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  Pharmaceutical Patents  

 

Title:  NF-.kappa.B peptides designed to disrupt NEMO oligomerization
United States Patent: 
7,390,872
Issued: 
June 24, 2008

Inventors: 
Agou; Fabrice (Paris, FR), Courtois; Gilles (Paris, FR), Israel; Alain (Paris, FR), Veron; Michel (Paris, FR), Traincard; Francois (Issy-les-Moulineaux, FR), Yamaoka; Shoji (Tokyo, JP)
Assignee: 
Institut Pasteur (Paris, FR)
Appl. No.:
 10/948,649
Filed:
 September 24, 2004


 

Outsourcing Guide


Abstract

The present invention relates to polypeptides that inhibit the NF-.kappa.B signaling pathway and polynucleotides encoding the same. The present invention further provides methods for the modulation of and/or treatment of inflammatory responses, oncogenesis, viral infection; the regulation of cell proliferation and apoptosis; and regulation of B or T lymphocytes in antigenic stimulation, by administering the polypeptides of the present invention to a subject in need thereof. Finally, the present invention provides a method of identifying polypeptides that modulate oligomerization of NEMO.

Description of the Invention

SUMMARY OF THE INVENTION

It is an object of the present invention to provide polypeptides derived from NEMO that are useful for the regulation and/or inhibition of the NF-.kappa.B signaling pathway.

To this end, the present invention provides NEMO-derived polypeptides that inhibit the NF-.kappa.B signaling pathway.

In one embodiment of the present invention, the NEMO-derived polypeptide is the CC2 domain (murine: SEQ ID NO: 3 or human: SEQ ID NO: 14).

In another embodiment of the present invention, the NEMO-derived polypeptide is the LZ domain (murine: SEQ ID NO: 7 or human: SEQ ID NO: 16).

In a preferred embodiment of the present invention, the NEMO-derived polypeptides are fused via a spacer sequence to a polypeptide having a high transduction potential.

Further, in another embodiment of the present invention are polynucleotides that encode for the NEMO-derived polypeptides either with or without the spacer sequence and the polypeptide having a high transduction potential.

In yet another embodiment of the present invention is methods of modulating or treating disorders regulated by the NF-.kappa.B signaling pathway by administering the NEMO-derived polypeptides to a subject in need thereof. The disorders regulated by the NF-.kappa.B signaling pathway include: inflammatory responses, oncogenesis, and viral infection.

The present invention also provides a method of regulating cell proliferation or apoptosis by administering the NEMO-derived polypeptides to a subject in need thereof.

In still another embodiment of the present invention is a method of regulating B or T lymphocytes in antigenic stimulation by administering the NEMO-derived polypeptides to a subject in need thereof.

In yet another embodiment, the present invention further provides a method of identifying polypeptides that modulate oligomerization of NEMO by

a) identifying a candidate polypeptide sequence;

b) creating a polypeptide fusion construct by linking said candidate polypeptide sequence to a polypeptide having a high transduction potential via a spacer sequence;

c) contacting a cell culture with the polypeptide fusion construct; and

d) monitoring the activity of the NF-.kappa.B signaling pathway;

e) comparing the activity of the NF-.kappa.B signaling pathway in the presence of said polypeptide fusion construct to the activity of the NF-.kappa.B signaling pathway in the absence of said polypeptide fusion construct to determine the relative inhibition by said polypeptide fusion construct; and

f) correlating relative inhibition by said polypeptide fusion construct to NEMO oligomerization.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in enzymology, biochemistry, cellular biology, molecular biology, and the medical sciences.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

In this application, the present inventors studied the inhibition of NF-.kappa.B activation by peptides designed to disrupt NEMO oligomerization. The present inventors have previously shown that the minimal trimerization domain comprises the CC2-LZ coiled-coil subdomain and that the isolated and/or purified CC2 and LZ domains bind to each other to form a stable trimer of heterodimers. This structural model is reminiscent of the fold of the gp41 ectodomain from HIV-1 (Traincard, 2003, J. Biol. Chem. submitted). It consist of a central three-stranded coiled coil (formed by the CC2 coiled coil motif of NEMO) which is surrounded by the LZ helical motif derived from the C-terminal end of NEMO, packed in an antiparallel manner around the outside of the CC2 coiled-coil. On the basis of this model, the present inventors rationally designed two cell-permeable peptides corresponding to optimal portions of CC2 or LZ subdomains that mimic the contact area between NEMO subunits. Peptide transduction was monitored by FACS and their effect on LPS-induced NF-.kappa.B activation was quantified using a NF-.kappa.B dependent .beta.-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. The present inventors have also demonstrated that the LZ peptide and, to a lesser extent the CC2 peptide, inhibit specifically NF-.kappa.B activation with IC.sub.50 values in the .mu.M range. The effects were specific because control peptides including mutated CC2 and LZ peptides as well as heterologous coiled-coil peptides (GCN4), had no inhibitory effect on NF-.kappa.B activation. Furthermore, the present inventors have shown that these NF-.kappa.B peptidic inhibitors induced the cell death in the human retinoblastoma cell lines Y79 that exhibit constitutive NF-.kappa.B activity. Collectively, the present inventors have provided a new and promising strategy to inhibit the NF-.kappa.B pathway by targeting NEMO's oligomerization.

The present inventors have proven that NEMO constitutes a preferential target for the search for drugs inhibiting the NF-.kappa.B signaling path, because this protein acts upstream from the NF-.kappa.B activation path. The role of NEMO and its various domains was partially studied and published in the following article, "NEMO trimerizes through its coiled-coil C-terminal domain." J Biol Chem, May 17, 2002;277(20):17464-75. Agou F. et al., a copy of which is incorporated by reference.

In the present invention, the inventors have synthesized peptides that mimic either the oligomerization domain (CC2 domain=approx. 40 residues), or the LZ motif (LZ domain=approx. 40 residues). The combination of these peptides alters either the oligomerization of NEMO or the combining thereof with the proteinic effector, in both cases inhibiting the NF-.kappa.B pathway.

In an aspect of the present invention, peptide drugs have been chemically combined with a peptide of 16 amino acids in length (penetratin/antennapedia), thereby enabling intracellular transport thereof possible. The resulting peptides also may be chemically coupled with a fluorescent tracer in order to monitor internalization into B lymphocyte cell lines through FACS.

The action of these peptides was tested directly on B lymphocytes having stably integrated the beta-galactosidase carrier gene also bearing upstream from its promoter several NF-.kappa.B transcription factor (Clone C3) activation sites.

The present inventors have successfully been able to monitor the inhibitory effect of these peptides by measuring the same following stimulation of the B lymphocytes by LPS.

The results as a whole reveal that the presence of the peptide mimicking the "CC2" motif reduces the NF-.kappa.B activity by 70% as compared with a control peptide at a relatively low dose of 20 .mu.M. At this concentration, the effect of the "Leucine zipper" peptide is still more significant, since its presence in the medium completely eliminates cell response.

These new inhibitors of the NF-.kappa.B cellular signalling path offer a major advantage as anti-inflammatory compounds and also as anti-tumor compounds, which may be used for the treatment and/or prevention of cancers and other disorders.

The present invention relates to compounds, peptides, or compositions that are used for modulating the oligomerization of NEMO. In particular, the peptide compounds described herein below may be in an isolated and/or purified or coupled form with or without a vectorizing agent. It is to be understood that the present invention also embraces peptides having at least 70% homology the NEMO-derived polypeptides, so long as the homologs possess said inhibitory activity. Methods for assessing inhibitory activity of the NEMO-derived polypeptides, and homologs thereof, are provided below and exemplified in the Examples of the present application. The peptides of the present invention and the doses thereof are deemed to possess inhibitory activity when the NF-.kappa.B activity is reduced by at least 50% as compared with a control peptide.

The present invention also relates to pharmaceutical compositions containing said peptides, especially for the preparation of medicines used for the treatment of inflammatory responses, oncogenesis, viral infection, the regulation of cell proliferation and apoptosis and antigenic stimulation. In a preferred embodiment, the pharmaceutical compositions containing said peptides are useful for the treatment of cancer.

Also embraced by the present invention are methods of obtaining, making, and identifying peptides and compounds that inhibit the NF-.kappa.B signaling pathway, in particular by means of the 70Z/3-C3 cell line filed with the CNCM.

As used herein, the term "reduced" or "inhibited" means decreasing the intracellular activity of one or more enzymes in the NF-.kappa.B pathway either directly or indirectly. The phrase "inhibiting the NF-.kappa.B pathway" preferably means that the NF-.kappa.B pathway is inhibited by disruption of NEMO oligomerization.

The term "enhanced" as used herein means increasing the intracellular activity or concentration of the NEMO derived peptides, which are encoded by the corresponding DNA. Enhancement can be achieved with the aid of various manipulations of the bacterial cell. In order to achieve enhancement, particularly over-expression, the number of copies of the corresponding gene can be increased, a strong promoter can be used, or the promoter- and regulation region or the ribosome binding site which is situated upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same manner. In addition, it is possible to increase expression by employing inducible promoters. A gene can also be used which encodes a corresponding enzyme with a high activity. Expression can also be improved by measures for extending the life of the mRNA. Furthermore, preventing the degradation of the enzyme increases enzyme activity as a whole. Moreover, these measures can optionally be combined in any desired manner. These and other methods for altering gene activity in a plant are known as described, for example, in Methods in Plant Molecular Biology, Maliga et al, Eds., Cold Spring Harbor Laboratory Press, New York (1995).

A gene can also be used which encodes a corresponding or variant NEMO derived peptide with a high activity of inhibiting the NF-.kappa.B pathway. Preferably the corresponding enzyme has a greater ability than the native form of the NEMO protein to inhibit the NF-.kappa.B pathway, more preferably at least in the range of 5, 10, 25% or 50% more inhibition. Most preferably the NEMO derived peptides of the present invention reduce the NF-.kappa.B pathway by at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, as compared to the pathway in the presence of the native NEMO protein.

In the context of the present Application, a polynucleotide sequence is "homologous" with the sequence according to the invention if at least 70%, preferably at least 80%, most preferably at least 90% of its base composition and base sequence corresponds to the sequence according to the invention. According to the invention, a "homologous protein" or "homologous peptide" is to be understood to comprise proteins (peptides) which contain an amino acid sequence at least 70% of which, preferably at least 80% of which, most preferably at least 90% of which, corresponds to the amino acid sequence of the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, wherein corresponds is to be understood to mean that the corresponding amino acids are either identical or are mutually homologous amino acids. It is further to be understood that, as evinced by the Examples of the present invention, the homologous peptide of CC2 preferably retains the coiled-coil motif structure and the homologous peptide of LZ preferably retains the helical motif structure. With the guidance proffered by the identification of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 14, and SEQ ID NO: 16 and the detailed description in the Examples (see Original Patent), screening of theoretical mutations within the scope of the present invention would require nothing more than a technicians level of skill in the art. More specifically, as is routine in the art, with the identification of a candidate sequence (i.e., the regions corresponding to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 14, and SEQ ID NO: 16) the artisan would assay and screen one or all possible permutations of the said sequence to identify mutants possessing the same or better therapeutic efficacy.

The expression "homologous amino acids" denotes those that have corresponding properties, particularly with regard to their charge, hydrophobic character, steric properties, etc.

Homology, sequence similarity or sequence identity of nucleotide or amino acid sequences may be determined conventionally by using known software or computer programs such as the BestFit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of identity or similarity between two sequences. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence alignment program such as BestFit, to determine the degree of sequence homology, similarity or identity, the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores. Similarly, when using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.

The present invention also relates to polynucleotides which encode the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof, and which can be obtained by screening by means of the hybridization of a corresponding gene bank with a probe which contains the sequence of said polynucleotide that encodes the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof, and isolation of said DNA sequence.

Polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate those cDNAs or genes that exhibit a high degree of similarity to the sequence that encodes the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof.

Polynucleotide sequences according to the invention are also suitable as primers for polymerase chain reaction (PCR) for the production of DNA, which encodes a NEMO-derived polypeptide having an ability to inhibit the NF-.kappa.B pathway.

Oligonucleotides such as these, which serve as probes or primers, can contain more than 30, preferably up to 30, more preferably up to 20, most preferably at least 15 successive nucleotides. Oligonucleotides with a length of at least 40 or 50 nucleotides are also suitable.

The term "isolated and/or purified" means separated from its natural environment.

The term "polynucleotide" refers in general to polyribonucleotides and polydeoxyribonucleotides, and can denote an unmodified RNA or DNA or a modified RNA or DNA.

The term "polypeptides" is to be understood to mean peptides or proteins that contain two or more amino acids that are bound via peptide bonds.

The polypeptides according to invention include polypeptides corresponding to the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof, particularly those with the biological activity of inhibition the NF-.kappa.B pathway, and also includes those, at least 70% of which, preferably at least 80% of which, are homologous with the polypeptide corresponding to the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof, and most preferably those which exhibit a homology of least 90% to 95% with the polypeptide corresponding to the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof, and which have the cited activity.

The invention also relates to coding DNA sequences that encode the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof, by degeneration of the genetic code. One of skill in the art would appreciate that the aforementioned DNA sequences may be based on the full-length DNA sequences for the murine-derived NEMO (SEQ ID NO: 11) and the human-derived NEMO (SEQ ID NO: 17) and thereby these sequences may be used to ascertain the scope of these sequences in accordance with the present invention.

In the same manner, the invention further relates to DNA sequences that hybridize with DNA sequences that encode the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof.

Moreover, one skilled in the art is also aware of conservative amino acid replacements such as the replacement of glycine by alanine or of aspartic acid by glutamic acid in proteins as "sense mutations" which do not result in any fundamental change in the activity of the protein, i.e. which are functionally neutral. It is also known that changes at the N-- and/or C-terminus of a protein do not substantially impair the function thereof, and may even stabilize said function.

In the same manner, the present invention also relates to DNA sequences that hybridize with the DNA sequence that encodes the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof. The present invention also relates to DNA sequences that are produced by polymerase chain reaction (PCR) using oligonucleotide primers that result from the DNA sequence that encodes the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof. Oligonucleotides of this type typically have a length of at least 15 nucleotides.

The terms "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree. C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60 to 65.degree. C.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA--DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): Tm=81.5.degree. C. +16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1.degree. C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity are sought, the Tm can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4.degree. C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20.degree. C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (2000).

Thus, with the foregoing information, the skilled artisan can identify and isolated and/or purified polynucleotides, which are substantially similar to the present polynucleotides. In so isolating such a polynucleotide, the polynucleotide can be used as the present polynucleotide in, for example, inhibiting the NF-.kappa.B pathway.

One embodiment of the present invention is methods of screening for polynucleotides, which have substantial homology to the polynucleotides of the present invention, preferably those polynucleotides encoding a protein having an ability of inhibiting the NF-.kappa.B pathway.

The polynucleotide sequences of the present invention can be carried on one or more suitable plasmid vectors, as known in the art for plants or the like.

In one embodiment, it may be advantageous for propagating the polynucleotide to carry it in a bacterial or fungal strain with the appropriate vector suitable for the cell type. Common methods of propagating polynucleotides and producing proteins in these cell types are known in the art and are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989).

The aforementioned embodiments are described in the context of SEQ ID NO: 3 (CC2 region of the NEMO protein; i.e., amino acids 246-286 of SEQ ID NO: 12) and SEQ ID NO: 7 (LZ region of the NEMO protein; i.e., amino acids 390-412 of SEQ ID NO: 12), where SEQ ID NO: 12 is murine-derived NEMO. The aforementioned embodiments have been further described based on SEQ ID NO: 14 and SEQ ID NO: 16, which are derived from SEQ ID NO: 18 (human-derived NEMO). However, it is understood that the present invention preferably provides peptide derivatives of the NEMO protein that may be internalized into eukaryotic cells. Internalization of the NEMO peptide derivatives may be imparted by fusing the NEMO peptide derivative(s), or homologues thereof, to a polypeptide having a high transduction potential. The skilled artisan would readily appreciate that the term "high transduction potential" as used herein means that the polypeptide, and the fusion protein thereof, readily transverses the cellular membrane resulting in the internalization of fusion peptide into the cellular milieu. Examples of peptides having a high transduction potential include: the third helix of the Antennapedia/penetratin protein (Ant) (Prochiantz, 2000, Curr. Opin. Cell Biol.), TAT derived peptides (Fawel, 1994, P.N.A.S.), VP22 from HSV-1 (Stroh C. 2003, Oncogene), Pep. 1 (Morris, 2001, Nature Biotech.).

To exemplify the present invention and the utility thereof; the present inventors have fused SEQ ID NO: 3 and SEQ ID NO: 7 to the internalization peptide Ant (SEQ ID NO: 1) separated by a short SKGMQ linker SEQ ID NO:40) or by a LKAQADI linker SEQ ID NO: 41). The resultant Ant-CC2 construct has the sequence: CRQIKIWFQNRRMKWKKSKG MQLEDLRQQLQQAEEALVAKQELIDKLKEEAEQHKIV (SEQ ID NO: 2), where the N-terminal cysteine has been added for coupling to a flurophore to facilitate detection of internalization and/or inhibition. The resultant Ant-LZ construct has the sequence: CRQIKIWFQNRRMKWKKLKAQADIYKADFQAERHAREKLVEKKEYLQEQLEQLQR EFNKL (SEQ ID NO: 6), where the N-terminal cysteine has been added for coupling to a flurophore to facilitate detection of internalization and/or inhibition.

In the present invention the N-terminal cysteine is an optional addition and, as such, this residue may be omitted from the final inhibitory peptide. Further, in the present invention the linker between the peptide having a high transduction potential (e.g., Ant) and the CC2 or LZ peptide can be of a variable sequence and/or length, so long as the linker sequence does not significantly diminish the inhibitory property of CC2 or LZ peptide. To this end, the linker may be of a length ranging from 1-35 amino acids, preferably 2-25 amino acids, more preferably 3-15 amino acids, most preferably 4-10 amino acids. In a particularly preferred embodiment, the linker sequence is that of SEQ ID NO: 40 or SEQ ID NO:41.

As set forth hereinabove, it is to be understood that the homologous peptide of CC2 preferably retains the coiled-coil motif structure and the homologous peptide of LZ preferably retains the helical motif structure, even when in the fusion construct set forth above. As such, the present invention embraces homologous peptides, within the homology constraints above, of SEQ ID NO: 2 and SEQ ID NO: 6 (murine-derived) and SEQ ID NO: 13 and SEQ ID NO: 15 (human-derived) with the caveat that said homologous peptides retain the structure of CC2 and LZ respectively, as well as the ability to inhibit the NF-.kappa.B pathway.

In an embodiment of the present invention, the inventors explored the N-terminal region of the wild-type NEMO, in particular the NLM conserved motif (residues 293-322 of SEQ ID NO: 12) appearing in FIG. 1A (see Original Patent). To this end, the following sequences were produced (see Table 1 (see Original Patent) for the corresponding sequence) -- see Original Patent.

NLM-DR is a 21 amino acid "motif" (and the corresponding wild type NLM covering the same amino acid range) derived from the larger 30 amino acid conserved NLM motif set forth in FIG. 1A. The NLM-DR has been mutated from the wild type NLM sequence in that the aspartic acid at residue 11 in the wild type sequence has been replaced by an arginine (see Table 1 and SEQ ID NO: 30). This mutation was selected because, as confirmed by structural studies, the resulting polypeptide would facilitate an intramolecular salt bridge allowing the stabilization of the peptide in its helicoidal form.

From circular dichroism studies (CD), it appears that CC2 and LZ peptides adopt a helicoidal structure, depending on their concentration. CC2 creates a helix more stable than LZ. RMN and Rayon X Diffraction studies have confirmed the structure of CC2. Always, by CD studies, the NLM-DR peptide is structured as a helix more stable than the wild-type peptide.

Although, the polypeptides utilized in this example are 21 amino acids long, it is contemplated in the present invention that the operable size of the NLM fragment may be as short as 15 amino acids. In addition, any mutation in the sequence of the NLM polypeptides that are able to reinforce the helicity and the intermolecular interactions between the peptides and their molecular target would be of particular interest and is within the scope of the present invention.

In the present invention it is speculated that Antennapedia mediated monomerization of peptides may be crucial. Specifically, it is speculated that monomerization allows them to interfer with NEMO oligomerization.

Nuclear factor-kB (NF-.kappa.B) signaling is an essential signal transduction pathway involved in inflammatory responses, oncogenesis, viral infection, the regulation of cell proliferation and apoptosis; and in the case of B and T lymphocytes in antigenic stimulation (Ghosh, 1998, Annu. Rev. Immunol.; Karin, 1999, J. Biol. Chem.; Israel, 2000, Trends Cell Biol.; Santoro, 2003, EMBO J.). As such, the inventive peptides are useful for the modulation of and/or treatment of inflammatory responses, oncogenesis, viral infection; the regulation of cell proliferation and apoptosis; and regulation of B or T lymphocytes in antigenic stimulation. Therefore, the present invention provides for a method of treating the same by administering to a subject in need thereof a peptide in accordance with the present invention.

In the present invention, the "subject in need thereof" may be a human, a domestic animal, a farm animal, or an animal that is generally found in the wild. For example, the subject may be selected from a human, a dog, a cat, a horse, a cow, a mouse, a guinea pig, a sheep, a pig, etc.

Clearly, the amount of the peptide to be administered will depend on the subject to which it is to be administered. In the case where the subject is a human, the amount of the peptide to be administered will depend on a number of factors including the age of the patient, the severity of the condition and the past medical history of the patient and always lies within the sound discretion of the administering physician. Generally, the total daily dose of the compounds of this invention administered to a human or other mammal in single or in divided doses can be in amounts, for example, of from 0.1 mg/Kg/day to 30 mg/Kg/day of the peptide, preferably from 0.1 mg/Kg/day to 20 mg/Kg/day of the peptide, more preferably from 2 mg/Kg/day to 10 mg/Kg/day of the peptide, in single or multiple doses. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. In a preferred embodiment, the preferred dose is 10 mg/patient to be administered twice a day. In a particularly preferred embodiment, the administration route is intravenous.

The peptides of the present invention may also be administered as a component of a pharmaceutically administrable composition. In other words, the peptide may be present in a formulation for administration to a subject in need thereof. The inventive peptide may be the sole active ingredient for NF-.kappa.B pathway inhibition or for treatment of inflammatory responses, oncogenesis, viral infection, the regulation of cell proliferation and apoptosis and antigenic stimulation. Alternatively, the composition may also contain one or more additional compounds that may be used to treat the same. In addition, the peptide of the present invention may be in a composition that contains one or more compounds that are useful for treatment of a disorder not caused by the NF-.kappa.B pathway.

A therapeutically effective amount of the peptides suitable for administration in the present invention may be administered alone or in combination with one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filer, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

The pharmaceutical compositions suitable for administration in the invention can be administered to humans and other animals orally, rectally, nasally, parenterally (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), intracisternally, intravaginally, intraperitoneally, sublingually, topically (e.g., as a powder, ointment, or drop), bucally, as an oral spray, or a nasal spray. The pharmaceutical compositions can be formulated in dosage forms appropriate for each route of administration.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active NEMO-derived polypeptides, the liquid dosage forms may contain inert diluents commonly used in the art. The inert diluents may include, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. The liquid dosage form for oral administration may also contain adjuvants, which include wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Other dosage forms for oral administration include, for example, aqueous suspensions containing the active NEMO-derived polypeptides in an aqueous medium in the presence of a non-toxic suspending agent such as sodium carboxy-methylcellulose, and oily suspensions containing a NEMO-derived polypeptides of the present invention in a suitable vegetable oil, for example arachis oil.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of the inventive NEMO-derived polypeptides, it is often desirable to slow the absorption of the peptides from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the NEMO-derived polypeptides then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, dissolving or suspending the drug in an oil vehicle accomplishes delayed absorption of a parenterally administered NEMO-derived polypeptides form. Injectable depot forms are made by forming microencapsulated matrices of the NEMO-derived polypeptides in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of NEMO-derived polypeptides to polymer and the nature of the particular polymer employed, the rate of NEMO-derived polypeptides release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides) Depot injectable formulations are also prepared by entrapping the NEMO-derived polypeptides in liposomes or microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the NEMO-derived polypeptides of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the peptide.

Solid dosage forms for oral administration include capsules, tablets, pills, prills, powders, and granules. In such solid dosage forms, the peptide is mixed with at least one inert, pharmaceutically acceptable excipient or carrier. In addition, the solid dosage form may contain one or more fillers, extenders, binders, humectants, disintegrating agents, retarding agents, absorption accelerators, wetting agents, absorbents, or lubricants. Examples of suitable fillers or extenders include, starches, lactose, sucrose, glucose, mannitol, and silicic acid, sodium citrate and dicalcium phosphate. Examples of suitable binders include, microcrystalline cellulose, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia. Glycerol is an example of a suitable humectant. Examples of suitable disintegrating agents include, agar-agar, calcium carbonate, potato or tapioca starch, maize starch, alginic acid, certain silicates, and sodium carbonate. Paraffin is an example of a suitable solution-retarding agent. As absorption accelerators, any quaternary ammonium compound may be used. Examples of suitable wetting agents include, cetyl alcohol and glycerol monostearate. Examples of suitable absorbents include, kaolin and bentonite clay. Examples of suitable lubricants include, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

The tablets may, if desired, be coated using known methods and excipients that may include enteric coating using for example hydroxypropylmethylcellulose phthalate. The tablets may be formulated in a manner known to those skilled in the art so as to give a sustained release of the NEMO-derived polypeptides of the present invention. Such tablets may, if desired, be provided with enteric coatings by known methods, for example by the use of cellulose acetate phthalate. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Similarly, capsules, for example hard or soft gelatin capsules, containing the active peptide with or without added excipients, may be prepared by known methods and, if desired, provided with enteric coatings in a known manner. The contents of the capsule may be formulated using known methods so as to give sustained release of the active NEMO-derived polypeptides. In such solid dosage forms the active NEMO-derived polypeptides may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

If desired, the NEMO-derived polypeptides of the present invention can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes and microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can dissolve in sterile water, or some other sterile injectable medium immediately before use.

The NEMO-derived polypeptides may be formulated into granules with or without additional excipients. The granules may be ingested directly by the patient or they may be added to a suitable liquid carrier (for example, water) before ingestion. The granules may contain disintegrates, e.g. an effervescent couple formed from an acid and a carbonate or bicarbonate salt to facilitate dispersion in the liquid medium.

Dosage forms for topical or transdermal administration of the peptide of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. Transdermal patches have the added advantage of providing controlled delivery of a peptide to the body. The rate can be controlled by either providing a rate controlling membrane or by dispersing the peptide in a polymer matrix or gel. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Dissolving or dispensing the peptide in the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of the peptide across the skin. Ophthalmic formulation, eardrops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

Dosage forms for topical administration may comprise a matrix in which the pharmacologically NEMO-derived polypeptides of the present invention are dispersed so that the peptides are held in contact with the skin in order to administer the peptides transdermally. A suitable transdermal composition may be prepared by mixing the pharmaceutically active NEMO-derived polypeptides with a topical vehicle, such as animal and vegetable fats, oils, petrolatum, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof, together with a potential transdermal accelerant such as dimethyl sulphoxide or propylene glycol. Alternatively the active NEMO-derived polypeptides may be dispersed in a pharmaceutically acceptable paste, cream, gel or ointment base. The amount of active NEMO-derived polypeptides contained in a topical formulation should be such that a therapeutically effective amount of the peptides are delivered during the period of time for which the topical formulation is intended to be on the skin.

Powders and sprays can contain, in addition to the NEMO-derived polypeptides of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons. The therapeutically active NEMO-derived polypeptides may be formulated into a composition, which is dispersed as an aerosol into the patient's oral or nasal cavity. Such aerosols may be administered from a pump pack or from a pressurized pack containing a volatile propellant.

The therapeutically active NEMO-derived polypeptides used in the method of the present invention may also be administered by continuous infusion either from an external source, for example by intravenous infusion or from a source of the NEMO-derived polypeptides placed within the body. Internal sources include implanted reservoirs containing the NEMO-derived polypeptides to be infused which is continuously released for example by osmosis and implants which may be (a) liquid such as an oily suspension of the peptides to be infused for example in the form of a very sparingly water-soluble derivative such as a dodecanoate salt or a lipophilic ester or (b) solid in the form of an implanted support, for example of a synthetic resin or waxy material, for the NEMO-derived polypeptides to be infused. The support may be a single body containing the entire quantity of the peptides or a series of several bodies each containing part of the quantity of the peptides to be delivered. The amount of active peptides present in an internal source should be such that a therapeutically effective amount of the peptides are delivered over a long period of time.

The present invention further provides a method of identifying polypeptides that modulate oligomerization of NEMO by

a) identifying a candidate polypeptide sequence;

b) creating a polypeptide fusion construct by linking said candidate polypeptide sequence to a polypeptide having a high transduction potential via a spacer sequence;

c) contacting a cell culture with the polypeptide fusion construct; and

d) monitoring the activity of the NF-.kappa.B signaling pathway;

e) comparing the activity of the NF-.kappa.B signaling pathway in the presence of said polypeptide fusion construct to the activity of the NF-.kappa.B signaling pathway in the absence of said polypeptide fusion construct to determine the relative inhibition by said polypeptide fusion construct; and

f) correlating relative inhibition by said polypeptide fusion construct to NEMO oligomerization.

In this method, the candidate polypeptide sequence preferably has a coiled-coil or helical structure. More preferably, the candidate polypeptide sequence has 20-60 amino acids. It is also preferred that the candidate polypeptide sequence be derived from NEMO.

As stated in the embodiments above, the spacer sequence may have a length ranging from 1-35 amino acids, but shorter lengths may also be employed (supra). Examples of the spacer sequence includes: SEQ ID NO: 9 and SEQ ID NO: 10. Additionally, an example of the polypeptide having a high transduction potential is a polypeptide having the amino acid sequence of SEQ ID NO: 1.

In a preferred embodiment, the cell culture contains pre-B 70Z/3 lymphocytes that have been transfected with NF-.kappa.B dependent .beta.-glactosidase reporter gene.

In order to ensure that the polypeptide fusion construct is actually incorporated into the cells contained in the cell culture it is desired that the polypeptide fusion construct have an N-terminal cysteine residue. In this manner, the polypeptide fusion construction may be labeled by chemically reacting the cysteine residue with a fluorophore (e.g., BODIPY) thus enabling monitoring of cellular uptake by a technique such as FACS.

Accordingly, the method of identifying polypeptides that modulate oligomerization of NEMO may also include the following steps:

b-1) labeling said polypeptide fusion construct; and

c-1) monitoring cellular uptake of the labeled polypeptide fusion construct.

Further, one of skill in the art may also be able to correlate NF-.kappa.B pathway inhibition with modulation of NEMO oligomerization by a pull down experiment with tagged peptides to show that NEMO associates in vivo with the peptides. The oligomeric state of this peptide associated NEMO protein could be characterized (cross-linking, gel filtration). In vitro, Inhibition of the anisotropy increase resulting from the association of fluorescent antennapedia labelled CC2 or LZ peptides with CC2 or LZ peptides, mimicking NEMO oligomerization, could be used to test compounds inhibiting NF-.kappa.B pathway in vivo.
 

Claim 1 of 8 Claims

1. A purified polypeptide consisting of an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, wherein the polypeptide inhibits the NF-.kappa.B pathway by disrupting the NF-kB essential modulator (NEMO) oligomerization.

 

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