WO2004094465A2 - Synthetic molecules that mimic chemokines - Google Patents

Synthetic molecules that mimic chemokines Download PDF

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WO2004094465A2
WO2004094465A2 PCT/US2004/012708 US2004012708W WO2004094465A2 WO 2004094465 A2 WO2004094465 A2 WO 2004094465A2 US 2004012708 W US2004012708 W US 2004012708W WO 2004094465 A2 WO2004094465 A2 WO 2004094465A2
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polypeptide
seq
nos
amino acid
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PCT/US2004/012708
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WO2004094465A3 (en
WO2004094465A8 (en
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Ziwei Huang
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The Board Of Trustees Of The University Of Illinois Office Of Technology Management University Of Illinois At Urbana-Champaign
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention is drawn to non-natural molecules that mimic or antagonize chemokine function.
  • Chemokines act as molecular beacons for the recruitment, activation, and directed migration of T lymphocytes, neutrophils and macrophages of the immune system, flagging pathogens and tumor masses for destruction.
  • Chemokine (chemoattractant cytokine) receptors are a group of membrane proteins and belong to the superfamily of G-protein-coupled receptors (GPCRs) that possess seven transmembrane helices and transmit signals (as evidenced by calcium flux) from extracellular ligands to intracellular biological pathways via heterotrimeric G-proteins (Murphy, 1994).
  • GPCRs G-protein-coupled receptors
  • chemokines are divided into four subfamilies: CC, CXC, CX3C and C (Rossi and Zlotnik, 2000).
  • the CXC and CC subfamilies selectively activate and recruit leukocytes to sites of inflammation.
  • CXC chemokines mostly act on neutrophils, while CC chemokines stimulate other leukocytes, such as monocytes, lymphocytes, and basophils.
  • chemokine signaling can either promote infections when not properly triggered (Forster et al., 1999) or lead to diseases associated with defective chemokine signaling, including asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, and atherosclerosis (Rossi and Zlotnick, 2000).
  • diseases associated with defective chemokine signaling including asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, and atherosclerosis (Rossi and Zlotnick, 2000).
  • Other diseases and disorders that are associated with chemokines include cancer (especially breast cancer), multiple myeloma and non- Hodgkin's lymphoma.
  • chemokine functions can betrayed by viruses, becoming unwitting collaborators in infection.
  • the human immunodeficiency virus is such a virus, appropriating chemokine receptors, such as CCR5 and CXCR4, as co-receptors.
  • CCR5 and CXCR4 chemokine receptors
  • Strains that co-opt CCR5 induce syncytia (masses of cells that have fused; these strains usually infect T cells), while those that appropriate CXCR4 do not induce syncytia and usually infect macrophages (Alkhatib, 1996; Deng et al., 1996; Dragic et al., 1996; Feng et al., 1996).
  • Some HIV strains can use both CCR5 and CXCR4.
  • the HIV gpl 20 envelope protein initially binds the CD4 receptor of a target cell. Following a conformational change, the gpl20-CD4 complex then binds to a chemokine receptor, such as CCR5 or CXCR4. The virus then takes over the cellular machinery to replicate itself, which progeny then infect more cells of the host.
  • a chemokine receptor such as CCR5 or CXCR4.
  • CXCR4 is essential for vascularization of the gastrointestinal tract as well as hematopoiesis (Tachibana et al., 1998).
  • SDF-1 stromal derived factor-1
  • Fetal cerebellar development requires CXCR4 function for proper neuronal cell migration and patterning.
  • the CC chemokine ligands of CCR5 include macrophage mflammatory protein ⁇ (MlP-l ⁇ ), macrophage inflammatory protein l ⁇ (MlP-l ⁇ ), and regulated on activation «o ⁇ nal Tcell expressed and secreted (R ANTES).
  • MlP-l ⁇ macrophage mflammatory protein ⁇
  • MlP-l ⁇ macrophage inflammatory protein l ⁇
  • R ANTES regulated on activation «o ⁇ nal Tcell expressed and secreted
  • the CXCR4 receptor can bind the .vtromal cell- ⁇ ferived /actor- 1 (SDF-1 ⁇ ), a CXC chemokine, its only known natural ligand, as well as the antagonistic viral macrophage inflammatory protein-Il (vMIP-II) encoded by the Kaposi's sarcoma-associated herpes virus (Moore et al., 1996).
  • SDF-1 ⁇ .vtromal cell- ⁇ ferived /actor- 1
  • vMIP-II antagonistic viral macrophage inflammatory protein-Il
  • vMIP-II binds with high affinity to many CC and CXC chemokine receptors, including CXCR4 and CCR5, and inhibits cell entry of strains of HIV that use CXCR4 and CCR5 as coreceptors (Boshoff et al., 1997; Kledal et al., 1997).
  • chemokines and their receptors play pivotal roles in inflammation, lymphocyte development and HIV entry and infection, the ability to specifically manipulate the activity of these molecules will have enormous impact on ameliorating and halting diseases that currently have no satisfactory treatment.
  • Most currently available antagonists and agonists of chemokine receptors suffer from diminished efficacy over time, as well as difficult and costly preparations.
  • CCR5 receptor antagonists that block HIV-1 infection are available (Arenzana-Seisdedos et al., 1996; Nardese et al., 2001 ; Simmons et al., 1997); however, their specificity for CCR5 is in doubt.
  • RANTES aminoxypentane
  • AOP aminoxypentane
  • RANTES binds to a number of chemokine receptors in addition to CCR5
  • treatment with RANTES analogs that block the natural chemokines from functioning results in complications.
  • the development of more specific inhibitors of CCR5 is indicated.
  • CXCR4 peptides and organic compounds which are unrelated to natural chemokines have antagonistic activity (Doranz et al., 1997; Murakami et al., 1997; Schols et al., 1997); but again, their specificity for CXCR4 are uncertain.
  • chemokine receptor antagonists and agonists are usually accomplished by brute force, an inefficient approach in which false positive and negative signals predominate.
  • high-throughput screening (HTS) methods for identifying antagonists of chemoattractant receptors, such as chemokine receptors rely on detecting perturbations of downstream events, such as leukocyte cell migration.
  • downstream events such as leukocyte cell migration.
  • compounds that disrupt cell membranes or block events downstream mimic these outcomes, masquerading as candidate antagonists.
  • Considerable effort is then required to distinguish the genuine antagonists from those compounds that yielded false positive signals. This task is daunting.
  • the invention is drawn to polypeptides having a sequence of SEQ ID NOs:2-9, 1 1 -17, and the dimers formed between SEQ ID NOs: 18 and 19 and between
  • the invention is drawn to polypeptides having a sequence at least 75% identical to a sequence of SEQ ID NOs:-9, 1 1 -17, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 ; the sequence identity may be 80%, 90%, 95%, 99% and 100%.
  • the invention is drawn to treating and preventing HIV infection and spread by administering a polypeptide having a sequence at least 75% identical to a sequence of SEQ ID NOs:2-9, 1 1 -17, and the dimers formed between SEQ ID NOs:18 and 19 and between SEQ ID NOs:20 and 21 ; the sequence identity may be 80%, 90%, 95%), 99% and 100%).
  • the invention is drawn to treating CXCR4- and CCR5-related diseases and conditions by administering a polypeptide having a sequence at least 75%> identical to a sequence of SEQ ID NOs:2-9, 1 1 -17, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 ; the sequence identity may be 80%,, 90%, 95%, 99%o and 100%.
  • the invention is drawn to polypeptides having a sequence at least 75% identical to a sequence of SEQ ID NOs: 1 , 2, 5-8, 10,1 1, 15, 16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 , having at least one D-amino acid; the sequence identity may be 80%>, 90%., 95%), 99%> and 100%.
  • the invention is drawn to treating and preventing HIV infection and spread by administering a polypeptide having a sequence at 75% with a sequence of SEQ ID NOs: l , 2, 5-8, 10, 11 , 15, 16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21, and having at least one D-amino acid; the sequence identity may be 80%., 90%, 95%, 99%> and 100%.
  • the invention is drawn to polypeptides having a sequence at least 75% identical to a sequence of SEQ ID NOs: 1-4, 8-18, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 , and having at least one N methylated-amino acid; the sequence identity may be 80%, 90%, 95%, 99%> and 100%.
  • the invention is drawn to polypeptides having at least 75% sequence identity to a sequence of SEQ ID NOs: 1 , 2, 8, 10, 15, 16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21., and having at least one D-amino acid and at least one N-methylated amino acid; the sequence identity may be 80%, 90%>, 95%, 99% and 100%.
  • the invention is drawn to a method of determining treatment efficacy, using a polypeptide having at least 75% sequence identity to a sequence of SEQ ID NOs: 2-9, 1 1 -16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 ; sequence identity may be 80%, 90%>, 95%, 99%> and 100%.
  • kits comprising a polypeptide having at least 75% sequence identity to a sequence of SEQ ID NOs:2-9 and 1 1 - 17, and the dimers formed between SEQ ID NOs:18 and 19 and between SEQ ID NOs:20 and 21 ; sequence identity may be 80%, 90%, 95%, 99% and 100%.
  • the invention is drawn to a method of determining CXCR4 and CCR5 expression, using a polypeptide having at least 75%> sequence identity to a sequence of SEQ ID Nos:2-9 and 1 1 -17, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 ; sequence identity may be 80%), 90%., 95%, 99%. and 100%.
  • the polypeptide may further contain a detectable label.
  • the present invention makes use of the discovery that synthetic molecules derived from SDF-1 ⁇ and vMIP-II can be used to mimic chemokine function and antagonize chemokine receptors.
  • the N-termini of SDF-1 ⁇ and vMIP-II are replaced by various synthetic sequences containing modified amino acids and amino acid enantiomers; in other modified polypeptides, the N terminal sequences are exchanged between SDF- 1 ⁇ and vMIP-II.
  • These synthetic SDF- 1 ⁇ and vMIP-II analogs are highly potent— and selective—I igands for chemokine receptors, such as CXCR4 and CCR5. They also effectively inhibit HIV entry and infection.
  • the DV1 peptide (SEQ ID NO:21), the D-amino acid form of SEQ ID NO:20, was synthesized to further study these complexes. Surprisingly, this peptide not only specifically binds to CXCR4 with higher affinity (3-32 nM concentrations) than the L- isomer, but it also blocks HIV entry and replication (Huang, 2001 ; Zhou et al., 2002).
  • the peptide in serum lasts at least three times longer than the VI form (24 hours vs. 72 hours). No cytotoxicity at concentrations as high as 200 ⁇ M was observed.
  • Peptides designed on natural chemokine ligand templates can therefore act as agonists and antagonists of CXCR4 (Crump et al., 1997; Heveker et al., 1998; Luo et al., 1999a; Luo et al., 1999b).
  • chemokines such as SDF-l ⁇ and vMIP-II
  • SDF-l ⁇ and vMIP-II are determinants for selective receptor binding. These sites are tolerant of changes in chirality; providing peptides with D-amino acids in the N-termini promises highly-specific, effective, and long-lasting therapeutics to treat chemokine-related disorders and to treat and prevent HIV infection and other diseases caused by infectious agents.
  • SDF-l ⁇ - and vMIP-II-based antagonists and agonists of CXCR4 and CCR5 are presented in Table 1 (SEQ ID NOs: l -9; 12-21). D-amino acids are indicated by underlining; N-methylated amino acids are indicated in boldface.
  • SDF-l ⁇ (SEQ ID NO: 1 ) and vMIP-II (SEQ ID NO: 10) sequences are not novel, but are given for comparison. Collectively, these novel polypeptides are referred to as ".synthetic chemokine mimic polypeptides" (SCMPs).
  • Val Cys lie Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys 50 55 60
  • Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys Ala Leu Asn Lys Xaa
  • Dimer is formed by a disulfide bridge between Cys75 of SEQ ID NO.18 and the Cys75 of SEQ ID NO: 19
  • the molecules based on the SDF-l ⁇ and vMIP-II template are suitable for treating any disease, disorder or condition that involves CXCR4 and/or CCR5.
  • these molecules may be used to block HIV entry and viral spread.
  • antagonists are administered to treat those diseases, disorders or conditions that result in part because of increased CXCR4 or CCR5 expression or activity.
  • agonists are administered to treat those conditions where the activity or expression of these receptors is decreased.
  • these analogs block receptor binding, they are useful in prophylactic methods that prevent infection and spread of HIN
  • the molecules are useful for modulating biological processes, such as vascularization of the gastrointestinal tract (during development), hematopoiesis, fetal cerebellar development, neuronal cell migration.
  • Diseases and conditions that the molecules can be used to treat include asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, atherosclerosis, cancer, multiple myeloma, non-Hodgkin's lymphoma and viral infection.
  • Isolated when referred to a molecule, refers to a molecule that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that interfere with diagnostic or therapeutic use.
  • a “polypeptide” is a protein having at least two amino acids.
  • the amino acids may be those usually incorporated into mammalian polypeptides (such as human SDF-l ⁇ or viral vMIP-II), or the amino acids may be modified in some way, including derivatives and enantiomers (such as SDF-l ⁇ containing D-amino acids, or ⁇ -methylated residues).
  • a “purified polypeptide” is one that is purified to homogeneity (1) by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain, or (2) by high performance liquid chromatography (HPLC).
  • An "active polypeptide” retains a biological and/or an immunological activity of an SCMP.
  • Immunological activity refers to the ability to induce antibody production against an antigenic epitope possessed by a SCMP; biological activity refers to a function possessed by the SCMP, excluding immunological activity.
  • Biologically active portions of SCMP include peptides comprising amino acid sequences sufficiently homologous to, or derived from, the amino acid sequence of an SCMP (SEQ ID NOs:3-9, 12-21) that include fewer amino acids than the given SCMP sequence and exhibit at least one activity of an SCMP.
  • Biologically active portions comprise a domain or motif with at least one activity of an SCMP.
  • a biologically active portion of an SCMP may have an amino acid sequence shown in SEQ ID NOs:3-9, 12-21, or substantially homologous to SEQ ID NOs:3-9, 12- 21 and retains the functional activity of an SCMP polypeptide, yet differs in amino acid sequence.
  • amino acid sequences of SCMPs can be varied, such that their function is not altered.
  • amino acid substitutions at "non-essential" residue positions can be made in SEQ ID NOs:3-9, 12-21.
  • a "non-essential” amino acid residue is one that can be altered from a given SCMP sequence without altering its biological activity; an "essential” amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the SCMPs are particularly non-amenable to alteration.
  • Residues are divided into groups based on common side-chain properties as denoted in Table B.
  • Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more preferably into non-conserved sites.
  • Table B Amino acid classes
  • an SCMP variant preserves SCMP-like function and includes any variant in which residues at a particular position in have been substituted by other amino acids, and further includes inserting an additional residue or residues between two residues of the parent polypeptide, as well as deleting one or more residues from the parent sequence.
  • the substitution is a conservative substitution (Table A).
  • SCMP polypeptide variant means an active SCMP having at least 75% amino acid sequence identity with a full-length SCMP sequence, taking into account amino acid enantiomers, where a D-isomer is not an identity to the corresponding L-isomer (e.g., D- Lys is not counted as an identity with L-Lys).
  • SCMP variants include those wherein one or more amino acid residues are added or deleted at the N- or C- terminus of the full-length amino acid sequence.
  • An SCMP polypeptide variant will have at least about 75% amino acid sequence identity, preferably at least about 80%) amino acid sequence identity, more preferably at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acid sequence identity and most preferably at least about 99%) amino acid sequence identity with a full- length sequence of an SCMP (SEQ ID NOs:2-9, 11-16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21).
  • Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues that are identical with amino acid residues in an SCMP sequence in a candidate sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and, if necessary, gaps are introduced to achieve the maximum %> sequence identity; conservative substitutions are not considered as part of the sequence identity. Publicly available computer software, such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) can be used to align polypeptide sequences. Those skilled in the art will determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B can be calculated as:
  • % amino acid sequence identity X/Y ' 100 where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and
  • Y is the total number of amino acid residues in B.
  • the %> amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
  • Fusion polypeptides are useful in, for example, cell-localization assays and bioassays.
  • An SCMP "chimeric polypeptide” or “fusion polypeptide” is a SCMP polypeptide fused to a non-SCMP polypeptide.
  • a non-SCMP polypeptide is not substantially homologous to SCMP.
  • An SCMP fusion polypeptide may include any portion of an entire SCMP, including any number of biologically active portions.
  • An SCMP may also be modified by N-methylation on at least one residue, as well as substituting the D-amino acid enantiomer for an L-amino acid enantiomer.
  • An SCMP may have both D-amino acids as well as N-methylated amino acids.
  • Antagonist includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of an endogenous SCMP.
  • Ant includes any molecule that mimics a biological activity of an endogenous SCMP. Examples of agonists include the polypeptides of SEQ ID NO: 15 (of CXCR4) and SEQ ID NO: 16 (of CCR5). Examples of antagonists include SEQ ID NOs:3-9 and 12 (of CXCR4) and SEQ ID NO: 12 (of CCR5).
  • terapéuticaally effective amount means the amount of the compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a clinician, or that is sufficient to prevent development of or alleviate to some extent one or more of the symptoms of the disease or condition being treated.
  • compositions can be incorporated into pharmaceutical compositions.
  • Such compositions typically comprise a SCMP and a pharmaceutically acceptable carrier.
  • a "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration (Remington and Gennaro, 2000).
  • Preferred examples of such carriers or diluents include water, saline, Finger's solutions, dextrose solution, and 5% human serum albumin.
  • Liposomes and non- aqueous vehicles, such as fixed oils, may also be used. Except when a conventional media or agent is incompatible with an active compound, use of these compositions is contemplated.
  • Supplementary active compounds can also be incorporated into the compositions. More than one SCMP may be incorporated into a composition.
  • a pharmaceutical composition is formulated to be compatible with the intended route of administration, including intravenous, intradermal, subcutaneous, oral, transdermal, transmucosal and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the composition must be sterile.
  • Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures.
  • Proper fluidity can be maintained, for example, by using a coating such as lecithin; by maintaining the required particle size in the case of dispersion, and by using surfactants.
  • Various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can control microorganism contamination.
  • Isotonic agents such as sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition.
  • compositions that delay absorption include agents such as aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an SCMP) in an appropriate solvent with one or more ingredient, followed by sterilization.
  • active compound e.g., an SCMP
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and any other required ingredients.
  • Sterile powders for the preparation of sterile injectable solutions include vacuum- and freeze-drying that yield a powder containing the active ingredient and any desired ingredient from a sterile solution.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included.
  • Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or STEROTES; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch
  • a lubricant such as magnesium stearate or STEROTES
  • a glidant such as colloidal silicon dioxide
  • compositions for inhalation For administration by inhalation, the compounds are delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, e.g., a gas such as carbon dioxide.
  • a suitable propellant e.g., a gas such as carbon dioxide.
  • Systemic administration can also be transmucosal or transdermal.
  • penetrants that can permeate the target barrier(s) are selected.
  • Transmucosal penetrants include detergents, bile salts and fusidic acid derivatives.
  • Nasal sprays or suppositories can be used for transmucosal administration.
  • the active compounds are formulated into ointments, salves, gels or creams.
  • the compounds can also be prepared as suppositories (with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid (ALZA Corporation; Mountain View, CA and NOVA Pharmaceuticals, Inc.; Lake Elsinore, CA).
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers (Eppstein, 1985).
  • Unit dosage form refers to physically discrete units suited as single doses for a subject to be treated, containing a therapeutically effective quantity of active compound in association with the required pharmaceutical carrier.
  • the specification for unit dosage forms are dictated by, and directly dependent on, the unique characteristics of the active compound and the particular desired therapeutic effect, and the inherent limitations of compounding the active compound.
  • compositions and methods of the present invention may further comprise other therapeutically active compounds that are usually applied in the treatment of chemokine (i.e., CXCR4 and CCR5) pathologies, such as HIV infection.
  • chemokine i.e., CXCR4 and CCR5 pathologies, such as HIV infection.
  • an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses.
  • the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day.
  • a suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day.
  • the compositions are preferably provided in the form of tablets containing 0.1 to 1000 milligrams of the active ingredient, particularly 0.1, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to a patient to be treated.
  • the compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.
  • the specific dose level and frequency of dosage for any particular patient may be varied and depends upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the subject undergoing therapy.
  • dosage may also depend on the aggressiveness of the particular HIV that has infected an individual.
  • Kits for pharmaceutical compositions Pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. When supplied as a kit, the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately permit long-term storage without losing activity of the components. Kits may also include reagents in separate containers that facilitate the execution of a specific test, such as diagnostic tests or tissue-typing. When kits are assembled for detection methods, a control may also be provided. Positive and negative controls may be included. For example, in the determining if an SCMP would be effective in treating a disorder, a kit could be provided that includes at a labeled SCMP and a polypeptide of SEQ ID NO: 1 or 10 that is also labeled.
  • a suitable negative control would include a scrambled SCMP or some other polypeptide known not to bind the target receptor.
  • kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container.
  • sealed glass ampules may contain lyophilized SCMP or buffer that have been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Containers include test tubes, vials, flasks, bottles, syringes, etc.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes may be glass, plastic, rubber, etc.
  • Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail. Prognostic assays
  • Diagnostic methods can be used to identify subjects having, or at risk of developing, a disease or disorder related to CXCR4 or CCR5, including HIV infection.
  • a CXCR4 or CCR5-related desease, disorder or condition refers to one which results from and/or is mediated, at least in some part, by an activity (or lack thereof) of CXCR4 or CCR5.
  • Prognostic assays can be used to determine whether a subject can be administered a polypeptide of SEQ ID NOs:2-9, 1 1 - 16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 to treat a disease or disorder related to CXCR4 or CCR5.
  • Such methods can be used to determine whether a subject can be effectively treated with SEQ ID NOs:2-9, 11 -16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21.
  • Methods for determining whether an HIV-infected subject can be effectively treated with a SCMP include obtaining a test sample having the HIV virus and testing the ability of the SCMPs to inhibit HIV entry.
  • the cells that are tested are also those of the subject; however, cells from cell lines may also be used to facilitate the tests, while using the infecting virus.
  • the class(es) of cells infected with HIV or responsible at least in part for the disorder, disease or condition are obtained in a sample from the subject. Purification of the target cell type(s) is not necessary if they are morphologically distinguishable or can be labeled with a cell-specific marker or stain.
  • the cells are then either directly incubated with a labeled polypeptide of SEQ ID NO:2-9, 1 1 -16, or the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21, such as with FAM or other label (e.g., SEQ ID NO: 17), washed and prepared for analysis, or preserved by applying a fixative, treating the samples to quench any background, applying the labeled polypeptide and washing the sample, and then preparing for analysis. Microscopic and spectrophotometric methods of observation are appropriate. Binding of the labeled polypeptide to the target cell type indicates that treatment with that polypeptide, without the label, will be effective.
  • a labeled polypeptide of SEQ ID NO:2-9, 1 1 -16, or the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21, such as with FAM or other label (e.g., SEQ ID NO: 17) washed and prepared for analysis, or
  • fluorescent moieties such as fluorescein isothiocyanate; fluorescein dichlorotriazine and fluorinated analogs of fluorescein; naphthofluorescein carboxylic acid and its succinimidyl ester; carboxyrhodamine 6G; pyridyloxazole derivatives; Cy2, 3 and 5; phycoerythrin; fluorescent species of succinimidyl esters, carboxylic acids, isothiocyanates, sulfonyl chlorides, and dansyl chlorides, including propionic acid succinimidyl esters, and pentanoic acid succinimidyl esters; succinimidyl esters of carboxytetramethylrhodamine; rhodamine Red-X succinimidyl ester; Texas Red sulfonyl chloride; Texas Red-X succinimidyl ester; Texas Red-X sodium tetrafluorophenol ester; Red-X; Texas Red dye
  • Suitable labels further include enzymatic moieties, such as alkaline phosphatase or horseradish peroxidase; radioactive moieties, including 35 S and l35 I- labels; avidin (or streptavidin)-biotin-based detection systems (often coupled with enzymatic or gold signal systems); and gold particles.
  • enzymatic-based detection systems the enzyme is reacted with an appropriate substrate, such as 3, 3'- diaminobenzidine (DAB) for horseradish peroxidase; preferably, the reaction products are insoluble.
  • DAB 3, 3'- diaminobenzidine
  • Gold-labeled samples if not prepared for ultrastructural analyses, may be chemically reacted to enhance the gold signal; this approach is especially desirable for light microscopy.
  • the choice of the label depends on the application, the desired resolution and the desired observation methods.
  • fluorescent labels the fluorophore is excited with the appropriate wavelength and the sample observed using a microscope, confocal microscope, or FACS machine.
  • radioactive labeling samples are contacted with autoradiography film, and the film developed; alternatively, autoradiography may also be accomplished using ultrastructural approaches. Alternatively, radioactivity may be quantified using a scintillation counter.
  • Prophylactic methods provides methods for preventing in a subject a disease or condition associated with aberrant CXCR4 or CCR5 expression or activity, by administering at least one SCMP.
  • Subjects at risk for such a disease or HIV infection are administered the SCMP occur prior to the manifestation of symptoms or to stave off HIV infection.
  • NCL native chemical ligation
  • TPCS total stepwise polypeptide chemical synthesis
  • preparation of a chemokine or analog consists of three main steps: (1) solid phase synthesis of two peptide sequences corresponding to the N- and C-terminal portions of the protein; (2) chemical ligation of these two peptide segments to form a complete protein sequence; and (3) purification and folding of the final protein product with proper tertiary structure and biological function.
  • the synthesis of wild-type vMIP-II is presented as a paradigm; specific syntheses for the polypeptides of SEQ ID NOs:2-9, 1 1 -16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 , can be found in Examples 1 -5.
  • N- (1 -34) and C-termini (35-71) of vMIP-II are synthesized.
  • the N-terminal region (1 -34) peptide is synthesized as a C-terminal thioester peptide using a sulfonamidobutyl resin.
  • the last C-terminal amino acid, 9-flourenylmethloxycarbonyl (Fmoc)-Leu is incorporated to the resin by using Benzotriazol-1 -yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) in the presence of N,N- di-isopropylethylamine (DIEA) as the coupling reagent.
  • DIEA N,N- di-isopropylethylamine
  • N-terminal Fmoc-protection is removed, and the peptidyl resin is activated with iodoacetonitrile to prepare cyanomethylsulfonamide peptidyl resin.
  • the peptide is cleaved from the resin as a thioester by adding benzylmercapten in tetrahydrofuran (THF).
  • THF tetrahydrofuran
  • the peptide is cleaved and purified by HPLC.
  • the peptide is synthesized in such a way that the thiol group of this Cys-35 is selected for the chemoselective ligation.
  • a thiophenol additive maintains a high concentration of highly reactive phenyl thioester peptides, whereas the benzyl mercaptan prevents the cysteine side chains from forming unreactive disulfide bonds during ligation. These additives also help the longer storage of the weakly activated thioester peptide until they are converted to more reactive phenyl thioester peptides during chemical ligation.
  • the final vMIP-II product is purified by HPLC and folded in 2 M guanidine HCl, 100 mM Tris, pH 8 at room temperature in the presence of air. The purity and identity are then determined by analytical HPLC and mass spectrometry.
  • TPCS Total stepwise polypeptide chemical synthesis
  • SCMPs can be readily chemically synthesized in vitro in their entirety using stepwise polypeptide chemistry.
  • polypeptide synthesis may be carried out in a stepwise manner on a solid phase support using an automated polypeptide synthesizer, such as a Rainin Symphony Peptide Synthesizer, Advanced Chemtech Peptide Synthesizer, Argonaut Parallel Synthesis System, and preferably, an Applied Biosystems Peptide Synthesizer.
  • an automated polypeptide synthesizer such as a Rainin Symphony Peptide Synthesizer, Advanced Chemtech Peptide Synthesizer, Argonaut Parallel Synthesis System, and preferably, an Applied Biosystems Peptide Synthesizer.
  • the peptide synthesizer instrument combines the Fmoc chemistry with 1 -Hydroxybenzotriazole/0-(1H- benzotriazole- 1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate/DIEA (HOBt/HBTU/DIEA) activation to perform solid-phase peptide synthesis.
  • Synthesis starts with the C-terminal amino acid, wherein the carboxyl terminus is covalently linked to an insoluble polymer support resin.
  • Useful resins can load 0.1 mmol to 0.7 mmol of C-terminal amino acid per gram of resin; display resistance to the various solvents and chemicals used during a typical synthetic cycle, such as dichloromethane (DCM), N,N-dimethylformamide (DMF), N- methylpyrrolidone ( ⁇ P), N,N-dimethylamine (DMA), 1 -Hydroxybenzotriazole (HOBt), 2-( 1 -H-Benzotriazol- 1-yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU), DIEA, methanol (MeOH), or water; and be amenable to continuous flow or batch synthesis applications.
  • DCM dichloromethane
  • DMF N,N-dimethylformamide
  • ⁇ P N
  • an acid labile and base stable resin such as an ether resin
  • Preferred resins include /?-Benzyloxybenzyl Alcohol resin (HMP resin), PEG co-Merrifield resin, ⁇ ovaSyn TGA ® resin ( ⁇ ovabiochem), 4-sulfamylbutyryl aminomethyl (AM) resin, and cross-linked ethoxylate acrylate resin (CLEAR) amide resin.
  • Amino acid- coupled resins are commercially available from a number of different sources, although such coupled resins may be prepared using known procedures in the art.
  • the ⁇ -terminus of the resin-coupled amino acid (or polypeptide) is chemically-protected by an Fmoc group that is removed prior to the addition of the next ⁇ -terminal amino acid reactant.
  • the Fmoc group is a base labile protecting group that is easily removed by concentrated solutions of amines, such as 20-55% piperidine, in a suitable solvent, such as N-methylpyrrolidone ( ⁇ MP) or NN- dimethylformamide (DMF).
  • amines for Fmoc deprotection include tris (2-aminoethyl) amine, 4-(aminomethyl)piperidine, tetrabutylammonium fluoride, and l,8-diazabicyclo[5.4.0]undec-7-ene (DBU).
  • DBU l,8-diazabicyclo[5.4.0]undec-7-ene
  • the side chains of many amino acids contain chemically reactive groups, such as amines, alcohols, or thiols. These side chains must be additionally protected to prevent undesired side-reactions during the coupling step.
  • Preferred side chain protecting groups are base-stable, more preferably, both base-stabile and acid-labile. Table D provides a preferred set of side chain protection groups for this category of amino acids that are used in one example.
  • the carboxylate group of the incoming Fmoc-protected amino acid is activated in order to achieve efficient chemical coupling to the N-terminus of the resin-bound polypeptide. Activation is typically accomplished by reacting an Fmoc- protected amino acid with a suitable reagent to yield a reactive ester.
  • Preferred activated esters include the pentafluorophenyl (OPfp) ester and the 3-hydroxy-2,3- dihydro-4-oxo-benzo-triazone (ODhbt) ester, more preferably the OBt ester, and even more preferably, the OAt ester derived from l -hydroxy-7-azabenzotriazole (HOAt).
  • the coupling reactions may be done in situ using activating reagents , such as DCC, BOP, BOP-CI, or TBTU, preferably HBTU or 0-(7-azabenzotrizol-l-yl)-l, 1,3,3, tetramethyluronium hexafluorophosphate (HATU).
  • activating reagents such as DCC, BOP, BOP-CI, or TBTU, preferably HBTU or 0-(7-azabenzotrizol-l-yl)-l, 1,3,3, tetramethyluronium hexafluorophosphate (HATU).
  • Preferred coupling reactions included a mixture comprising HOBt and HBTU, and more preferably, HOBt, HBTU, and DIEA.
  • the preferred coupling conditions use bromo- tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) as the only coupling reagent, and the coupling reaction is performed manually in DCM with DIEA present under N .
  • the Fmoc-protected amino acid is present in molar excess to the polypeptide coupled to the resin, preferably, in a five-fold molar excess.
  • the coupling reactions are repeated one or more times (double or multiple coupling) to ensure that all resin-bound polypeptide has undergone a successful addition reaction with the activated Fmoc-amino acid.
  • any unreacted N-terminal residues are capped using a suitable capping reagent.
  • the resin support is washed to remove the unreacted Fmoc-amino acids and coupling reagents.
  • the resin is then subjected to a new cycle of base-catalyzed removal of the N-terminal Fmoc group to prepare the polypeptide for another amino acid addition.
  • the resin is subjected to base-catalyzed removal of the remaining Fmoc protection group.
  • the polypeptide-coupled resin is washed to remove the base and subsequently treated with acid to remove any amino acid side chain protecting groups and to release the polypeptide chain from the resin support.
  • the preferred acid is a strong acid, such as trifluoroacetic acid (TFA) in the presence of suitable scavengers, such as reagent K [TFA: thioanisole: ethanedithiol: phenol: water (82.5:5:2.5:5:5)].
  • TFA trifluoroacetic acid
  • the polypeptide is subsequently separated from the resin by filtration and optionally washed repeatedly with a suitable solvent, such as DCM/DMF.
  • a suitable solvent such as DCM/DMF.
  • the polypeptide may be optionally desalted through ultrafiltration using a membrane with a suitable MW cutoff.
  • the polypeptide may be precipitated from solution using a suitable solvent, such as cold methyl t-butyl ether or t-butylethylether, and the precipitate optionally washed with a suitable solvent, such as cold ether and dried.
  • the polypeptide may be further purified using a suitable chromatographic means, such as hydrophobic chromatography using a CI 8 resin and an appropriate chromatographic buffer system, such as TFA/water/acetonitrile.
  • the purity of the peptide optionally may be analyzed by mass spectrometry, such as MALDI-MS, analytical HPLC, amino acid analysis or sequencing.
  • reagents were from Acros-Fisher Scientific (Pittsburgh, PA) and Sigma-Aldrich (St. Louis, MO); only highest purity grade preparations were used.
  • HMP 4-(hydroxymethyl)phenoxymethylpolystyrene
  • Fmoc-Lys(Boc)- NovaSyn ® TGA resin 4-(hydroxymethyl)phenoxymethylpolystyrene (HMP) resin, Fmoc-Lys(Boc)- NovaSyn ® TGA resin, N-(9-fluorenylmethoxycarbonyl) (Fmoc)-L-amino acids, solvents and other reagents for SPPS were from Novabiochem (San Diego, CA), polypeptide International (Louisville, KY) or Applied Biosystems (Foster City, CA). Fmoc-8-aminocaprylic acid was obtained from Ana Spec (San Jose, CA). CLEAR amide support was procured from polypeptide International (Louisville, KY). I- SDF- l ⁇ (Perkin Elmer Life Science; Boston, MA) had a specific activity of 2200 Ci/mmol.
  • 293 cells gifts from Dr. R. Doms, University of Pennsylvania (also available from American Type Tissue Collection (ATCC); Manassas, VA) and Sup-Tl cells (provided by the National Institutes of Health AIDS Reagent Program; Rockville, MD; also available from ATCC) were maintained in Dulbecco's Modified Eagle's Medium (DMEM)/10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • polypeptides were synthesized in a stepwise manner on an Applied Biosystems model 433A automated polypeptide synthesizer using commercially available solid phase support resins and Fmoc chemistry with HOBt/HBTU/DIEA activation chemistry.
  • Applied Biosystems model 433A automated polypeptide synthesizer using commercially available solid phase support resins and Fmoc chemistry with HOBt/HBTU/DIEA activation chemistry.
  • MALDI-MS Matrix-assisted laser desorption/ioni ⁇ ation mass spectrometry
  • Example 1 Main chain modified SDF-1 a analogs SEQ ID NOs:4-9 by TSPCS
  • PyBroP was a preferred coupling reagent because it was more efficient than HBTU/HOBt.
  • polypeptides Upon completion of SDF-l ⁇ and its N-methyl derivatives, the polypeptides were cleaved from the resin and side chains deprotected by reagent K in the presence of 2.5% of TIPS. HPLC-purified linear polypeptides were then oxidized in air, and the folded polypeptides were re-purified by HPLC. Fractions were analyzed by RP- HPLC and MALDI-MS, and those containing the pure folded polypeptide were pooled and freeze-dried. The polypeptides were then dissolved in water, their concentration determined using UV spectrometer, and stored at -20° C. TSPCS
  • Valine-39 was then coupled to the resin in a flask under inert atmosphere over 3 days using the same coupling reagents as above, except that DCM)/DMF (1/1, v/v) was used as a solvent instead of NMP/DMF.
  • the remaining non-N-methyl amino acids were condensed to the resin, using the same conditions as for the C-terminal 29 amino acids segment.
  • PyBroP was used as the only coupling reagent, and the coupling reaction was manually performed in DCM in the presence of DIEA overnight under N 2 (ref).
  • the following side chain protections were used in this synthesis (Table E):
  • polypeptide-resin 100 mg was treated with TFA (10 ml) in the presence of reagent K (1.5 g phenol, 0.5 ml thioanisole, 0.25 ml ethane-1,2- dithiol (EDT), 0.5 ml water), and triisopropylsilane (0.5 ml) for 2 hours at room temperature (22-24° C).
  • reagent K 1.5 g phenol, 0.5 ml thioanisole, 0.25 ml ethane-1,2- dithiol (EDT), 0.5 ml water
  • triisopropylsilane 0.5 ml
  • polypeptides were further purified using semi-preparative HPLC on a C18 column. Analytical RP-HPLC and MALDI-MS were used to identify pure linear polypeptides.
  • the 4-sulfamylbutyryl AM resin (1 .12 mmol/g, 200 mg, 0.224 mmol) was swelled in the presence of CHCI 3 (2.5 ml; pre-filtered through activated basic A1 0 3 ), and DIEA (0.3 ml; 1.72 mmol) and Fmoc-Arg(Pbf)-OH (0.65 g; 1 mmol) were added.
  • the amino acid was dissolved by stirring at room temperature (22-24° C) and then cooled and incubated for 20 minutes at -20° C. PyBoP (0.52 g; 1 mmol) was then added as a solid, and the coupling reaction was conducted for 6 hours at a temperature of -20° C to -30° C.
  • the resin was filtered, washed three times with DMF (2 ml) and three times with DCM (2 ml), and dried over P 2 ⁇ 5. Since a single coupling reaction only loads the available sulfonamide groups to a 53% yield, the coupling reaction was repeated to increase amino acid loading onto the sulfonamide groups to 70% yield.
  • the unreacted sulfonamide groups were acetylated by treating the resin with acetic anhydride (0.1 ml; 1.06 mmol) in the presence of a catalytic amount of 4- dimethylaminopyridine (DMAP) and DIEA (0.4 ml; 2.29 mmol) for 2.5 hours at room temperature (22-24° C).
  • Polypeptide chain synthesis was performed on Fmoc-Arg(Pbf)-sulfonamide resin (0.1 mmol) as described in TSPCS, using the same protecting groups for the side chains of amino acids, except for the N-terminal Lys, which was di-Boc protected in dicyclohexylamine (DCHA) salt.
  • DCHA dicyclohexylamine
  • Polypeptide-bound sulfonamide resin (100 mg; about 25 ⁇ mol) was activated by iodoacetonitrile (0.16 ml; 2.2 mmol), distilled and pre-filtered through a plug of basic alumina in NMP (1 ml) in the presence of DIEA (0.18 ml; 1.0 mmol) overnight in the dark at room temperature. The resin was washed three times with DMF (2 ml), subsequently washed three times with DCM (2 ml), and then transferred to a 5 ml round-bottom flask.
  • the side chain protected polypeptide was cleaved from the resin with benzyl mercaptan (0.12 ml) in DMF/THF (1 ml; 1 : 1 [v/v]) in the presence of DIEA (0.21 ml) for 39 hours at room temperature (22-24° C).
  • the polypeptide was separated from the resin by filtration and washed five times with DCM/DMF (2 ml;
  • the polypeptide was purified using semi-preparative HPLC on a CI 8 column using a gradient of 20% to 60% B over 60 minutes with a flow rate of 3 ml/min.
  • the product was characterized by analytical RP-HPLC and MALDI-MS. The observed molecular mass was 1068.8 Da (calculated MW, 1069.1 Da).
  • the reaction mixture was desalted by repeated dilution of the reaction mixture with water (about 13 ml), followed by volume reduction of the diluted reaction mixture to its original volume using a polypeptide concentrator (MWCO: 5 KDa, Millipore; Billerica, MA) and centrifugation (4000 rpm for 50 min).
  • MWCO polypeptide concentrator
  • the recovered product was purified by semi-preparative HPLC on a C18 column.
  • the polypeptide (0.75 mg, 43% yield) was characterized by analytical RP-HPLC (about 90% pure) and MALDI-MS. The observed molecular mass was 7971.9 Da (calculated MW, 7973.5 Da).
  • DTNB 5'-dithiobis(2-nitrobenzoic acid)
  • the polypeptide yields no absorption at 412 nm, indicating the absence of free thiol groups in the polypeptide and thereby confirming its folded state.
  • the polypeptides were purified using semi-preparative HPLC on a C18 column with a gradient 10-50% B over 60 minutes at a flow rate of 3 ml/min. The fractions were analyzed by analytical RP- HPLC and MALDI-MS. After freeze-drying the pure polypeptides, the polypeptides were dissolved in water and the concentration determined by UV absorption spectrometer, using an extinction coefficient of 8730 M "1 cm " ' at 280 nm, calculated on the basis of the number of tryptophane, tyrosine and the disulfide bond in the polypeptides (Pace et al., 1995).
  • the linear sequence of NT (D Lys) SDF- l ⁇ was synthesized in 0.1 mmol scale using a CLEAR amide resin. Prior to incorporation of the C-terminal amino acid, the resin was swelled in DCM for 2 hours. Individual amino acids were coupled to the C- terminal amino acid-coupled resin at room temperature (22-24° C) using a five-fold excess of activated Fmoc-amino acids and double coupling each residue. For the first twenty amino acids from the C-terminus, the first and the second coupling reactions were individually performed for 1 hour. For all residues incorporated beyond the twentieth residue, the second coupling time was increased to 2 hours.
  • the polypeptide was cleaved from the support by incubating with a cleavage solution (0.2 % phenol (w/v), 5 % thioanisol (v/v), 5% water (v/v), 2.5% ethanedithiol (v/v), 1.5% triisopropylsilane (v/v) and 86% TFA (v/v) for four hours at room temperature (22-24° C).
  • a cleavage solution 0.2 % phenol (w/v), 5 % thioanisol (v/v), 5% water (v/v), 2.5% ethanedithiol (v/v), 1.5% triisopropylsilane (v/v) and 86% TFA (v/v) for four hours at room temperature (22-24° C).
  • the cleaved polypeptide was filtered and concentrated to one-third of its original volume under reduced pressure.
  • the polypeptide was precipitated with ice-cold t-butylethylether, the precipitate washed twice with ice-cold ether and then dried.
  • the crude product was then dissolved in water and purified by semi-preparative HPLC using a C18 column.
  • the solvent systems were 0.1% TFA/water (A) and 0.1% TFA/20%water/acetonitrile (B).
  • the chromatography was performed in 35-75% B in 70 minutes at a flow rate of 3 ml/min. Chromatography fractions containing the target polypeptide were identified by MALDI-MS, which were subsequently pooled and lyophilized.
  • the estimated purity of the linear polypeptide was greater than 96%.
  • the molecular weight as determined by MALDI-MS was 7963.38 Da (calculated MW, 7962.47).
  • the linear polypeptide was resuspended in 2M guanidine HCI-0.1 M Tris (pH-8.5) to a final concentration of 1 mg/ml and air- oxidized at room temperature.
  • the folding reaction was monitored by analytical
  • NT (D Lys) SDF-1 ⁇ was further purified by semi- preparative HPLC using Vydac C18 column. Chromatography fractions containing the target polypeptide were tested for folded NT (D Lys) SDF-l ⁇ by analytical HPLC and MALDI-MS.
  • the 71 amino acid residue-long polypeptide was initially synthesized by native chemical ligation. Leu 34 was selected as the ligation site. Because a Cys side chain sulfhydryl group was required for ligation chemistry, a Cys residue at position 35 (in the middle of the polypeptide) was available for the ligation reaction. Residues 1 -34 (from the N-terminus) of vMIP II was synthesized as polypeptide thioester using a SLilfamylbutyryl AM resin (Novabiochem).
  • the N-terminal Leu was incorporated as Boc-Leu to protect the amino terminus during subsequent activation and cleavage steps.
  • the polypeptide thioester was released from the resin by activating the thioester with iodoacetonitrile in the presence of DIEA in the dark, and then by treating the resin with benzyl mercapten and DIEA in DMF. The released polypeptide was obtained in a yield greater than 80%.
  • Residues 35-71 were synthesized on a CLEAR amide resin.
  • the native chemical ligation was initiated by mixing equimolar quantities of polypeptide with phosphate buffer containing 15% TFE and 6M guanidine HCl in the presence of 2% thiophenol and 1 % benzyl mercapten (final pH 7 at 37° C).
  • the ligation reaction was monitored by HPLC and MALDI-MS analysis. Though the ligation reaction was 60%-70% complete within the first two hours, the reaction mixture was incubated overnight at 37° C to maximize yield.
  • the sample was then desalted by adding water to the mixture, followed by concentrating the solution in a filter device (Ultrafree-15, with high-flux Biomax' M ultrafiltration membrane, MW cutoff 5,000; Millipore) using centrifugation at 2000 g for 30 minutes. The process was repeated twice, adding 20% acetonitrile in water to the concentrated solution.
  • the polypeptide was purified by semi-preparative HPLC using a C 18 column with a linear gradient of 30- 70% B over 60 minutes at a flow rate of 3 ml/min. Chromatography fractions (1 ml) containing the target polypeptide were identified by MALDI-MS, which were subsequently pooled and lyophilized.
  • TSPCS For the TSPCS of vMIP II, CLEAR amide resin was used. For coupling reactions with a slow rate (53D, 54K, 37K, 38P, 39G, 22Q, 2 I P, 18R, 17K, 16Q, 2G), a double coupling followed by capping of the unreacted amino functionality was performed. After the incorporation of residue 50, 2% dimethyl sulfoxide (DMSO) was added to the coupling solution. After removing the N-terminal Fmoc protecting group, the polypeptide was cleaved from the resin support by incubating the resin with the cleavage solution for four hours.
  • DMSO dimethyl sulfoxide
  • the polypeptide was precipitated with ice cold t-butylethylether, and the precipitate was washed repeatedly (at least twice) with cold ether.
  • the washed pellet was dissolved in 25% acetonitrile in water containing 0.1 % TFA and then lyophilized.
  • the lyophilized polypeptide (108 mg) was dissolved in water (4 ml) and purified by semi-preparative HPLC, which yielded the linear polypeptide (22 mg).
  • the purified polypeptide was characterized by MALDI-MS; the observed mass was 8128.68 Da (calculated MW, 8127.68 Da).
  • the purified polypeptide was folded by resuspending the polypeptide in 1 M guanidine hydrochloride-100 mM Tris-HCI (pH 8.5; 1 ml buffer/mg polypeptide).
  • the polypeptide was then subjected to analytical HPLC using a CI 8 column (0.46 x 15 cm, 5 ⁇ m) with a linear gradient 30-70% B over 30 minutes at a flow rate of I ml/min. Within half an hour after buffer addition, over 90% of the polypeptide was folded; however, incubation continued for four hours to maximize folding.
  • the analytical HPLC profile of the folded polypeptide showed a two-unit shift toward the hydrophilic region of the solvent mixture as compared to the spectrum of the linear polypeptide. After the polypeptide was folded, desalting and purification were performed as described above, yielding 12 mg of folded polypeptide.
  • FAM-vMlP II was prepared wherein an Fmoc-8-aminocaprylic acid (Aca) was incorporated to the N-terminal Lys residue as a spacer.
  • the primary sequence of vMIP II (SEQ ID NO: 10) was then incorporated to the support by stepwise synthesis.
  • the N-terminal residue Leu was incorporated as Boc-Leu to the peptidyl resin.
  • the C-terminal Lys side chain protection ivDde was removed from the peptidyl resin using the following wash protocol: (a) twice with 2% hydrazine monohydrate (v/v) in DMF (5 ml; first wash 5 minutes; second, 7 minutes);(b) five times with 10% DIEA (v/v) in DMF (5 ml), (c) five times with DCM (5 ml); and (d) five times with diethyl ether (5 ml).
  • the fluorescent tag 5-carboxyfluorescene was incorporated into the side chain amino group of the resin-bound N-terminal Lys residue using a three-fold excess of 5-carboxyfluorescenesuccinimidyl ester (5-FAM-SE), HOBt and a five-fold excess of diisopropylethylamine.
  • the reaction vessel was mixed using a multi-pulse vortexer overnight in the dark.
  • the FAM polypeptide was cleaved from the support by incubating the polypeptide-coupled resin in cleavage solution for four hours in the dark at room temperature (22-24° C). The sample was then filtered, concentrated to one-third of its original volume in the dark under reduced pressure.
  • FAM (1 - I OD)vM IP II was precipitated with ice-cold t-butylethylether, and the precipitate then washed twice with ice-cold ether before drying.
  • the product was subsequently dissolved in water and purified by semi-preparative HPLC using a C 18 column (1.0 x 25 cm, 5 ⁇ m), with solvents A and B and a linear gradient 35-75% B over 70 minutes at a flow rate of 3 ml/min. Chromatography fractions (2 ml) containing the target polypeptide were identified by MALDI-MS, which were subsequently pooled and lyophilized. The purity of the linear polypeptide was >96%.
  • the molecular weight obtained from MALDI TOF MS was 8759.43 Da (calculated MW, 8758.65).
  • the linear polypeptide was resuspended in 2 M guanidine FICI-0.1 M Tris (pH 8.5) to a final concentration of 1 mg/ml, and air-oxidized at room temperature.
  • the folding reaction was monitored by analytical HPLC. More than 60% folding was completed within the first two hours; however, the solution was stirred overnight to assure complete folding.
  • the solution was transferred to a filter device (Ultrafree-15, with high-flux BiomaxTM ultrafiltration membrane, MW cutoff 5,000; Millipore) and centrifuged at 2000 g for 30 minutes. The concentrate was diluted with water and centrifuged two more times.
  • the concentrated solution containing the FAM (l -l OD)vMIP II was further purified by semi-preparative HPLC using a CI 8 column as described above. Chromatography fractions (1 ml) containing polypeptide were assayed for the presence of FAM ( l -l OD)vMIP II by analytical HPLC and MALDI-MS. Fractions containing the folded polypeptide were pooled and lyophilized; 2.3 mg of pure material was obtained.
  • the polypeptide vMIP II (Aca) ⁇ C(Acm) was prepared wherein Cys(Acm) was incorporated as a C-terminal amino acid coupled to a CLEAR amide support.
  • Coupling reactions were performed using a five-fold excess of Fmoc-protected amino acid. A second coupling was also performed to drive the reaction to completion. Six 8-aminocaprylic acid molecules were incorporated to the N-terminal Cys(Acm) residue to provide sufficient spacer effect. The primary sequence of vMIP II was then incorporated onto the support by stepwise synthesis. The sequence was subsequently removed from the resin by incubating the resin in cleavage solution in the dark for four hours at room temperature. The cleaved polypeptide mixture was filtered from the resin and concentrated to one-third volume.
  • the polypeptide was precipitated with ice-cold t-butylethylether, and the precipitate washed twice with ice-cold ether and then dried.
  • the crude product was subsequently dissolved in water and purified by semi-preparative HPLC using a CI 8 column (1 .0 x 25 cm, 5 ⁇ m).
  • the solvents systems were 0.1% TFA/water (A) and 0.1% TFA/20% water/acetonitrile (B), with a gradient 35%-75% B in 70 minutes at a flow rate of 3 ml/min. Chromatography fractions (2 ml) containing polypeptides were tested by MALDI-MS to identify those factions that contain the target polypeptide.
  • the lyophilized material was resuspended in 10% DMSO in the dark.
  • the folding reaction was monitored by analytical HPLC, revealing that greater than 68% of the polypeptide was folded within 28 hours.
  • the solution was transferred to a filter device (Ultrafree- 15, with high-flux Biomax' M ultrafiltration membrane, MW cutoff 5,000; Millipore) and centrifuged at 2000 g for 30 minutes.
  • the concentrate was diluted with water and centrifuged two more times.
  • the polypeptide was further purified by semi-preparative HPLC using a C I 8 column as above. Chromatography fractions ( I ml) containing polypeptides were tested for VMIP II (Aca) 6 C(Acm) by analytical HPLC and MALDI-MS.
  • Chromatography fractions containing the folded polypeptide were pooled and lyophilized, obtaining 12 mg of polypeptide. Purity, as tested by analytical HPLC using a C I 8 column (0.46 x 15 cm, 5 ⁇ m) with a gradient of 0.1% TFA/20%water/acetonitrile in 0.1 %TF A/water at a flow rate of 0.5 ml/min, was shoparwn to be greater than 98%. The observed molecular mass, as obtained from MALDI-MS analysis, was 8745.34 Da (calculated MW, 9146.28 Da).
  • Example 6 Competitive binding of SDF-1 a, vMIP-II and analogs to CXCR4
  • SDF- l ⁇ and vMIP-II analogs were assayed by incubating CXCR4-expressing cells with the analogs in the presence of labeled SDF- l ⁇ or vMIP-II.
  • CEM-T4 cells (a T cell, CD-4 expressing lymphoblastoid cell line) were harvested and washed twice with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • vMIP-II analogs to compete as a ligand for CCR5 was assayed by incubating CCR5-expressing cells with the analogs in the presence of labeled vMIP-II. The better the analog as a competitor, the weaker the observed signal.
  • 293 cells transfected with CCR5 Zhou et al., 2002 were harvested and washed twice with phosphate buffered saline (PBS). 2 x 10 5 cells were incubated with 0.2 nM of l25 I-vMIP-II in 100 ⁇ l of binding buffer with increasing concentrations of unlabelled vMIP-H.
  • SUP-T1 cells (a human T cell lymphoma cell line; 10 7 /ml) were loaded with 2 ⁇ M of Fura-2/AM (Molecular Probes; Eugene, OR) and 0.01% Pluronic F-127 in Hank's balanced salt saline (HBSS; 140 mM NaCl, 5 mM KC1, 10 mM HEPES (pH 7.4), 1 mM CaCl 2 , 1 mM MgCl 2 , 1 mg/ml glucose and 0.025% BSA) for 20 minutes at room temperature. The cells were then washed twice with HBSS and resuspended to 10 6 cells/ml.
  • HBSS Hank's balanced salt saline
  • Fura-2 fluorescence was measured at room temperature with a fluorescence spectrophotometer (ISA SPEX FIuoroMax-2) using excitation wavelengths of 340 nm and 380 nm, and an emission wavelength of 510 nm.
  • Various concentrations of peptides were first added to the cell suspension, and after 5 minutes of incubation, 50 nM of SDF-l ⁇ was added.
  • Polypeptides (SEQ ID NOs:2-9, 1 1 -16, and the dimers formed between SEQ ID NOs:18 and 19 and between SEQ ID NOs:20 and 21) were dissolved in the serum solution to a concentration of 10 mM. Samples were collected at different times during incubation at room temperature and subjected to HPLC analysis by injecting 10 ⁇ l of the peptide samples (Microsorb-MV C I 8 5 ⁇ m, 25 cm x 4.6 mm, 80% CH 3 CN with 0.1% trifluoroacctic acid, UV 220 nm, 1 ml/minute). The stability of the peptides was calculated based on the changes in the intensity of UV absorbance of the peptides.
  • Recombinant viruses were generated by cotransfecting the human 293 T cell line with pSVIIIenv plasmids expressing either HXBc2 or 89.6 envelope glycoproteins and pHXBH10_envCAT (Thali et al., 1992). Recombinant viruses were normalized for reverse transcriptase activity and used to infect (i) human PBMC activated with phytohemagglutinin (PHA) and interleukin- 2 or (ii) canine thymocytes expressing human CD4 or human CD4 plus human CCR5 (Choe et al., 1998).
  • the target cells were preincubated with the peptides (SEQ ID NOs:2-9, 1 1-16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21) at varying concentrations for 1 hour prior to co-culture with the recombinant viruses.
  • Infected cells were harvested three days later, and chloramphenical acetyl transferase (CAT) activity was measured in cell lysates (Helseth et al., 1990).
  • CAT chloramphenical acetyl transferase
  • CC CKR5 a RANTES, MIP- 1 alpha, MlP-l beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science. 272: 1955-1958.
  • Boshoff C, Y. Endo, P.D. Collins, Y. Takeuchi, J.D. Reeves, V.L. Schweickart, M.A. Siani, T. Sasaki, T.J. Williams, P. W. Gray, P.S. Moore, Y. Chang, and
  • Clark-Lewis I., Vo, L., Owen, P., Anderson, J. 1997. Chemical synthesis, purification, and folding of C-X-C and C-C chemokines. Methods Enzymol. 287. Crump, M.P., J.H. Gong, P. Loetscher, K. Rajarathnam, A. Amara, F. Arenzana- Seisdedos, J.L. Virelizier, M. Baggiolini, B.D. Sykes, and I. Clark-Lewis.
  • HIV- 1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.
  • HIV-1 entry cofactor functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 272:872-7.
  • CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell. 99:23-33.
  • Tachibana K., S. Hirota, H. Iizasa, H. Yoshida, K. Kawabata, Y. Kataoka, Y.
  • chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature. 393:591 -4. Thali, M., C. Furman, D.D. Ho, J. Robinson, S. Tilley, A. Pinter, and J. Sodroski.

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Abstract

The invention is directed to synthetic polypeptides that mimic chemokine function. These polypeptides are useful for the treatment of chemokine-related disorders, as well as the treatment and prevention of HIV infection. Novel synthetic methods for these polypeptides are also provided.

Description

SYNTHETIC MOLECULES THAT MIMIC CHEMOKINES
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
The subject matter of this application may in part have been funded by the National Institutes of Health, ROl GM57761. The government may have certain rights in this invention
FIELD OF THE INVENTION
The invention is drawn to non-natural molecules that mimic or antagonize chemokine function.
BACKGROUND
Chemokines, a group of more than 40 small peptides (generally 7-10 kDa in size), act as molecular beacons for the recruitment, activation, and directed migration of T lymphocytes, neutrophils and macrophages of the immune system, flagging pathogens and tumor masses for destruction. Chemokine (chemoattractant cytokine) receptors are a group of membrane proteins and belong to the superfamily of G-protein-coupled receptors (GPCRs) that possess seven transmembrane helices and transmit signals (as evidenced by calcium flux) from extracellular ligands to intracellular biological pathways via heterotrimeric G-proteins (Murphy, 1994). Based on the positions of two conserved cysteine residues in their N-termini, chemokines are divided into four subfamilies: CC, CXC, CX3C and C (Rossi and Zlotnik, 2000). The CXC and CC subfamilies selectively activate and recruit leukocytes to sites of inflammation. CXC chemokines mostly act on neutrophils, while CC chemokines stimulate other leukocytes, such as monocytes, lymphocytes, and basophils.
While defending the individual from invading pathogens and tumors, the immune system can also be the source of disease when improperly regulated. Inappropriate chemokine signaling can either promote infections when not properly triggered (Forster et al., 1999) or lead to diseases associated with defective chemokine signaling, including asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, and atherosclerosis (Rossi and Zlotnick, 2000). Other diseases and disorders that are associated with chemokines include cancer (especially breast cancer), multiple myeloma and non- Hodgkin's lymphoma. However, chemokine functions can be betrayed by viruses, becoming unwitting collaborators in infection. The human immunodeficiency virus (HIV) is such a virus, appropriating chemokine receptors, such as CCR5 and CXCR4, as co-receptors. Strains that co-opt CCR5 induce syncytia (masses of cells that have fused; these strains usually infect T cells), while those that appropriate CXCR4 do not induce syncytia and usually infect macrophages (Alkhatib, 1996; Deng et al., 1996; Dragic et al., 1996; Feng et al., 1996). Some HIV strains can use both CCR5 and CXCR4.
The HIV gpl 20 envelope protein initially binds the CD4 receptor of a target cell. Following a conformational change, the gpl20-CD4 complex then binds to a chemokine receptor, such as CCR5 or CXCR4. The virus then takes over the cellular machinery to replicate itself, which progeny then infect more cells of the host.
In addition to bodily defense functions, chemokines and chemokine receptors serve in other diverse roles. For example, CXCR4 is essential for vascularization of the gastrointestinal tract as well as hematopoiesis (Tachibana et al., 1998). When CXCR4 is prevented from binding ligand, such as stromal derived factor-1 (SDF-1 ), lethal deficiencies in these processes are observed. Fetal cerebellar development requires CXCR4 function for proper neuronal cell migration and patterning.
The CC chemokine ligands of CCR5 include macrophage mflammatory protein \ (MlP-l α), macrophage inflammatory protein l β (MlP-l β), and regulated on activation «oπnal Tcell expressed and secreted (R ANTES). Among these CCR5 ligands, RANTES and MlP-lα can bind to other CC chemokine receptors while MlP-lβ is most specific for CCR5 (Rossi and Zlotnik, 2000).
The CXCR4 receptor can bind the .vtromal cell-ύferived /actor- 1 (SDF-1 α), a CXC chemokine, its only known natural ligand, as well as the antagonistic viral macrophage inflammatory protein-Il (vMIP-II) encoded by the Kaposi's sarcoma-associated herpes virus (Moore et al., 1996). vMIP-II binds with high affinity to many CC and CXC chemokine receptors, including CXCR4 and CCR5, and inhibits cell entry of strains of HIV that use CXCR4 and CCR5 as coreceptors (Boshoff et al., 1997; Kledal et al., 1997).
Because chemokines and their receptors play pivotal roles in inflammation, lymphocyte development and HIV entry and infection, the ability to specifically manipulate the activity of these molecules will have enormous impact on ameliorating and halting diseases that currently have no satisfactory treatment. Most currently available antagonists and agonists of chemokine receptors suffer from diminished efficacy over time, as well as difficult and costly preparations. For example, several CCR5 receptor antagonists that block HIV-1 infection are available (Arenzana-Seisdedos et al., 1996; Nardese et al., 2001 ; Simmons et al., 1997); however, their specificity for CCR5 is in doubt. An example is the chemically modified analog of RANTES, aminoxypentane (AOP)-RANTES (Simmons et al., 1997). Because RANTES binds to a number of chemokine receptors in addition to CCR5, treatment with RANTES analogs that block the natural chemokines from functioning results in complications. The development of more specific inhibitors of CCR5 is indicated. For CXCR4, peptides and organic compounds which are unrelated to natural chemokines have antagonistic activity (Doranz et al., 1997; Murakami et al., 1997; Schols et al., 1997); but again, their specificity for CXCR4 are uncertain.
Identifying chemokine receptor antagonists and agonists is usually accomplished by brute force, an inefficient approach in which false positive and negative signals predominate. For example, high-throughput screening (HTS) methods for identifying antagonists of chemoattractant receptors, such as chemokine receptors, rely on detecting perturbations of downstream events, such as leukocyte cell migration. However, compounds that disrupt cell membranes or block events downstream mimic these outcomes, masquerading as candidate antagonists. Considerable effort is then required to distinguish the genuine antagonists from those compounds that yielded false positive signals. This task is formidable.
SUMMARY
In a first aspect, the invention is drawn to polypeptides having a sequence of SEQ ID NOs:2-9, 1 1 -17, and the dimers formed between SEQ ID NOs: 18 and 19 and between
SEQ ID NOs:20 and 21.
In a second aspect, the invention is drawn to polypeptides having a sequence at least 75% identical to a sequence of SEQ ID NOs:-9, 1 1 -17, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 ; the sequence identity may be 80%, 90%, 95%, 99% and 100%.
In a third aspect, the invention is drawn to treating and preventing HIV infection and spread by administering a polypeptide having a sequence at least 75% identical to a sequence of SEQ ID NOs:2-9, 1 1 -17, and the dimers formed between SEQ ID NOs:18 and 19 and between SEQ ID NOs:20 and 21 ; the sequence identity may be 80%, 90%, 95%), 99% and 100%).
In a fourth aspect, the invention is drawn to treating CXCR4- and CCR5-related diseases and conditions by administering a polypeptide having a sequence at least 75%> identical to a sequence of SEQ ID NOs:2-9, 1 1 -17, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 ; the sequence identity may be 80%,, 90%, 95%, 99%o and 100%..
In a fifth aspect, the invention is drawn to polypeptides having a sequence at least 75% identical to a sequence of SEQ ID NOs: 1 , 2, 5-8, 10,1 1, 15, 16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 , having at least one D-amino acid; the sequence identity may be 80%>, 90%., 95%), 99%> and 100%.
In a sixth aspect, the invention is drawn to treating and preventing HIV infection and spread by administering a polypeptide having a sequence at 75% with a sequence of SEQ ID NOs: l , 2, 5-8, 10, 11 , 15, 16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21, and having at least one D-amino acid; the sequence identity may be 80%., 90%, 95%, 99%> and 100%.
In a seventh aspect, the invention is drawn to polypeptides having a sequence at least 75% identical to a sequence of SEQ ID NOs: 1-4, 8-18, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 , and having at least one N methylated-amino acid; the sequence identity may be 80%, 90%, 95%, 99%> and 100%..
In an eighth aspect, the invention is drawn to polypeptides having at least 75% sequence identity to a sequence of SEQ ID NOs: 1 , 2, 8, 10, 15, 16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21., and having at least one D-amino acid and at least one N-methylated amino acid; the sequence identity may be 80%, 90%>, 95%, 99% and 100%.
In a ninth aspect, the invention is drawn to a method of determining treatment efficacy, using a polypeptide having at least 75% sequence identity to a sequence of SEQ ID NOs: 2-9, 1 1 -16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 ; sequence identity may be 80%, 90%>, 95%, 99%> and 100%.. In a tenth aspect, the invention is drawn to kits comprising a polypeptide having at least 75% sequence identity to a sequence of SEQ ID NOs:2-9 and 1 1 - 17, and the dimers formed between SEQ ID NOs:18 and 19 and between SEQ ID NOs:20 and 21 ; sequence identity may be 80%, 90%, 95%, 99% and 100%. In an eleventh aspect, the invention is drawn to a method of determining CXCR4 and CCR5 expression, using a polypeptide having at least 75%> sequence identity to a sequence of SEQ ID Nos:2-9 and 1 1 -17, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 ; sequence identity may be 80%), 90%., 95%, 99%. and 100%. The polypeptide may further contain a detectable label.
Other systems, methods, features, and advantages of the present invention will be or will become apparent to one with skill in the art upon examination of the detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims.
DETAILED DESCRIPTION
The present invention makes use of the discovery that synthetic molecules derived from SDF-1 α and vMIP-II can be used to mimic chemokine function and antagonize chemokine receptors. In some of these modified polypeptides, the N-termini of SDF-1 α and vMIP-II are replaced by various synthetic sequences containing modified amino acids and amino acid enantiomers; in other modified polypeptides, the N terminal sequences are exchanged between SDF- 1 α and vMIP-II. These synthetic SDF- 1 α and vMIP-II analogs are highly potent— and selective—I igands for chemokine receptors, such as CXCR4 and CCR5. They also effectively inhibit HIV entry and infection. In the course of performing computer modeling studies to determine the three - dimensional structure of both CCR5 and CXCR4 and their complexes with their respective natural ligands, a panel of site-specific mutants of CCR5 and CXCR4 were developed to determine functionally important residues critical for ligand binding, receptor signaling or co-receptor function in mediating HIV- 1 entry. Specific functional residues, particularly those clustered around the N-terminus and the second extracellular loop 2 (ECL2) of CCR5, were determined to be important in HIV-1 entry (Dragic et al., 1998; Farzan et al., 1998; Rabut et al., 1998; Zhou et al., 2000). Chimeras of CXCR4 and site-specific mutants were used to determine those functional residues of the N- terminus, ECL2 and ECL3 of CXCR4 required for HIV-1 co-receptor activity (Zhou et al., 2001). These studies led to the development of the VI peptide, derived from 16 residues of the N-terminus of vMIP-II (SEQ ID NO:20; Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly Tyr Gin), which also effectively blocks HIV entry (Huang, 2001 ).
The DV1 peptide (SEQ ID NO:21), the D-amino acid form of SEQ ID NO:20, was synthesized to further study these complexes. Surprisingly, this peptide not only specifically binds to CXCR4 with higher affinity (3-32 nM concentrations) than the L- isomer, but it also blocks HIV entry and replication (Huang, 2001 ; Zhou et al., 2002).
Because of the D-configuration, the peptide in serum lasts at least three times longer than the VI form (24 hours vs. 72 hours). No cytotoxicity at concentrations as high as 200 μM was observed. Peptides designed on natural chemokine ligand templates can therefore act as agonists and antagonists of CXCR4 (Crump et al., 1997; Heveker et al., 1998; Luo et al., 1999a; Luo et al., 1999b).
The N-termini of chemokines, such as SDF-lα and vMIP-II, are determinants for selective receptor binding. These sites are tolerant of changes in chirality; providing peptides with D-amino acids in the N-termini promises highly-specific, effective, and long-lasting therapeutics to treat chemokine-related disorders and to treat and prevent HIV infection and other diseases caused by infectious agents.
Novel SDF-1 a- and vMIP-II-based antagonists and agonists ofCXCR4 and CCR5
The sequences of the novel SDF-lα- and vMIP-II-based antagonists and agonists of CXCR4 and CCR5 are presented in Table 1 (SEQ ID NOs: l -9; 12-21). D-amino acids are indicated by underlining; N-methylated amino acids are indicated in boldface. The SDF-l α (SEQ ID NO: 1 ) and vMIP-II (SEQ ID NO: 10) sequences are not novel, but are given for comparison. Collectively, these novel polypeptides are referred to as ".synthetic chemokine mimic polypeptides" (SCMPs).
Table 1 Sequences of novel SDF-lα- and vMIP-II-based antagonists and agonists
Figure imgf000008_0001
Lys Pro Val Ser Leu Ser Tyr Arq Cys Pro Cys Arg Phe Phe Glu Ser
1 5 10 15
His Val Ala Arg Ala Asn Val Lys His Leu Lys He Leu Asn Thr Pro 20 25 30
D(l-8)-SDF-lα 3 Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin 35 40 45
Val Cys lie Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys 50 55 60
Ala Leu Asn Lys
65
Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser
1 5 10 15
His Val Ala Arg Ala Asn Val Lys His Leu Lys He Leu Asn Thr Pro 20 25 30
Dl-SDF-lα 4 Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin 35 40 45
Val Cys He Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys 50 55 60
Ala Leu Asn Lys
65
Lys Ala Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser
1 5 10 15
His Val Ala Arg Ala Asn Val Lys His Leu Lys He Leu Asn Thr Pro 20 25 30
NMeA-2-SDF-lα 5 Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin 35 40 45
Val Cys He Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys 50 55 60
Ala Leu Asn Lys
65
Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser
1 5 10 15
His Val Ala Arg Ala Asn Val Lys His Leu Lys He Leu Asn Thr Pro 20 25 30
NMeV-3-SDF-lα 6 Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin 35 40 45
Val Cys He Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys 50 55 60
Ala Leu Asn Lys
65
Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser
1 5 10 15
His Val Ala Arg Ala Asn Val Lys His Leu Lys He Leu Asn Thr Pro 20 25 30
NMeL-5-SDF-lα 7 Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin 35 40 45
Val Cys He Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys 50 55 60
Ala Leu Asn Lys
65
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Pro Cys Arg Phe Phe
1 5 10 15
Glu Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys He Leu Asn 20 25 30 vMIP-II(l-10)-SDF-lα 8 Thr Pro Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn 35 40 45
Arg Gin Val Cys He Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu 50 55 60
Glu Lys Ala Leu Asn Lys
65 70
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Pro Cys Arg Phe Phe 1 5 10 15
Glu Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys He Leu Asn 20 25 30
D- Pro vMIP-II(l-10)-SDF-lα 9 Thr Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn 35 40 45
Arg Gin Val Cys He Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu 50 55 60
Glu Lys Ala Leu Asn Lys 65 70
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly Tyr Gin 1 5 10 15
Lys Arg Pro Leu Pro Gin Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30 vMIP-II1 10 Gin Leu Cys Ser Lys Pro Gly Val He Phe Leu Thr Lys Arg Gly Arg 35 40 45
Gin Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu Met Gin 50 55 60
Gin Leu Pro Val Thr Ala Arg
65 70
Cys Cys Leu Gly Tyr Gin Lys Arg Pro Leu Pro Gin Val Leu Leu Ser
1 5 10 15
Ser Trp Tyr Pro Thr Ser Gin Leu Cys Ser Lys Pro Gly Val He Phe 20 25 30
Δ(l- 10)-vMIP-II 11
Leu Thr Lys Arg Gly Arg Gin Val Cys Ala Asp Lys Ser Lys Asp Trp
35 40 45
Val Lys Lys Leu Met Gin Gin Leu Pro Val Thr Ala Arg 50 55 60
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly Tyr Gin
1 5 10 15
Lys Arg Pro Leu Pro Gin Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30
D-(l -10)-vMIP-II 12 Gin Leu Cys Ser Lys Pro Gly Val He Phe Leu Thr Lys Arg Gly Arg 35 40 45
Gin Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu Met Gin 50 55 60
Gin Leu Pro Val Thr Ala Arg
65 70
Lys Pro Val Ser Leu Ser Tyr Arg Gly Gly Cys Cys Leu Gly Tyr Gin
1 5 10 15
Lys Arg Pro Leu Pro Gin Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30
D-'SDF1α(1 -8)-GG-vMIP-
13 Gin Leu Cys Ser Lys Pro Gly Val He Phe Leu Thr Lys Arg Gly Arg II 35 40 45
Gin Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu Met Gin 50 55 60
Gin Leu Pro Val Thr Ala Arg
65 70
Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Leu Gly Tyr Gin 1 5 10 15
Lys Arg Pro Leu Pro Gin Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30
D-MIP-lβ(l-10)-vMIP-II 14 Gin Leu Cys Ser Lys Pro Gly Val He Phe Leu Thr Lys Arg Gly Arg 35 40 45
Gin Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu Met Gin
50 55 60
Gin Leu Pro Val Thr Ala Arg 65 70
Lys Pro Val Ser Leu Ser Tyr Arg Gly Gly Cys Cys Leu Gly Tyr Gin
1 5 10 15
Lys Arg Pro Leu Pro Gin Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30
SDF-1α(l-8)-GG-vMIP-II 15 Gin Leu Cys Ser Lys Pro Gly Val He Phe Leu Thr Lys Arg Gly Arg 35 40 45
Gin Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu Met Gin 50 55 60
Gin Leu Pro Val Thr Ala Arg 65 70
Ala Pro Met Gly Ser Asp Pro Pro Thr Ala Cys Cys Leu Gly Tyr Gin 1 5 10 15
Lys Arg Pro Leu Pro Gin Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30
MIP-lβ(l-10)-vMIP-II 16 Gin Leu Cys Ser Lys Pro Gly Val He Phe Leu Thr Lys Arg Gly Arg 35 40 45
Gin Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu Met Gin 50 55 60
Gin Leu Pro Val Thr Ala Arg 65 70
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly Tyr Gin
1 5 10 15
Lys Arg Pro Leu Pro Gin Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30
FAM-D(l-10)-vMIP-II 17 Gin Leu Cys Ser Lys Pro Gly Val He Phe Leu Thr Lys Arg Gly Arg 35 40 45
Gin Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu Met Gin 50 55 60
Gin Leu Pro Val Thr Ala Arg Ala Cys Ala Lys-Aca-K ( FAM) -COCH2 3 65 70 75
Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser
1 5 10 15
His Val Ala Arg Ala Asn Val Lys His Leu Lys He Leu Asn Thr Pro
20 25 30
Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin 35 40 45
Val Cys He Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys 50 55 60
Ala Leu Asn Lys Xaa Xaa Xaa Xaa Xaa Xaa Cys Lys Pro Val Ser Leu
65 70 75 80
SDF-lα dimer23 18,19
Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser His Val Ala Arg Ala 85 90 95
Asn Val Lys His Leu Lys He Leu Asn Thr Pro Asn Cys Ala Leu Gin 100 105 110
He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin Val Cys He Asp Pro 115 120 125
Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys Ala Leu Asn Lys Xaa
130 135 140
Xaa Xaa Xaa Xaa Xaa Cys
145 150
Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser 1 5 10 15
His Val Ala Arg Ala Asn Val Lys Hi s Leu Lys He Leu Asn Thr Pro 20 25 30
Asn Cys Ala Leu Gin He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin 35 40 45
Val Cys He Asp Pro Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys 50 55 60
Ala Leu Asn Lys Xaa Xaa Xaa Xaa Xaa Xaa Cys Lys Pro Val Ser Leu 65 70 75 80 vMIP-II dimer24 20, 21
Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu Ser His Val Ala Arg Ala 85 90 95
Asn Val Lys Hi s Leu Lys He Leu Asn Thr Pro Asn Cys Ala Leu Gin 100 105 110
He Val Ala Arg Leu Lys Asn Asn Asn Arg Gin Val Cys He Asp Pro 115 120 125
Lys Leu Lys Trp He Gin Glu Tyr Leu Glu Lys Ala Leu Asn Lys Xaa 130 135 140
Xaa Xaa Xaa Xaa Xaa Cys 145 150
2Xaa. 8-aminocaprylιc acid
3Dimer is formed by a disulfide bridge between Cys75 of SEQ ID NO.18 and the Cys75 of SEQ ID NO: 19
"Dimer is formed by a disulfide bridge between Cys75 of SEQ ID NO:20 and the Cys75 of SEQ ID NO:21
These novel molecules, based on the chemokines SDF-lα and vMIP-II are biologically active, specifically binding their targeted receptors, CXCR4 and CCR5, with high affinity and affecting signaling (Tables 2 and 3).
Table 2 Novel SDF-1 α-based antagonists and agonists of CXCR4
Figure imgf000019_0001
Table 3 Novel vMIP-II based antagonists and agonists of CXCR4 and CCR5
Figure imgf000020_0001
Uses for SDF-1 a- and vMIP-II-based D-amino acid and N-methylated analogs
Because of their biological function, the molecules based on the SDF-lα and vMIP-II template are suitable for treating any disease, disorder or condition that involves CXCR4 and/or CCR5. Significantly, these molecules may be used to block HIV entry and viral spread. To treat those diseases, disorders or conditions that result in part because of increased CXCR4 or CCR5 expression or activity, antagonists are administered. In those conditions where the activity or expression of these receptors is decreased, agonists are administered. Furthermore, because these analogs block receptor binding, they are useful in prophylactic methods that prevent infection and spread of HIN In addition to the treatment and prevention of HIV infection, the molecules are useful for modulating biological processes, such as vascularization of the gastrointestinal tract (during development), hematopoiesis, fetal cerebellar development, neuronal cell migration. Diseases and conditions that the molecules can be used to treat include asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, atherosclerosis, cancer, multiple myeloma, non-Hodgkin's lymphoma and viral infection.
Definitions
"Isolated," when referred to a molecule, refers to a molecule that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that interfere with diagnostic or therapeutic use.
A "polypeptide" is a protein having at least two amino acids. The amino acids may be those usually incorporated into mammalian polypeptides (such as human SDF-lα or viral vMIP-II), or the amino acids may be modified in some way, including derivatives and enantiomers (such as SDF-lα containing D-amino acids, or Ν-methylated residues). A "purified polypeptide" is one that is purified to homogeneity (1) by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain, or (2) by high performance liquid chromatography (HPLC). An "active polypeptide" retains a biological and/or an immunological activity of an SCMP. Immunological activity refers to the ability to induce antibody production against an antigenic epitope possessed by a SCMP; biological activity refers to a function possessed by the SCMP, excluding immunological activity. Biologically active portions of SCMP include peptides comprising amino acid sequences sufficiently homologous to, or derived from, the amino acid sequence of an SCMP (SEQ ID NOs:3-9, 12-21) that include fewer amino acids than the given SCMP sequence and exhibit at least one activity of an SCMP. Biologically active portions comprise a domain or motif with at least one activity of an SCMP. A biologically active portion of an SCMP may have an amino acid sequence shown in SEQ ID NOs:3-9, 12-21, or substantially homologous to SEQ ID NOs:3-9, 12- 21 and retains the functional activity of an SCMP polypeptide, yet differs in amino acid sequence.
Variants
The amino acid sequences of SCMPs can be varied, such that their function is not altered. For example, amino acid substitutions at "non-essential" residue positions can be made in SEQ ID NOs:3-9, 12-21. A "non-essential" amino acid residue is one that can be altered from a given SCMP sequence without altering its biological activity; an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the SCMPs are particularly non-amenable to alteration.
Useful conservative substitutions are shown in Table A. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the invention so long as the substitution does not materially alter the biological activity of the compound. If such substitutions result in a change in biological activity, then more substantial changes, indicated in Table B as exemplary, are introduced and the products screened for SCMP activity. Table A Preferred substitutions
Figure imgf000023_0001
Residues are divided into groups based on common side-chain properties as denoted in Table B. Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more preferably into non-conserved sites. Table B Amino acid classes
Figure imgf000024_0001
In general, an SCMP variant preserves SCMP-like function and includes any variant in which residues at a particular position in have been substituted by other amino acids, and further includes inserting an additional residue or residues between two residues of the parent polypeptide, as well as deleting one or more residues from the parent sequence. Preferably, the substitution is a conservative substitution (Table A).
"SCMP polypeptide variant" means an active SCMP having at least 75% amino acid sequence identity with a full-length SCMP sequence, taking into account amino acid enantiomers, where a D-isomer is not an identity to the corresponding L-isomer (e.g., D- Lys is not counted as an identity with L-Lys). For example, SCMP variants include those wherein one or more amino acid residues are added or deleted at the N- or C- terminus of the full-length amino acid sequence. An SCMP polypeptide variant will have at least about 75% amino acid sequence identity, preferably at least about 80%) amino acid sequence identity, more preferably at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acid sequence identity and most preferably at least about 99%) amino acid sequence identity with a full- length sequence of an SCMP (SEQ ID NOs:2-9, 11-16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21). "Percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues that are identical with amino acid residues in an SCMP sequence in a candidate sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and, if necessary, gaps are introduced to achieve the maximum %> sequence identity; conservative substitutions are not considered as part of the sequence identity. Publicly available computer software, such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) can be used to align polypeptide sequences. Those skilled in the art will determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
When amino acid sequences are aligned, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as:
% amino acid sequence identity = X/Y ' 100 where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and
Y is the total number of amino acid residues in B.
If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the %> amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
Chimeric and fusion polypeptides
Fusion polypeptides are useful in, for example, cell-localization assays and bioassays. An SCMP "chimeric polypeptide" or "fusion polypeptide" is a SCMP polypeptide fused to a non-SCMP polypeptide. A non-SCMP polypeptide is not substantially homologous to SCMP. An SCMP fusion polypeptide may include any portion of an entire SCMP, including any number of biologically active portions. Some exemplary fusions are presented in Table C.
Figure imgf000026_0001
An SCMP may also be modified by N-methylation on at least one residue, as well as substituting the D-amino acid enantiomer for an L-amino acid enantiomer. An SCMP may have both D-amino acids as well as N-methylated amino acids.
Therapeutic applications of SCMP Antagonists and agonists
"Antagonist" includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of an endogenous SCMP. "Agonist" includes any molecule that mimics a biological activity of an endogenous SCMP. Examples of agonists include the polypeptides of SEQ ID NO: 15 (of CXCR4) and SEQ ID NO: 16 (of CCR5). Examples of antagonists include SEQ ID NOs:3-9 and 12 (of CXCR4) and SEQ ID NO: 12 (of CCR5).
The term "therapeutically effective amount" means the amount of the compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a clinician, or that is sufficient to prevent development of or alleviate to some extent one or more of the symptoms of the disease or condition being treated.
Pharmaceutical compositions The SCMPs and their derivatives, fragments, analogs and homologs, can be incorporated into pharmaceutical compositions. Such compositions typically comprise a SCMP and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration (Remington and Gennaro, 2000). Preferred examples of such carriers or diluents include water, saline, Finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non- aqueous vehicles, such as fixed oils, may also be used. Except when a conventional media or agent is incompatible with an active compound, use of these compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. More than one SCMP may be incorporated into a composition. General considerations
A pharmaceutical composition is formulated to be compatible with the intended route of administration, including intravenous, intradermal, subcutaneous, oral, transdermal, transmucosal and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
Injectable formulations Injection provides direct access to the immune system. Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile. Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures. Proper fluidity can be maintained, for example, by using a coating such as lecithin; by maintaining the required particle size in the case of dispersion, and by using surfactants. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can control microorganism contamination. Isotonic agents, such as sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition. Compositions that delay absorption include agents such as aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an SCMP) in an appropriate solvent with one or more ingredient, followed by sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and any other required ingredients. Sterile powders for the preparation of sterile injectable solutions include vacuum- and freeze-drying that yield a powder containing the active ingredient and any desired ingredient from a sterile solution.
Oral compositions Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or STEROTES; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
Compositions for inhalation For administration by inhalation, the compounds are delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, e.g., a gas such as carbon dioxide.
Systemic administration Systemic administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, penetrants that can permeate the target barrier(s) are selected. Transmucosal penetrants include detergents, bile salts and fusidic acid derivatives. Nasal sprays or suppositories can be used for transmucosal administration. For transdermal administration, the active compounds are formulated into ointments, salves, gels or creams.
The compounds can also be prepared as suppositories (with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
Carriers
In one embodiment, the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid (ALZA Corporation; Mountain View, CA and NOVA Pharmaceuticals, Inc.; Lake Elsinore, CA). Liposomal suspensions can also be used as pharmaceutically acceptable carriers (Eppstein, 1985).
Unit dosage
Oral formulations or parenteral compositions in unit dosage form can be created to facilitate administration and dosage uniformity. Unit dosage form refers to physically discrete units suited as single doses for a subject to be treated, containing a therapeutically effective quantity of active compound in association with the required pharmaceutical carrier. The specification for unit dosage forms are dictated by, and directly dependent on, the unique characteristics of the active compound and the particular desired therapeutic effect, and the inherent limitations of compounding the active compound.
Dosage
The pharmaceutical compositions and methods of the present invention may further comprise other therapeutically active compounds that are usually applied in the treatment of chemokine (i.e., CXCR4 and CCR5) pathologies, such as HIV infection. In the treatment or prevention of conditions which require modulation of a chemokine receptor, an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Because the half-life of D-amino acids, many of these compounds can be supplied in even lower doses, such as 0.01 μg to 1 μg/kg per day; preferably 0.1 to 0.5 μg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 0.1 to 1000 milligrams of the active ingredient, particularly 0.1, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to a patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.
The specific dose level and frequency of dosage for any particular patient may be varied and depends upon a variety of factors, including the activity of the specific compound, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the subject undergoing therapy. In the case of HIV infection, dosage may also depend on the aggressiveness of the particular HIV that has infected an individual.
Kits for pharmaceutical compositions Pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. When supplied as a kit, the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately permit long-term storage without losing activity of the components. Kits may also include reagents in separate containers that facilitate the execution of a specific test, such as diagnostic tests or tissue-typing. When kits are assembled for detection methods, a control may also be provided. Positive and negative controls may be included. For example, in the determining if an SCMP would be effective in treating a disorder, a kit could be provided that includes at a labeled SCMP and a polypeptide of SEQ ID NO: 1 or 10 that is also labeled. A suitable negative control would include a scrambled SCMP or some other polypeptide known not to bind the target receptor. When the test is executed, if a signal is observed in the test sample and in the positive control and not in the negative control, then the user can have confidence in the result.
Containers or vessels
The reagents included in kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container. For example, sealed glass ampules may contain lyophilized SCMP or buffer that have been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, etc.. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, etc.
Instructional materials
Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail. Prognostic assays
Diagnostic methods can be used to identify subjects having, or at risk of developing, a disease or disorder related to CXCR4 or CCR5, including HIV infection. As used herein, a CXCR4 or CCR5-related desease, disorder or condition refers to one which results from and/or is mediated, at least in some part, by an activity (or lack thereof) of CXCR4 or CCR5. Prognostic assays can be used to determine whether a subject can be administered a polypeptide of SEQ ID NOs:2-9, 1 1 - 16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 to treat a disease or disorder related to CXCR4 or CCR5. Such methods can be used to determine whether a subject can be effectively treated with SEQ ID NOs:2-9, 11 -16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21. Methods for determining whether an HIV-infected subject can be effectively treated with a SCMP include obtaining a test sample having the HIV virus and testing the ability of the SCMPs to inhibit HIV entry. Preferably, the cells that are tested are also those of the subject; however, cells from cell lines may also be used to facilitate the tests, while using the infecting virus.
In another test to predict treatment efficacy, the class(es) of cells infected with HIV or responsible at least in part for the disorder, disease or condition are obtained in a sample from the subject. Purification of the target cell type(s) is not necessary if they are morphologically distinguishable or can be labeled with a cell-specific marker or stain. The cells are then either directly incubated with a labeled polypeptide of SEQ ID NO:2-9, 1 1 -16, or the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21, such as with FAM or other label (e.g., SEQ ID NO: 17), washed and prepared for analysis, or preserved by applying a fixative, treating the samples to quench any background, applying the labeled polypeptide and washing the sample, and then preparing for analysis. Microscopic and spectrophotometric methods of observation are appropriate. Binding of the labeled polypeptide to the target cell type indicates that treatment with that polypeptide, without the label, will be effective.
Other suitable labels include fluorescent moieties, such as fluorescein isothiocyanate; fluorescein dichlorotriazine and fluorinated analogs of fluorescein; naphthofluorescein carboxylic acid and its succinimidyl ester; carboxyrhodamine 6G; pyridyloxazole derivatives; Cy2, 3 and 5; phycoerythrin; fluorescent species of succinimidyl esters, carboxylic acids, isothiocyanates, sulfonyl chlorides, and dansyl chlorides, including propionic acid succinimidyl esters, and pentanoic acid succinimidyl esters; succinimidyl esters of carboxytetramethylrhodamine; rhodamine Red-X succinimidyl ester; Texas Red sulfonyl chloride; Texas Red-X succinimidyl ester; Texas Red-X sodium tetrafluorophenol ester; Red-X; Texas Red dyes; tetramethyl rhodamine; lissamine rhodamine B; tetramethylrhodamine; tetramethyl rhodamine isothiocyanate; naphthofluoresceins; coumarin derivatives; pyrenes; pyridyloxazole derivatives; dapoxyl dyes; Cascade Blue and Yellow dyes; benzofuran isothiocyanates; sodium tetrafluorophenols; 4,4-difluoro-4-bora-3a,4a- diaza-s-indacene. Suitable labels further include enzymatic moieties, such as alkaline phosphatase or horseradish peroxidase; radioactive moieties, including 35S and l35I- labels; avidin (or streptavidin)-biotin-based detection systems (often coupled with enzymatic or gold signal systems); and gold particles. In the case of enzymatic-based detection systems, the enzyme is reacted with an appropriate substrate, such as 3, 3'- diaminobenzidine (DAB) for horseradish peroxidase; preferably, the reaction products are insoluble. Gold-labeled samples, if not prepared for ultrastructural analyses, may be chemically reacted to enhance the gold signal; this approach is especially desirable for light microscopy. The choice of the label depends on the application, the desired resolution and the desired observation methods. For fluorescent labels, the fluorophore is excited with the appropriate wavelength and the sample observed using a microscope, confocal microscope, or FACS machine. In the case of radioactive labeling, samples are contacted with autoradiography film, and the film developed; alternatively, autoradiography may also be accomplished using ultrastructural approaches. Alternatively, radioactivity may be quantified using a scintillation counter.
Prophylactic methods The invention provides methods for preventing in a subject a disease or condition associated with aberrant CXCR4 or CCR5 expression or activity, by administering at least one SCMP. Subjects at risk for such a disease or HIV infection are administered the SCMP occur prior to the manifestation of symptoms or to stave off HIV infection.
Syntheses ofSMCPs (see Examples 1-5) Synthesis exemplified by vMIP-II
Two methods of synthesis were applied to the generation of the chemokines and analogs: (1) native chemical ligation (NCL) of fragments (Dawson, 2000), and (2) total stepwise polypeptide chemical synthesis (TSPCS).
Chemical ligation of fragments
For chemical ligation, preparation of a chemokine or analog consists of three main steps: (1) solid phase synthesis of two peptide sequences corresponding to the N- and C-terminal portions of the protein; (2) chemical ligation of these two peptide segments to form a complete protein sequence; and (3) purification and folding of the final protein product with proper tertiary structure and biological function. The synthesis of wild-type vMIP-II is presented as a paradigm; specific syntheses for the polypeptides of SEQ ID NOs:2-9, 1 1 -16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21 , can be found in Examples 1 -5. First, the N- (1 -34) and C-termini (35-71) of vMIP-II are synthesized. The N-terminal region (1 -34) peptide is synthesized as a C-terminal thioester peptide using a sulfonamidobutyl resin. The last C-terminal amino acid, 9-flourenylmethloxycarbonyl (Fmoc)-Leu, is incorporated to the resin by using Benzotriazol-1 -yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) in the presence of N,N- di-isopropylethylamine (DIEA) as the coupling reagent. After incorporating all amino acids, N-terminal Fmoc-protection is removed, and the peptidyl resin is activated with iodoacetonitrile to prepare cyanomethylsulfonamide peptidyl resin. The peptide is cleaved from the resin as a thioester by adding benzylmercapten in tetrahydrofuran (THF). For the preparation of the C-terminal part (35-71), after incorporating the first N-terminal residue Cys (position 35), the peptide is cleaved and purified by HPLC. The peptide is synthesized in such a way that the thiol group of this Cys-35 is selected for the chemoselective ligation. Using native chemical ligation, which allows a mutual chemoselective reaction of certain uniquely reaction functionalities incorporated chemically to each peptide (Clark-Lewis, 1997; Dawson, 1994), the unprotected 1 -34 and 35-71 peptides undergo a chemoselective reaction that results in the formation of the native peptide bond at the ligation site. The thiol ligation is initiated by a chemoselective reaction between the N-terminal Cys-35 thiol of the 35-71 peptide and the thioester functionality of the C-terminal Leu-34 residue of the 1 -34 peptide. Exchange of the peptide thioester bond with the thiol side change yields a thioester-linked intermediate as the initial covalent product. This thioester-linked intermediate then spontaneously rearranges, involving an S- to N-acyl transfer, to generate a native peptide bond between Leu-34 and Cys-35. The three other Cys residues can produce thioesters that are capable of further thiol exchange; however, these do not undergo further reaction because only the N-terminal Cys residue can rearrange to form an amide bond. This reversible exchange can be facilitated by adding thiol additives, such as a thiophenol and benzyl mercaptan mixture. A thiophenol additive maintains a high concentration of highly reactive phenyl thioester peptides, whereas the benzyl mercaptan prevents the cysteine side chains from forming unreactive disulfide bonds during ligation. These additives also help the longer storage of the weakly activated thioester peptide until they are converted to more reactive phenyl thioester peptides during chemical ligation. The final vMIP-II product is purified by HPLC and folded in 2 M guanidine HCl, 100 mM Tris, pH 8 at room temperature in the presence of air. The purity and identity are then determined by analytical HPLC and mass spectrometry.
Total stepwise polypeptide chemical synthesis (TSPCS) of chemokines and analogs
SCMPs can be readily chemically synthesized in vitro in their entirety using stepwise polypeptide chemistry. For example, polypeptide synthesis may be carried out in a stepwise manner on a solid phase support using an automated polypeptide synthesizer, such as a Rainin Symphony Peptide Synthesizer, Advanced Chemtech Peptide Synthesizer, Argonaut Parallel Synthesis System, and preferably, an Applied Biosystems Peptide Synthesizer. In a preferred embodiment, the peptide synthesizer instrument combines the Fmoc chemistry with 1 -Hydroxybenzotriazole/0-(1H- benzotriazole- 1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate/DIEA (HOBt/HBTU/DIEA) activation to perform solid-phase peptide synthesis.
Synthesis starts with the C-terminal amino acid, wherein the carboxyl terminus is covalently linked to an insoluble polymer support resin. Useful resins can load 0.1 mmol to 0.7 mmol of C-terminal amino acid per gram of resin; display resistance to the various solvents and chemicals used during a typical synthetic cycle, such as dichloromethane (DCM), N,N-dimethylformamide (DMF), N- methylpyrrolidone (Ν P), N,N-dimethylamine (DMA), 1 -Hydroxybenzotriazole (HOBt), 2-( 1 -H-Benzotriazol- 1-yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate (HBTU), DIEA, methanol (MeOH), or water; and be amenable to continuous flow or batch synthesis applications. When using Fmoc-protected α-amino acids, an acid labile and base stable resin, such as an ether resin, is preferred. Preferred resins include /?-Benzyloxybenzyl Alcohol resin (HMP resin), PEG co-Merrifield resin, ΝovaSyn TGA® resin (Νovabiochem), 4-sulfamylbutyryl aminomethyl (AM) resin, and cross-linked ethoxylate acrylate resin (CLEAR) amide resin. Amino acid- coupled resins are commercially available from a number of different sources, although such coupled resins may be prepared using known procedures in the art.
The Ν-terminus of the resin-coupled amino acid (or polypeptide) is chemically-protected by an Fmoc group that is removed prior to the addition of the next Ν-terminal amino acid reactant. The Fmoc group is a base labile protecting group that is easily removed by concentrated solutions of amines, such as 20-55% piperidine, in a suitable solvent, such as N-methylpyrrolidone (ΝMP) or NN- dimethylformamide (DMF). Other useful amines for Fmoc deprotection include tris (2-aminoethyl) amine, 4-(aminomethyl)piperidine, tetrabutylammonium fluoride, and l,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Complete removal of the Fmoc group from the Ν-terminus is important so that all resin-coupled polypeptide chains effectively participate in each coupling cycle; otherwise, polypeptide chains of heterogeneous length and sequence will result. Following base-catalyzed removal of the Fmoc group, the resin is extensively washed with a suitable buffer to remove the base catalyst.
The side chains of many amino acids contain chemically reactive groups, such as amines, alcohols, or thiols. These side chains must be additionally protected to prevent undesired side-reactions during the coupling step. Preferred side chain protecting groups are base-stable, more preferably, both base-stabile and acid-labile. Table D provides a preferred set of side chain protection groups for this category of amino acids that are used in one example.
Table D Side chain protection reagents
Figure imgf000039_0001
The carboxylate group of the incoming Fmoc-protected amino acid is activated in order to achieve efficient chemical coupling to the N-terminus of the resin-bound polypeptide. Activation is typically accomplished by reacting an Fmoc- protected amino acid with a suitable reagent to yield a reactive ester. Preferred activated esters include the pentafluorophenyl (OPfp) ester and the 3-hydroxy-2,3- dihydro-4-oxo-benzo-triazone (ODhbt) ester, more preferably the OBt ester, and even more preferably, the OAt ester derived from l -hydroxy-7-azabenzotriazole (HOAt). The coupling reactions may be done in situ using activating reagents ,such as DCC, BOP, BOP-CI, or TBTU, preferably HBTU or 0-(7-azabenzotrizol-l-yl)-l, 1,3,3, tetramethyluronium hexafluorophosphate (HATU). Preferred coupling reactions included a mixture comprising HOBt and HBTU, and more preferably, HOBt, HBTU, and DIEA. For N-methyl amino acids, the preferred coupling conditions use bromo- tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) as the only coupling reagent, and the coupling reaction is performed manually in DCM with DIEA present under N . The Fmoc-protected amino acid is present in molar excess to the polypeptide coupled to the resin, preferably, in a five-fold molar excess. For coupling reactions that proceed with a slow rate, the coupling reactions are repeated one or more times (double or multiple coupling) to ensure that all resin-bound polypeptide has undergone a successful addition reaction with the activated Fmoc-amino acid. For incomplete coupling reactions, any unreacted N-terminal residues are capped using a suitable capping reagent.
Following the coupling reaction, the resin support is washed to remove the unreacted Fmoc-amino acids and coupling reagents. The resin is then subjected to a new cycle of base-catalyzed removal of the N-terminal Fmoc group to prepare the polypeptide for another amino acid addition. After the desired polypeptide has been synthesized, the resin is subjected to base-catalyzed removal of the remaining Fmoc protection group. The polypeptide-coupled resin is washed to remove the base and subsequently treated with acid to remove any amino acid side chain protecting groups and to release the polypeptide chain from the resin support. The preferred acid is a strong acid, such as trifluoroacetic acid (TFA) in the presence of suitable scavengers, such as reagent K [TFA: thioanisole: ethanedithiol: phenol: water (82.5:5:2.5:5:5)].
The polypeptide is subsequently separated from the resin by filtration and optionally washed repeatedly with a suitable solvent, such as DCM/DMF. The polypeptide may be optionally desalted through ultrafiltration using a membrane with a suitable MW cutoff. The polypeptide may be precipitated from solution using a suitable solvent, such as cold methyl t-butyl ether or t-butylethylether, and the precipitate optionally washed with a suitable solvent, such as cold ether and dried. The polypeptide may be further purified using a suitable chromatographic means, such as hydrophobic chromatography using a CI 8 resin and an appropriate chromatographic buffer system, such as TFA/water/acetonitrile. The purity of the peptide optionally may be analyzed by mass spectrometry, such as MALDI-MS, analytical HPLC, amino acid analysis or sequencing.
EXAMPLES
Materials for all Examples
Unless otherwise specified, reagents were from Acros-Fisher Scientific (Pittsburgh, PA) and Sigma-Aldrich (St. Louis, MO); only highest purity grade preparations were used.
4-(hydroxymethyl)phenoxymethylpolystyrene (HMP) resin, Fmoc-Lys(Boc)- NovaSyn® TGA resin, N-(9-fluorenylmethoxycarbonyl) (Fmoc)-L-amino acids, solvents and other reagents for SPPS were from Novabiochem (San Diego, CA), polypeptide International (Louisville, KY) or Applied Biosystems (Foster City, CA). Fmoc-8-aminocaprylic acid was obtained from Ana Spec (San Jose, CA). CLEAR amide support was procured from polypeptide International (Louisville, KY). I- SDF- l α (Perkin Elmer Life Science; Boston, MA) had a specific activity of 2200 Ci/mmol.
293 cells (gift from Dr. R. Doms, University of Pennsylvania (also available from American Type Tissue Collection (ATCC); Manassas, VA) and Sup-Tl cells (provided by the National Institutes of Health AIDS Reagent Program; Rockville, MD; also available from ATCC) were maintained in Dulbecco's Modified Eagle's Medium (DMEM)/10% fetal bovine serum (FBS). CEM-T4 cells were available from the American Type Culture Collection (Manassas, VA).
Unless otherwise specified, all polypeptides were synthesized in a stepwise manner on an Applied Biosystems model 433A automated polypeptide synthesizer using commercially available solid phase support resins and Fmoc chemistry with HOBt/HBTU/DIEA activation chemistry. Although the particular resins chosen and variations in the synthetic routes that were used in the preferred embodiments were described in Examples 1 -4, the particular selections were not meant to limit the present invention.
High Performance Liquid Chromatography (HPLC)
Both analytical and semi-preparative reversed-phase HPLC were performed on a Waters 600 system using Vydac C4 or CI 8 columns (5 μm, 0.46 x 15 cm for analytical preparations, and 10 μm, 1.0 x 25 cm for semi-preparative preparations). Linear gradients of B (80% CH3CN, 20% H20, 0.1 % TFA) in A (0.1 % TFA in H20) were used in both analytical and semi-preparative HPLC. The flow rate for analytical preparations was 1.0 ml/min (analytical) and 3.0 ml/min for semi-preparative preparations. Detection was set at 214 nm and 280 nm. Unless otherwise stated, all polypeptides were purified on either an analytical or semi -preparative scale according to these chromatographic programs.
Matrix-assisted laser desorption/ioni∑ation mass spectrometry (MALDI-MS) Mass spectra were obtained with a Voyager™ Workstation (Applied Biosystems, Foster City, CA). Polypeptide masses were determined from experimental mass to charge ratios (m/z) of all observed charge states of a polypeptide. Theoretical masses were calculated using SynthAssistI M software (Applied Biosystems). Unless otherwise stated, all polypeptides were analyzed according to this procedure.
Example 1 Main chain modified SDF-1 a analogs SEQ ID NOs:4-9 by TSPCS
Summary
When TSPCS was carried out on a 0.1 mmol scale using HMP as the coupling resin, HPLC and Maldi-MS analyses of the resultant polypeptide reveal the absence of deletions after assembling the C-terminal 14 amino acids onto the resin. Several deletions were evident, however, in the synthesis of the next 11 amino acids, significantly in the triple N region. Increasing the coupling reaction time to permit improved coupling efficiency in this region reduced these deletions.
When the synthesis was carried out using PEG resin with longer coupling reaction times, a product of molecular mass of 3652.3 Da was observed. The polypeptides of the desired length were obtained following the completion of coupling reactions that resulted in the addition Cys-34, Lys-24, and the ultimate N- terminal residue. The 3652.3 Da product results from its cyclization, involving carboxyl group from a side chain-protecting group, most likely lysine. To avoid cyclization, Fmoc-(Hmb)Ala-OH was incorporated at position 40 during the synthesis.
For coupling reactions that link an amino acid next to an N-methyl amino acid in the preparation of N-terminal thioester segments in NCL synthesis, PyBroP was a preferred coupling reagent because it was more efficient than HBTU/HOBt.
Upon completion of SDF-lα and its N-methyl derivatives, the polypeptides were cleaved from the resin and side chains deprotected by reagent K in the presence of 2.5% of TIPS. HPLC-purified linear polypeptides were then oxidized in air, and the folded polypeptides were re-purified by HPLC. Fractions were analyzed by RP- HPLC and MALDI-MS, and those containing the pure folded polypeptide were pooled and freeze-dried. The polypeptides were then dissolved in water, their concentration determined using UV spectrometer, and stored at -20° C. TSPCS
Assembly of the C-terminal 29 amino acids of SDF-lα was carried out using 0.05 mmol of Fmoc-Lys(Boc)-NovaSyn® TGA resin (0.18 mmol/g). Alanine-40 was coupled to the resin as an N-Hmb derivative. A 20-fold excess (1 mmol) of Fmoc- protected amino acids was coupled in NMP/DMF in the presence of HBTU/HOBt, using a 170 min/cycle coupling time. CondMonPrevPk software (as provided by Applied Biosystems) was used to monitor the synthesis. Valine-39 was then coupled to the resin in a flask under inert atmosphere over 3 days using the same coupling reagents as above, except that DCM)/DMF (1/1, v/v) was used as a solvent instead of NMP/DMF. The remaining non-N-methyl amino acids were condensed to the resin, using the same conditions as for the C-terminal 29 amino acids segment. For N- methyl amino acids, PyBroP was used as the only coupling reagent, and the coupling reaction was manually performed in DCM in the presence of DIEA overnight under N2 (ref). The following side chain protections were used in this synthesis (Table E):
Table E Side chain protection reagents
Figure imgf000044_0001
To simultaneously cleave the polypeptide from the resin and the protecting groups from the side chains, polypeptide-resin (100 mg) was treated with TFA (10 ml) in the presence of reagent K (1.5 g phenol, 0.5 ml thioanisole, 0.25 ml ethane-1,2- dithiol (EDT), 0.5 ml water), and triisopropylsilane (0.5 ml) for 2 hours at room temperature (22-24° C). The resin was filtered and the TFA removed under reduced pressure. The polypeptide was then precipitated with cold methyl t-butyl ether (-20° C) and pelleted by centrifugation (4000 rpm/min for 10 minutes). The residue was dissolved in TFA (2 ml), precipitated with cold methyl t-butyl ether (-20° C) and re- pelleted. The pellet was then dissolved in 10% CH3CN and freeze-dried. The polypeptides were further purified using semi-preparative HPLC on a C18 column. Analytical RP-HPLC and MALDI-MS were used to identify pure linear polypeptides.
Preparation of 5NMe-(' K-sR)SDF-la-COSBn by TSPCS
The 4-sulfamylbutyryl AM resin (1 .12 mmol/g, 200 mg, 0.224 mmol) was swelled in the presence of CHCI3 (2.5 ml; pre-filtered through activated basic A1 03), and DIEA (0.3 ml; 1.72 mmol) and Fmoc-Arg(Pbf)-OH (0.65 g; 1 mmol) were added. The amino acid was dissolved by stirring at room temperature (22-24° C) and then cooled and incubated for 20 minutes at -20° C. PyBoP (0.52 g; 1 mmol) was then added as a solid, and the coupling reaction was conducted for 6 hours at a temperature of -20° C to -30° C. The resin was filtered, washed three times with DMF (2 ml) and three times with DCM (2 ml), and dried over P2θ5. Since a single coupling reaction only loads the available sulfonamide groups to a 53% yield, the coupling reaction was repeated to increase amino acid loading onto the sulfonamide groups to 70% yield. The unreacted sulfonamide groups were acetylated by treating the resin with acetic anhydride (0.1 ml; 1.06 mmol) in the presence of a catalytic amount of 4- dimethylaminopyridine (DMAP) and DIEA (0.4 ml; 2.29 mmol) for 2.5 hours at room temperature (22-24° C).
Polypeptide chain synthesis was performed on Fmoc-Arg(Pbf)-sulfonamide resin (0.1 mmol) as described in TSPCS, using the same protecting groups for the side chains of amino acids, except for the N-terminal Lys, which was di-Boc protected in dicyclohexylamine (DCHA) salt.
Polypeptide-bound sulfonamide resin (100 mg; about 25 μmol) was activated by iodoacetonitrile (0.16 ml; 2.2 mmol), distilled and pre-filtered through a plug of basic alumina in NMP (1 ml) in the presence of DIEA (0.18 ml; 1.0 mmol) overnight in the dark at room temperature. The resin was washed three times with DMF (2 ml), subsequently washed three times with DCM (2 ml), and then transferred to a 5 ml round-bottom flask. The side chain protected polypeptide was cleaved from the resin with benzyl mercaptan (0.12 ml) in DMF/THF (1 ml; 1 : 1 [v/v]) in the presence of DIEA (0.21 ml) for 39 hours at room temperature (22-24° C). The polypeptide was separated from the resin by filtration and washed five times with DCM/DMF (2 ml;
1 : 1 [v/v]). The filtrates were combined and concentrated to dryness before treating with reagent K (with 5 ml 2.5% TIPS) for two hours at room temperature (22-24° C).
The polypeptide was purified using semi-preparative HPLC on a CI 8 column using a gradient of 20% to 60% B over 60 minutes with a flow rate of 3 ml/min. The product was characterized by analytical RP-HPLC and MALDI-MS. The observed molecular mass was 1068.8 Da (calculated MW, 1069.1 Da).
Folded 5NMe-SDF-l a synthesized by native chemical ligation (NCL) Two polypeptides obtained by stepwise synthesis, 5NMe-('K-8R)SDF-lα-
COSBn (0.444 mg, 0.3 15 μmol, based on the molecular mass of TFA salt) and linear H-(9C-68K)SDF-l α (1.535 mg, 0.178 μmol), were dissolved in 6 M guanidine HC1- 100 mM sodium phosphate solution (178 μl; buffered to pH 7.5). Thiophenol (7.2 μl, 4% final v/v) was added, and the reaction mixture was vortexed briefly to saturate the ligation buffer with thiophenol. After incubation of the reaction mixture for 16 hours at 37° C, the reaction mixture was desalted by repeated dilution of the reaction mixture with water (about 13 ml), followed by volume reduction of the diluted reaction mixture to its original volume using a polypeptide concentrator (MWCO: 5 KDa, Millipore; Billerica, MA) and centrifugation (4000 rpm for 50 min). The recovered product was purified by semi-preparative HPLC on a C18 column. The polypeptide (0.75 mg, 43% yield) was characterized by analytical RP-HPLC (about 90% pure) and MALDI-MS. The observed molecular mass was 7971.9 Da (calculated MW, 7973.5 Da).
To confirm folding, 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used to determine whether any free thiol group exists in the polypeptide. The purified polypeptide (0.15 mg) was dissolved in water to give a final concentration of about 50 μM, and an aliquot (100 μl) was added to a cuvette that contains an assay solution (900 μl, comprising: 0.2 mM DTNB, 100 mM Tris-HCl (pH 8.0), 2.5 mM sodium acetate). The assay permits detection of free cysteine groups within a polypeptide by virtue of the absorption of the TNB chromophore at 412 nm (ε = 13600 M"1 cm"') following reaction with thiol nucleophiles. The polypeptide yields no absorption at 412 nm, indicating the absence of free thiol groups in the polypeptide and thereby confirming its folded state.
Folding of SDF-1 a and analogs
Purified linear polypeptides were dissolved to a concentration of 0.1-0.3 mg/ml in 1 M guanidine hydrochloride- 100 mM Tris-HCl (pH 8.5) at 4° C and stirred vigorously overnight in open air (Clark-Lewis et al., 1991). Disulfide bond formation was monitored by analytical HPLC and MALDI-MS. Upon completion of disulfide bond formation (generally within 24 hours), the solution was desalted in a polypeptide concentrator (MWCO: 5 kDa, Millipore), by repeatedly (three times) diluting the solution with water (12 ml), followed by centrifugation at 4000 rpm for 50 minutes for each dilution. The sample was then lyophilized. The polypeptides were purified using semi-preparative HPLC on a C18 column with a gradient 10-50% B over 60 minutes at a flow rate of 3 ml/min. The fractions were analyzed by analytical RP- HPLC and MALDI-MS. After freeze-drying the pure polypeptides, the polypeptides were dissolved in water and the concentration determined by UV absorption spectrometer, using an extinction coefficient of 8730 M"1 cm"' at 280 nm, calculated on the basis of the number of tryptophane, tyrosine and the disulfide bond in the polypeptides (Pace et al., 1995).
Example 2 Synthesis of Dl -SDF-1 a (SEQ ID NO:3) by TSPCS
The linear sequence of NT (D Lys) SDF- lα was synthesized in 0.1 mmol scale using a CLEAR amide resin. Prior to incorporation of the C-terminal amino acid, the resin was swelled in DCM for 2 hours. Individual amino acids were coupled to the C- terminal amino acid-coupled resin at room temperature (22-24° C) using a five-fold excess of activated Fmoc-amino acids and double coupling each residue. For the first twenty amino acids from the C-terminus, the first and the second coupling reactions were individually performed for 1 hour. For all residues incorporated beyond the twentieth residue, the second coupling time was increased to 2 hours. In the case of 41 -48 residues from the C-terminus, a third coupling reaction was performed to drive the overall coupling reaction to completion. Fmoc deprotection was carried out for 20-30 minutes, depending on the percent cleavage of the Fmoc group from the peptidyl resin. After incorporating the C-terminal 35 amino acid residues to the resin, half of the peptidyl resin was removed and the synthesis was continued with the other half. The N-terminal Lys was incorporated as D-Lys. The coupling was performed three times to achieve a negative Kaiser test.
After removing the N-terminal Fmoc group, the polypeptide was cleaved from the support by incubating with a cleavage solution (0.2 % phenol (w/v), 5 % thioanisol (v/v), 5% water (v/v), 2.5% ethanedithiol (v/v), 1.5% triisopropylsilane (v/v) and 86% TFA (v/v) for four hours at room temperature (22-24° C). The cleaved polypeptide was filtered and concentrated to one-third of its original volume under reduced pressure. The polypeptide was precipitated with ice-cold t-butylethylether, the precipitate washed twice with ice-cold ether and then dried. The crude product was then dissolved in water and purified by semi-preparative HPLC using a C18 column. The solvent systems were 0.1% TFA/water (A) and 0.1% TFA/20%water/acetonitrile (B). The chromatography was performed in 35-75% B in 70 minutes at a flow rate of 3 ml/min. Chromatography fractions containing the target polypeptide were identified by MALDI-MS, which were subsequently pooled and lyophilized.
The estimated purity of the linear polypeptide was greater than 96%. The molecular weight as determined by MALDI-MS was 7963.38 Da (calculated MW, 7962.47). After lyophilization, the linear polypeptide was resuspended in 2M guanidine HCI-0.1 M Tris (pH-8.5) to a final concentration of 1 mg/ml and air- oxidized at room temperature. The folding reaction was monitored by analytical
HPLC; 60% folding was observed within the first 2 hours. The solution was stirred overnight to complete folding. The solution was transferred to a filter device (Ultrafree-15, with high-flux Biomax™ ultrafiltration membrane, MW cutoff 5,000; Millipore) and reconcentrated by centrifugation at 2000 g for 30 minutes. The concentrate was diluted with water and reconcentrated two more times by centrifugation as before. The NT (D Lys) SDF-1 α was further purified by semi- preparative HPLC using Vydac C18 column. Chromatography fractions containing the target polypeptide were tested for folded NT (D Lys) SDF-l α by analytical HPLC and MALDI-MS. Fractions containing the folded polypeptide were pooled and lyophilized to obtain 6.3 mg of product. The purity of the product was tested by analytical HPLC using a CI 8 column with a 0.5%/min gradient of 0.1 % TFA/20%water/acetonitrile in 0.1 %TF A/water. The estimated purity of NT (D Lys) SDF-1 α was greater than 98.5%. Molecular weight obtained from MALDI-MS analysis was 7859.63 Da (calculated MW, 7858.47 Da).
Example 3 Synthesis ofvMIP-II and related analogs (SEQ ID NOs: 12- 16) by NCL and TSPCS
NCL synthesis
The 71 amino acid residue-long polypeptide was initially synthesized by native chemical ligation. Leu 34 was selected as the ligation site. Because a Cys side chain sulfhydryl group was required for ligation chemistry, a Cys residue at position 35 (in the middle of the polypeptide) was available for the ligation reaction. Residues 1 -34 (from the N-terminus) of vMIP II was synthesized as polypeptide thioester using a SLilfamylbutyryl AM resin (Novabiochem). Though the Fmoc-amino acids were used for the synthesis, the N-terminal Leu was incorporated as Boc-Leu to protect the amino terminus during subsequent activation and cleavage steps. The polypeptide thioester was released from the resin by activating the thioester with iodoacetonitrile in the presence of DIEA in the dark, and then by treating the resin with benzyl mercapten and DIEA in DMF. The released polypeptide was obtained in a yield greater than 80%.
Residues 35-71 were synthesized on a CLEAR amide resin. The native chemical ligation was initiated by mixing equimolar quantities of polypeptide with phosphate buffer containing 15% TFE and 6M guanidine HCl in the presence of 2% thiophenol and 1 % benzyl mercapten (final pH 7 at 37° C). The ligation reaction was monitored by HPLC and MALDI-MS analysis. Though the ligation reaction was 60%-70% complete within the first two hours, the reaction mixture was incubated overnight at 37° C to maximize yield. The sample was then desalted by adding water to the mixture, followed by concentrating the solution in a filter device (Ultrafree-15, with high-flux Biomax'M ultrafiltration membrane, MW cutoff 5,000; Millipore) using centrifugation at 2000 g for 30 minutes. The process was repeated twice, adding 20% acetonitrile in water to the concentrated solution. The polypeptide was purified by semi-preparative HPLC using a C 18 column with a linear gradient of 30- 70% B over 60 minutes at a flow rate of 3 ml/min. Chromatography fractions (1 ml) containing the target polypeptide were identified by MALDI-MS, which were subsequently pooled and lyophilized.
TSPCS For the TSPCS of vMIP II, CLEAR amide resin was used. For coupling reactions with a slow rate (53D, 54K, 37K, 38P, 39G, 22Q, 2 I P, 18R, 17K, 16Q, 2G), a double coupling followed by capping of the unreacted amino functionality was performed. After the incorporation of residue 50, 2% dimethyl sulfoxide (DMSO) was added to the coupling solution. After removing the N-terminal Fmoc protecting group, the polypeptide was cleaved from the resin support by incubating the resin with the cleavage solution for four hours. The polypeptide was precipitated with ice cold t-butylethylether, and the precipitate was washed repeatedly (at least twice) with cold ether. The washed pellet was dissolved in 25% acetonitrile in water containing 0.1 % TFA and then lyophilized. The lyophilized polypeptide (108 mg) was dissolved in water (4 ml) and purified by semi-preparative HPLC, which yielded the linear polypeptide (22 mg). The purified polypeptide was characterized by MALDI-MS; the observed mass was 8128.68 Da (calculated MW, 8127.68 Da).
The purified polypeptide was folded by resuspending the polypeptide in 1 M guanidine hydrochloride-100 mM Tris-HCI (pH 8.5; 1 ml buffer/mg polypeptide). The polypeptide was then subjected to analytical HPLC using a CI 8 column (0.46 x 15 cm, 5 μm) with a linear gradient 30-70% B over 30 minutes at a flow rate of I ml/min. Within half an hour after buffer addition, over 90% of the polypeptide was folded; however, incubation continued for four hours to maximize folding. The analytical HPLC profile of the folded polypeptide showed a two-unit shift toward the hydrophilic region of the solvent mixture as compared to the spectrum of the linear polypeptide. After the polypeptide was folded, desalting and purification were performed as described above, yielding 12 mg of folded polypeptide.
This same purification and folding procedure was performed for (1 -10)D-(1 1- 71 ) vMIP II, obtaining 5.6 mg. The purified polypeptide was shown by MALDI-MS to have a mass of 8124.68 Da (calculated MW, 8123.68 Da). Example 4 Synthesis of FAM-vMIP-II (SEQ ID NO: 17) by TSPCS
FAM-vMlP II was prepared wherein an Fmoc-8-aminocaprylic acid (Aca) was incorporated to the N-terminal Lys residue as a spacer. The primary sequence of vMIP II (SEQ ID NO: 10) was then incorporated to the support by stepwise synthesis. The N-terminal residue Leu was incorporated as Boc-Leu to the peptidyl resin. The C-terminal Lys side chain protection ivDde was removed from the peptidyl resin using the following wash protocol: (a) twice with 2% hydrazine monohydrate (v/v) in DMF (5 ml; first wash 5 minutes; second, 7 minutes);(b) five times with 10% DIEA (v/v) in DMF (5 ml), (c) five times with DCM (5 ml); and (d) five times with diethyl ether (5 ml). The fluorescent tag 5-carboxyfluorescene was incorporated into the side chain amino group of the resin-bound N-terminal Lys residue using a three-fold excess of 5-carboxyfluorescenesuccinimidyl ester (5-FAM-SE), HOBt and a five-fold excess of diisopropylethylamine. The reaction vessel was mixed using a multi-pulse vortexer overnight in the dark. The FAM polypeptide was cleaved from the support by incubating the polypeptide-coupled resin in cleavage solution for four hours in the dark at room temperature (22-24° C). The sample was then filtered, concentrated to one-third of its original volume in the dark under reduced pressure. FAM (1 - I OD)vM IP II was precipitated with ice-cold t-butylethylether, and the precipitate then washed twice with ice-cold ether before drying. The product was subsequently dissolved in water and purified by semi-preparative HPLC using a C 18 column (1.0 x 25 cm, 5 μm), with solvents A and B and a linear gradient 35-75% B over 70 minutes at a flow rate of 3 ml/min. Chromatography fractions (2 ml) containing the target polypeptide were identified by MALDI-MS, which were subsequently pooled and lyophilized. The purity of the linear polypeptide was >96%. The molecular weight obtained from MALDI TOF MS was 8759.43 Da (calculated MW, 8758.65).
After lyophilization, the linear polypeptide was resuspended in 2 M guanidine FICI-0.1 M Tris (pH 8.5) to a final concentration of 1 mg/ml, and air-oxidized at room temperature. The folding reaction was monitored by analytical HPLC. More than 60% folding was completed within the first two hours; however, the solution was stirred overnight to assure complete folding. The solution was transferred to a filter device (Ultrafree-15, with high-flux Biomax™ ultrafiltration membrane, MW cutoff 5,000; Millipore) and centrifuged at 2000 g for 30 minutes. The concentrate was diluted with water and centrifuged two more times. The concentrated solution containing the FAM (l -l OD)vMIP II was further purified by semi-preparative HPLC using a CI 8 column as described above. Chromatography fractions (1 ml) containing polypeptide were assayed for the presence of FAM ( l -l OD)vMIP II by analytical HPLC and MALDI-MS. Fractions containing the folded polypeptide were pooled and lyophilized; 2.3 mg of pure material was obtained. Purity was tested by analytical HPLC using a CI 8 column (0.46 x 15 cm, 5 μm) using a gradient of 0.1% TFA/20%water/acetonitrile in 0.1 %TF A/water for 30 minutes at a flow rate of 0.5 ml/min. The purity of FAM (l -l OD)vMIP II was greater than 98.5%. The molecular mass as ascertained by MALDI-MS was 8755.34 Da (calculated MW, 8754.64 Da).
Example 5 Synthesis ofvMlP-II and SDF-1 a dimers (SEQ ID NOs: 18-21) by TSPCS
The polypeptide vMIP II (Aca)ήC(Acm) was prepared wherein Cys(Acm) was incorporated as a C-terminal amino acid coupled to a CLEAR amide support.
Coupling reactions were performed using a five-fold excess of Fmoc-protected amino acid. A second coupling was also performed to drive the reaction to completion. Six 8-aminocaprylic acid molecules were incorporated to the N-terminal Cys(Acm) residue to provide sufficient spacer effect. The primary sequence of vMIP II was then incorporated onto the support by stepwise synthesis. The sequence was subsequently removed from the resin by incubating the resin in cleavage solution in the dark for four hours at room temperature. The cleaved polypeptide mixture was filtered from the resin and concentrated to one-third volume. The polypeptide was precipitated with ice-cold t-butylethylether, and the precipitate washed twice with ice-cold ether and then dried. The crude product was subsequently dissolved in water and purified by semi-preparative HPLC using a CI 8 column (1 .0 x 25 cm, 5 μm). The solvents systems were 0.1% TFA/water (A) and 0.1% TFA/20% water/acetonitrile (B), with a gradient 35%-75% B in 70 minutes at a flow rate of 3 ml/min. Chromatography fractions (2 ml) containing polypeptides were tested by MALDI-MS to identify those factions that contain the target polypeptide. Chromatography fractions containing the target polypeptide were pooled and lyophilized. Purity of the linear polypeptide was greater than 96%. The molecular mass, as determined by MALDI-MS, was 9149.38 Da (calculated MW, 9150.28 Da).
The lyophilized material was resuspended in 10% DMSO in the dark. The folding reaction was monitored by analytical HPLC, revealing that greater than 68% of the polypeptide was folded within 28 hours. The solution was transferred to a filter device (Ultrafree- 15, with high-flux Biomax' M ultrafiltration membrane, MW cutoff 5,000; Millipore) and centrifuged at 2000 g for 30 minutes. The concentrate was diluted with water and centrifuged two more times. The polypeptide was further purified by semi-preparative HPLC using a C I 8 column as above. Chromatography fractions ( I ml) containing polypeptides were tested for VMIP II (Aca)6C(Acm) by analytical HPLC and MALDI-MS. Chromatography fractions containing the folded polypeptide were pooled and lyophilized, obtaining 12 mg of polypeptide. Purity, as tested by analytical HPLC using a C I 8 column (0.46 x 15 cm, 5 μm) with a gradient of 0.1% TFA/20%water/acetonitrile in 0.1 %TF A/water at a flow rate of 0.5 ml/min, was shoparwn to be greater than 98%. The observed molecular mass, as obtained from MALDI-MS analysis, was 8745.34 Da (calculated MW, 9146.28 Da).
Folded VMIP II (Aca)6C(Acm) was stirred in MeOH:H20 (4: 1) (300 μl) containing I2 (2.1 mg). The Cys oxidation was monitored at 15, 30, 45 minutes using analytical HPLC. The reaction was quenched after 45 minutes by adding water (2 ml). Excess iodine was removed by extracting the aqueous solution with an excess of CC14 (4 x 15 ml) in a separatory funnel. The aqueous solution was lyophilized and analyzed using analytical HPLC and MALDI-MS. The dimer was further purified by semi-preparative HPLC using a C18 column as described above.
Example 6 Competitive binding of SDF-1 a, vMIP-II and analogs to CXCR4 The ability of the SDF- l α and vMIP-II analogs to compete as a ligand for CXCR4 was assayed by incubating CXCR4-expressing cells with the analogs in the presence of labeled SDF- lα or vMIP-II. The better the analog as a competitor, the weaker the observed signal. CEM-T4 cells (a T cell, CD-4 expressing lymphoblastoid cell line) were harvested and washed twice with phosphate buffered saline (PBS). 2 x 105 CEM-T4 cells were incubated with 0.2 nM of l 25I-SDF-lα in 100 μl of binding buffer (50 mM 4-(2-Hydroxyethyl)piperazine-l-ethanesulfonic acid (HEPES; pH 7.4) 1 mM CaCl2, 5 mM MgCl , 0.1 % bovine serum albumin) with increasing concentrations of unlabelled SDF-l α. The samples were incubated for 60 minutes at room temperature before washing the cells twice with 500 ml of cold binding buffer. After counting γ emissions, specifically bound counts per minute (cpm) were calculated by subtracting the non-specifically bound cpm (the cpm bound in the presence of 500-fold molar excess of unlabeled SDF-l α or vMIP-II) from the total cpm that was bound to the cells. The results were shown in Tables 2 and 3.
Example 7 Competitive binding ofvMIP-11 and analogs to CCR5
The ability of vMIP-II analogs to compete as a ligand for CCR5 was assayed by incubating CCR5-expressing cells with the analogs in the presence of labeled vMIP-II. The better the analog as a competitor, the weaker the observed signal. 293 cells transfected with CCR5 (Zhou et al., 2002) were harvested and washed twice with phosphate buffered saline (PBS). 2 x 105 cells were incubated with 0.2 nM of l25I-vMIP-II in 100 μl of binding buffer with increasing concentrations of unlabelled vMIP-H. The samples were incubated for 60 minutes at room temperature before washing the cells twice with 500 ml of cold binding buffer. After counting γ emissions, specifically bound counts per minute (cpm) were calculated by subtracting the non-specifically bound cpm (the cpm bound in the presence of 500- fold molar excess of unlabeled vMIP-II) from the total cpm that was bound to the cells. The results were shown in Table 3.
Example 8 Intracellular calcium signaling
SUP-T1 cells (a human T cell lymphoma cell line; 107/ml) were loaded with 2 μM of Fura-2/AM (Molecular Probes; Eugene, OR) and 0.01% Pluronic F-127 in Hank's balanced salt saline (HBSS; 140 mM NaCl, 5 mM KC1, 10 mM HEPES (pH 7.4), 1 mM CaCl2, 1 mM MgCl2, 1 mg/ml glucose and 0.025% BSA) for 20 minutes at room temperature. The cells were then washed twice with HBSS and resuspended to 106 cells/ml. Fura-2 fluorescence was measured at room temperature with a fluorescence spectrophotometer (ISA SPEX FIuoroMax-2) using excitation wavelengths of 340 nm and 380 nm, and an emission wavelength of 510 nm. Various concentrations of peptides were first added to the cell suspension, and after 5 minutes of incubation, 50 nM of SDF-l α was added.
Example 9 Stability of peptides in human serum (Prophetic; (Zhou et al, 2002))
Pure human serum was diluted with PBS to 80% serum solution. Polypeptides (SEQ ID NOs:2-9, 1 1 -16, and the dimers formed between SEQ ID NOs:18 and 19 and between SEQ ID NOs:20 and 21) were dissolved in the serum solution to a concentration of 10 mM. Samples were collected at different times during incubation at room temperature and subjected to HPLC analysis by injecting 10 μl of the peptide samples (Microsorb-MV C I 8 5 μm, 25 cm x 4.6 mm, 80% CH3CN with 0.1% trifluoroacctic acid, UV 220 nm, 1 ml/minute). The stability of the peptides was calculated based on the changes in the intensity of UV absorbance of the peptides.
Example 10 Virus neutralization assay (Prophetic; (Zhou et al, 2002))
Recombinant viruses were generated by cotransfecting the human 293 T cell line with pSVIIIenv plasmids expressing either HXBc2 or 89.6 envelope glycoproteins and pHXBH10_envCAT (Thali et al., 1992). Recombinant viruses were normalized for reverse transcriptase activity and used to infect (i) human PBMC activated with phytohemagglutinin (PHA) and interleukin- 2 or (ii) canine thymocytes expressing human CD4 or human CD4 plus human CCR5 (Choe et al., 1998). The target cells were preincubated with the peptides (SEQ ID NOs:2-9, 1 1-16, and the dimers formed between SEQ ID NOs: 18 and 19 and between SEQ ID NOs:20 and 21) at varying concentrations for 1 hour prior to co-culture with the recombinant viruses. Infected cells were harvested three days later, and chloramphenical acetyl transferase (CAT) activity was measured in cell lysates (Helseth et al., 1990).
While various embodiments of the present invention have been described, it will be apparent to those of skill in the art that many more embodiments and implementations are possible that are within the scope of this invention. Accordingly, the present invention is not to be restricted except in light of the attached claims and their equivalents. REFERENCES
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Claims

1 . A polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs:2-9; 1 1 - 17, a dimer formed between SEQ ID NOs: 18 and 19 and a dimer between SEQ ID NOs:20 and 21.
2. The polypeptide of claim 1 , wherein the polypeptide is an antagonist of CXCR4.
3. The polypeptide of claim 2, wherein the polypeptide is selected from the group consisting of SEQ ID NOs:3-9 and 12.
4. The polypeptide of claim 1, wherein the polypeptide is an antagonist of CCR5.
5. The polypeptide of claim 4, wherein the polypeptide comprises SEQ
ID NO: l l or 12.
6. The polypeptide of claim 1 , wherein the polypeptide is an agonist of CCR5.
7. The polypeptide of claim 6, wherein the polypeptide comprises SEQ ID NO: 16.
8. The polypeptide of claim 1 , wherein the polypeptide is an agonist of CXCR4.
9. The polypeptide of claim 8, wherein the polypeptide is SEQ ID NO: 15.
10. A method of treating HIV infection, comprising administering a pharmaceutical composition comprising a polypeptide of claim 1.
1 1. The method of claim 10, wherein the administering comprises oral administration, inhalation or injection.
12. A method of preventing HIV infection, comprising administering a pharmaceutical composition comprising a polypeptide of claim 1.
13. The method of claim 12, wherein the administering comprises oral administration, inhalation or injection.
14. A method of treating a CXCR4- or CCR5-related disease, disorder or condition, comprising administering a polypeptide of claim 1 .
15. The method of claim 14, wherein the disease, disorder or condition is selected from the group consisting of asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, atherosclerosis, cancer, multiple myeloma, non-Hodgkin's lymphoma and viral infection.
16.. A method of inhibiting HIV from entering a cell, comprising contacting the cell with a polypeptide of claim 1
17. A method of preventing HIV from spreading in a population of cells, comprising contacting the cells with a polypeptide of claim 1.
18. The method of claim 16 or 17, wherein the cells express CXCR4 or CCR5.
19. A polypeptide comprising at least one D-amino acid of an amino acid sequence having at least 75% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 , 2, 5-8, 10, 1 1 , 15, 16-17, a dimer formed between SEQ ID NOs: 18 and 19 and a dimer between SEQ ID NOs:20 and 21.
20. The polypeptide of claim 19, wherein the sequence identity is at least 85%.
21 . The polypeptide of claim 19, wherein the sequence identity is at least 90%.
22. The polypeptide of claim 19, wherein the sequence identity is at least 95%.
23. A method of treating HIV infection, comprising administering a pharmaceutical composition comprising a polypeptide of claim 19.
24. A method of treating a CXCR4- or CCR5-related disease, disorder or condition, comprising administering a polypeptide of claim 19.
25. The method of claim 24, wherein the disease, disorder or condition is selected from the group consisting of asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, atherosclerosis, cancer, multiple myeloma, non-Hodgkin's lymphoma and viral infection.
26. A method of inhibiting HIV from entering a cell, comprising contacting the cell with a polypeptide of claim 19.
27. A method of preventing HIV from spreading in a population of cells, comprising contacting the cells with a polypeptide of claim 19.
28. The method of claim 26 or 27, wherein the cells express CXCR4 or CCR5.
29. A polypeptide comprising a sequence modified to have at least one D- amino acid having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 , 2, 5-8, 10, 1 1 , 15, 16, 18-20 and 21.
30. A polypeptide comprising at least one N-methylated amino acid having an amino acid sequence having at least 75% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 -4, 8-20 and 21.
31. The polypeptide of claim 30, wherein the sequence identity is at least 85%.
32. The polypeptide of claim 30, wherein the sequence identity is at least 90%.
33. The polypeptide of claim 30, wherein the sequence identity is at least 95%.
34. A method of treating HIV infection, comprising administering a pharmaceutical composition comprising a polypeptide of claim 30.
35. A method of treating a CXCR4- or CCR5-related disease, disorder or condition, comprising administering a polypeptide of claim 30.
36. The method of claim 35, wherein the disease, disorder or condition is selected from the group consisting of asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, atherosclerosis and viral infection.
37. A method of inhibiting HIV from entering a cell, comprising contacting the cell with a polypeptide of claim 30.
38. A method of preventing HIV from spreading in a population of cells, comprising contacting the cells with a polypeptide of claim 30.
39. The method of claim 37 or 38, wherein the cells express CXCR4 or CCR5.
40. A polypeptide comprising a sequence modified to have at least one N- methylated amino acid selected from the group consisting of SEQ ID NOs: 1 -4, 8-20 and 21.
41. A polypeptide comprising at least one D-amino acid and at least one N-methylated amino acid of an amino acid sequence having at least 75% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 , 2, 8, 10, 15, 16, 1 8-20 and 21 .
42. A method of treating HIV infection, comprising administering a pharmaceutical composition comprising a polypeptide of claim 41.
43. A method of treating a CXCR4- or CCR5-related disease, disorder or condition, comprising administering a polypeptide of claim 41.
44. The method of claim 41 , wherein the disease, disorder or condition is selected from the group consisting of asthma, allergic diseases, multiple sclerosis, rheumatoid arthritis, atherosclerosis, cancer, multiple myeloma, non-Hodgkin's lymphoma and viral infection.
45. A method of inhibiting HIV from entering a cell, comprising contacting the cell with a polypeptide of claim 41.
46. A method of preventing HIV from spreading in a population of cells, comprising contacting the cells with a polypeptide of claim 41.
47. The method of claim 45 or 46, wherein the cells express CXCR4 or CCR5.
48. A polypeptide comprising a label and comprising a sequence having at least 75% identity with a sequence selected from the group consisting of SEQ ID NOs:2-9, 1 1 - 16, 18 and 19.
49. The polypeptide of claim 48, wherein the sequence identity is at least
90%.
50. The polypeptide of claim 48,wherein the label is selected from the group consisting of a fluorescent, radio and enzymatic label.
51. A method of determining treatment efficacy of polypeptide for a disease, disorder, or condition, comprising: isolating a target cell from a subject; contacting the cell with a labeled polypeptide of SEQ ID NO:2-9, 1 1-16, a dimer formed between SEQ ID NOs: 18 and 19 or a dimer between SEQ ID NOs:20 and 21 ; and analyzing the signal from the label, such that the observation of a signal indicates treatment efficacy.
52. A kit, comprising a polypeptide comprising a sequence with at least
75% identity with a polypeptide of a sequence selected from the group consisting of SEQ ID Os:2-9, 1 1 -17, a dimer formed between SEQ ID NOs: 18 and 19 and a dimer between SEQ ID NOs:20 and 21.
53. The kit of claim 52, wherein the polypeptide comprises a label.
54. The kit of claim 52, comprising a polypeptide selected from the group consisting of SEQ ID NOs:2-9, 1 1 -17, a dimer formed between SEQ ID NOs: l 8 and 19 and a dimer between SEQ ID NOs:20 and 21..
55. The kit of claim 52, further comprising a control sample.
56. A method of detecting CXCR4 or CCR5 expression, comprising applying to a sample a polypeptide selected from the group consisting of SEQ ID NOs:2-9, 1 1 -17, a dimer formed between SEQ ID NOs: 18 and 19 and a dimer between SEQ ID NOs:20 and 21.
57. The method of claim 56, wherein the polypeptide further comprises a label.
58. The method of claim 56, wherein the polypeptide is selected from the group consisting of SEQ ID NOs:3-9, 15 and 17, and CXCR4 is detected.
59. The method of claim 56, wherein the polypeptide is selected from the group consisting of SEQ ID NO: 16, and CCR5 is detected.
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WO2006108594A1 (en) * 2005-04-08 2006-10-19 Lonza Ag Peptide synthesis of alpha-helixes on peg resin
US8628750B2 (en) 2006-02-27 2014-01-14 Technische Universitat Munchen Cancer imaging and treatment
US7696309B2 (en) 2006-10-23 2010-04-13 The Brigham And Women's Hospital, Inc. Protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage
US10774124B2 (en) 2006-10-23 2020-09-15 The Brigham And Women's Hospital, Inc. Protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage
US7999067B2 (en) 2006-10-23 2011-08-16 The Brigham And Women's Hospital, Inc. Protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage
US9631005B2 (en) 2006-10-23 2017-04-25 The Brigham And Women's Hospital, Inc. Protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage
EP2676674A1 (en) * 2006-10-23 2013-12-25 The Brigham and Women's Hospital, Inc. Protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage
EP2053060A1 (en) * 2007-10-24 2009-04-29 Protaffin Biotechnologie AG SDF-1-based glyocosaminoglycan antagonists and methods of using same
WO2009053064A1 (en) * 2007-10-24 2009-04-30 Protaffin Biotechnologie Ag Sdf-1-based glycosaminoglycan antagonists and methods of using same
US9453024B2 (en) 2009-01-29 2016-09-27 Mapi Pharma Ltd. Polymorphs of darunavir
US8921415B2 (en) 2009-01-29 2014-12-30 Mapi Pharma Ltd. Polymorphs of darunavir
US8829208B2 (en) 2010-01-28 2014-09-09 Mapi Pharma Ltd. Process for the preparation of darunavir and darunavir intermediates
CN102725295A (en) * 2010-01-28 2012-10-10 Mapi医药公司 Process for the preparation of darunavir and darunavir intermediates
WO2011092687A1 (en) * 2010-01-28 2011-08-04 Mapi Pharma Hk Limited Process for the preparation of darunavir and darunavir intermediates
US9308277B2 (en) 2010-02-25 2016-04-12 Mesoblast International Sàrl Protease-resistant mutants of stromal cell derived factor-1 in the repair of tissue damage
US10456451B2 (en) 2010-02-25 2019-10-29 Mesoblast International Sàrl Protease-resistant mutants of stromal cell derived factor-1 in the repair of tissue damage
US10662234B2 (en) 2011-06-07 2020-05-26 Mesoblast International Sàrl Methods for repairing tissue damage using protease-resistant mutants of stromal cell derived factor-1
CN111875670A (en) * 2013-06-12 2020-11-03 法瑞斯生物技术有限公司 Peptides having antagonistic activity against native CXCR4
CN107325187A (en) * 2017-07-19 2017-11-07 黄子为 A kind of polypeptide and its application and pharmaceutical composition with CXCR4 albumen agonist activities

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