WO2002057313A2 - Composes de liaison et procedes d'identification de ces derniers - Google Patents

Composes de liaison et procedes d'identification de ces derniers Download PDF

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WO2002057313A2
WO2002057313A2 PCT/US2001/051165 US0151165W WO02057313A2 WO 2002057313 A2 WO2002057313 A2 WO 2002057313A2 US 0151165 W US0151165 W US 0151165W WO 02057313 A2 WO02057313 A2 WO 02057313A2
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Prior art keywords
binding
molecule
chemokine receptor
cxc chemokine
seq
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PCT/US2001/051165
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English (en)
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WO2002057313A8 (fr
WO2002057313A3 (fr
Inventor
John J. Nestor, Jr.
Carol J. Wilson
Christina A. Tan Hehir
Steven A. Kates
John Krstenansky
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Consensus Pharmaceuticals, Inc.
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Priority claimed from US09/813,653 external-priority patent/US20020064770A1/en
Priority claimed from US09/813,448 external-priority patent/US20020142346A1/en
Priority claimed from US09/813,651 external-priority patent/US20030018438A1/en
Application filed by Consensus Pharmaceuticals, Inc. filed Critical Consensus Pharmaceuticals, Inc.
Priority to AU2002246912A priority Critical patent/AU2002246912A1/en
Publication of WO2002057313A2 publication Critical patent/WO2002057313A2/fr
Publication of WO2002057313A3 publication Critical patent/WO2002057313A3/fr
Publication of WO2002057313A8 publication Critical patent/WO2002057313A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/08—Antibacterial agents for leprosy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

  • the invention generally relates to Cysteine-X-Cysteine Chemokine Receptor 4
  • CXCR4 or CXC chemokine receptor 4" and more particularly, to binding compounds for CXC chemokine receptor 4.
  • Methods of the invention are useful for the treatment of disease by identifying and preparing binding compounds for CXC chemokine receptor 4.
  • Chemokines comprise a family of structurally related secreted proteins of about 70-110 amino acids that share the ability to induce migration and activation of specific types of blood cells. See Proost P., et al. (1996) Int. J. Clin. Lab. Rse. 26: 211-223; Premack, et al. (1996) Nature Medicine 2: 1174 178; Yoshie, et al. (1997) J.
  • Leukocyte Biol. 62: 634-644 Over 30 different human chemokines have been described to date. While they are primarily responsible for the activation and recruitment of leukocytes, they vary in their specificities for different leukocyte types (neutrophils, monocytes, eosinophils, basophils, lymphocytes, dendritic cells, etc.), and in the types of cells and tissues where the chemokines are synthesized. Further analysis of this family of proteins has shown that it can be divided up into two further subfamilies of proteins. These have been termed CXC or ⁇ - chemokines, and the CC or ⁇ -chemokines based on the spacings of two conserved cysteine residues near the amino terminus of the proteins.
  • Chemokines are typically produced at sites of tissue injury or stress, where they promote the infiltration of leukocytes into tissues and facilitate an inflammatory response. Some chemokines act selectively on immune system cells such as subsets of T-cells or B lymphocytes or antigen presenting cells, and may thereby promote immune responses to antigens. In addition, some chemokines have the ability to regulate the growth or migration of hematopoietic progenitor and stem cells that normally differentiate into specific leukocyte types, thereby regulating leukocyte numbers in the blood.
  • chemokines are mediated by cell surface receptors that are members of a family of seven transmembrane ("7TM"), G-protein coupled receptors ("GPCR"). At least twelve different human chemokine receptors are known, including CCRl, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR1, CXCR2, CXCR3, and CXCR4. These receptors vary in their specificities for specific chemokines. Some receptors bind to a single known chemokine, while others bind to multiple chemokines.
  • 7TM transmembrane
  • GPCR G-protein coupled receptors
  • Binding of a chemokine to its receptor typically induces intracellular signaling responses such as a transient rise in cytosolic calcium concentration, followed by cellular biological responses such as chemotaxis.
  • some chemokine receptors such as CXCR4, serve as co-receptors for Human Immunodeficiency Virus (HIN), such that they interact with HIV and with the cellular CD4 receptor to facilitate viral entry into cells.
  • HIN Human Immunodeficiency Virus
  • Chemokines are important in medicine because they regulate the movement and biological activities of leukocytes in many disease situations, including, but not limited to: allergic disorders, autoimmune diseases, ischemia/reperfusion injury, development of atherosclerotic plaques, cancer (including mobilization of hematopoietic stem cells for use in chemotherapy or myeloprotection during chemotherapy), chronic inflammatory disorders, chronic rejection of transplanted organs or tissue grafts, chronic myelogenous leukemia, and infection by HIV and other pathogens
  • CXCR4 in particular, has been implicated in diseases such as glioblastoma multiforme tumor, hepatocellular carcinoma, colon cancer, esophageal cancer, gastric cancers, breast cancer metastasis, pancreatic cancer and in renal allograft rejection.
  • Antagonists of chemokine receptors may be of benefit in many of these diseases by reducing excessive inflammation and immune system responses.
  • chemokines and antagonists that bind to HIV co-receptors may have utility in inhibiting viral entry into cells.
  • HIV causes Acquired Immune Deficiency Syndrome ("AIDS"), which is one of the leading causes of death in the United States and throughout the world.
  • AIDS Acquired Immune Deficiency Syndrome
  • According to the Center for Disease Control at least 30.6 million people world-wide have been infected with HIV. HIV attacks the immune system and leaves the body vulnerable to a variety of life-threatening illnesses. Common bacteria, yeast, and viruses that would not cause disease in people with a fully functional immune system often cause these illnesses in people affected with HIV.
  • CD4 + lymphocyte count a patient having a count of less than 200 cells/ ⁇ l is considered to have AIDS.
  • the CD4 protein is a glycoprotein of approximately 60,000 molecular weight and is expressed on the cell membrane of mature, thymus-derived (T) lymphocytes, and to a lesser extent on cells of the monocyte/macrophage lineage.
  • CD4 cells appear to function by providing an activating signal to B cells, by inducing T lymphocytes bearing the reciprocal CD8 marker to become cytotoxic/suppressor cells, and/or by interacting with targets bearing major histocompatibility complex (MHC) class II molecules.
  • MHC major histocompatibility complex
  • the host immune system will produce antibodies targeted against various antigenic sites, or determinants, of gpl20. Some of those antibodies will have a neutralizing effect and will inhibit HIV infectivity. It is believed that this neutralizing effect is due to the antibodies' ability to interfere with HIV's cellular attachment. It is also believed that this effect may explain in part, the rather long latency period between the initial seroconversion and the onset of clinical symptoms.
  • T-tropic T cell tropic
  • M-tropic macrophage tropic
  • CXCR4 CXCR4
  • the second extracellular loop is the region most required for the entry of HIV into the cell; however, the N- terminus and the third extracellular loop are also involved. Several charged residues on the.
  • CXCR4 Several inhibitors of CXCR4 have been reported. These include positively charged peptides such as T22 and T140 and small molecule inhibitors such as ALX40-4C and T134.
  • AMD 3100 a heterocyclic bicyclam (one positive charge on each of two rings) has been reported as well. See e.g., Tamamura H, et. al, Bioorg Med Chem 6(7):1033-41(1998); Tamamura H, et. al, Biochem. Biophys. Rs. Comm. 252:877-82 (1998); Doranz BJ, et. al, J Exp Med 186(8): 1395-400 (1997); Arakaki R, et. al, J. Virol.
  • PBMCs Peripheral Blood Monocytes
  • bicyclams may interfere with other CXCR4-like receptors. See e.g., Schols D, et. al, J. Exp. Med. 186(8):1383-1388 (1997).
  • T22 inhibits calcium mobilization, therefore interfering with CXCR4's natural required signaling.
  • the present invention provides binding compounds for CXCR4 and methods for identifying those binding compounds.
  • screening methods are provided to identify binding motifs for CXCR4, as well as ligands capable of binding to CXCR4.
  • the invention comprises the design and identification of therapeutic peptides, peptidomimetics, or small molecules suitable for use in the prevention or treatment of HIV and AIDS.
  • methods of the invention provide for the synthesis and purification of linear and cyclic peptide libraries useful for screening and identifying a binding motif for CXCR4, as well as screening for potential ligands thereof.
  • Methods of the invention provide for the incorporation of unnatural amino acids and amino acids of the D configuration into linear or cyclic peptides for use in such libraries. Libraries comprising peptides having such amino acids demonstrate enhanced binding affinity and duration of action in vivo resulting from resistance to proteolysis.
  • the invention provides for the use of highly diverse libraries of peptide (linear and cyclic, natural and unnatural amino acids), peptidomimetic, and small molecule compounds for the lead ligand identification step.
  • Such ligands may be directly or indirectly agonistic or antagonistic to CXCR4 binding activity.
  • the invention provides for the use of phage display methods for the identification of preliminary motif information, followed by additional rounds of affinity purification with purified receptor preparations of the invention and highly diverse libraries.
  • phage display technology is combined with the use of cyclic peptide and/or peptidomimetic libraries.
  • computer-aided design technology is used to virtually screen, identify, design, or validate lead compounds for agonistic or antagonistic potential with regard to CXCR4 activity.
  • Such technology uses computer- generated, three-dimensional images based upon molecular and structural information of both the CXCR4 and the potential binding partners by virtually aligning the protein with the binding partners.
  • a great deal of the information necessary for lead optimization is obtained directly from the library design.
  • potential leads are identified by prior screening of an actual library or through some other means.
  • One embodiment of the invention involves the screening of biologically appropriate drugs that relies on structure based rational drug design. In such cases, a three dimensional structure of the protein (or similar family member), peptide or molecule is determined and potential agonists and/or antagonists are designed with the aid of computer modeling.
  • the drug is contacted with CXCR4, whereby a binding complex is formed between the potential drug and CXCR4.
  • Methods of contacting the drug to CXCR4 are generally understood by anyone having skill in the art of drug development.
  • the present invention provides for the use of partially purified CXCR4 receptor protein as the agent for carrying out the selection, identification, and improvement of tight binding ligands in identifying therapeutically useful compounds.
  • the invention comprises the use of tagging methods to generate a modified CXCR4 receptor protein that functions to facilitate purification and identification steps involved in the screening methods.
  • the invention comprises a nucleic acid sequence corresponding to the receptor CXCR4 fused to tag sequences (i.e., GST, FLAG, 6xHis, dual tagged with FLAG-GST, C-MYC, MBP, V5, Xpress, CBP, HA) with appropriate specific protease sites engineered into the vector.
  • methods of the invention provide for solubilization or immobilization of CXCR4 to facilitate ligand selection methods provided herein.
  • CXCR4 may be derived from any source, including without limitation: inactive, precipitated protein preparations; cell membrane preparations; and, whole cell preparations.
  • the invention provides for a method of screening combinatorial libraries directly for general affinity determination using membranes from baculovirus expression systems or any other appropriate expression system.
  • partially purified CXCR4 is used in carrying out the selection, identification, and improvement of tight binding ligands.
  • partially purified, tagged CXCR4 is used in a sequestered form to screen diverse libraries (focused or highly diverse) for the affinity purification of a tight binding ligand.
  • the conditions for solubilization or immobilization of the appropriate ligand provide for the use of low salt, such as, for example, low magnesium or calcium concentrations; and no sodium chloride ("NaCl") (O.OnM NaCl).
  • the invention further comprises the step of eluting bound components of the libraries from the immobilized protein with specific N-terminally blocked peptides or other non-sequencable analogs.
  • the invention comprises the optional step of binding combinatorial libraries to a resin-immobilized protein.
  • the invention comprises a purified polypeptide with tag sequences, which may be optionally immobilized onto an appropriate affinity resin for assay.
  • a further embodiment comprises the step of releasing or eluting tagged protein with its bound library with specific N- terminally blocked peptides or other non-sequencable analogs.
  • a method of the invention comprises the step of cleaving a tag from a protein of interest using a specific protease (as designed into the protein/vector) after immobilization onto an affinity resin and after the combinatorial library is bound to release the complex.
  • the target ligand is selected from a linear peptide library, a peptidomimetic library, a cyclic peptide library, or a focused library developed using an initial motif identified by phage display techniques or a library combining any of the foregoing.
  • a target ligand is eluted from the receptor preparation using a peptide or other ligand, or by using pH change or chaotropic agents, such as urea or guanidine hydrochloride, that can disrupt the hydrogen bonding structure of water and denature proteins in concentrated solutions by reducing the hydrophobic effect.
  • ligands for CXCR4 identified using the methods disclosed herein.
  • protein sequencing techniques are used for the determination of the structure of the ligand identified by the affinity purification step.
  • the invention comprises therapeutic agents, such as, for example, a small molecule antagonist of CXCR4 binding that are identified using methods of the invention appropriate for the treatment of a disease or disorder, such as, for example, HIV infection or AIDS.
  • a patient infected with HIV is treated with a therapeutic agent comprising a compound identified using methods of the invention, or a small molecule antagonist of CXCR4 binding.
  • a patient infected with HIV is treated through the use of combinations of therapeutics that include, for example, CXCR4 inhibitors and reverse transcriptase and protease inhibitors.
  • Figure 1 shows an exemplary peptide library with a fixed, non-degenerate lysine or arginine and eight degenerate positions consisting of eighteen amino acids in approximately equal proportion.
  • Figure 2 shows an exemplary peptide library screening using binding domains.
  • Figure 2a shows a SDS-PAGE of cell lysate containing CXCR4 and of purified
  • Figure 3 shows an isolated human CXCR4 cDNA sequence (SEQ ID NO: 125).
  • Figure 4 shows an exemplary baculovirus transfer vector for CXCR4-HIS.
  • Figure 5 shows an exemplary baculovirus transfer vector for CXCR4-FLAG.
  • Figure 6 shows an exemplary baculovirus transfer vector for CXCR4-GST.
  • Figure 7a is a chart showing representative radioligand saturation binding studies using membrane preparations of GST-CXCR4 (High Five).
  • Figure 7b is a chart showing representative displacement curves for radioligand binding studies using membrane preparations of GST-CXCR4 (High Five).
  • Figure 8 shows the immobilization of GPCRs for affinity purification from libraries.
  • Figure 9 depicts representative displacement curves for CXCR4.
  • Figure 10 is a bar graph showing representative high-throughput ligand-binding inhibition for CXCR4.
  • Figure 11 depicts IC 50 's for certain exemplary CXCR4 analogs of the present invention.
  • Figure 12 depicts the inhibition of HIV infection by certain exemplary CXCR4 peptide inhibitors of the present invention.
  • Figure 13 depicts the inhibition of HIV infection by an exemplary CXCR4 peptide inhibitor of the present invention.
  • methods of the invention provide for the determination of a binding motif for CXCR4. Further, methods of the invention provide for the identification of agonists or antagonists of the interaction of CXCR4 with its natural ligand, thereby providing for the identification of therapeutic lead compounds.
  • Methods for library design and synthesis, and library screening that are particularly useful in the invention are described in the following patent and patent applications, the disclosure of each of which is incorporated by reference herein: Cantley et al., U.S. Patent No. 5,532,167; Cantley, et al, U.S.S.N. 08/369,643, filed December 17, 1998; Cantley, et al, U.S.S.N.
  • CXCR4 is cloned and expressed, and tested for activity.
  • the CXCR4 may be tagged on the C-terminus or on the N-terminus to facilitate the determination of the character of the CXCR4's ligand-binding properties.
  • Exemplary tags include, without limitation, 6xHis, FLAG, GST, V5, Xpress, c-myc, HA, CBD, and MBP.
  • the tagged CXCR4 is used in screening of libraries comprising, for example, linear and/or cyclic peptides having natural and/or unnatural amino acids, peptidomimetics and/or small molecules.
  • Such peptidomimetics and small molecules may comprise any natural or synthetic compound, composition, chemical, protein, or any combination or modification of any of the foregoing that is used to screen for binding compounds of CXCR4.
  • an oriented degenerate peptide library useful in methods of the invention employs soluble peptide libraries consisting of one or more amino acids in non- degenerate positions, known or suspected to be important for ligand binding, and eighteen amino acids in approximately equal proportions in degenerate positions.
  • Cysteine and tryptophan may be omitted to avoid certain analytical difficulties on sequencing.
  • a library is shown in Figure 1 , where X represents a degenerate position consisting of any of eighteen amino acids and a lysine or arginine is fixed at a non-degenerate position.
  • arginine or lysine as an orienting residue is based on the fact that basic residues of gpl20 are important determinants in binding to CXCR4.
  • Another aspect of the invention involves the selection of any amino acid as an orienting residue. Additional residues can be added to the N-terminal of the sequence shown in Figure 1 because there are often interfering substances present in the first and second sequencing cycles.
  • Another aspect of the invention provides for the use of highly diverse libraries of peptide (linear and cyclic, natural and unnatural amino acids), peptidomimetic, and small molecule compounds for the lead identification step.
  • these ligands can be agonistic or antagonistic in their function on the receptor.
  • the invention uses partially purified CXCR4 as the agent for carrying out the selection, identification, and improvement of tight binding ligands as a route to therapeutically useful compounds.
  • the invention provides for the development and use of solubilization as well as immobilization procedures that facilitate the efficient ligand selection methods as provided herein.
  • the optimal conditions for solubilization and/or immobilization for efficient ligand selection comprise the use of low salt, such as, for example, low or no magnesium or calcium concentrations, and no NaCl concentrations (O.OnM NaCl).
  • Low salt such as, for example, low or no magnesium or calcium concentrations, and no NaCl concentrations (O.OnM NaCl).
  • Ligand selection methods using, for example, inactive, precipitated protein, cell membrane preparations, and whole cell preparations are further provided herein.
  • the screening step may comprise phage display technology.
  • phage display systems have been used to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. More recent improvements of the display approach have made it possible to express enzymes as well as antibody fragments on the bacteriophage surface thus allowing for selection of specific properties by selecting with specific ligands. See e.g., Smith SF, et. al, Methods Enzym. 217:228-257 (1993).
  • Phage display methods may be used for the identification of preliminary motif information, and can be followed by additional rounds of affinity purification with purified receptor preparations of the invention and highly diverse libraries, especially cyclic peptide and peptidomimetic libraries.
  • the phage display methods allow the identification of motifs of natural amino acids.
  • Information derived from phage display can be applied to affinity purification methods using, for example, synthetic libraries containing novel amino acid analogs or cyclic peptides to select ligands that have enhanced pharmaceutical characteristics.
  • the use of initial, secondary and tertiary libraries allows a more complete definition of the specificity of the binding site. Secondary libraries may be sequenced incorporating information from the initial library.
  • some degenerate positions may yield high preferences for specific amino acids and these may become non- degenerate positions consisting of the preferred amino acid in a second library. See e.g., Wu R, J Biol Chem 271(27): 15934-41 (1996).
  • computer-aided design technology may be used in the screening and/or designing of peptides, peptidomimetics, and small molecules.
  • computer aided design technology may virtually screen, identify, design and validate potential compounds with regards to their CXCR4 activity.
  • Computer programs that may be used to aid in the design of appropriate peptides, peptidomimetics and small molecules include, for example, DOCK (which may be obtained, for example, from University of California, San Francisco), FRODO (which may be obtained, for example, from University of Alberta) and INSIGHT (which may be obtained, for example, from Accelrys, San Diego, CA).
  • DOCK which may be obtained, for example, from University of California, San Francisco
  • FRODO which may be obtained, for example, from University of Alberta
  • INSIGHT which may be obtained, for example, from Accelrys, San Diego, CA.
  • An example of a method for screening of biologically appropriate drugs relies on structure based rational drug design. In such cases, a three dimensional structure of the protein, peptide or molecule is determined (or modeled after a close family member) and potential agonists and/or antagonists are designed with the aid of computer modeling.
  • the screening step may be performed in solution phase, or with the CXCR4 immobilized on affinity columns.
  • receptor and bound library components can be separated from non-bound library components using equilibrium dialysis.
  • the tagged receptor can be bound to specific affinity membranes, which are in the form of plates or are separate.
  • the libraries can then be incubated with the membrane and easily washed to remove non-specific binding components. Size exclusion methodology can be used to separate a purified receptor bound library complex from unbound components after pre-incubating the receptor with the library.
  • micellar complex containing the receptor (which may or may not incorporate lipids as well as detergent) can be separated after binding select affinity components from a library by differential centrifugation.
  • the high affinity ligand can be released using low pH or high salt conditions and the structure identified by sequencing as described herein.
  • peptide libraries were screened to determine each library's respective inhibition binding. In general, a greater than 10% inhibition at 100 ⁇ M was significant for continued evaluation of the sequence via affinity purification.
  • specific peptides are synthesized by the same methods as employed for library synthesis.
  • a high preference value is greater than 1. The value is determined by subtracting the control value from the sample value and dividing by the reference value. In a preferred embodiment of the invention, the preference value is greater than 1.2.
  • the preference value is greater than 2.
  • HPLC High Performance Liquid Chromatography
  • MALDI-TOF MS Matrix-Assisted Laser Desorption Ionization- Time of Flight Mass Spectrometer
  • Edman Sequencing Generally, relative affinities may be measured by modifying the radiolabel binding assay used in receptor purification.
  • the bound components of the libraries can be eluted from the immobilized protein with specific N-terminally blocked peptides or other non-sequencable analogs.
  • the release/elution of the tagged CXCR4 with its bound library can be accomplished using specific N-terminally blocked peptides or other non-sequencable analogs. This can be done using acetylated FLAG peptide to elute CXCR4-FLAG receptor from the resin.
  • the tag from CXCR4 may be cleaved using a specific protease (as designed into the protein/vector; either enterokinase or thrombin) after immobilization onto an affinity resin and after the combinatorial library is bound to release the complex.
  • libraries can be prescreened for their ability to bind to the receptor (using significantly less protein) by a binding assay using CXCR4-containing membranes from, for example, Sf9 (Spodoptera frugiperda) or High Five (Trichoplusia ni) cells (both obtained from Invitrogen, Carlsbard, CA) in a single assay or in an array assay.
  • Methods of the invention further comprise the design of therapeutic agents comprising peptides, peptidomimetics, and/or small molecules that are antagonistic to CXCR4 activity appropriate for the treatment of patients with a disease, such as AIDS.
  • Binding compounds for CXCR4 and the identification of optimal synthesis and purification thereof provides for an effective treatment of AIDS and HIV infection.
  • the small peptide ligand binding compounds of the invention, both cyclic and linear peptide ligands demonstrate enhanced binding affinity and anti-viral activity, and are resistant to proteolysis as identified, for example, in Table 1.
  • EXAMPLE 1 Preparation of Tagged CXCR4 and Screening of Peptide Libraries
  • CXCR4 vectors were prepared for the baculovirus expression system containing epitope tags that allowed for easier purification of the receptor.
  • Tags may be incorporated at the N- or C-terminus of proteins.
  • tags were incorporated at the C-terminus of the receptor to determine the receptor's ligand-binding properties at the N-terminal region of the molecule to allow easier purification of the receptor.
  • tags were placed at the N- terminus of proteins.
  • tags may be incorporated either at the N- or C-termini of the receptor.
  • C terminal 6xHis tagged and C-terminal FLAG constructs are provided below as examples.
  • Alternative tags may include, for example, GST, V5, Xpress, c- myc, HA, CBD, and MBP. These constructs were made using standard techniques known by those skilled in the art.
  • the 6xHis tag enables a one-step purification using nickel chelation.
  • the cDNA for CXCR4 was isolated from a spleen cDNA library using Polymerase Chain Reaction ("PCR") and primers for the 3' and 5' ends of CXCR4, as well as to the middle of the gene. To create a C- terminal 6xHis tag, CXCR4 was subcloned into an E.
  • coli vector pET30a, with a C-terminal 6xHis tag.
  • the newly created CXCR4-6xHis was then excised and ligated into pBlueBac, a baculovirus transfer vector (Invitrogen, Carlsbad, CA).
  • the construct was analyzed using both restriction digest and sequencing, and transfected into Sf9 insect cells (Pharmingen, San Diego, CA) for expression as typically done by those skilled in the art of protein expression.
  • a C-terminal bacterial FLAG construct was available from Sigma (St. Louis, MO) (pFLAG-CTC). A similar strategy using standard techniques was employed for the construction of this vector.
  • the CXCR4 was subcloned into the pFLAG-CTC plasmid, excised with the C-terminal FLAG tag and then ligated into the digested pBlueBac vector. The construct was analyzed using both restriction digest and sequencing, and transfected into Sf9 or High Five insect cells for expression. [0059] To express the CXCR4 gene in Sf9 or High Five cells, the pBlueBac vector containing the CXCR4 insert was cotransfected with Bac-N-Blue DNA using cationic liposome mediated transfection using standard techniques. The CXCR4 was inserted into the baculovirus genome by homologous recombination. Cells were monitored from 24 hours posttransfection to 4-5 days.
  • the transfection supernatant was assayed for recombinant plaques using a standard plaque assay.
  • Cells which have the recombinant virus produce blue plaques when grown in the presence of X-gal (5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside). These plaques were purified and the isolate was verified by PCR for correctness of recombination using standard techniques. From this, a high-titer stock was generated and infection performed from this stock for expression work using standard techniques. Controls for transfection include cells only and transfer vector.
  • Sf9 or High Five cells were maintained both as adherent and suspension cultures using standard techniques known to those skilled in the art.
  • the adherent cells were grown to confluence and passaged using the sloughing technique at a ratio of 1 :5.
  • Suspension cells were maintained in spinner flasks with 0.1% pluronic F-68 (to minimize shearing) for 2-3 months by sub-culturing to a density of 1 x 10 6 cells/ml.
  • FIG. 1 A time course after infection with recombinant virus was used to define optimal growth conditions for expression using standard techniques. Aliquots of cells from spinner flasks were taken for this time course, centrifuged at 800 x g for 10 minutes at 4°C and both supernatant and pellet assayed by SDS-PAGE/Western blot analysis.
  • Figure 2a shows the SDS- PAGE/Western Blots of cell lysate containing CXCR4 and purified CXCR4 stained with coomassie. The CXCR4 was expected to be in the membrane fraction (pellet). All viable systems were assayed in this fashion for levels of expression.
  • the membrane fraction was isolated by first pelleting the whole Sf9 cells (800 x g for 10 minutes at 4°C), then resuspending the pellet in a lysis buffer with homogenization.
  • Typical lysis buffer is around neutral pH and contains a cocktail of protease inhibitors, all of which are standard techniques for those skilled in the art. Membranes were pelleted.
  • Solubilization was conducted using varying NaCl concentrations. Despite conventional thinking, the step of solubilization using low salt, for example, low calcium and magnesium concentrations substantially in the absence of NaCl provided unexpected optimal conditions for solubilization when compared for quantity and activity.
  • CXCR4 was purified from the membrane fraction.
  • the exact purification scheme will depend on the construct chosen, which is subject to activity and ease of solubilization. The skilled artisan can readily construct a purification scheme using only routine experimentation.
  • the membrane fraction was loaded onto a Ni-NTA column (Qiagen, Valencia, CA) in the presence of detergent, washed extensively, and eluted with imidazole. Purification of the FLAG-tagged CXCR4 was performed using the anti-FLAG M2 affinity matrix (Sigma, St.
  • tBu was used for the protection of side-chains of Asp, Glu, Ser, Thr, and Tyr, tert.- butyloxycarbonyl ("Boc") for Lys and Tip, 2,2,4,6J-pentamethyldihydrobenzofuran-5-sulfonyl ("Pbf ') for Arg, and triphenylmethyl ("trityl", “Trt”) for Cys, His, Asn and Gin.
  • the scale of the synthesis was 0.20 mmol.
  • the resin was initially washed with N-methylpyrrolidinone (" ⁇ MP") followed by a 1 x 3 minutes and 1 x 7.6 minutes treatment of piperidine: ⁇ MP (1:4) for N°-Fmoc removal.
  • ⁇ BTU N-[(lH-benzotriazol-l- yl)(dimethylamino)methylene] -N-methylmethanaminium hexafluorophosphate N-oxide
  • DMF N,N-dimethylformamide
  • DIEA N,N-diisopropylethylamine
  • Fmoc-Asp(OH)-ODmab (Dmab, 4-[N-(l-(4,4- dimethyl-2,6-dioxoxcyclohexylidene)-3-methylbutyl)amino]-benzyl) was side-chain anchored to Rink amide resin followed by chain elongation as described above. Following linear assembly, removal of the Dmab and Fmoc group was accomplished by treatments with hydrazine:DMF (1 :49) for 7 minutes and piperidine: ⁇ MP (1 :4) for 6 x 3 minutes, respectively. The resin was transferred to a syringe containing a polypropylene frit for manual cyclization.
  • On-resin head- to-tail cyclization was performed using 7-azabenzotriazol-l-yloxy)- tris(pyrrolidino)phosphonium hexafluorophosphate ("PyAOP"):DIEA (1 :2, 4 equiv) in a solution containing 1% Trition X in NMP:DMF:dichloromethane, methylene chloride, DCM) (1 : 1 : 1) for 2 hours at 55°C.
  • the unreacted linear precursor was treated with Fmoc-Nva-OH/PyAOP/DIEA ("Nva", "norvaline”)(l :l :2, 4 equiv) in DMF for 1 x 18 hours and 1 x 3 hours.
  • Peptides and peptide libraries were characterized by HPLC, MALDI-TOF MS and Edman degradation.
  • MALDI-TOF MS analysis is capable of detecting the presence of high- molecular weight impurities due to incomplete deprotection, deblocking, or re-alkylation.
  • Edman degradation provides quantitative information about the amount of each amino acid in each degenerate position in a library.
  • Cyclic libraries (head-to-tail) were also prepared with single, non-degenerate orienting amino acids. Through the use of these initial libraries, the optimal residues at some degenerate positions become defined and secondary libraries were made fixing these positions.
  • the head to tail cyclized library cyclo(M-X-X-X-X-R-X-X-X-N) indicated that the -4 position (from the fixed R) should be lysine, the -2 position should be aspartate, the -1 position should be histidine, and the +3 position should be lysine so the secondary library was cyclo(M-K-X-D-H-R-X-X-K-N) (SEQ ID NO: 61).
  • An oriented linear peptide library was applied to a column containing immobilized CXCR4 and a small fraction of isolated high affinity peptides.
  • a schematic diagram showing the peptide library using binding domains can be seen in Figure 2.
  • the initial library used a single, non-degenerate basic amino acid (/ ' . e. , M-A-X-X-
  • M-A-X-X-X-X-W-X-X-X-X-A-K- K-K may indicate that the -4 position should be arginine, -1 should be isoleucine, and +1 should be arginine so the secondary library would be M-A-R-X-X-I-W-R-X- X-X-A-K-K-K (SEQ ID NO: 70).
  • the receptor was exposed to the library, and separation of free and bound peptides was accomplished by pelleting the membranes by centrifugation.
  • the 6xHis-tagged CXCR4 purified receptor was incubated with a peptide library, about 1 ⁇ mole of peptide and about 1 nmole of binding sites. After incubation, receptor with bound peptide was separated from unbound peptides by centrifugation (receptor*peptide complex in the pellet, unbound peptide in the supernatant). Nonspecifically bound peptides were removed by exhaustive washing, and resuspension of the pellet in low pH ( ⁇ 2.5) was used to remove the bound peptide.
  • This peptide was sequenced to determine the consensus sequence.
  • the receptor was immobilized on an anti-FLAG M2 affinity matrix (St. Louis, MO).
  • An additional purification approach used the CXCR4-GST construct and immobilized glutathione (Pierce, Rockford, IL).
  • Both the bound peptide mixture and the starting peptide library were sequenced using standard techniques. The amounts of each amino acid, as a function of position, were determined. Preference values for each amino acid at each position were calculated by comparing the amounts of amino acids present in the starting library and bound fraction of peptides. These procedures were used to generate preferred sequences of peptides interacting with many binding domains and have been described in Table 2. [0078] Also, secondary libraries were sequenced incorporating information from the initial library. For the first round of characterization, phage display technology is also used to identify preliminary binding motifs. The phage display method provides for the identification of motifs of natural amino acids. Phage display technology involves the insertion of DNA sequences into a gene coding for one of the phage coat proteins.
  • each phage can express a different peptide sequence ("a phage library"). Incubation of this phage library with the immobilized receptor can be used to identify sequences which specifically bind to the receptor. Even weak signals can detected because they can be amplified by growing the isolated phage.
  • Information derived from phage display is applicable to affinity purification methods using synthetic libraries containing novel amino acid analogs or cyclic peptides to select ligands that have enhanced pharmaceutical characteristics.
  • CXCR4 was isolated from a spleen cDNA library in two halves and spliced together. These two fragments were isolated using PCR technology and primers to the 3' and 5' ends and the middle of the CXCR4 gene. A full-length clone was not isolated with the 3' and 5' primers; however, two halves were isolated and ligated together using a unique BamHl site in the gene. The identity of the construct was confirmed by sequencing. The sequence of the isolate is in Figure 3. An alternate splice shorter form was also isolated, which is called CXCR4s. Tags were added to the C-terminus of the receptor for use in immobilizing them for affinity purification assays using standard techniques. The following are specific examples from experiments using the tagging method. [0084] Construction ofCXCR4 with C-Terminal Histidine Tag (Insect Select Expression
  • CXCR4 construct A previous construct containing the gene for GnRHR (gonadotropin releasing hormone receptor) was used to make the first CXCR4 construct.
  • the gene for GnRHR was spliced out and replaced by the isolated cDNA for CXCR4.
  • This vector was originally the pet30a vector with the 6xHis tag at the C-terminus.
  • Construction ofCXCR4 construct with C-terminal FLAG tag PCR was performed using the primers 5' BspEl CXCR4 and 3' Bgl CXCR4 engineered with unique sites for ligation of CXCR4 in frame with the FLAG tag of pFLAG-CTC (a bacterial expression vector) from Sigma. This construct is called CXCR4-FLAG-CTC.
  • CXCR4-FLAG was then removed by digestion and filled in with Klenow fragment.
  • the fragment containing CXCR4- FLAG was ligated into pBluebac 4.5 that was first digested then blunted with Klenow. This final construct is called CXCR4-FLAG.
  • the construct was confirmed by restriction digestion and sequencing using standard techniques. This construct has been used for expression and has been determined to be expressed sufficiently and in active form for use in affinity purification screening.
  • CXCR4-FLAG cDNA was removed from the CTC vector and subcloned into another construct, CCR5-GST, in place of the CCR5 (using Bgl and BspEl). This created the vector for CXCR4-GST using one step.
  • the construct was confirmed by restriction digestion and sequencing using standard techniques. This construct has been used for expression and has been determined to be expressed sufficiently and in active form for use in affinity purification screening.
  • CXCR4 with N -terminal 6xHis tag This construct was prepared by subcloning the CXCR4 into the commercially available vector, pBluebacHis2b (Invitrogen, Carlsbad, CA). The construct was confirmed by restriction digestion and sequencing using standard techniques. [0088] Plasmid maps for these vectors are found in Figures 4-6. [0089] The vectors for the three new constructs (for CXCR4-FLAG, CXCR4-GST, and
  • CXCR4-HIS were used to co-transfect Sf9 cells for the production of a viral stock of each. These viral stocks were purified using a standard plaque assay and then used in experiments to infect for the optimization of expression of CXCR4 with its various C-terminal tags. High Five cells (Invitrogen, Carlsbad, CA) were also transfected with these CXCR4 tagged constructs and tested for expression of CXCR4. All constructs were determined to express the appropriately tagged receptor. Expression levels after 72 hours were as much as 5 times greater in High Five cells than those for Sf9 cells. All of the above described experiments were done using standard techniques known to those skilled in the art.
  • a fourth construct for the expression of CXCR4 was made from the starting vector pBlueBac 4.5 (Invitrogen, Carlsbad, CA) to remove the thrombin and enterokinase cleavage sites in the previously described vectors.
  • the GST tag was added into the multiple cloning site by using PCR to generate the GST tag, then ligating into the digested vector (Smal/EcoRI) using standard procedures known to those skilled in the art.
  • the vector was made compatible with the Gateway cloning technology from Lifetech for ease of manipulation. This was done by ligating into the Smal site the cassette containing the recombination sites required for this technology (from Lifetech, Rockville, MD).
  • CXCR4 was amplified using PCR with primers to extend the gene to contain the attachment sites for recombination. Then, the PCR product was incorporated into the baculovirus vector using BP clonase (the enzyme required for homologous recombination) to make a vector for baculovirus expression containing CXCR4 with a C-terminal GST tag without the enterokinase or thrombin cleavage sites. This vector was cotransfected into Sf9 cells for preparation of the virus stock necessary for expression. The virus was plaque purified, and a PCR and sequence checked clone was used for expression of CXCR4. A time course with this construct showed that less proteolysis of the protein was observed and less time was necessary to obtain maximal expression of the receptor. [0091] Activity
  • HIS HIS
  • the radioligand [ I]-SDF-l ⁇ was incubated with membranes (0.5 ⁇ g) in binding buffer at 27°C for 1 hour with and without unlabelled SDF-1 : For filtration and washing, the reaction was transferred to Millipore Multiscreen filter plates (HATF 0.45 ⁇ m; pre-blocked with 10% BSA) (Bedford, MA), filtered using vacuum, washed 4-5 times with 200 ⁇ L ice-cold buffer, and radioactive counts bound were detected using scintillation counting. All points were done in triplicate. Uninfected cells were used as a control for this experiment. To determine the K D , saturation binding was measured using increasing concentrations of [ I]-SDF-l ⁇ (from 0.5 nM to 10 nM).
  • Nonspecific binding was measured in the presence of 400 nM unlabelled SDF-1 ⁇ .
  • Competitive binding assays were performed by incubating CXCR4-containing membranes with 0.5 nM [ I]-SDF-l ⁇ and serial dilutions of unlabelled SDF-l ⁇ or peptide ligand.
  • Analyses of K D and IC 50 were performed using non-linear curve fitting in Kaleidagraph (software program sold by Synergy Software, Reading, PA).
  • Figure 7 exemplifies radioligand binding studies using membrane preparations of GST-CXCR4 (High Five). Saturation binding demonstrated a K D of 3.27 nM as seen in Figure 7A.
  • Solubilization of the tagged versions of CXCR4 have been performed using many different combinations of detergents (NP- 40, Triton X-100, ⁇ -D-maltoside, n-octylglucoside, CYMAL, Zwittergents, Tween-20, lysophosphatidyl choline, CHAPS, etc.), salts (NaCl, CaCl 2 , MgCl 2 , MnCl 2 , KC1, etc.), buffers (Tris, Hepes, Hepps, Pipes, Mes, Mops, acetate, phosphate, imidazole, etc.), and various pH's (range 6.8-8.2).
  • detergents NP- 40, Triton X-100, ⁇ -D-maltoside, n-octylglucoside, CYMAL, Zwittergents, Tween-20, lysophosphatidyl choline, CHAPS, etc.
  • salts Na
  • CXCR4-GST were immobilized onto affinity columns for purification and as an active protein ready for use in screening of peptide libraries.
  • a schematic diagram showing the immobilization of GPCRs for affinity purification from libraries is shown in Figure 8.
  • CXCR4-GST was bound and immobilized onto glutathione-agarose (Pierce, Rockford, IL) and glutathione-sepharose (Amersham Pharmacia Biotech, Piscataway, NJ).
  • the immobilization of the functional protein was accomplished by first solubilizing the receptor in 0.3%) NP-40, 10 mM Hepes (or Pipes), pH 7.5, then binding it to the glutathione-sepharose resin in 0.3% NP-40, 10 mM Hepes (or Pipes), pH 7.5, 3 mM CaCl 2 , 15 mM MgCl 2 .
  • the activity of the immobilized receptor was determined by incubation for 1 hour at 4°C with the radiolabeled SDF-1 ⁇ (as with the membrane assay above) and competition with cold SDF-1 ⁇ . Uninfected cells were used as controls for this activity, as well as the column alone.
  • the initial libraries used a single, non-degenerate basic amino acid (i.e., M-X-X-
  • FIG. 10 is a bar graph depicting the high-throughput ligand-binding inhibition for CXCR4. CXCR4-containing membranes were incubated in the presence of 100 ⁇ M library and radiolabeled SDF- 1. Percent inhibition was calculated to determine the effect of each library. Peptide libraries with the highest percent inhibition were assayed first.
  • Eluted peptide was filtered using a Centricon-10 (Millipore, Bedford, MA) to remove any protein that might have co-eluted with acetic acid. The filtrate was dried under vacuum, dissolved in water and subjected to peptide sequencing.
  • CPI-1221 W-R-P-A-K-A-K-K-K-K (CPI-1221) (SEQ ID NO: 1).
  • the affinity for the receptor was determined using standard radioligand displacement methodology.
  • This peptide CPI-1221 was determined to have an IC 50 of -60 ⁇ M.
  • Figure 11 depicts the IC 50 for certain CXCR4 analogs represented by the symbols closed circles (•), open circles (o), open triangles ( ⁇ ) and open squares (D).
  • CPI-1221, CPI-1336, CPI-1289, and CPI-1365 correspond to the symbols closed circles (•), open circles (o), open triangles ( ⁇ ) and open squares (D), respectively.
  • certain analogs increased binding inhibition as described herein.
  • CPI-1221, a 15-mer was the original peptide. Analogs of this peptide were selected having increased specificity as demonstrated by the binding curves in Figure 11.
  • CXCR4 inhibitors were also tested for specificity at other receptors, such as, for example, CXCR2, CCRl, Angiotensin II ATI, and neuropeptide Y.
  • the CXCR4 inhibitors demonstrated no specificity for any of these receptors.
  • Rational analoging of this peptide was performed as follows. Minimal length of the peptide sequence was determined by deleting from both the N- and C-termini. Approaches to protect against metabolism were conducted using synthetic analogs.
  • Insertion of a rigid segment of the peptide was accomplished with cyclic amino acids such as azetidine-2-carboxylic acid, tetrahydroisoquinoline (Tic), pipecolic acid (Pip), thiazolidine-4-carboxylic acid (Thz), 1 - amino- 1-cyclopentane-carboxylic acid and 1 -amino- 1-cyclohexanecarboxylic acid (Sawyer, 1995) as well as ⁇ , ⁇ -dialkyl residues such as aminoisobutyric acid (Aib) and diethylglycine (Deg).
  • cyclic amino acids such as azetidine-2-carboxylic acid, tetrahydroisoquinoline (Tic), pipecolic acid (Pip), thiazolidine-4-carboxylic acid (Thz), 1 - amino- 1-cyclopentane-carboxylic acid and 1 -amino- 1-cyclohexanecarbox
  • Table 3 is a summary of the substitutions made at the various positions in the peptide CPI-1221 which were synthesized and screened for activity using inhibition of [ I]-SDFl ⁇ binding and toxicity on Jurkat and CEM cells.
  • all positions were individually substituted with Ala, several positions were substituted with D-amino acids, the N-terminus of several peptides were acetylated, deletions of the N- and C-termini were made, and several retro-inverso peptides were synthesized.
  • CPI-1500 (ARSLI(2-Nal)R(Tic)ARR(2-Nal)RR) (SEQ ID NO: 72) demonstrated specificity for the HIV Illb over the R5 virus 9881.
  • Figure 12B shows that other CXCR4 peptide inhibitors also demonstrated inhibition of HIV Illb with specificity. Table 3:
  • G' P, H, Tic, A, Hyp, azetidine-2-carboxylic acid
  • Phage Display As an alternative or additional method useful in screening for binding compounds and analogs thereof, immobilized functional CXCR4 was used to isolate phage which bind to CXCR4 using standard techniques known to those skilled in the art. Subtraction of the background from the glutathione-sepharose beads, BSA, and SDFl ⁇ used in the assay was performed by incubation of the phage library with the mixture of these components. After incubating subtracted phage libraries (i.e., NEB PhD C7C) with the receptor, bound phage were eluted both with the natural CXCR4 ligand (SDFl ⁇ ) and with glycine, pH 2.2.
  • subtracted phage libraries i.e., NEB PhD C7C
  • PhD C7C is a particular phage library with 7 random amino acids between disulfides, and may be obtained from New England Biolabs ("NEB", Beverly, MA). Multiple rounds of screening were performed. Both conditions have provided specific sequences (see Table 4) which bind to CXCR4. Table 4:
  • Example 4 Prevention or Treatment of HIV Infection or AIDS
  • Methods of preventing and treating AIDS and HIV infection comprise the step of administering a composition comprising such a compound capable of inhibiting CXCR4 binding as described herein.
  • Administration may be by any compatible route.
  • administration may include oral or parenteral, including intravenous and intraperitoneal routes of administration.
  • a particularly preferred method is by controlled-release injection of a suitable formulation.
  • administration may be by periodic injections of a bolus of a composition, or may be made more continuous by intravenous or intraperitoneal administration from a reservoir that is external (e.g., an intravenous bag) or internal (e.g., a bioerodable implant).
  • a reservoir that is external (e.g., an intravenous bag) or internal (e.g., a bioerodable implant).
  • compositions contemplated by the present invention may be provided to an individual by any suitable means, directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e.g., parenterally or orally).
  • parenterally such as by intravenous, subcutaneous, intramolecular, ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, intranasal or by aerosol administration
  • the composition may comprise part of an aqueous or physiologically compatible fluid suspension or solution.
  • the carrier or vehicle is physiologically acceptable so that in addition to delivery of the desired composition to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance.
  • Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, A., ed.), Mack Pub., 1990.
  • Formulations of the therapeutic agents of the invention may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like.
  • Formulations for direct administration in particular, may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus.
  • Biocompatible, preferably bioresorbable, polymers including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide, and glycolide polymers and lactide/glycolide copolymers, may be useful excipients to control the release of the agent in vivo.
  • the concept of a controlled release injectable formulation for peptide drugs is well-accepted and offers several advantages.
  • bioavailabilities are high.
  • treatment regimens can consist of once per month or per three months (like Abbott's Leupron®), or once per year (e.g. Alza's Viadur®).
  • controlled release injectable formulations substantially reduces the doses that can be used (the Leupron injection dose is 1 mg/day but the 90 day formulation uses is 11.25 mg total). Also, increased efficacy can be achieved if the therapeutic is present continuously to prevent infectivity. This consideration is particularly important in view of the need to approach a cure for this disease by preventing the reformation of slow-to-clear deposits of infection such as the memory T cell compartment. See e.g., Lee, V., ed. Peptide and Protein Drug Delivery. Marcel Dekker, Inc., NY (1991).
  • parenteral delivery systems for these agents include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid for vaginal administration.
  • Example 5 Exemplary CXCR4 Binding Compounds
  • Methods of the invention further contemplate the design of therapeutic agents comprising peptides, peptidomimetics, and/or small molecules which are antagonistic to CXCR4 activity and which are appropriate for the treatment of patients with a disease, such as AIDS.
  • Table 5 further provides both linear and cyclic exemplary peptides that demonstrate enhanced binding affinity and anti-HIV activity, and are resistant to proteolysis.
  • the bolded text represents any changes from the amino acid sequence of CPI-1221, the sequence for which is provided in Table 1.
  • Certain exemplary peptides in Table 5 contain a number in superscript. The supercript represents the position number of the amino acid that corresponds to the respective position in the formula of the general structure of exemplary peptides provided in Table 6.
  • the amino acids corresponding to each of the two position numbers are connected to form a cyclic structure within the peptide.
  • the Glutamine at position 8 (based upon the formula provided in Table 6) is connected to the Lysine at position 12 to form a cyclic structure within the peptide.
  • exemplary peptide sequences are depicted from N-terminal amino acid to the C-terminal amino acid, with the C terminal amino acid having a C- terminal amido group (optionally depicted herein by the # symbol), such as, for example, -NH 2 .
  • Example 6 More Exemplary Peptides and Peptide Analogs of CXCR4
  • Table 6 is a summary of the substitutions made at the various positions in certain of the exemplary peptides (cyclic and linear) that are antagonistic to CXCR4 activity.
  • the bolded text represents the peptide sequence identified by CPI-1221 having an amino acid sequence with the appropriate amino acid substitutions based on the formula provided below.
  • Example 7 Assay for Inhibition of HIV Infectivity
  • the anti-HIV activity of exemplary compounds was tested using established adherent HELA cells or Magi cell-based (Magi cell line incorporating the reporter genes) cell line infected with a variety of HIV-1 isolates. Inhibitory activity was determined using a reporter system.
  • the cell line was modified to contain the gene for the HIV-LTR (long terminal repeat) promoter which is used by HIV after infection. This gene was coupled to an enzyme, ⁇ - galactosidase, which can cleave a substrate, CPRG (Chlorophenol red ⁇ -D-galactoside) which gives rise to a colored signal or FDG (Fluorescein di-Galactoside) which gives rise to a fluorescent signal.
  • HIV-LTR long terminal repeat
  • CPRG Chlorophenol red ⁇ -D-galactoside
  • FDG Fluorescein di-Galactoside
  • the cell becomes infected with HIV, it couples to the HIV-LTR promoter, producing the enzyme ⁇ -galactosidase, which then cleaves the CPRG substrate giving rise to a measurable absorbance signal, for example, at or about 595nm.
  • the signal is thus proportional to the amount of infection by HIV, giving a specific measure of how much HIV gets into a cell.
  • Toxicity was measured with the same cell system by measuring the effect of the inhibitor on cellular metabolism.
  • a standard measure of the toxicity uses the ability of cells to convert the tetrazolium salt MTS (5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazol)-3-(4- sulphophenyl) tetrazolium, inner salt) (Promega, Madison, Wl) to a colored substance, formazan, giving a direct measurement of the number of viable cells remaining (by measuring the absorbance at or about 450nm).
  • Measurements were taken four days after infection. A well-characterized HIV inhibitor, AZT, was used as a control.
  • EC 5 o's were determined graphically using Kaleidagraph, a software program sold by Synergy Software, Reading, PA. Representative data for infectivity and viral specificity of CPI-1500 is depicted, for example in Figure 12A and Figure 13. Other exemplary compounds which can inhibit HIV infection are also listed in Table 7.
  • Table 7 Inhibition of HIV infection by CXCR4 inhibitors.

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Abstract

L'invention concerne des composés de liaison pour le récepteur de chimiokine CXC 4 et des procédés d'identification desdits composés. L'invention concerne également des agents thérapeutiques renfermant ces composés.
PCT/US2001/051165 2000-10-27 2001-10-26 Composes de liaison et procedes d'identification de ces derniers WO2002057313A2 (fr)

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WO2014177021A1 (fr) * 2013-05-03 2014-11-06 南京工业大学 Petit motif peptidique renforçant un peptide signal de sécrétion forte et son utilisation
EP3301106A1 (fr) * 2013-05-03 2018-04-04 Nanjing University of Technology Petit motif peptidique renforçant un peptide signal de sécrétion forte et son utilisation
US10626147B2 (en) 2014-05-21 2020-04-21 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
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US11225506B2 (en) 2014-05-21 2022-01-18 Entrada Therapeutics, Inc. Cell penetrating peptides and methods of making and using thereof
US10456443B2 (en) 2014-08-27 2019-10-29 Ohio State Innovation Foundation Peptidyl calcineurin inhibitors
US11576946B2 (en) 2018-01-29 2023-02-14 Ohio State Innovation Foundation Peptidyl inhibitors of calcineurin-NFAT interaction
US11168310B2 (en) 2018-02-22 2021-11-09 Entrada Therapeutics, Inc. Compositions and methods for treating mitochondrial neurogastrointestinal encephalopathy
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US11987647B2 (en) 2018-05-09 2024-05-21 Ohio State Innovation Foundation Cyclic cell-penetrating peptides with one or more hydrophobic residues

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