mammalian subject. Exemplary subjects include, but are not necessarily limited to subjects susceptible to HCV infection, particularly with development of hepatocellular carcinoma (HCC).
The disclosure provides methods and compositions for screening agents for activity in modulating a neoplastic cellular phenotype mediated by the NS4B nucleotide binding motif (NBM) of hepatitis C virus (HCV). In general, the methods involve contacting a candidate agent with a mammalian cell expressing an NS4B NBM polypeptide of an HCV virus, wherein expression of the NS4B NBM polypeptide in the absence of candidate agent promotes a neoplastic cellular phenotype, and detecting the presence or absence of an effect of said candidate agent on the NS4B-mediated neoplastic cellular phenotype.
The disclosure also provides methods for identifying an agent that modulates a neoplastic cellular phenotype of cells infected with hepatitis C virus (HCV). In general the methods involve contacting an NS4B nucleotide binding motif (NBM) polypeptide with a candidate agent; determining an effect of the candidate agent on a nucleotide binding activity, a nucleotide hydrolyzing activity, or a nucleotide-dependent RNA binding activity of said polypeptide; and assessing the effect of said candidate agent on a neoplastic cellular phenotype of a mammalian cell expressing an NS4B NBM polypeptide.
Compositions related to the above methods are also disclosed. Generally, these compositions comprise cells comprising a polynucleotide encoding an NS4B polypeptide, which when expressed promote a neoplastic cellular phenotype; and a culture medium suitable for detecting a neoplastic cellular phenotype of the cells. The disclosed methods and compositions find use in a variety of applications.
The role of the nucleotide binding motif (NBM) of hepatitis C virus (HCV) NS4B polypeptide in promoting a neoplastic cellular phenotype can be exploited in assays designed to identify agents capable of modulating a neoplastic cellular phenotype.
In one embodiment, methods and compositions for screening agents for activity in modulating a neoplastic cellular phenotype mediated by an NS4B nucleotide binding motif (NBM) polypeptide of a virus are provided, with HCV viruses being of particular interest. Such agents can be applied to the treatment of conditions characterized by a neoplastic cellular phenotype (e.g. to inhibit the cellular proliferation characteristic of hepatocellular carcinoma (HCC)). In general, the methods involve contacting a candidate agent with a mammalian cell expressing an NS4B NBM polypeptide of an HCV virus, wherein expression of the NS4B NBM polypeptide in the absence of candidate agent promotes a neoplastic cellular phenotype, and detecting the presence or absence of an effect of said candidate agent on the neoplastic cellular phenotype.
In another embodiment, methods for identifying agents that modulate a neoplastic cellular phenotype of cells infected with hepatitis C virus (HCV) are disclosed. In general these methods involve contacting an NS4B nucleotide binding motif (NBM) polypeptide with a candidate agent; determining an effect of the candidate agent on a nucleotide binding activity, a nucleotide hydrolyzing activity, or a nucleotide-dependent RNA binding activity of said polypeptide; and assessing the effect of said candidate agent on a neoplastic cellular phenotype of a mammalian cell expressing an NS4B NBM polypeptide.
Also disclosed, is a composition comprising a recombinant mammalian cell exhibiting a neoplastic cellular phenotype which includes anchorage-independent growth when cultured in soft agar, wherein said recombinant mammalian cell comprises an expression construct encoding an NS4B nucleotide binding motif (NBM) polypeptide, and wherein expression of the NS4B NBM promotes the neoplastic cellular phenotype of the recombinant mammalian cell, with the proviso that the recombinant mammalian cell does not express an exogenous Ha-ras oncogene.
The mammalian cell of the disclosed composition may be an NIH3T3 cell or other cell capable of exhibiting a neoplastic cellular phenotype in connection with the expression of NS4B NBM.
The disclosure further provides an NS4B polypeptide having a modified nucleotide binding motif, e.g., a nucleotide binding motif that is impaired in nucleotide binding, and/or the ability to promote a neoplastic cellular phenotype, and a polynucleotide encoding this polypeptide. The disclosure also provides modified viral genomes encoding a modified NBM. The disclosed methods and compositions find use in a variety of applications.
The disclosed HCV may be of any genotype (genotype 1, 2, 3, 4, 5, 6, and the like), as well as subtypes of an HCV genotype (e.g., 1a, 1b, 2a, 2b, 3a, etc.)). Because currently HCV genotype 1 is normally the most difficult to treat, and thus most commonly associated with chronic infection, HCV genotype 1 and genotype 1 subtypes are of particular interest.
In further describing the invention in greater detail than provided in the Summary and as informed by the Background and Definitions provided above, the compositions for use in the disclosed methods are described first, followed by a discussion of methods for screening agents for activity in modulating a neoplastic cellular phenotype and methods for identifying agents that modulate a neoplastic cellular phenotype of cells infected with an HCV virus, a review of representative applications in which the disclosed methods find use, and disclosed kits provided for practicing the disclosed methods.
NS4B NBM Polypeptides
An “NS4B NBM polypeptide” is a polypeptide that contains an NS4B NBM and that, when expressed in a mammalian cell in the absence of an exogenous Ha-ras oncogene gene product, promotes a neoplastic cellular phenotype. Thus, an NS4B NBM polypeptide encompasses, for example, a polypeptide that contains an NBM from an HCV NS4B protein (e.g., a naturally-occurring NS4B protein), a fusion protein containing an NBM-containing fragment of an NS4B polypeptide or full-length NS4B polypeptide, operably linked to a fusion partner, a polypeptide that comprises a variant NS4B NBM (including fusion proteins thereof) that retains activity in promoting a neoplastic cellular phenotype, or the like, provided that the NS4B NBM polypeptide is capable of promoting a neoplastic cellular phenotype in a mammalian cell in the absence of an exogenous Ha-ras oncogene gene product. Such polypeptides find use in, for example, screening assays for agents with activity in inhibiting a neoplastic cellular phenotype.
The NS4B NBM polypeptide referenced above may be a naturally-occurring NS4B protein or a variant thereof, or in other embodiments, a non-naturally occurring NS4B protein or a variant thereof, provided that the protein or variant thereof is capable of promoting a neoplastic cellular phenotype in a mammalian cell in the absence of an exogenous Ha-ras oncogene gene product.
In certain embodiments the fusion partner may be a reporter protein, e.g., a light emitting reporter such as a fluorescent or luminescent polypeptide (for example GFP or luciferase), may contain sequences from another nucleotide-binding polypeptide (e.g., a G-protein), or may contain sequence from any other polypeptide. In particular embodiments, an NS4B NBM polypeptide is a fusion protein between an NS4B NBM and a partner such as GST, poly-histidine, or avidin. These fusions are convenient for assay formats using glutathione, nickel, or biotin coupled to solid supports (using beads or a microtiter plate well, etc.).
The NS4B NBM polypeptides referenced above contain either an NS4B NBM or a variant NS4B NBM. An “NS4B NBM” is generally defined as having a sequence conforming to an NS4B NBM consensus amino acid sequence comprising: an A motif consensus sequence of G-S/G-I/V-G-L/I-G-K/R: SEQ ID NO. 16, a G motif consensus F (Phenylalanine) residue, a PM2 motif consensus T (Threonine) residue, and a B motif consensus sequence of DAAA: SEQ ID NO:17. In certain embodiments, the NS4B NBM has an A motif of the sequence GSIGLGK: (SEQ ID NO: 18), a G motif with an F (Phenylalanine) residue, a PM2 motif with a T (Threonine) residue, and a B motif with the sequence DAAA: (SEQ ID NO: 17).
A polypeptide comprising a variant NS4B NBM, and having activity in promoting a neoplastic cellular phenotype, generally has one or more amino acid substitutions relative to the NBM consensus sequences set out above, but retains the ability to promote a neoplastic cellular phenotype when expressed in a mammalian cell in the absence of an exogenous Ha-ras oncogene gene product. For example, substitution of an “N” in the (I/V) position of the A motif results in a decrease in the promotion of a neoplastic cellular phenotype but does not abolish the ability of the NS4B NBM polypeptide comprising a variant NS4B NBM to promote a neoplastic cellular phenotype. A double mutation including the substitution of an “N” in the (I/V) position of the A motif and an “L” in the D position of the B motif results in a further decrease in the promotion of a neoplastic cellular phenotype but also does not abolish the ability of the NS4B NBM polypeptide comprising a variant NS4B NBM to promote a neoplastic cellular phenotype.
Those of ordinary skill in the art will be able to readily identify NS4B NBM polypeptides having the ability to promote a neoplastic cellular phenotype, by performing assays similar to those described in the Examples below.
NS4B NBM polypeptides that retain nucleotide-binding activity usually have a sequence conforming to the NS4B NBM consensus sequence: G-S/G-I/V-G-L/I-G-K/R (SEQ ID NO:16) or, in other embodiments G-S/G-I-G-L-G-K/R (SEQ ID NO:39), and in addition, may have NS4B NBM motifs G, PM2 and B, spaced from the consensus sequence at an appropriate distance, as discussed in Dever et al., (Proc Natl Acad Sci U S A. 1987 84:1814-8) although their precise spacing distance is not as important as their relationship to each other in the polypeptide tertiary structure.
Nucleic Acids Encoding NS4B NBM Polypeptides
Since the genetic code and recombinant techniques for manipulating nucleic acid are known, and the amino acid sequences of NS4B NBM polypeptides are described above, the design and production of nucleic acids encoding a NS4B NBM polypeptide is well within the skill of an artisan. In certain embodiments, standard recombinant DNA technology (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.) methods are used. For example, naturally occurring NS4B NBM polypeptide coding sequences may be isolated from a library of nucleic acids using any one or a combination of a variety of recombinant methods that do not need to be described herein. Subsequent substitution, deletion, and/or addition of nucleotides in the nucleic acid sequence encoding a protein may also be done using standard recombinant DNA techniques.
For example, site directed mutagenesis and subcloning may be used to introduce/delete/substitute nucleic acid residues in a polynucleotide encoding a NS4B NBM polypeptide. In other embodiments, PCR may be used. Nucleic acids encoding a NS4B NBM polypeptide may also be made by chemical synthesis entirely from oligonucleotides (e.g., Cello et al., Science (2002) 297:1016-8).
In certain embodiments, the codons of the nucleic acids encoding an NS4B NBM polypeptide are optimized for expression in cells of a particular species, particularly a mammalian, e.g., human or mouse, species.
Vectors (also referred to as “constructs”) comprising a nucleic acid encoding a NS4B NBM polypeptide are also disclosed. In many embodiments, a nucleic acid sequence encoding a NS4B NBM polypeptide may be expressed in a host after the sequence has been operably linked to an expression control sequence, including, e.g. a promoter. The NS4B NBM polypeptide encoding nucleic acids are typically placed in an expression vector that can replicate in a host cell either as an episome or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein by reference). Vectors, including single and dual expression cassette vectors are well known in the art (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Suitable vectors include viral vectors, plasmids, cosmids, artificial chromosomes (human artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, etc.), mini-chromosomes, and the like. Retroviral, adenoviral and adeno-associated viral vectors may be used. In certain embodiments, however, the disclosed nucleic acids are contained in the genome of HCV, where the genome has been genetically modified such that it contains a non-naturally occurring NS4B polypeptide-encoding sequence.
A variety of expression vectors are available to those in the art for purposes of producing a polypeptide of interest in a cell. One suitable vector is pCMV, which used in certain embodiments. This vector is publicly available from the American Type Culture Collection (ATCC) and was deposited on Oct. 13, 1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be viable. The ATCC has assigned the following deposit number to pCMV: ATCC #203351.
The nucleic acids encoding a NS4B NBM polypeptide usually contain a single open reading frame, however, in certain embodiments, since the host cell for expression of the polypeptide of interest may be a eukaryotic cell, e.g., a mammalian cell, such as a human cell, the open reading frame may be interrupted by introns. Nucleic acids encoding a NS4B NBM polypeptide are typically part of a transcriptional unit which may contain, in addition to the disclosed nucleic acids 3′ and 5′ untranslated regions (UTRs) which may direct RNA stability, translational efficiency, etc. The disclosed nucleic acids may also be part of an expression cassette which contains, in addition to the disclosed nucleic acid a promoter, which directs the transcription and expression of a polypeptide of interest, and a transcriptional terminator.
Eukaryotic promoters can be any promoter that is functional in a eukaryotic host cell, including viral promoters and promoters derived from eukaryotic genes. Exemplary eukaryotic promoters include, but are not limited to, the following: the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-310, 1981); the yeast gall gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-59SS, 1984), the CMV promoter, the EF-1 promoter, Ecdysone-responsive promoter(s), tetracycline-responsive promoter, and the like. Viral promoters may be of particular interest as they are generally particularly strong promoters. In certain embodiments, a promoter is used that is a promoter of the target pathogen. Promoters for use in the present invention are selected such that they are functional in the cell type (and/or animal) into which they are being introduced. In certain embodiments, the promoter is a CMV promoter.
In certain embodiments, a vector may also provide for expression of a selectable marker. Suitable vectors and selectable markers are well known in the art and discussed in Ausubel, et al, (Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995) and Sambrook, et al, (Molecular Cloning: A Laboratory Manual, Third Edition, (2001) Cold Spring Harbor, N.Y.). A variety of different genes have been employed as selectable markers, and the particular gene employed in the disclosed vectors as a selectable marker is chosen primarily as a matter of convenience. Known selectable marker genes include: the thimydine kinase gene, the dihydrofolate reductase gene, the xanthine-guanine phosphoribosyl transferase gene, CAD, the adenosine deaminase gene, the asparagine synthetase gene, the antibiotic resistance genes, e.g. tetr, ampr, Cmr or cat, kanr or neor (aminoglycoside phosphotransferase genes), the hygromycin B phosphotransferase gene, and the like.
As mentioned above, NS4B NBM polypeptides may be fusion proteins. Methods for making fusions between two or more nucleic acids encoding a fusion protein, are well within the skill of one of skill in the art and will not be described any further.
Nucleic acids encoding NS4B NBM polypeptides may also contain restriction sites, multiple cloning sites, primer binding sites, ligatable ends, recombination sites etc., usually in order to facilitate the construction of a nucleic acid encoding a polypeptide of interest.
In certain embodiments, a nucleic acid of interest may be a modified viral (e.g., HCV) genome, or model thereof, that encodes an NS4B polypeptide having altered nucleotide binding activity and/or altered activity in promoting a neoplastic cellular phenotype, as described above. In these embodiments, the nucleic acid, either RNA or DNA, may be part of a virus particle. In certain embodiments, viral nucleic acids encoding a wild-type NS4B NBM are replaced by nucleic acids encoding an NS4B NBM with modified nucleotide binding activity and/or altered activity in promoting a neoplastic cellular phenotype.
Host Cells
The invention further provides host cells, including isolated in-vitro host cells (e.g., for construct production and/or use in screening assays) and in-vivo host cells of a non-human animal, that comprise a disclosed nucleic acid and/or polypeptide.
E. coli is a prokaryotic host useful for cloning nucleic acid sequences in connection with the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
Other microbes, such as yeast, may also be used for expression. Saccharomyces or Pichia are preferred hosts, with suitable vectors having expression control sequences, such as promoters, including the 3-phosphoglycerate kinase promoter or those of other glycolytic enzyme genes, and an origin of replication, termination sequences and the like as desired.
In addition to microorganisms, mammalian tissue cell culture may also be used to express and produce NS4B NBM polypeptides in connection with the present invention (see, Winnacker, “From Genes to Clones,” VCH Publishers, New York, N.Y. (1987). Suitable mammalian cells include CHO cell lines, various COS cell lines, HeLa cells, and myeloma cell lines, etc.
Modified polypeptides may be expressed in prokaryotes or eukaryotes in accordance with conventional ways, depending upon the purpose for expression. For large scale production of the protein, a unicellular organism, such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, particularly mammals, e.g. COS 7 cells, may be used as the expression host cells. In some situations, it is desirable to express the gene in eukaryotic cells, where the encoded protein will benefit from native folding and post-translational modifications. Polypeptides can also be synthesized in the laboratory. Polypeptides that are subsets of the complete sequences of the disclosed proteins may be used to identify and investigate parts of the protein important for function.
Other embodiments of particular interest include host cells useful in the screening methods disclosed herein. In certain embodiments, the host cell is a mammalian cell which exhibits a neoplastic cellular phenotype when an NSB4 NBM polypeptide is expressed therein. In certain further embodiments, a mammalian cell which exhibits a neoplastic cellular phenotype when an NSB4 NBM polypeptide is expressed therein is an NIH3T3 cell, or a primary cell susceptible to cellular transformation (e.g., primary embryo fibroblast, e.g., a primary rat embryo fibroblast). In some embodiments, a mammalian cell which exhibits a neoplastic cellular phenotype when an NSB4 NBM polypeptide is expressed therein is a cell which does not express an exogenous Ha-ras oncogene.
The disclosure also contemplates host cells suitable for use in the assays disclosed herein. For example, where it may be desirable to provide for HCV replication, the host cell is a mammalian (e.g.) human cell that may be infected with HCV (including models thereof). In certain embodiments the human cell is a CHOP cell, a huh7 cell or a H9C2 cell, B-cell lines including B-cell lymphoma cells, and the cell is chosen because it is susceptible to infection by HCV or can support the replication of HCV or HCV replicons. The latter include a wide variety of cells (including but not limited to primary human, mouse, or rat liver cells, or cell lines originally derived from primary cells such as HeLa, Hepa-6, MDCK, etc.) where each cell type has its own set of preferred adaptive mutations.
Animal Models
In addition, the disclosure contemplates use of animal models of viral infection, particularly HCV infection. Mouse models, in particular the mouse models for HCV, described in PCT publication WO01/67854, may be used. The disclosure further contemplates the use of non-human animal models of oncogenesis. In certain embodiments these non-human animal models comprise a recombinant mammalian cell, wherein said recombinant mammalian cell comprises a polynucleotide encoding an exogenous NS4B nucleotide binding motif (NBM) polypeptide, wherein expression of the exogenous NS4B NBM polypeptide promotes oncogenesis in the non-human animal. In certain further embodiments, a non-human animal model comprising a recombinant mammalian cell, wherein said recombinant mammalian cell comprises a polynucleotide encoding an exogenous NS4B nucleotide binding motif (NBM) polypeptide, and wherein expression of the exogenous NS4B NBM polypeptide promotes oncogenesis in the non-human animal, is an immuno-compromised non-human animal, e.g. a nude mouse, scid (severe combined immune deficiency) mouse, or Rag1 mouse.
Screening Assays
The disclosure provides methods of screening an agent for activity in modulating a neoplastic cellular phenotype. In general, the methods involve contacting a candidate agent with a mammalian cell expressing an NS4B NBM polypeptide, wherein expression of the NS4B NBM polypeptide in the absence of candidate agent promotes a neoplastic cellular phenotype, and detecting the presence or absence of an effect of said candidate agent on the NS4B NBM polypeptide-mediated neoplastic cellular phenotype. In one embodiment, the “mammalian cell” is an NIH3T3 cell which does not express an exogenous Ha-ras oncogene, and the modulation of a neoplastic cellular phenotype is an inhibition of a neoplastic cellular phenotype.
The disclosure also provides methods for identifying an agent that modulates a neoplastic cellular phenotype of cells infected with hepatitis C virus (HCV). In general the methods involve contacting a NS4B nucleotide binding motif (NBM) polypeptide with a candidate agent; determining an effect of the candidate agent on a nucleotide binding activity, a nucleotide hydrolyzing activity, or a nucleotide-dependent RNA binding activity of said polypeptide; and assessing the effect of said candidate agent on a neoplastic cellular phenotype of a mammalian cell expressing an NS4B NBM polypeptide. In one embodiment, the effect of said candidate agent on a neoplastic cellular phenotype is an inhibitory effect on a neoplastic cellular phenotype.
In many embodiments, a candidate agent that inhibits a nucleotide binding activity of the polypeptide is an agent which inhibits a neoplastic cellular phenotype of a cell in which the polypeptide is expressed. What is meant by “inhibits a nucleotide binding activity” is reducing an activity related to nucleotide binding, such as, for example, the affinity of the polypeptide to a nucleotide, the specificity of the polypeptide to a nucleotide, or, in certain embodiments, a conformation change in the polypeptide that is induced by nucleotide binding or the ability of the polypeptide to catalyze a reaction of said nucleotide (e.g., hydrolysis, etc), where the nucleotide can be dGTP, dATP, dTTP or dCTP, including analogs and/or variants thereof, including ribonucleotides, etc., and polymers thereof.
In general, agents identified using the disclosed screening assays will inhibit an activity (i.e., a nucleotide binding activity, a nucleotide hydrolyzing activity, a nucleotide-dependent RNA binding activity, or a neoplastic cellular phenotype promoting activity) of an NS4B NBM polypeptide by more than about 20%, more than about 40%, more than about 60%, more than about 80%, more than about 90%, or more than about 95%, or more than about 98%, usually up to about 100%, as compared to the same activity of a NS4B NBM polypeptide in the absence of a candidate agent.
The portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly.
Prior to or following cell-based assays to assess the effect of an agent upon a neoplastic cellular phenotype, assays may be performed in a cell free system, using NS4B NBM polypeptide that is in solution or immobilized in a solid support to identify agents that bind the NBM. In other embodiments, NBM binding can be assessed by using a cell containing an NS4B NBM polypeptide within the cell or on its surface.
Assays for nucleotide binding are generally very well known in the art (for example, as described in Feig, Mol Endocrinol. 1987 February; 1(2):127-36; Sigal, Anticancer Drug Des. 1987 October; 2(2):107-15; Colman, Adv Exp Med Biol. 1990; 281:257-63; Ali, J Pharmacol Toxicol Methods. 1994 December; 32(4):187-96; and Farr, Natl. Acad. Sci. USA. 1990 Jul. 1; 87 (13): 5041-5045), and generally involve producing a nucleotide binding polypeptide bound to a solid support, incubating the polypeptide with a labeled nucleotide, washing the solid support, and determining if the nucleotide is associated with the solid support. Other assays may involve assays for nucleotide hydrolysis, which are also well known in the art (e.g., Wilkes, Biochem Biophys Res Commun. 2002 Aug. 16; 296(2):388-94; Krumins, Methods Enzymol. 2002; 344:673-85; and Tisdale, Mol Biol Cell. 1999 June; 10(6):1837-49).
Assays for RNA binding are also well known in the art, and may be adapted for use in the disclosed methods. In many embodiments, a polypeptide disclosed herein is contacted with a nucleotide (e.g., G, A, T or C) and candidate agent in the presence of RNA, and RNA binding to the polypeptide is evaluated. Exemplary assays for evaluating RNA binding are well known in the art (see, e.g., Blair et al, RNA 1998 4:215-225; Gallinari et al, J Virol. 1998 72:6758-69; Cheng et al, J Virol. 1999 73:7044-9). An RNA binding assay may employ any RNA, although an RNA derived from the genome of a virus containing an nucleotide-binding NS4B protein (e.g., an HCV genome or any NS4B-binding fragment thereof) may be used.
A variety of different test compounds may be screened using the above methods. Test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Test compounds comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The test compounds often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Test compounds are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Test compounds may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
In particular, candidate agents that are nucleotide variants, e.g., variants of nucleotides, nucleosides, ribonucleotides, etc., such as dGTP, dATP, dTTP and dCTP or variants thereof are of particular use. For example, non-hydrolysable nucleotides, GTPγS, GppNHp, GMPPCP, 5′-adenylylimidodiphosphate, guanosine 5′-3-O-(thio)triphosphate, 5′-O-(thio)triphosphate and adenosine 5′-(βγ-imino)triphosphate, Gpp(NH)p, etc may be used. In some variants, the ribose moiety can be replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics. Conformational constraints such as fused cyclopropane and cyclopentane rings in place of ribose can also be built into the ribose rings of nucleoside and nucleotide ligands. Phosphate analogs may also be used. Further examples of nucleotide variants may be found in Jacobson et al., (Nucleic Acids. 2001 20:333-41) and Plunkett et al., (Cancer Chemother Biol Response Modif. 2001; 19:21-45).
In particular embodiments, agents that bind to the NS4B NBM but not NBMs from host (e.g. human) proteins are desirable because they may inhibit GTP binding activity of the NS4B, but do not inhibit GTP binding activity of human proteins. Such agents, e.g., monoclonal antibodies, phage display peptides, etc, may be identified using a number of approaches. In one embodiment, monoclonal antibodies that specifically bind a NS4B NBM are produced and tested against a number of host protein NBMs, or a protein having a consensus host NBM. In another embodiment, a phage display library is screened for phage that bind to a NS4B NBM but not host NBMs, or a consensus thereof.
An agent, which may be identified using the above assays, may be assayed in a variety of cellular and animal models, for example, models of NS4B mediated hepatocellular carcinoma (HCC).
In-Vitro Cell Based Assays
In some embodiments of the invention, an agent's ability to modulate a neoplastic cellular phenotype is determined by means of in-vitro cell based assays.
Focus Formation Assay
Generally, an appropriate cell line, which does not express an exogenous Ha-ras oncogene, such as NIH3T3, is transfected with a vector which encodes an NS4B nucleotide binding motif (NBM) polypeptide. After a period of growth under appropriate conditions colonies larger than a predetermined size (e.g., which may be selected as a colony size that is formed in the presence of a neoplastic agent) are counted. The colonies are then analyzed microscopically in order to determine whether a modulation of a neoplastic cellular phenotype has occurred.
In test samples, cells expressing an NS4B NBM polypeptide are treated with a candidate agent prior to colony count and analysis. Where treated cells exhibit a modulation of a neoplastic cellular phenotype as compared with untreated cells expressing an NS4B NBM polypeptide there is an indication that the candidate agent is one which has activity in modulating a neoplastic cellular phenotype.
In certain embodiments, treated cells which exhibit a modulation of a neoplastic cellular phenotype exhibit density-dependent growth inhibition as compared with untreated control cells expressing an NS4B NBM polypeptide. In other embodiments, treated cells exhibit an inhibition in cellular proliferation as compared with untreated control cells expressing an NS4B NBM polypeptide. Standard procedures for carrying out focus formation assays are described in Clark et al. Methods in Enzymology 255:395-412 (1995).
Assays for Growth Rate, Saturation Density, and Serum Dependence
In order to analyze growth rate, saturation density and serum dependence, standard methods such as those disclosed in Clark et al. Methods in Enzymology 255:395-412 (1995) may be used. Generally, an appropriate cell line which does not express an exogenous Ha-ras oncogene, such as NIH3T3, is transfected with a vector which encodes a NS4B nucleotide binding motif (NBM) polypeptide. In one embodiment, cells are seeded at an appropriate density in growth medium and incubated at an appropriate temperature. Cells are then rinsed, trypsinized and counted daily for an appropriate period of time. Growth rates are then determined from the slope of the logarithmic curve during exponential growth. In another embodiment, saturation densities are determined, and in yet another embodiment, serum dependence is determined.
In test samples, cells expressing an NS4B NBM polypeptide are treated with a candidate agent prior to the determination of growth rate, saturation density, or serum dependence. Where treated cells exhibit a modulation of a neoplastic cellular phenotype as compared with untreated cells expressing an NS4B NBM polypeptide there is an indication that the candidate agent is one which has activity in modulating a neoplastic cellular phenotype.
In certain embodiments, treated cells which exhibit a modulation of a neoplastic cellular phenotype exhibit decreased growth rate, decreased saturation density, or serum dependence as compared with untreated control cells expressing an NS4B NBM polypeptide.
Anchorage-Independent Growth Potential
An agent's ability to modulate a neoplastic cellular phenotype can be assessed by determining the anchorage-independent growth potential of cells expressing an NS4B NBM polypeptide in the presence or absence of a candidate agent. Anchorage-independent growth potential may be determined using soft agar assays. Generally, cells expressing an NS4B NBM polypeptide, but not an exogenous Ha-ras oncogene, are suspended in a 0.25% agar mixture and overlayed onto 0.75% agar in cell culture dishes. Cells are fed with growth medium at appropriate intervals. After an appropriate incubation period, colony morphology is assessed microscopically and the number of colonies is counted. Where treated cells exhibit a modulation of a neoplastic cellular phenotype as compared with untreated cells expressing an NS4B NBM polypeptide there is an indication that the candidate agent is one which has activity in modulating a neoplastic cellular phenotype.
In certain embodiments, treated cells which exhibit a modulation of a neoplastic cellular phenotype exhibit decreased anchorage independent growth potential as compared with untreated control cells expressing an NS4B NBM polypeptide. The use of soft agar assays to determine anchorage-independent growth potential are described generally in Clark et al. Methods in Enzymology 255:395-412 (1995).
In-Vivo Assays
In certain embodiments, the disclosed assays include for example, administering candidate agent to a non-human animal model. An agent's ability to modulate a neoplastic cellular phenotype can be assessed by monitoring tumor growth and/or development in non-human animals comprising a recombinant mammalian cell, wherein said recombinant mammalian cell comprises a polynucleotide encoding an exogenous NS4B nucleotide binding motif (NBM) polypeptide, wherein expression of the exogenous NS4B NBM polypeptide promotes tumor growth and/or development in the non-human animal.
The non-human animal of the above model may be treated with a candidate agent and monitored for tumor growth and/or development. Tumor growth and/or development in these animals may be compared with animals of the above model which are not treated with a candidate agent. Where treated animals exhibit a modulation of a neoplastic cellular phenotype as compared with untreated animals there is an indication that the candidate agent is one which has activity in modulating a neoplastic cellular phenotype.
In further embodiments, treated animals which exhibit a modulation of a neoplastic cellular phenotype exhibit decreased tumor growth and/or development as compared with untreated control animals.
In certain embodiments, a non-human animal model suitable for use in the above in-vivo assay is an immuno-compromised non-human animal, e.g. a nude mouse, scid (severe combined immune deficiency) mouse, or Rag1 mouse
High-Throughput Assays
The disclosure contemplates that many of the assays disclosed herein may be adapted for use in high-throughput screening. For example, assays for focus formation, growth rate, saturation density, serum-growth dependence, and anchorage-independent growth potential may be performed in a multiple well plate format. In other embodiments, automated or semi-automated robotic systems may be used to perform the steps of the respective assays.
For example, the disclosure contemplates the use of an array comprising a plurality of discrete locations, wherein at least two of said discrete locations comprise a recombinant mammalian cell comprising an expression construct encoding an NS4B nucleotide binding motif (NBM) polypeptide with the proviso that the recombinant mammalian cell does not express an exogenous Ha-ras oncogene; and different candidate anti-oncogenic agents in said at least two of said discrete locations. In certain embodiments the recombinant mammalian cell is an NIH3T3 cell.
The disclosure also contemplates the use of various reporters of cellular proliferation, which include reporters of cellular metabolism, such as agents or indicators used in calorimetric assays and radio-labeling assays. These reporters find use in the above arrays as well as other contemplated embodiments. The MTT Cell Proliferation Assay available from ATCC is an example of a suitable cellular proliferation assay which may be performed in a high throughput manner. In this assay, the yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometric means. The XTT Cell Proliferation Kit available from PromoKine provides another example of a colorimetric cellular proliferation assay. The XTT Cell Proliferation Assay is a colorimetric assay system which measures the reduction of a tetrazolium component (XTT) into soluble formazan product by the mitochondria of viable cells. The samples are read using an ELISA plate reader at a wavelength of 450 nm. The amount of color produced is directly proportional to the number of viable cells. The ALAMAR BLUE™ dye available from US Biological provides another means of measuring cellular proliferation. Proliferation measurements may be made either spectrophotometrically by monitoring the absorption of ALAMAR BLUE™ supplemented cell culture media at two wavelengths. Alternatively, proliferation measurements with ALAMAR BLUE™ may be made fluorometrically. Radio-labeling assay techniques for measuring cellular proliferation are also available, e.g., 3H-Thymidine incorporation assays.
Kits
Also provided are kits for practicing the disclosed methods. The disclosed kits at least include one or more of: a recombinant NS4B NBM polypeptide or a nucleic acid encoding the same; a recombinant NS4B NBM polypeptide that has reduced nucleotide binding activity as compared to a native NS4B, or a nucleic acid encoding the same; or virus particles containing any of the disclosed compositions. Other optional components of the kit include: adjuvant, carrier, syringes, other anti-viral drugs, reporters of cellular proliferation (such as agents for use in colorimetric assays and radio-labeling assays), etc. Any nucleic acids in the kit may also have restrictions sites, multiple cloning sites, primer sites, etc to facilitate their ligation to other plasmids. The various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired. In many embodiments, kits with unit doses of the active agent, e.g. in oral or injectable doses, are provided.
In addition to above-mentioned components, the disclosed kits typically further include instructions for using the components of the kit to practice the disclosed methods. The instructions for practicing the disclosed methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
Materials and Methods
Plasmids. Standard recombinant DNA technology was used to construct and purify all plasmids. All regions that were amplified by PCR were analyzed by automated DNA sequencing. Plasmid DNAs were prepared from large-scale bacterial cultures and purified by a Maxiprep kit (Marligen Biosciences). Restriction enzymes were purchased from New England Bio Labs (Massachusetts).
The plasmid Bart79I is described in Elazar et al. J Virol 2003 77(10), 6055-6061. Briefly, it was made by PCR mutagenesis of HCVrep1bBartMan/AvaII such that nucleotide 5336 was changed from a G to T resulting in a change in NS5A codon 1179 from serine to isoleucine. This mutation results in a dramatic increase in replication efficiency of the HCV subgenomic replicon.
Additional materials and methods are described in the following examples.
Inspection of the NS4B primary sequence revealed the presence of a nucleotide binding motif (NBM) within the middle of NS4B. This motif consists of a set of conserved amino acids found in both the GTP-binding members of the G-protein family, as well as in the superfamily of viral proteins with nucleotide-binding domains. The most highly-conserved elements within these nucleotide-binding domains are the so-called A motif and B motif (
The properties associated with the wild type and mutated versions of NS4B's NBM were determined. A plasmid was constructed, termed NS4B-GFP, which encodes a NS4B protein with a C-terminal, in frame green fluorescent protein (GFP) tag. The latter allows for visualization in live cells and provides a convenient epitope outside of any future field of mutagenesis within NS4B. Importantly, GFP fusions to NS4B have been previously reported to have no difference in intracellular localization patterns from those described for wild type NS4B.
To demonstrate that NS4B can bind GTP, GTP-binding experiments using Huh-7 cells infected with a T7RNAP-expressing vaccinia virus and transfected with plasmids encoding NS4B-GFP, GFP, or mock transfected, were performed. Membrane preparations were prepared and aliquots incubated with 32P-labeled GTP-γ-4-azidoanilide (a UV-photoactivatable non-hydrolyzable GTP analog). Following a brief pulse of UV-irradiation to activate covalent attachment of any bound GTP, pelleted membranes were washed and subjected to immunoprecipitation with a rabbit anti-GFP antibody, SDS-PAGE, transfer to nitrocellulose, autoradiography and western blot.
As shown in
The specificity of NS4B's GTP binding was further evaluated by performing binding experiments in the presence of excess unlabeled ligand. Membrane aliquots of Huh-7 cells expressing NS4B-GFP were incubated with 32P-labeled GTP analog as described above, except that cold GTPγS was added to the incubation mixture. As shown in
Binding assays were performed using Huh-7 cells expressing NS4B-GFP as in
Mutations within the NS4B NBM can impair GTP binding. Four mutants each harboring a single amino acid mutation within the NS4B NBM were utilized. Ile131Asn (or “IN”) has a single amino acid change in the second position of the A motif of the NS4B NBM, a position that has been shown to be critical for NBM function in other proteins. The Lys135Ser (“KS”) and Lys135Arg (“KR”) mutants both have a single amino acid change at position 135. The Gly129Val (“GV”) is a single amino acid mutation at the highly conserved first position of the NBM A-motif consensus sequence. These mutations were introduced into NS4B-GFP and GTP binding assays were performed. As shown in the top panel of
The NS4B NBM mediates GTP hydrolysis. The wild type NS4B and the four NBM mutants were fused in frame with N-terminal Glutathione-S Transferase (GST) tags and assayed. The resultant fusion proteins, termed GST-NS4B, GST-NS4B-(IN), GST-NS4B-(GV), GST-NS4B-(KS) and GST-NS4B-(KR) were expressed in E. coli BL21 and purified with glutathione beads. The purified proteins were then tested for their ability to hydrolyze GTP by a standard GTPase assay wherein release of phosphate from [γ32P]GTP was monitored by quantitative thin-layer chromatography, essentially as previously described. Not only does NS4B have GTPase activity, but the latter is sensitive to disruption of the NBM (
To test whether the con1 isolate of genotype 1b NS4B (Blight et al, 2000 Science 290:1972-4, Lohmann et al, 1999 Science 42 285:110-113) is able to transform NIH3T3 cells when co-expressed with the activated Ha-ras gene, a plasmid termed pcDNA3.1-NS4B was constructed which encodes the con1 NS4B sequence. For this, a PCR fragment of the NS4B gene amplified from the Bart79I plasmid with primers containing BamH1 and EcoR1 restriction sites (primers 1 and 2;
Briefly, single stock NIH 3T3 cells were propagated in Dulbecco's modified Eagle's medium (Gibco), supplemented with 10% calf serum (Colorado Serum Co., Denver, Colo.), 1% penicillin, 1% streptomycin, 1% L-Glutamine (Gibco), and maintained at 37° in a humidified 10% CO2 incubator. Cells were discarded after 3 passages. Cells were plated in 6 well plates (2.5×105 cells per well), grown for 24 hours, and then transfected with 0.5 μg of the pcDNA3.1-NS4B plasmid, 0.5 μg pEJ6.6, or both, using Lipofectamine 2000 (Invitrogen). Empty pcDNA3.1 plasmid was co-transfected with pEJ6.6 samples as a source for neomycin-resistance. The total amount of transfected DNA was kept constant at 2 pgs per well by the addition of the empty vector, pUC19, as a carrier. 24 hours following transfection, cells were split at a ratio of 1:20, transferred into 10-cm plates and grown under G418 selection (400 μg/ml) (Invitrogen) for two weeks. G418-18 resistant colonies were stained with crystal violet and colonies larger than 2 mm were counted using ImageJ analysis (NIH) on scanned plates. The experiment was repeated four times, each time with duplicates.
As shown in
To determine whether the transfected NS4B and/or Ha-ras were successfully expressed in transfected cells, at least 3 representative colonies from plates transfected with NS4B, Ha-ras or both, and passaged them 3 times in the presence of G418. Cell lysates prepared from these stable clones were then subjected to western blot analyses using mouse anti-NS4B (Virostat) and rabbit anti-ras (Santa Cruz) antibodies. As shown in
Confirmation that NS4B-transfected clones are indeed transformed was obtained using several phenotypic in-vitro assays. Standard methods were used to determine the growth rate, saturation density, and serum dependence of the stable clones (Clark et al, 1995 Methods in Enzymology 255:395-412). Briefly, 105 cells were seeded in a 10-cm dish in growth medium and incubated at 37°. Cells were rinsed with PBS, trypsinized and counted daily for seven days. Growth rates were determined from the slope of the logarithmic curve during exponential growth and saturation densities were measured. As shown in
To determine the anchorage-independent growth potential of the G418-resistant clones, soft agar assays were performed essentially as described in Clark et al, 1995 Methods in Enzymology 255:395-412. In brief, cells (5,000 or 20,000 of NS4B, ras and empty pcDNA3.1 stable transfectants) were suspended in 0.25% agar mixture and overlayed onto 0.75% agar in 10 cm dishes. Cells were fed with growth medium weekly. After a 2 week incubation, colony morphology was assessed microscopically and the number of colonies was counted after staining with crystal violet. As shown in
To examine whether NS4B transfectants are able to form tumors in-vivo, 106 cells of NS4B stable transfectants were resuspended in 0.1 ml of phosphate buffered saline and injected subcutaneously into the flanks of 4- to 6-week old nude mice (Balb/c nude male, Taconic, Hudson, N.Y.). Five clonal lines of NS4B cells were injected, each into 5 different mice. Three stable clones of ras transfectants were used as a positive control (each injected into 3-5 mice). Non-transfected NIH3T3 cells and neomycin vector alone stably transfected NIH3T3 cells were used as negative controls. Tumor size was measured bi-weekly with linear calipers and cross sectional area was calculated using the formula 3.14×0.5×length×0.5×width—shown to correlate best with tumor mass when tumor size is less than 6 gr (Tomayko et al, 1989 Cancer Chemotherapy and Pharmacology 24: 148-154). Tumors were defined as a growth with a section area larger than 1.5 cm2. Mice were sacrificed before tumors reached a cross-sectional area of 2 cm2.
As shown in
In contrast to Park et al, 2000 Biochemical and Biophysical Research Communications 267: 581-587, NS4B did not require co-transfection with Ha-ras in order to achieve NS4B-mediated transformation. Although both of genotype 1b, the NS4B sequence of the con-1 isolate used in the Examples above differs in 7 amino acids from the K isolate used by Park et al. To test the hypothesis that these sequence variations account for the different transforming phenotypes of the two HCV clones, these 7 amino acid changes were introduced by using site directed mutagenesis (using primers 9-18,
Similarly to Park et al., there was an increase in the number of foci induced by the K isolate with Ha-ras co-transfection. The number of transformed foci induced by the K isolate, however, was significantly (about 5 fold) lower than the number induced by the Con1 NS4B (p value=0.0087 in student t-test) (
To test whether the NBM of NS4B mediates NS4B's role in transformation, the I131IN mutation (IN) was introduced in the A-motif of the NS4B NBM—a mutation previously shown to significantly impair GTPase activity (Einav et al, 2004 J. Virol. 78: 11288-95)—into the pcDNA3.1-NS4B vector by site directed mutagenesis (using primers 3 and 4;
Mutations in the NS4B B-(D228L) and G-(F211A) motifs were also introduced by site-directed mutagenesis (using primers 5-8;
These findings were not a result of differences in transfection efficiency (monitored by SEAP reporter gene assay) or simply the result of an obvious gross effect of the mutations on folding, as the apparent intracellular distribution patterns and expression levels of mutant and wild-type GFP-fused proteins appeared similar by fluorescence microscopy and western blotting, respectively (
Together the above results demonstrate that NS4B can robustly transform NIH3T3 cells in the absence of exogenous Ha-ras. Moreover, the transforming activity of NS4B is mediated by its NBM. Thus, while the data confirm a transforming activity of NS4B described by others (Park et al, 2000 Biochemical and Biophysical Research Communications 267:581-587), the current results extend their findings and differ in a significant way. Specifically, the results described herein did not show a requirement for co-transfection with Ha-ras in order to achieve Con 1 NS4B-mediated transformation. These findings indicate that this difference in requirement for activate HA-ras co-transfection can be accounted for by the sequence variations between the Con 1 and K genotype 1b isolates.
Interestingly, none of the 7 amino acid mismatches between the K and the Con1 isolate are within the NBM. It is possible that these amino acid mismatches affect NS4B in a way that affects the 3-dimensional conformation of the NBM.
These results reveal that the NBM within NS4B represents an attractive new target for anti-HCC therapy. Because the amino acid sequence immediately adjacent to either side of the NBM region is highly conserved among HCV isolates yet very different from that contained in known host cell GTP-binding proteins, highly selective inhibitors can be readily screened for.
It is evident from the above results and discussion that the assays disclosed herein provide an important new tool for discovery of anti-HCC agents. In particular, the disclosure provides a system for identifying anti-HCC agents based on their ability to inhibit a neoplastic cellular phenotype. As such, the disclosed methods and systems find use in a variety of different applications, including research, medical, therapeutic and other applications.
This application claims the benefit of U.S. Provisional Patent Application No. 60/946,684 (expired), filed Jun. 27, 2007, which is incorporated herein by reference in its entirety.
This invention was made with Government support under Federal Grant No. RO1-DK066793 awarded by the National Institutes of Health. The Government has certain rights in this invention.
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| Number | Date | Country | |
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| 20090083869 A1 | Mar 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60946684 | Jun 2007 | US |
