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WO2009095500A1 - Inhibiteurs de la réplication lentivirale - Google Patents

Inhibiteurs de la réplication lentivirale Download PDF

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Publication number
WO2009095500A1
WO2009095500A1 PCT/EP2009/051150 EP2009051150W WO2009095500A1 WO 2009095500 A1 WO2009095500 A1 WO 2009095500A1 EP 2009051150 W EP2009051150 W EP 2009051150W WO 2009095500 A1 WO2009095500 A1 WO 2009095500A1
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WIPO (PCT)
Prior art keywords
peptide
fragment
sequence
seq
hiv
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PCT/EP2009/051150
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English (en)
Inventor
Zeger Debyser
Frauke Christ
Johannes P.M. Langedijk
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Katholieke Universiteit Leuven, K.U.Leuven R & D
Pepscan Therapeutics Bv
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Application filed by Katholieke Universiteit Leuven, K.U.Leuven R & D, Pepscan Therapeutics Bv filed Critical Katholieke Universiteit Leuven, K.U.Leuven R & D
Publication of WO2009095500A1 publication Critical patent/WO2009095500A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention provides peptides having lentiviral replication inhibiting properties, more in particular having antiviral activities with respect to HIV (Human Immunodeficiency Virus).
  • the invention further relates to the use of said peptides as a medicine and in the manufacture of a medicament useful for the treatment of subjects suffering from a HIV infection, as well as for treatment of other lentiviral infections and to the treatment of animals suffering from FIV or lentiviral infections.
  • the invention also relates to pharmaceutical compositions comprising said peptides.
  • the invention also provides methods of treatment or prevention of a lentiviral infection in a mammal.
  • the invention also relates to corresponding nucleic acid sequences encoding said peptides, to constructs comprising said corresponding nucleic acid sequences and to compositions thereof useful for the prevention or treatment of lentiviral, more in particular HIV infections in mammals.
  • a retrovirus designated human immunodeficiency virus is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system.
  • AIDS immunodeficiency syndrome
  • RNA ribonucleic acid
  • Coexisting with RNA are reverse transcriptase, integrase, a protease, a ribonuclease and other enzymes.
  • RT nucleoside reverse transcriptase
  • AZA azidothyraidine
  • lamivudine non- nucleoside reverse transcriptase inhibitors
  • non- nucleoside reverse transcriptase inhibitors such as nevirapine (Boehringer Ingelheim), efavirenz (DuPont) or protease inhibitors such as nelfinavir (Agouron), saquinavir (Roche), ritanovir (Abbott laboratories) and amprenavir (GlaxoWellcome).
  • a new reverse transcriptase drug recently added in the batlle against HIV is tenofovir (PMPA, Gilead Sciences).
  • PMPA Gilead Sciences
  • a relatively new target that is focussed on lately is the integrase enzyme of HIV.
  • Mainly two classes of integrase inhibitors have already been described, namely the diketo acids and the pyranodipyrimidines (M. Witvrouw et al., Current Drug Metabolism, 2004, 5 (4), 2004, pp. 291 -304).
  • LEDGF/p75 Lens Epithelial Derived Growth Factor
  • LEDGF/p75 binds to the integrases of HIV-1 , HIV-2, SIV mac and FIV but not to MoMLV, RSV or HTLV-2 integrase proving the lentiviral specificity of the interaction.
  • LEDGF/p75 as a protein co-purifying with the transcriptional co-activator PC4 and as a growth factor.
  • LEDGF/p75 is a weak co-activator of general transcription, but plays a protective role against cellular stress.
  • LEDGF/p75 consists of 530 amino acids and contains an N-terminal PWWP motif, involved in chromatin binding. Accordingly, the nuclear accumulation of HIV-1 integrase is apparently due to chromosomal tethering by LEDGF/p75.
  • HIV-1 human immunodeficiency virus type 1
  • Replication of the human immunodeficiency virus type 1 can be drastically reduced in infected patients by combining potent antiviral drugs targeted at multiple viral targets, as reviewed by Vandamme et al. in Antiviral Chem. Chemother. (1998) 9:187-203.
  • Multiple-drug combination regimens can reduce viral load below the detection limit of the most sensitive tests. Nevertheless low level ongoing replication has been shown to occur, possibly in sanctuary sites, leading to the emergence of drug-resistant strains, according to Perelson et al. in Nature (1997) 387:123-124.
  • the selectivity index of many anti-viral agents is rather low, thereby they are possibly responsible for side-effects and toxicity.
  • HIV can develop resistance to most, if not all, currently approved antiviral drugs, according to Schmit et al. in J. Infect. Dis. (1996) 174:962-968. It is well documented that the ability of HIV to rapidly evolve drug resistance, together with toxicity problems requires the development of additional classes of antiviral drugs. It has also been shown that IBD expression is able to inhibit the replication of HIV in cells.
  • a goal of the present invention is to satisfy this urgent need by identifying efficient and non- harmful active ingredients and combination of ingredients for the treatment of lentiviral infections, and more particularly HIV infections, in mammals and in humans. Furthermore, there is also a need for compounds which either complement existing drugs such that the resulting cocktail has improved activity or resistance to virus mutation or compounds which are themselves effective against many or all viable mutations of a virus.
  • the present invention relates to the inhibition of HIV replication by interacting with the interaction of LEDGF/p75 and integrase, more in particular by targeting the interaction of the integrase helix 3 (amino acids 124 - 132) with a shallow depression between the loops of p75, more in particular by using peptides comprising fragments of the helix 3 of integrase.
  • the present invention relates to the antiviral, more in particular anti-lentiviral, yet more in particular the anti-HIV properties of peptides which comprise the amino acids, or a part thereof, of integrase helix 3 involved in the interaction between LEDGF/p75 and integrase.
  • the present invention relates to helical and/or dimeric peptide mimics of the integrase helix 3.
  • the present invention demonstrates that said peptides are potent inhibitors of the interaction between LEDGF/p75 and integrase, an interaction which is crucial for lentiviral replication, more in particular of HIV, which is the etiological agent of Acquired Immune Defiency Syndrome (AIDS) in humans, and consequently may be useful for the treatment of individuals infected by HIV.
  • AIDS Acquired Immune Defiency Syndrome
  • a first aspect of the present invention thus relates to peptides which comprise the amino acid sequence or part thereof of the integrase helix 3 domain which interacts with LEDGF/p75.
  • said peptides comprise the amino acid sequence TTXKAXXWXX [SEQ ID NO:1] wherein each X is selected from any amino acid.
  • said helical peptide template is the Heptad repeat (LXXXIXXLXXXIXX [SEQ ID NO:2] or more in particular LXXXIXXLXXXIXXLXXIXXLXXXI [SEQ ID NO:3]), wherein each X is independently selected from any amino acid.
  • said peptides comprise the amino acid sequence TTXKAXXWXX [SEQ ID NO:1 ], more in particular the sequence TTXKAXXWWX [SEQ ID NO:4], and more in particular TTXKAXAWWX [SEQ ID NO:5] , wherein each X is selected from any natural amino acid.
  • said peptides furthermore have an amino acid sequence which has 70% sequence identity with a helical peptide template.
  • said helical peptide template is the Heptad repeat (LXXXIXXLXXXIXX [SEQ ID NO:2] or more in particular LXXXIXXLXXXIXXLXXIXXLXXXI [SEQ ID NO:3]), wherein each X is independently selected from a natural amino acid.
  • the present invention relates to peptides comprising the amino acid sequence TTXKAXXWXX [SEQ ID NO:1], more in particular the sequence TTXKAXXWWX [SEQ ID NO:4], and more in particular TTXKAXAWWX [SEQ ID NO:5], wherein each X is selected from any natural amino acid, wherein the peptide has a helical form or wherein the peptide further comprises structural motifs that are able to present the sequence TTXKAXXWXX [SEQ ID NO:1] (more in particular TTXKAXXWWX [SEQ ID NO:4] and TTXKAXAWWX [SEQ ID NO:5]) in a helical format.
  • the present invention relates to a peptide and/or fragments thereof, comprising the Heptad repeat sequence LXXX I XX LXXX I XX [SEQ ID NO:2], more in particular LXXIXXLXXXIXXLXXIXXLXXXI [[SEQ ID NO:3]], wherein each X is selected from any natural amino acid, characterized in that the Heptad repeat sequence also comprises the integrase helix 3 derived sequence TTXKAXXWXX [SEQ ID NO:1] wherein each X is selected from any natural amino acid.
  • said peptide and/or fragments thereof comprise the sequence LXXXITTLKAAIWWLXXXIXXLXXXI [SEQ ID NO:6]. In a more particular embodiment, said peptide and/or fragments thereof have the sequence CAALEDRITTLKAAIWWLENEIARLAAAIRRRR [SEQ ID NO:7]. In another embodiment, said peptide and/or fragments thereof comprises the sequence LXXXIXXLXTTIKALXWWIXXLXXXI [SEQ ID NO:8]. In a more particular embodiment, said peptide and/or fragments thereof have the sequence CAALEDRIAALSTTIKALAWWIARLAAAIRRRR [SEQ ID NO:9].
  • said peptide and/or fragments thereof comprise the sequence LXTTIKALXWWIXXLXXXIXXLXXXI [SEQ ID NO:10].
  • said peptide and/or fragments thereof comprise or generally consist of the sequence CAALSTTIKALAWWIYHLENEIARLAAAIRRRR [SEQ ID NO:1 1].
  • the peptides of the embodiments above are further coupled to a transport peptide as are known in the art.
  • said transport peptide is selected from those described in European patent appl. 00202255.6 or WO0200882.
  • the transport peptide is GRQLRIAGRRLRGRSR [SEQ ID NO:12].
  • the transport peptide is coupled to the C-terminal end of the peptide.
  • said peptides and/or fragments thereof according to the embodiments above are dimeric and/or helical. Said peptides and/or fragments thereof can be of different lengths (as long as they show their inhibitory activity or provided they have inhibitory activity), but preferably range between 100 and 10 amino acids, more in particular comprise between 60, 50, 40, 30, 20 or 15 amino acids and 10 amino acids.
  • the invention also relates to modified versions of said peptides and/or fragments which share at least 70%, 80%, 90% or 95 % with the amino acid sequences mentioned.
  • the peptides and/or fragments thereof comprise the integrase helix 3 derived sequence TTXKAXXWXX [SEQ ID NO:1] which is a sequence TTX 1 KAX 2 X 3 WXX 1 , wherein X, X 1 , X 2 , and X 3 are any amino acids, and Xi is either L or I, and either X 2 or X 3 is L or I (TT[LI]KA[LI]X 3 WX[LI] (SEQ ID NO:13) and TT[LI]KAX 2 [LI]WX[LI] (SEQ ID NO:14)).
  • a second aspect of the present invention relates to the use of the peptides and/or fragments thereof according to the first aspect of the invention in an assay to measure the inhibitory activity of a test substance on the interaction between HIV integrase and LEDGF/p75.
  • the present invention also relates to the use of the three-dimensional structure of the peptides and/or fragments of the first aspect of the invention to design new anti-HIV drugs.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the pepides and/or fragments thereof according to the first aspect of the invention, in a mixture with a pharmaceutically acceptable carrier.
  • Yet another aspect of the invention relates to the peptides and/or fragments thereof according to the first aspect of the invention for use as a medicine, more in particular an antiviral medicine, yet more in particular an anti-lentiviral or an anti-HIV medicine.
  • Another aspect of the invention relates to the use of the peptides and/or fragments thereof according to the first aspect described herein for the manufacture of a medicament for the prevention or treatment of lentiviral infections in mammals.
  • Yet another aspect of the invention relates to a method for the prevention or treatment of a lentiviral infection in a mammal in need of said prevention or treatment, comprising administering to said mammal a peptide and/or fragment thereof according to the first aspect of the invention.
  • polynucleotide or nucleic acid encoding the peptides and/or fragments thereof according the first aspect of the invention.
  • said polynucleotide or nucleic acid is a vector comprising a coding sequence which encodes the peptides and/or fragments thereof according to the first aspect of the invention and wherein said coding sequence is operatively linked to an expression control system or promotor sequence.
  • said nucleic acid sequences are part of a construct such as a vector, more in particular such as a plasmid, allowing the translation of the nucleic acid sequence into the desired peptide and/or fragments thereof as described herein. These constructs can be used in gene therapy.
  • Another aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising said polynucleotides or nucleic acids in a mixture with a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of said polynucleotides in an assay to measure the inhibitory activity of a test substance on the interaction between HIV integrase and LEDGF/p75.
  • the present invention also relates to said polynucleotides for use as a medicine.
  • the present invention also relates to the use of said polynucleotide or nucleic acid sequences as a medicine, more in particular for the treatment or prevention of viral disorders, such as HIV infections.
  • the present invention relates therefore to the use of said nucleic acid sequences for the manufacture of a medicament for the prevention or treatment of viral diseases, more in particular of HIV.
  • the present invention also provides methods of preventing or treating viral disorders by using said polynucleotides or nucleic acid sequences. Said nucleic acid sequences can be used in gene therapy.
  • FIG. 1 Schematic drawing of the AlphaScreen interaction assay.
  • FIG. 1 A representative experiment for the inhibition of the LEDGF/p75-IN interaction by PEA-64 (light grey bars at the right part of the figure) is shown. The signal reached by incubating HiS 6 -IN [30OnM] and flag-LEDGF/p75 [10OnM], maximum inhibition by competing with LEDGF/p75 and the background signal are shown in the dark grey bars at the left part of the figure. The signal to noise ration is >100 fold. Background levels of signal are reached by addition of 30 ⁇ M PAE-64 to the interaction assay.
  • LEDGF/p75 refers to the human natural protein referred to as lens epithelium-derived growth factor p75 with 530 amino acids, such as described in Singh, D. P. et al. Gene, 2000, 242 (1 -2), 265-273.
  • the protein is also known as PC4- and SFRS1 -interacting protein 2; PSIP 2; transcriptional coactivator p75.
  • HlV-integrase refers to the enzyme of HIV responsible for integration of the genetic code of HIV into host cells, among other things.
  • Modified version of a peptide relates to a peptide which has in total at least 70%, more in particular 80%, 90 % or 95% sequence identity with the sequence referred to, which still has the capacity of interfering with the interaction between LEDGF/p75 and integrase or inhibiting the viral replication.
  • the relation between a protein and a modified version thereof can be expressed by the percentage of amino acid sequence similarity or amino acid sequence identity between two sequences and this is also so for nucleic acids i.e. DNA or RNA and their nucleotide sequence (polynucleotides or oligonucleotieds).
  • the percentage of amino acid or nucleotide sequence identity/similarity is determined by alignment of the two sequences and identification of the number of positions with identical/similar amino acids or nucleotides divided by the number of nucleotides or amino acids in the shorter of the sequences x100.
  • the alignment of two nucleotide sequences is performed by the algorithm as described by Wilbur and Lipmann (1983) Proc. Natl. Acad. Sci. U.S.A. 80:726, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4.
  • Two amino acids in a sequence alignment are similar when they belong to a same class of amino acids in Table 1 above.
  • amino acids are referred to herein with their full name, their three letter abbreviation or their one letter abbreviation (Table 1 ). If variations in amino acids are referred to, we refer to any amino acid, including both natural L-form or their D-form and unnatural amino acids such as amino acid analogues or other linker molecules that can be introduced in an amino acid chain to substitute amino acids.
  • Nonpolar / hydrophobic glycine GIy G alanine Ala A valine VaI V leucine Leu L isoleucine Ne I methionine Met M phenylalanine Phe F tryptophan Trp W proline Pro P
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • DNA molecule or “DNA” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and integration termination sequence will usually be located 3' to the coding sequence.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 1 direction) coding sequence.
  • the promoter sequence is bound at its 3' terminus by the transcription initiation site and extends upstream (5 1 direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease S1 ), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • oligonucleotide as used herein is defined as a molecule comprised of two or more (deoxy)ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • a "polynucleotide” as used herein comprises at least 20 (deoxy)ribonucleotides.
  • nucleic acids refers to the same as polynucleotides.
  • a cell has been "transformed" by exogenous or heterologous polynucleotide when such polynucleotides have been introduced inside the cell.
  • the transforming polynucleotide may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming polynucleotide may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming polynucleotide has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • HIV integrase catalyzes the insertion and integration of viral DNA into the host genome which is required for viral replication.
  • Human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) protein was identified as a binding partner for HIV-1 integration in human cells. Protein/protein interactions are very attractive targets for a chemotherapeutical intervention of various infectious diseases. Compounds capable of blocking such interactions are likely to be highly specific for a given target and less prone to the development of resistance.
  • the present invention relates to peptides and/or fragments of said peptides which combine the Heptad repeat sequence LXXXIXXLXXXIXXLXXXIXXLXXXI [SEQ ID NO:3] and the integrase helix 3 derived sequence TTXKAXXWXX [SEQ ID NO:1] in their peptide sequence and wherein X is selected from any natural amino acid.
  • the present invention relates to peptides and/or fragments of said peptides which combine the Heptad repeat sequence LXXXIXXLXXXIXXLXXXIXXLXXXI [SEQ ID NO:3] and the integrase helix 3 derived sequence TTXKAXXWXX [SEQ ID NO:1] in their peptide sequence and wherein X is selected from any amino acid.
  • sequences comprise the sequence LXXXITTLKAAIWWLXXXIXXLXXXI [SEQ ID NO:6], or the sequence LXXIXXLXTTIKALXWWIXXLXXXI [SEQ ID NO:8] or the sequence LXTTIKALXWWIXXLXXIXXLXXI [SEQ ID NO:10].
  • sequences are :
  • CAALEDRITTLKAAIWWLENEIARLAAAIRRRR [SEQ ID NO:7] CAALEDRIAALSTTIKALAWWIARLAAAIRRRR [SEQ ID NO:9], or CAALSTTIKALAWWIYHLENEIARLAAAIRRRR [SEQ ID NO:1 1].
  • the peptides of the invention are furthermore coupled to transport peptides in order to obtain a better cell permeability.
  • Many peptides with cell membrane permeating capacity are known in the art. Examples include GRQLRIAGRRLRGRSR [SEQ NO:12], (C-terminal) E rns peptide and analogs thereof. Examples are also described in European patent appl. 00202255.6, PCT/N L01/00484 or WO02/00882.
  • the peptides of the invention preferably have a dimeric and helical structure. Since the peptides yield a strong inhibition of the LEDGF/p75 - integrase interaction, said peptides can be used for different purposes.
  • the peptides of the invention can be used in an assay to study the inhibition of the interaction between HIV integrase and LEDGF/p75. Via a competition assay, for examlpe small molecule LEDGF/p75 - integrase inhibitors can be identified.
  • the information obtained from the structure of said inhibitory peptides could furthermore serve as basis for the design of new inhibitory agents, such as small molecules and at the end to design new anti-HIV drugs.
  • the peptides of the present invention can be used for the manufacture of medicaments for the prevention or treatment of lentiviral disorders. They can serve as a basis for a method for the prevention, treatment or alleviation of a lentiviral infection in a mammal in need of said prevnetion, treatment or alleviation, comprising administering to said mammal a peptide according to the invention.
  • polynucleotides or nucleic acids encoding the peptides can be applied.
  • the present invention relates to the use of nucleic acids encoding the peptides described herein for gene transfer, including gene therapy and to deliver the above mentioned peptides with antiviral activity.
  • Gene therapy means the treatment by the delivery of therapeutic nucleic acids to patient's cells. This is extensively reviewed in Lever and Goodfellow 1995; Br. Med Bull. ,51 , 1 -242; Culver1995; Ledley, F. D. 1995. Hum. Gene Ther. 6, 1 129.
  • a nucleic acid encoding an antiviral peptide as described herein is introduced into cells in a subject to express the peptide and inhibit the viral replication.
  • To achieve gene therapy there must be a method of delivering genes to the patient's cells and additional methods to ensure the effective production of any therapeutic genes.
  • nucleic acids encoding a peptide according to the invention is introduced in a subject in order to express the peptide and prevent or treat a viral infection.
  • the key steps are 1 ) to select the mode of delivery, e.g. a proper vector for delivery of the inhibitor peptide to the subject, 2) administer the nucleic acid to the subject; and 3) achieve appropriate expression of the transferred sequence for satisfactory durations.
  • Methods for gene transfer are known in the art. Most of the techniques to construct delivery vehicles such as vectors and the like are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions, reagents and procedures which are described in the literature.
  • Peptides as described herein can easily be obtained and can be tested for their inhibitory or antiviral activity with the methods described herein. Fragments or peptides can be prepared by using standard peptide synthesis chemistry including solid phase chemistry as is generally known in the art. Certainly peptides up to 70 ot 80 amino acids can be synthesised with an automated peptide synthesiser.
  • the preventive or treatment methods with the peptides or polynucleotides as described herein can also be used in combination with any therapy or compound known in the art for the prevention or treatment of viral infections, more in particular HIV infections.
  • the invention also relates to combinations, especially combinations with other classes of inhibitors.
  • the composition of the invention can also contain drugs having a general beneficial activity for virally infected mammals, such as interferon, antibiotics or corticosteroids, next to pharmaceutcially acceptable carriers.
  • the invention further relates to the use of a composition
  • a composition comprising (a) one or more peptides or polynucleotides as described herein, and (b) one or more viral inhibitors as biologically active agents in respective proportions, such as to provide an inhibitory activity against a viral infection, preferably a lentiviral infection and more preferably an HIV infection in a mammal, for instance in the form of a combined preparation for simultaneous, separate or sequential use in lentiviralinfection therapy.
  • the lentiviral inhibitors used as a therapeutically active ingredients (b) may belong to categories already known in the art and include, among others,
  • HIV integrase inhibitors such as are known in the art
  • nucleoside, non-nucleoside and nucleotide reverse transcriptase inhibitors such as for instance, dideoxyadenosine, stavudine, zalcitabine, zidovudine, lamivudine, didanosine, nevirapine, delavirdine, efavirenz, tenofovir, foscamet sodium and the like,
  • HIV protease inhibitors such as for instance saquinavir, ritonavir, indinavir, nelfinavir, amprenavir and the like, - HIV fusion inhibitors such as enfevurtide.
  • the invention thus relates to a pharmaceutical composition or combined preparation having effects against a lentiviral infection and containing either A) (a) a combination of two or more of peptides of the present invention, and (b) optionally one or more pharmaceutical excipients or pharmaceutically acceptable carriers, for simultaneous, separate or sequential use in the treatment or prevention of a viral infection, or B)
  • the invention relates to the peptides as described herein being useful as agents having antiviral activity or as diagnostic agents. Any of the uses mentioned with respect to the present invention may be restricted to a non-medical use, a non- therapeutic use, a non-diagnostic use, or exclusively an in vitro use, or a use related to cells remote from an animal.
  • the active ingredients may be administered to the mammal (including a human) to be treated by any means well known in the art, i.e. orally, intranasally, subcutaneously, intramuscularly, intradermal ⁇ , intravenously, intra-arterially, parenterally or by catheterization.
  • the therapeutically effective amount of the preparation especially for the treatment of viral infections in humans and other mammals, particularly is a HIV inhibiting amount. More particularly for combination preparations, it is a HIV replication inhibiting amount of derivative (a) and a HIV enzyme inhibiting amount of inhibitor (b). Still more particularly when the said HIV enzyme inhibitor (b) is a reverse transcriptase inhibitor, its effective amount is a reverse transcriptase inhibiting amount. When the said HIV enzyme inhibitor (b) is a protease inhibitor, its effective amount is a protease inhibiting amount.
  • ingredients (a) and (b) may be administered simultaneously but it is also beneficial to administer them separately or sequentially, for instance within a relatively short period of time (e.g. within about 24 hours) in order to achieve their functional fusion in the body to be treated.
  • the present invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefore.
  • Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.
  • the formulations both for veterinary and for human use, of the present invention comprise at least one active ingredient, as above described, together with one or more pharmaceutically acceptable carriers therefore and optionally other therapeutic ingredients.
  • the carrier(s) optimally are "acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions used in these therapies may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, liposomes, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art and which will be selected in accord with ordinary practice. Tablets will contain excipients, glidants, fillers, binders and the like.
  • Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic.
  • Formulations optionally contain excipients such as those set forth in the "Handbook of Pharmaceutical Excipients" (1986).
  • pharmaceutically acceptable carrier means any material or substance with which the active ingredient is formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness.
  • the compositions of the invention are in the form of a unit dose and will usually be administered to the patient one or more times a day.
  • Peptides of the invention may be administered to the patient in any pharmaceutically acceptable dosage form, including intravenous, intramuscular, intralesional, or subcutaneous injection.
  • compositions and methods of this invention may be used in combination with other therapies, once improvement of the patient's condition has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained, When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • Peptides of the invention can be used to provide controlled release pharmaceutical formulations containing as active ingredient one or more compounds of the invention ("controlled release formulations") in which the release of the active ingredient can be controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given invention compound.
  • the correct primary ion molecular weights of the peptides was confirmed by electron-spray ionization mass spectrometry on a Micromass ZQ (Micromass, The Netherlands) or a VG Quattro Il (VG Organic, U.K.) mass spectrometer.
  • Oxidative folding of purified peptides was performed as following: 0.1 mg/ml peptide was placed in Tris-HCI buffer ( 0.1 M, pH 8.4) comprising 1 mM GSH and 1 mM GSSG. This mixture was quenched after 16 h with an equal volume of 0.5 % TFA. The peptides were purified by RPC as described above.
  • the GCN4-based Heptad repeat helical template (pdb code: 1 UOI) for mimicking the structure of IN helix 3 was used.
  • the IN helix 3 sequence TTVKAACWWA [SEQ ID:
  • SEQ ID NO:15 (Integrase helix 3) :
  • SEQ ID NO 1 1 Hybrid (peptide PEQ-82) CAALSTTIKALAWWIYHLENEIARLAAAIRRRR
  • the inhibitory activity of the peptides of the invention was evaluated in a direct LEDGF/p75 - HIV-1 lntegrase interaction assay (AlphaScreen) ( Figure 1 ). Therefore both proteins (LEDGF/p75 and integrase) were expressed and purified from E.coli BL21. LEDGF/p75 was fused to an N-terminal triple flag-tag whereas Integrase was expressed as a His 6 -tag fusion protein. The recombinant proteins were bound to anti- flag donor and Ni-chelat acceptor beads (Perkin Elmer, Belgium). Interaction of both proteins with each other and subsequent excitation of the donor bead resulted in transfer of singlet oxygen and emission from the acceptor bead.
  • the plasmid pKB-IN6H was used for the expression of the C-terminally tagged form of HIV-1 IN.
  • the IN gene (derived from the NL4-3 HIV-1 clone) was PCR-amplified from plNSD using the primers ⁇ '-AATACGACTCACTATAGGG (T7 promoter primer [SEQ ID NO:17]) and ⁇ '-GCGCGTCGACATCCTCATCCTGTCTAC (INSaII primer [SEQ ID NO:18]); the resulting PCR fragment was digested with Nde ⁇ and Sal ⁇ and subcloned into the pET-20b(+) vector (Novagen).
  • the Flag-tagged LEDGF/p75 expression plasmid, pCPNatFlag was cloned by ligating an adaptor (5TACTAGTATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGATATCGAT TACAAGGATGACGATGACAAGGCTTCTA-3')[SEQ ID NO:19] encoding a triple Flag peptide, N-terminally to the LEDGF/p75 coding region into the Ndel site of the pCP-Nat plasmid.
  • the IBD mutant expression plasmids were cloned by site-directed mutagenesis using appropriate primers introducing the mutation into the coding region of the full length LEDGF/p75 protein.
  • PC1 E. coli cells harboring pKB-IN6H were grown in LB medium to an optical density of 0.8 and induced by addition of 0.5 mM isopropyl-1 -thio-P-D-galactopyranoside, at 29 0 C for 3 h.
  • the protein was purified as follows: In brief, cells were lysed using a French press in 1 M NaCI, 7.5 mM CHAPS, 30 mM Tris, pH 7.4, and the soluble His 6 -tagged IN protein was enriched by batch adsorption to Ni-NTA-agarose (Qiagen, Hilden, Germany).
  • Triple flag-tagged LEDGF/p75 was produced from the plasmid pCP-Nat75-flag, respectively, in the Endo l-free PC1 Escherichia coli host strain (E. coli B, BL21 (DE3), ⁇ encM::Tc R , pLysS). Expression was induced in LB medium at 29 0 C by addition of 0.5 mM isopropyl-1 -thio- ⁇ -D-galactopyranoside. Cells harvested 3 h after induction were disrupted using a French press in 450 mM NaCI, 30 mM Tris, pH 7.0.
  • the supernatant obtained by centrifugation of the lysate was passed through a 1 -ml HiTrap heparin column (Amersham Biosciences, Uppsala Sweden) to capture flag-LEDGF/p75, and the protein was eluted by a linear gradient of NaCI concentration in 30 mM Tris, pH 7.0.
  • the fractions containing flag-LEDGF/p75 were pooled and further purified by cation exchange chromatography on a 1 -ml HiTrap SP Sepharose column (Amersham Biosciences, Uppsala Sweden).
  • the inhibitory activity of fragments of LEDGF/p75 of the invention can be tested for their potential to inhibit the replication of HIV and SIV in a cell culture model for acute infection.
  • Compounds can be tested against HIV-1 strains (HE, NL43, MN, III B ), HIV-2 strains (ROD, EHO, RF), and SIV (MAC251 ) for inhibition of virus-induced cytopathicity in MT-4 cells (or CEM or C8166 or Molt4/C8 cells), using the colorimetric test described by Pauwels et al. in J. Virol. Methods (1988) 20:309-321 or a microscopic investigation of the cytopathogenic effect, evaluation being made 4 to 5 days post-infection.
  • microtiter 96-well plates containing ⁇ 3 x 10 5 CEM cells/ml, infected with 100 CCID 50 of HIV per ml and containing appropriate dilutions of the test compounds can be used.
  • a rapid and automated assay procedure can be used for the in vitro evaluation of anti- HIV agents.
  • An HTLV-1 transformed T4-cell line MT-4 which was previously shown to be highly susceptible to and permissive for HIV infection, can serve as the target cell line. Inhibition of the HIV-induced cytopathogenic effect is used as the end point. The viability of both HIV- and mock-infected cells is also assessed spectrophotometrically via in situ reduction of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
  • MTT 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • Methods comprise for example the microscopic examination of CEM, C8166 or Molt4/C8 giant (syncytium) cell formation, after 4 to 5 days of incubation at 37 ⁇ € in a CO 2 -controlled humidified atmosphere.
  • the 50 % cytotoxic concentration (CC 50 in ⁇ g/ml) is defined as the concentration of compound that reduces the absorbance of the mock-infected control sample by 50 %.
  • the percent protection achieved by the compound in HIV-infected cells is calculated by the following formula:
  • (OD ⁇ )mv is the optical density measured with a given concentration of the test compound in HIV-infected cells
  • (OD c ) H ⁇ v is the optical density measured for the control untreated HIV-infected cells
  • (OD C ) M OC K is the optical density measured for the control untreated mock-infected cells
  • all optical density values are determined at 540 nm.
  • the dose achieving 50 % protection according to the above formula is defined as the 50 % inhibitory concentration (IC 50 in ⁇ g/ml).
  • the ratio of CC 50 to IC 50 is defined as the selectivity index (Sl).
  • MT-4 cells (Miyoshi et al., 1982) are grown and maintained in RPMI 1640 medium supplemented with 10 % heat-inactivated fetal calf serum, 2 mM 1 -glutamine, 0.1 % sodium bicarbonate, and 20 ⁇ g of gentamicin per ml.
  • HIV-1 The HIV-1 (NIB, NL4.3) strain (Adachi et al., 1986) is a molecular clone obtained from the National Institutes of Health (Bethesda, MD).
  • the HIV-1 strain SO561945 is a strain resistant to non-nucleoside reverse transcriptase inhibitors.
  • the HIV-2 (ROD, EHO) (Barr-Sinoussi et al., 1983) stock is obtained from culture supernatant of HIV-2 infected cell lines.
  • Mac251 is a SIV strain.
  • All assays are performed in 96-well microtiter plates. To each well are added 5 - 7.5 x 10 4 cells and a given amount of the test fragments. The cells are allowed to proliferate for 48 h (murine leukemia L1210) or 72 h (human lymphocyte CEM and Molt4/clone 8) at 37 ⁇ € in a humidified CO 2 -controlled atmosphere. At the end of the incubation period, the cells can be counted in a Coulter counter. The IC 50 (50% inhibitory concentration) was defined as the concentration of the fragments that reduced the number of cells by 50%.
  • Peptides based on the heptad repeat scaffold showed the highest inhibition in a direct interaction assay as described herein (AlphaScreen).
  • the first heptad repeat-based peptide (PEA-63) is structurally very similar to the second heptad repeat - based peptide with a similar surface.
  • the orientation of the helix dimer in PEA-64 is very different compared with PEA-63.
  • the other helix in the PEA-64 dimer superimposes on the other integrase helix in the integrase - p75 interface (helix 1 , residues 94 - 107).

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Abstract

La présente invention concerne des peptides présentant des propriétés d'inhibition de la réplication lentivirale, et notamment une activité antivirale dirigée contre le VIH (virus de l'immunodéficience humaine). L'invention concerne également l'utilisation de ces peptides comme médicament et dans la fabrication d'un médicament utile pour le traitement de sujets souffrant d'une infection à VIH, ainsi que pour le traitement d'autres infections lentivirales et le traitement d'animaux souffrant d'infections lentivirales ou à VIF. Par ailleurs, l'invention concerne des compositions pharmaceutiques comprenant ces peptides. Elle se rapporte en outre à des méthodes de traitement ou de prévention d'une infection lentivirale chez un mammifère. L'invention concerne également des séquences d'acides nucléiques correspondantes codant lesdits peptides, des constructions comprenant ces séquences d'acides nucléiques correspondantes et des compositions à base de celles-ci utiles pour prévenir ou traiter des infections lentivirales, et notamment une infection à VIH, chez des mammifères.
PCT/EP2009/051150 2008-02-01 2009-02-02 Inhibiteurs de la réplication lentivirale WO2009095500A1 (fr)

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US8987250B2 (en) 2012-04-20 2015-03-24 Gilead Sciences, Inc. Therapeutic compounds
US9006229B2 (en) 2011-04-21 2015-04-14 Gilead Sciences, Inc. Benzothiazole compounds and their pharmaceutical use
US9102614B2 (en) 2010-07-02 2015-08-11 Gilead Sciences, Inc. Naphth-2-ylacetic acid derivatives to treat AIDS
US9284323B2 (en) 2012-01-04 2016-03-15 Gilead Sciences, Inc. Naphthalene acetic acid derivatives against HIV infection
US9296758B2 (en) 2010-07-02 2016-03-29 Gilead Sciences, Inc. 2-quinolinyl-acetic acid derivatives as HIV antiviral compounds
US9376392B2 (en) 2012-01-04 2016-06-28 Gilead Sciences, Inc. 2-(tert-butoxy)-2-(7-methylquinolin-6-yl) acetic acid derivatives for treating AIDS

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9102614B2 (en) 2010-07-02 2015-08-11 Gilead Sciences, Inc. Naphth-2-ylacetic acid derivatives to treat AIDS
US9296758B2 (en) 2010-07-02 2016-03-29 Gilead Sciences, Inc. 2-quinolinyl-acetic acid derivatives as HIV antiviral compounds
US9006229B2 (en) 2011-04-21 2015-04-14 Gilead Sciences, Inc. Benzothiazole compounds and their pharmaceutical use
US9284323B2 (en) 2012-01-04 2016-03-15 Gilead Sciences, Inc. Naphthalene acetic acid derivatives against HIV infection
US9376392B2 (en) 2012-01-04 2016-06-28 Gilead Sciences, Inc. 2-(tert-butoxy)-2-(7-methylquinolin-6-yl) acetic acid derivatives for treating AIDS
US8987250B2 (en) 2012-04-20 2015-03-24 Gilead Sciences, Inc. Therapeutic compounds
US9096586B2 (en) 2012-04-20 2015-08-04 Gilead Sciences, Inc. Therapeutic compounds

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