EP0935608A1 - Peptides cytotoxiques - Google Patents

Peptides cytotoxiques

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Publication number
EP0935608A1
EP0935608A1 EP97941730A EP97941730A EP0935608A1 EP 0935608 A1 EP0935608 A1 EP 0935608A1 EP 97941730 A EP97941730 A EP 97941730A EP 97941730 A EP97941730 A EP 97941730A EP 0935608 A1 EP0935608 A1 EP 0935608A1
Authority
EP
European Patent Office
Prior art keywords
nef
nef2
peptide
myr
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97941730A
Other languages
German (de)
English (en)
Other versions
EP0935608A4 (fr
Inventor
Ahmed Azad
Melinda Lowe
Cyril Curtain
Jonathan Baell
Barry Matthews
Ian Macreadie
Chinniah Arunagiri
Don Rivett
Raymond Norton
Kevin Barnham
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biomolecular Research Institute Ltd
Original Assignee
Biomolecular Research Institute Ltd
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Filing date
Publication date
Priority claimed from AUPO2659A external-priority patent/AUPO265996A0/en
Priority claimed from AUPO2680A external-priority patent/AUPO268096A0/en
Application filed by Biomolecular Research Institute Ltd filed Critical Biomolecular Research Institute Ltd
Publication of EP0935608A1 publication Critical patent/EP0935608A1/fr
Publication of EP0935608A4 publication Critical patent/EP0935608A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C07K5/06113—Asp- or Asn-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • 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
    • 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
    • G01N33/5014—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 toxicity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G01N2333/163—Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx

Definitions

  • This invention relates to peptides derived from the amino-terminus of the Nef protein and which exhibit lytic or cytotoxic activities.
  • it relates to sequences which are myristylated at a site proximal to hydrophobic and/or basic amino acid residues.
  • the invention also relates to a method of screening for inhibitors of cytotoxic activities and to the synthesis of such inhibitors.
  • nef a highly conserved gene that overlaps the 3' LTR.
  • nef gene product a myristylated protein of 205 amino acids.
  • SIVmac nef Genetically-engineered point and deletion mutations in molecularly- cloned SIVmac nef also implicate Nef in AIDS pathogenesis in rhesus monkeys (Kestler et al . , 1991).
  • Nef is implicated in a diverse range of activities within the cell. However, there has been little consideration of an extracellular mode of action, even though there is some evidence that Nef exists in an extracellular form. Antibodies to Nef appear early after HIV-1 infection (Cul ann et al., 1989, 1991; Reiss et al . , 1989, 1991;
  • Nef was reported to be 'secreted' from cells into the medium (Guy et al., 1990), and in a yeast expression system Nef was released from the cells under conditions of stress (Macreadie et al 1995) .
  • the Nef protein of HIV-1 is a 27 kDa cytoplasmic protein targeted to the plasma membrane and to other cellular membranes by the sequence at the N-terminus which has an N-myristyl group added post-translationally at glycine 2 (Franchini et al, 1986, Guy et al , 1987, Ka inchik et al, 1991, Yu et al, 1992) . It is encoded by the nef gene and its mRNA accounts for more than 75% of all the viral mRNA (Schwartz et al , 1990) .
  • Nef appears to play a number of roles in HIV infection and pathogenesis. Several of these roles involve cell membrane interactions. Nef downregulates the HIV-1 receptor, CD4 (Guy et al 1987), as well as IL-2 mRNA and the surface expression of the IL-2 receptor (Luria et al 1991) . Nef has also been shown to interact with proteins involved in signal transduction (Saksela et al 1995, Lee et al 1995) and with a cellular kinase (Bodeus et al 1995) where membrane targeting appears to be essential for association. Such targeting also appears to be essential for the association of Nef with a range of other membrane- bound proteins (Sawai et al 1995) .
  • Residues 2-7 appear to be essential for CD4 downregulation (Salghetti et al 1995) , while residues 11-18 appear to furnish a binding site for a serine kinase (Baur et al 1997) .
  • Nef expressed in a variety of eukaryotic cells is cytotoxic, particularly under conditions of stress. Since there is evidence that Nef produced during HIV-1 infection can be exported to the external medium, it has been suggested that such cytotoxicity might be a cause of the killing of bystander cells observed in AIDS pathology.
  • myristylated N-terminal peptides of Nef are lytic for cells such as human leucocytes, erythrocytes and yeast.
  • the lytic activity is not merely a result of the presence of the myristyl chain, and appears to be sequence and structure specific.
  • the invention provides a cytotoxic peptide comprising a myristylated Nef-amino terminal sequence, wherein the site of myristylation is proximal to hydrophobic.
  • the myristylation site is proximal to a hydrophobic and basic amino acid residues.
  • the peptide is haemolytic.
  • the Nef-amino terminal sequence comprises a first flexible domain and a second a-helical domain.
  • the myristylation site is in the first flexible domain and the peptide comprises Myr-Nef2-20, Myr-Nef2-22 and Myr-Nef2-26.
  • the cytotoxic peptide comprises a flexible domain Nef2 -8, which is myristylated at Gly2 , and an a-helical Nef9-21.
  • the invention relates to a cytotoxic or lytic peptide comprising a myristylated Nef-amino terminal sequence, wherein the sequence is positively charged. More preferably, the proximal region of the N terminus eg. the first seven amino acid residues of sequence, has a net positive charge, and the succeeding residues form an ⁇ - helical region, preferably spanning residues 9 to 21.
  • the flexible site of myristylation is preferably proximal to a hydrophobic amino acid residue, preferably Trp, lie or Phe, most desirably Trp5.
  • the positive charge can be provided by one or more of Lys and/or Arg residues.
  • Preferred myristylated peptides of the invention include but are not limited to:
  • the first flexible domain appears to be involved in lytic or cytotoxic activities.
  • the invention provides cytotoxic or lytic peptides which may be targetted to unwanted cells such as malignant, cancer or pathogenic cells.
  • the peptides of the invention may be administered as selective toxins for treatment of conditions such as cancer or for reducing or ameliorating tumours.
  • the invention also provides a mehtod of inducing selective cell death.
  • Nef activity may be attained by targeting one or both of these domains.
  • Compounds which interfere with membrane localisation and perturbation may be used to inhibit lysis of cells and those which form complexes with the second domain to result in non- amphipathic entities may be used to produce non-fusogenic structures .
  • the invention provides a method of screening for inhibitors of cytotoxic, lytic and/or fusogenic activities, comprising the step of measuring the effect of the presence of one or more putative inhibitors on the activity of the cytotoxic peptide of the invention, or of a domain thereof.
  • the invention provides novel compounds which block the activity of HIV or Nef; these compounds include but are not limited to those listed in Tables 3, 4, 5, and 6. Preferably, the compounds are those listed in Table 6.
  • Cytotoxic or lytic activities of a compound, or derivatives or analogues of those given in the Tables can be assayed using cultured cells or artificial membranes. Fusogenic activities can be measured by using artificial membranes or phospholipid vesicles. Observations on the regulation of receptors such as CD4 and IL2-R may also be made. The person skilled in the art will be able to determine suitable forms of assays to be employed.
  • Compounds which exhibit inhibitory activities on the first domain preferably contain acidic groups capable of forming salt bridges with the basic amino-acid residues of the flexible domain to enhance binding and to block binding of Nef to the negatively-charged phospholipids of the cell membrane and also preferably have a specificity- endowing moiety such as an aromatic moiety capable of reacting with the indole ring system of the tryptophan residue 5.
  • the activity of these compounds may be assayed by inhibition of the cytolytic activity of myristylated Nef2-26 peptide or variants thereof.
  • Compounds which may be used include, but are not limited to lipophilic, aromatic, anionic or zwitterionic or polar moieties.
  • Bis- inhibitors made up of mono inhibitors linked by long chain di-acids, or inhibitors comprising multiple peptide sequences are also contemplated.
  • Non-limiting examples of compounds with the activities described above are one or more of those listed in Tables 3 to 6, preferably the compounds in Table 6.
  • the fusogenic activity of the second domain may be targeted with compounds that block the amphiphilic nature of this domain.
  • Such compounds may be amphiphilic themselves and complementary to the hydrophobic phase of the helix. Alternatively, they may be complementary to the hydrophilic face. In both cases, specific interaction would result in a complex with a second domain, in which the complex is non-amphipathic, and therefore is non- fusogenic.
  • Putative inhibitors of fusogenic activity include, but are not limited to, negatively charged amphiphathic helical peptides, coil/coil inhibitors or anti-sense peptide nucleic acid (PNA) inhibitors.
  • Preferred compounds which inhibit fusogenic activity as described above include but are not limited to those described in Tables 2 to 6, most preferably those in Table 6.
  • Derivatives or analogues of the peptide and compounds described herein are also contemplated by this invention. A person skilled in the art will be able to obtain such compounds on the basis of the sequence, structure and chemical or biological characteristics of the peptide and compounds described herein.
  • the invention provides a method of modulating interaction of Nef protein with a cell membrane, comprising the step of administering a compound as described above to prevent the Nef protein interacting with membrane bound components.
  • the components are preferably information or energy transducers, and may include proteins, peptides, hormones, ions or the like.
  • the invention provides a method of reducing or eliminating the cytotoxicity of Nef or related sequences, comprising the step of administering an inhibitor or compound as described above.
  • the compound inhibits the down regulation of CD4 receptors and IL-2 receptors by HIV or a part thereof, such as the Nef protein. More preferably, the compound inhibits interaction of Nef with intracellular proteins such as lck and/or serine kinase. Most preferably, the compounds inhibit Nef-induced cytotoxicity in cells of the lymphoid tissue, particularly non-infected lymphoid cells, and/or inhibit Nef-induced killing of bystander cells.
  • the invention in a sixth aspect, relates to a method of treating HIV infection, comprising the step of administering a compound of the invention to a subject in need of such treatment.
  • the invention also relates to pharmaceutical compositions comprising one or more compounds of the invention, together with a pharmaceutically acceptable carrier.
  • the composition of the invention may be formulated and administered in a manner known to a person skilled in the art.
  • the composition may be in the form of a liquid formulation, aerosols, micronised particles, liposomes, tablets, capsules or powdered form for oral, ip, iv or parenteral administration. It may also be administered via the mucosa using, eg. nasal sprays or nose drops.
  • the dosages and frequency of administration will be easily determined by a person skilled in the art in accordance with the condition to be treated.
  • Modifications may be made to the peptides or compunds described herein without deleteriously affecting the biologically activity thereof. Such modifications would be within the knowledge and expertise of the person skilled in the art and include, for example, conservative or non-conservative amino-acid substitutions, insertions, deletions or derivatisation which do not substantially modify the activity of the molecules.
  • Figure 1 shows the haemolytic effects of Nef peptides on human erthrocytes .
  • the extent of haemolysis was calculated by dividing the OD540nm at each point by the OD540nm of cells exposed to 2% Tween 80. Cell concentrations were 2% (v/v) and duration of exposure to lytic agent for each point was 5 in . Assays were performed in duplicate and intra-assay variation was within 5%.
  • Figure 2 shows the kinetics of lysis of human erythrocytes by Nef peptides. The extent of haemolysis was calculated as in Figure 1. Cell concentrations were 2% and the concentration of each peptide was mg/ l. Assays were performed in duplicate and intra-assay variation was within 5%.
  • Figure 3 comprises photomicrographs of cultured cells treated with Nef peptides (10X magnification). CEM cells were treated with lOmg peptide, or PBS for 30 minutes.
  • Figure 4 shows LDH release by Nef-treated CEM cells.
  • Figure 4C shows the kinetics of release of LDH from CEM cells incubated with Myr-Nef2-26 (squares) , Nef2- 26 (triangles), or Myr-Nef31-50 (circles) at a final concentration of 6.25 ⁇ M for up to 4 hr. Values represent the mean ⁇ SEM for three separate experiments, performed in triplicate, and are expressed as the percentage of maximum LDH release induced by 1% Triton X-100.
  • Figure 5 shows a fluorescence emission spectra for Nef2-26(Serl3) (solid curve) and Myr-Nef2-26 (Serl3 ) (dotted curve) in PBS. The excitation wavelength was 290 nm, temperature-22°C. The concentration of each peptide was 5mM.
  • Figure 6 shows Stern-Vollmer plots of
  • Figure 7 illustrates the effect of Myr-Nef2-22 on yeast colony formation.
  • the photograph shows the colonies formed after one day of the peptide treatment for C. albicans and C. glabrata, two days for K lactis and S. cerevisiae, and three days for Sz . without peptide treatment. With peptide treatment all plates were identical - no colonies formed.
  • Figure 8 shows the effect of myristylation of Nef peptides on yeast mortality.
  • A. S. cerevisiae cells were treated with a 1 mM concentration of the Nef peptides: Myr-Nef31-50, Myr-Nef2-22 and Nef2-22, B Dose responses for treatments of 30 minutes with Myr-Nef2-22 and Nef2-22.
  • Figure 9 represent cellular propidium iodide (PI) uptake as analysed by flow cytometric analysis.
  • the peptides were Myr-Nef31-50 (Myr control), Myr-Nef2-22 (Myr- Nef), and Nef2-22 (Nef).
  • An arbitrary gate was set such that 5% of untreated cells were classified as stained.
  • Figure 10 shows the effect of peptides on E coli colony formation. Peptides were added to E. coli cells suspended in water. After 30 minutes cells were spread onto solidified 2 x YT and plates were examined after overnight incubating at 37°C.
  • Figure 11 shows that exposure to myristylated N- terminal Nef peptide reduces the rate of extracellular acidification in CEM CD4+ T cells.
  • the arrows denote the time of peptide addition and tracings are representative of a minimum of three separate experiments.
  • Figure 12 is the region of the 2D IH TOCSY spectrum at 500 MHz of Nef2-26 in 90% H2O/10% 2H20 at pH 4.5 and 281 K, showing the Ala NH-CbH3 connectivities and the effect of cis-trans isomerism at Prol4. Peaks A and B were assigned to Ala26, Peaks C and D are due to Ala23, peak E is due to Alal5 when Prol4 is in the trans configuration, peak F arises from Alal5 when Prol4 is in the cis configuration. Assignment of peaks A, B, C or D to a particular conformation was not possible as spectral overlap prevented an unambiguous assignment of the spectrum. Small cross-peaks were also observed for Ala26 due to the cis-trans isomerism of Pro25, but these peaks are too weak to observe at the contour level of this figure . Figure 13 shows chemical shift analysis for Nef2-
  • D Deviation of Ca chemical shift from random coil values for Nef2-26 in C2H302H at pH 4.8 and 281 K.
  • E Deviation of CdH chemical shift from random coil values for Nef2-26 in SDS micelles at pH 5.1 and 298 K.
  • Figure 14 shows regions of the 300-ms mixing time NOESY spectrum of Nef2-26 in C2H30H at pH 4.5 and 281 K.
  • A CaH-NH region. Intra-residue NH-CaH cross-peaks are labelled with a single number. Sequential NOE cross-peaks are not labelled but are indicated by the line joining cross-peak due to consecutive residues (the CaH chemical shifts of Alal5 and Prol4 are quire close; as a result the aN connectivity between Prol4 and Alal5 overlapped with the NH-CaH cross-peak of Alal5) . Medium-range cross-peaks are indicated by two numbers indicating the residue contributing the CaH and NH protons respectively.
  • B NH-NH region. The NH-NH cross-peaks are labelled with two numbers identifying the sequence position of the interacting residues; numbers beside the cross-peak identify the contributing residue from the FI dimension while the numbers above or below the cross-peak identify the contributing residue from the F2 dimension.
  • Figure 15 is a summary of the NMR data for Nef2- 26 in C2H30H at pH 4.5 and 281 K.
  • the intensities of daN, dbN connectivities are represented as strong, medium or weak by the height of the bars. Shaded lines indicate dad connectivities to Pro residues. Asterisks indicate that the presence of a NOE could not be confirmed unambiguously due to peak overlap. Values of 3JHNCaH ⁇ 6 Hz are indicated by " , while those > 8 Hz are indicated by -. Those left blank could not be measured due to overlap, or were between 6 and 8 Hz.
  • the relative exchange rates of backbone NH protons are indicated in the row labelled NH, based on the strength of cross-peaks in 2H20 exchange TOCSY experiments; slowly exchanging amides are indicated by filled circles, while open circles indicate intermediate exchange.
  • the row labelled aH shows the chemical shift index of the CaH, if the CaH deviation from random coil is >0.1 ppm higher field than the random coil value it is given a value of -1, if the CaH value is > 0.1 ppm to lower field than the random coil value it is given a value of 1.
  • Figure 16 represents parameters characterising the final twenty structures of Nef2-26 in methanol, plotted as function of residue number.
  • A Upper-bound distance restraints used in the final round of structure refinement; medium-range (2 ⁇ i-j ⁇ 5), sequential and intra-residue NOEs are shown in black, hatched and white shading respectively, the one long-range NOE (Trpl3 C(4)H to Glul ⁇ NH) is not shown. NOEs are counted twice, once for each proton involved.
  • B RMS deviations from the mean structure for the backbone heavy atoms (N, Ca, C) following superposition over the whole molecule.
  • C-F Angular order parameters (S) (Hyberts et al, 1992; Pallaghy et al, 1993) for the backbone (f and y) and side-chain (cl andc2 ) dihedral angles. Gaps in the cl plot are due to Gly and Ala residues. Gaps in the c2 plot, in addition to Gly and Ala, are due to Ser, Pro, and Val residues.
  • Figure 17 is a stereo view of the final 20 structures of Nef2-26 in methanol superimposed over the backbone heavy atoms (N, Ca, C) of the well-defined 9Sf and Sy> 0.9) region of the molecule, encompassing residues 9- 22.
  • A Backbone heavy atoms.
  • B Orthogonal view showing side chains of residues with well-defined cl angles (Scl> 0.9) are shown.
  • Figure 18 shows the dose-dependent release of calcein from CEM cells treated with Nef peptides. Calcein- loaded CEM cells were incubated with serial dilutions of Myr-Nef2-26 (squares), Nef2-26 (circles) or Myr-Nef31-50 (triangles) for 30 min and calcein release determined as described in Materials and Methods. Data is representative of at least three separate experiments.
  • Figure 19 shows the Nef inhibitory activity and cytotoxicity of synthetic compounds in CEM cells. Synthetic compounds were incubated with (solid bars, % Nef inhibition) and without (shaded bars, % cytotoxicity) Nef N-terminal peptide at a molar ratio of 10:1 compound:Nef peptide for 30 min at 37oC prior to incubation with calcein-loaded CEM cells for 1.5 hr . Data represent the mean ⁇ SEM for at least two separate experiments .
  • Figure 20 shows the Nef inhibitory activity of synthetic compounds in PBMC .
  • BRI6209 closed squares
  • AP13 closed triangles
  • DER-AP3 closed circles
  • DER45 open squares
  • Figure 21 represents the Far-UV circular dichroism spectra of Nef peptides in methanol.
  • Figure 22 shows the conformational probabilities for different Nef N-terminal peptides calculated by discrete state-space theoretical analysis (Stultz et al 1993, White et al, 1994) performed on the Boston University Biomolecular Engineering Research Center Protein Sequence Analysis server.
  • Figure 23 shows the pressure/surface area isotherms for DPPC monolayers on a subphase containing Myr-
  • Nef2-22 ( ), Myr-Nef 31-50 ( — - — - ) or Nef2-22
  • Figure 24 represents the dequenching of the fluorescence of the fluorochrome octadecyl-rhodamine in the presence of non-myristylated Nef2-22 and Nef 9-22, indicating the occurrence of membrane-mixing fusion.
  • Figure 25 represents the dose response curves for the lysis of sheep red blood cells by Nef N-terminal peptides. Peptides were incubated with cells at 20oC for 3 min.
  • Figure 26 shows the kinetics of lysis of sheep red blood cells by Nef N-terminal peptides at 20oC. The concentration of each peptide was 32 mM.
  • Figure 27 represents the dose response curves of peptide cytotoxicity for CD4+ T cells.
  • CEM cells were incubated with peptides at 37oC for 2 hr and LDH release quantitated as described in Materials and Methods.
  • Figure 28 shows that the Nef N-terminal peptide induces LDH release in a range of leukocytic cell lines .
  • Figure 29 shows the Nef N-terminal peptide- induced LDH release uncultured PBMC .
  • Cells (1 x 105/well) were incubated with 12.5 ⁇ M Myr-Nef2-26, Ne 2-26 or Myr- Nef31-50 for 30 min and LDH release was measured. Values represent the mean ⁇ SEM for three separate experiments, performed in triplicate, and are expressed as the percentage of maximum LDH release induced by 1% Triton X- 100.
  • Figure 30 shows the Nef N-terminal peptide- induced LDH release from cells isolated from tonsil lymphoid tissue. Tonsillar cells (1 x 105/well) were incubated with 10 ⁇ M Myr-Nef2-26, Nef2-26 or Myr-Nef31-50 for 30 min (solid bars) or 2 hr (shaded bars) and LDH release was measured. Values represent the mean ⁇ SEM for three separate experiments, performed in triplicate, and are expressed as the percentage of maximum LDH release induced by 1% Triton X-100. GENERAL METHODS Peptide Synthesis
  • the peptides were myristylated on the synthesiser by applying myristic acid to the N-terminal amine of the peptide/resin (ie Fmoc group removed) using the protocol normally used for recoupling serine (ie longest dissolving time) .
  • the myristylated peptides were coded Myr-Nef2 -22, Myr-Nef2-22a, Myr-Nef2-22b, Myr-Nef2-22c, Myr-Nef2-26, Myr-Nef2-26a, Myr-Nef2-22 (Valll ) , Myr ef2-10, and My - Nef10-26. Myr-Nef2-26(Serl3) and Myr-Nef31-50.
  • the peptides were purified using a Vydac C18 column, and their purity and composition confirmed by HPLC, amino acid analysis and electrospray mass spectroscopy .
  • sequences of the peptides are as follows :-
  • the Nef2-22 and Ne 2 -26 sequences were selected because we had found earlier that Myr-Nef2-22 could provoke the formation of non-lamellar structures in lipid bilayer membranes (Curtain et al 1994) .
  • Myr-Nef31-50 was chosen as a potential control sequence to evaluate the effect of myristylation on the membrane activity of the Nef2 -22 and Nef2-26 peptides because discrete statespace theoretical analysis (Stultz et al, 1993, White et al 1994) performed on the Boston University Biomolecular Engineering Research Center Protein Sequence Analysis server showed that it would form a rigid a-helix in contrast to Nef2-22 and Nef2- 26 in which an a-helix was only probable from residues 14 on, leaving considerable flexibility at the N-terminus of the peptide.
  • the predicted overall a-helical content of the peptides was confirmed by circular dichroism measurements made in ethanol/water (60/40).
  • LUV Large unilamellar vesicles
  • EYPC EYPC in buffer (5 mM Hepes, 100 mM NaCl, pH7.4 ) , freezing and thawing the dispersion 10 times and extruding 10 times through two stacked 0.4 mm pore size polycarbonate filters (Nucleopore, Pleasanton CA) as described by Hope et al (Hope et al 1985) .
  • the lipid spin labels were added in the desired molar ratio to the EYPC in chloroform/methanol (70/30) before the preparation of the dried films.
  • PBMC peripheral blood mononuclear cells
  • Tonsil cells were isolated from tonsils surgically removed during therapeutic tonsillectomy . Tonsils were stored on ice in PBS pH7.4 containing 250 ⁇ g/ml gentamicin and processed within 3-6 hr. The epithelial layer was removed and the tissue mechanically disrupted by sieving through a 200 mesh in PRMI 1640 supplemented with 10% (v/v) heat-inactivated foetal bovine serum (GIBCOBRL) . Dead cells, epithelial cells and debris were removed by centrifugation over an isotonic metrizamide gradient at 1600 x g at room temperature for 10 min. Cells with a density of between 1.04 and 1.065 were collected and washed before use.
  • GIBCOBRL heat-inactivated foetal bovine serum
  • the human leukocyte cell lines CEM (CD4+T-cell ) , Jurkat (CD4+T-cell) , RPMI 8226 (B-cell), U266 (B-cell), THP-1 (monocyte) and U-937 (monocyte, Sundstrom and Nilsson, 1976) were obtained from American Type Culture Collection, Rockville, Maryland, USA.
  • the RC2a human monocytic cell line was from the Macfarlane Burnet Centre for Medical Research, Melbourne, Australia.
  • yeast strains used were Saccharomyces cerevisiae strain DY150 (MATa ura3-52 leu2-3,112 trpl-1 ade2-l his3-ll canl-100), Candida glabrata strain L5 (leu), Candida albicans clinical isolate JRW#5, Kluyveromyces lactis strain MW98-8c (MAT-a uraA arg lys ) and
  • Schizosaccharomyces pombe (h- ade ura leul-32). Strains were grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) . The required number were resuspended in 700-1000 ml modified buffer, mixed with molten agarose and stirred at 39°C until required.
  • Peptides were purified to homogeneity by HPLC and then dissolved in distilled water at a concentration of 2mg/ml and stored at 4°C until required. Aliquots of peptide solutions were added to cells suspended in a final volume of 100ml water, and after 1 hour or other specified time, the mixture was plated onto the appropriate solidified medium. Plates were incubated for one to three days, depending on the strain, and the number of viable colonies was determined.
  • PI stained cells were analysed by forward angle and 90° light scatter as well as PI fluorescence using a Coulter EPICS® elite flow cytometer. PI was added to 25 mg/ml and dye penetration was measured by the presence of fluorescence emission at 520 nm. Gating was adjusted such that the population defined as "PI stained" comprised 5% of the control cell population.
  • the packed cells were washed three times and then diluted to a 1% or 3% suspension in phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • the peptides were dispersed by sonication in PBS to a final concentration of 100 mM.
  • Each was added by micropipette to 1 ml of the cell suspension over the desired concentration range (0.05 - 20.0 mM) and the mixture was shaken for 30 seconds, allowed to stand for the desired time with gentle inversion of the tube every 30 seconds and then centrifuged at 1500 x g for 2 minutes.
  • the optical density (OD)540nm of the supernatant was determined spectrophotometrically .
  • a blank tube containing 3% cells only was used as a control.
  • CEM CD4+ T cells were washed once in PBS, pH 7.4 and once in assay buffer (0.96 mM KH2P04, 5.28 mM Na2HP04, 1.74 mM KC1, 143 mM NaCl, pH 7.4), and resuspended at lxl07/mL in assay buffer. 100 mL cells were added to 10 L peptide (1 mg/mL in PBS) , or PBS and incubated at room temperature for up to 6 hours. At various time intervals, 10 mL of cell suspension was removed, placed on a glass slide and examined by light microscopy.
  • Nef peptides were assayed for cytotoxic activity against human cells using a commercially available kit (Cytotoxicity Detection Kit (LDH) , Boehringer Mannheim,
  • Cells were prepared as described above and resuspended at 2xl05/mL (cell lines) and lxl06/ml (PBMC and tonsil cells) in assay buffer. The cell concentrations were determined in preliminary experiments to give optimal maximum versus spontaneous calcein release for each cell type.
  • the peptides were dissolved in water at a concentration of 1 mM. 100ml cells were mixed with 100 ml peptide diluted to the desired concentration in assay buffer in a microtitre plate and incubated at 37°C in 5% C02, 90% humidity for 30 minutes.
  • the plate was centrifuged at 250xg for 10 minutes and 100 mL supernatant transferred into a EIA plate (Dynatech, Chantilly, Virginia, USA) .
  • Lactate dehydrogenase (LDH) activity was determined by adding 100 ml reaction mixture and incubating at room temperature, protected from light, for 30 minutes.
  • the OD490nm on an EIA plate reader (Dynatech DIAS ; Dynatech, Chantilly, Virginia, USA; or Nunc, Roskilde, Denmark), using a reference wave-length of 630 ran. Plates were blanked on wells containing buffer alone.
  • I and 10 are the intensities of the tryptophan fluorescence in the presence and absence of the buffer soluble quencher tempo choline chloride, (TCC)
  • kq is the bimolecular rate constant
  • [Q]t is the total concentration of TCC
  • t is the fluorescence lifetime.
  • concentration of [Q] may be taken to be the mole fraction added because electron spin resonance spectroscopy studies on bilayer membrane-associated spin-labelled phospholipids have shown that the spin labels at all positions are almost entirely confined to the lipid phase
  • palmitoyl-stearoyl phosphatidyl choline probes for use in ultraviolet fluorescence quenching studies, palmitoyl-stearoyl phosphatidyl choline probes, doxyl spin-labelled at the 5 (5NP), 12 (12NP) and 16 (16NP) carbons of the stearoyl chain, were obtained from Avanti Polar Lipids Ine, Pelham AL .
  • the water soluble spin probe, tempo choline chloride (TCC) was obtained from Molecular Probes Ine, (Junction City, OR) . Spin labels from both sources were checked for purity and to ensure that their number of spins/M were >90% of theory, as described by Gordon and Curtain (1988) .
  • Egg yolk phosphatidyl choline (EYPC, Type XVI_E) was obtained from Sigma St Louis MO and used without further purification.
  • NMR Spectroscopy NMR samples were prepared by dissolving 2.4 mg of lyophilized Nef2-26 peptide in 0.55 ml of the appropriate solvent (final concentration 1.5 mM) ; solvents used were C2H30H, C2H302H, 90% H2O/10% 2H20 and 2H20. The pH was adjusted with small additions of 0.5 M Na02H or 2HC1. The peptide was also examined in SDS micelle solution; a 400 mM solution of SDS-2H25 was prepared in 1 ml 90% H20/ 10% 2H20 then 2.4 mg of Nef2-26 was dissolved in 0.6 ml of this solution and the pH was adjusted to 5.1.
  • NMR spectra were recorded at 298 K; attempts to accumulate spectra at lower temperatures resulted in the crystallization of SDS from solution. Reported pH values were recorded at room temperature and were not corrected for isotope or solvent effects.
  • the IH chemical shifts were referenced to 2,2- dimethyl-2-silapentane-5-sulfonate (DSS) at 0 ppm, via the chemical shift of residual CH20H at dCH3 3.35 ppm (W ⁇ thrich, 1976) or the H20 resonance (Wishart et al, 1995a) .
  • Spectra were recorded on Br ⁇ ker AMX-500 or AMX- 600 spectrometers. Unless stated otherwise, all spectra were recorded at 281 K; probe temperatures were calibrated according to the method of van Geet (1970) . All 2D spectra were recorded in phase-sensitive mode using time- proportional phase incrementation (Marion & W ⁇ thrich, 1983) . Solvent suppression was achieved by selective, low power irradiation of the water signal during the relaxation delay (typically 2s) and during the mixing time in NOESY experiments. For some spectra obtained in 90% H2O/10% 2H20, water suppression was also achieved by pulsed field gradients using the WATERGATE method of Sklenar et al (1993) . 2D homonuclear NOESY spectra (Anil-Kumar et al,
  • the 3JNHCaH coupling constants were measured from the DQF-COSY spectra at 500 MHz.
  • the appropriate rows were extracted from the spectrum, inverse courier transformed, zero-filled to 32 K, and multiplied by a Gaussian window function prior to Fourier transformation.
  • the antiphase peak shapes were simulated to take account of the effect of broad line widths on small coupling constants, using an in- house program COUPLING.
  • Slowly- exchanging amide protons were identified by dissolving the lyophilized peptide in the appropriate deuterated solvent (C2H302 or 2H20) and recording and series of ID and TOCSY spectra immediately after dissolution.
  • Simulated annealing was performed using 20,000 steps at 1000 K and 10,000 steps as the molecule was gradually cooled to 300 K. A time step of 1 fs was employed throughout.
  • the 100 structures were then subject to further simulated annealing which they were gradually cooled from 300 to 0 K in 20,000 steps and then energy minimized using 100 steps of Powell conjugate gradient minimization. For each structure this procedure was carried out 10 times and the best of these 10 in terms of total energy and NOE energies was selected.
  • the structures were then energy minimized in the empirical CHARMm force field (Brooks et al, 1983), with all explicit charges neutralized (Monks et al, 1995) and with a distance- dependent dielectric instead of explicit water molecules.
  • Example 1 Cytotoxic activity of the amino-terminal region of HIV Nef protein on human erythrocytes and human leukocyte cell lines.
  • a dose-response study showed that significant haemolysis only occurred with the non-myristylated N- terminal peptides at concentrations of 20 mM as shown in Figure 1, whereas the myristylated peptides were active at concentrations as low as 0.5 mM.
  • the control non N- terminal peptide Myr-Nef31-50 was virtually non-lytic over the concentration range 0.5-20 mM.
  • the four myristylated N-terminal peptides, regardless of sequence or length gave approximately the same dose-response curve.
  • Fluorescence quenching studies were undertaken to determine the localisation of the tryptophan residue at position 5 in relation to the lipid bilayer in the myristylated and unmyristylated peptide. Because the haemolysis results had shown that substitution of the distal position tryptophan 13 with serine did not affect haemolytic activity of the myristylated peptide, Myr-Nef2- 26(Serl3) and Nef2-26 (Serl3 ) were used in these experiments. This strategy meant that ultraviolet fluorescence derived only from the proximal tryptophan, which made interpretation of the results simpler.
  • Example 2 Cytotoxic effect of the amino-terminal regions of HIV Nef protein on yeast.
  • Myr-Nef2-22 (lO M) and control Nef31-50 (lOmM) were added to the five yeast strains as described above. All yeast species responded similarly to Myr-Nef2-22.
  • Figure 7 shows that treatment with Myr-Nef2-22 caused the loss of colony formation of the entire cell population.
  • yeast provides an advantage for this kind of examination because the cell wall remains intact even when the membranes are permeabilized.
  • peptide treatment cells were examined for staining with propidium iodide ( PI ) .
  • PI staining is useful to distinguish unstained viable cells or cells with an intact membrane from those that are dead or have a compromised membrane (Shapiro, 1994) . Quantitation of the peptide-induced permeabilization was investigated by flow cytometry ( Figure 9).
  • Nef2-22 caused about a third of cells to be permeabilized, while the treatment with the control peptide, Myr-Nef31-50 produced about 5% Pi-staining, similar to the untreated cells.
  • the peptide was labelled with FITC. The resulting labelled peptide did not associate with cells (as judged by flow cytometry and fluorescence microscopy) and had no cell killing activity. This suggests that the Lys residue (s) are important for the killing activity. Further studies to examine the cellular localization will require alternate labelling strategies. Nef peptides kill E. coli cells
  • Nef peptides were added to a suspension of E. coli cells in water to determine whether they would be capable of killing prokaryotic microbial cells.
  • the Nef peptides killed E. coli cells in a manner comparable to yeast ( Figure 10) .
  • Peptide cytotoxicity could be delayed by suspending the cells in buffer (50 mM HEPES/2% glucose) so that complete killing was not observed until 20 hours incubation at 30°C.
  • Cytosensor Microphysiometer The Cytosensor Microphysiometer system is an extremely sensitive assay system and was used to examine the effects of extracellular Nef peptides on human CD4+ cells. This system is also used to develop assays to quantitatively assess and compare the effect of inhibitors of these Nef peptides.
  • the Cytosensor Microphysiometer (Molecular Devices Inc., CA) is a light addressable potentiometric sensor-based device that can be used to indirectly measure the metabolic rate of cells in vitro (Parce et al . , 1989; McConnell et al . , 1992). Metabolism is determined by measuring the rate of acid metabolite production from cells immobilised inside a microvolume flow chamber. Human CEM cells were centrifuged and resuspended in low-buffered serum-free/bicarbonate-free RPMI 1640 medium (Molecular Devices; hereafter referred to as modified medium) .
  • the cells were seeded at a density of 30,000 - 63,000 cells/capsule on to the polycarbonate membrane (3um porosity) of cell capsule cups (Molecular Devices) . Cells were immobilised using an agarose entrapment medium (Molecular Devices) . The seeded capsule cups were transferred to sensor chambers containing the silicon sensor which detects changes in pH (and thus cellular metabolism) . The Cytosensor system used for this set of experiments contained four separate chambers for the measurement of acidification rates . Modified medium was pumped across the cells at a rate of 100 - 120 ⁇ l/min. Each cell chamber was served by fluid from either of two reservoirs, which could be alternated using a software command .
  • Basal acidification rates were monitored (in the absence of any treatment) for at least 20 min. After this time, the peptides were exposed to the cells for periods of no less than 1 hour. In all experiments, at least one chamber was not exposed to any of the compounds, providing a negative control. The results are shown in Figure 11.
  • Example 4 Solution structure of the amino-terminal region of HIV Nef protein.
  • the solution structure of a 25-residue polypeptide (Nef2-26) was investigated by 1H-NMR spectroscopy as described above.
  • Peaks A and B were due to Ala26, and C and D to Ala23, while peaks E and F were due to Alal5 with Prol4 in the trans and cis conformations, respectively. Peaks A, B, C and D were not assigned to particular conformers as the limited dispersion in IH chemical shifts resulted in highly overlapped spectra, preventing unambiguous assignments for the two conformers. Only one set of signals was observed for the residues 2-7, indicating that conformational differences associated with cis-trans isomerism at Prol4 had negligible effects on the chemical shifts of these residues.
  • Cis-trans isomerism was also observed for Pro25, but only a small proportion ( ⁇ 5%) was present in the cis form. The presence of the cis isomer was confirmed by observation of a weak NOESY cross-peak from Glu24 CaH to Pro25 CaH. Only the flanking residues Glu24 and Ala26 gave rise to separate NMR cross-peaks due to this conformation, indicating that the structural differences between the two forms were limited, in contrast to those due to Prol4 isomerism.
  • TFE 2-trifluoroethanol
  • polypeptides melittin (Bazzo et al, 1988; Brown & W ⁇ thrich, 1981; Brown et al, 1982; Inagaki et al , 1989; Ikura et al, 1991) and d-haemolysin (Lee et al, 1987, Tappin et al, 1988) have very similar structures in methanol and lipid micelles, implying that methanol is an appropriate solvent for mimicking structures of peptides in membrane-like environments.
  • Figure 15 summarises the sequential and medium- range NOE connectivities, 3JNHCaH coupling constants, and slowly exchanging amide protons observed in methanol .
  • the presence of helical structure encompassing residues 7 to 23 is indicated by the low 3JNHCaH coupling constants ( ⁇ 6 Hz), numerous daN(i,i+3), daN(i,i+4) and dab(i,i+3) NOE connectivities and the presence of slowly exchanging backbone amide protons in this region of the molecule.
  • the low coupling constants indicate that under these conditions the peptide is not undergoing significant conformational averaging .
  • Nef2-26 The overall conformation of Nef2-26 in methanol is shown in Figure 17A, where the backbone heavy atoms of the 20 best structures (those with the lowest overall energies, excluding the electrostatic term) have been superimposed over residues 10-23 (where the molecule is a- helical).
  • Figure 17B An orthogonal view of the structures, which also shows the well-defined side chains, is presented in Figure 17B. Structure in Detergent Micelles.
  • Nef2-26 in methanol has been described in detail because the good quality of the spectra in this solvent permitted an extensive set of NMR restraints to be obtained.
  • the hydrophobic face of the helical region Nef2- 26 consists of residues Trpl3, Vall5, Met20 and Ala23. This allows a peptide to form potential coiled-coil interactions over two turns of the helix, with Trpl3 and Vall5 in the a and d positions of the first turn, VallO and Glyl2 occupy the interface border positions e and g.
  • Met20 and Ala23 are in the a and d positions while the e and g positions are occupied by Argl7 and Argl9.
  • a coiled-coil with the peptides in an antiparallel arrangement would give a favourable alignment of the helical dipoles .
  • a suitable coiled-coil interaction would be similar to that shown below, with amino acid residues denoted by standard single letter codes.
  • the peptide AXXLEXEAXXAAXL represents a starting position from which to design the peptide with the most efficient coiled-coil interactions.
  • the residues labelled X in this peptide could be occupied by any helix-enhancing residues such as alanine, although for solubility and spectroscopic reasons, some of these residues should be more hydrophilic in nature, such as Gin, Asn, and Ser at position f .
  • the ability to form Glu to Lys lactam bridges i to i+4 residues apart could also be used in these positions to increase the helical propensity of the peptide (Houston et al . , 1996).
  • Variations of this peptide include replacing some or all alanine residues (1, 11, 12) with other hydrophobic residues such as valine, leucine or isoleucine, to maximise hydrophobic interactions.
  • the residue at position 8 will interact with Trpl3 on Nef2-26 and it is possible that an aromatic residue such as Phe or Tyr may give a stronger interaction, although steric restrictions may rule this possibility out.
  • Other variations include extending the peptide to allow it to interact with Lys4 and 7 of Nef2-26. This could be achieved by adding one or more Glu residues to the C-terminus of the peptide, a variable length spacer, made of Gly or Ser residues may be required to achieve the best interaction.
  • Example 6 Inhibitors that interact with the N-terminal region of the Nef protein
  • Lip is a lipophilic moiety necessary for complexing with the hydrophobic regions of the Nef terminus. This is exemplified by a number of different structures. These include cholic acid, the hydrophobic tripeptide Ile-Val- He, peptoids and polyglycols.
  • “Ar” is a link which contains an aromatic ring and is a point at which conformational constraint may be used to effect. Encompassed moieties range from phenylalanine derivatives to heterocyclic compounds.
  • “Pol” is a link with a polar sidechain, typically aspartic acid or asparagine.
  • AA is typically a neutral amino acid residue with a small sidechain, such as 2-aminobutyric acid, cysteine, proline or 3-chloroalanine .
  • sidechain such as 2-aminobutyric acid, cysteine, proline or 3-chloroalanine .
  • attachment of bulky groups, such as 4-nitrobenzyl was compatible with good inhibitory potency.
  • inspection of inhibitor models docked onto the Nef structure indicate the possibility of extending the sidechain such as in Lysine to interact with a glutamate residue on the Nef structure.
  • N-terminal tripeptide sequence IVI with a hydrophobic but more soluble moiety such as cholic acid or a polyglycol or a tripeptoid (N-alkylation) .
  • Suitable inhibitors include combinations of : - a) Lipophilic moieties are steroids; ethylene oxide fatty acids; multi-fatty acid chains eg phosphatidylcholine esters; branched fatty acids; branched polymers with acid terminal group. b) Aromatic moieties: Different aromatic groups may be used to increase the interaction with Trp groups eg naphthalene or indane groups.
  • Multi-presentation of inhibitors or peptide sequences may also be utilised.
  • d) Complementary, negatively charged amphiphatic helical peptides or coil/coil inhibitors.
  • Small synthetic cationic helical peptides made up of repeating hexamer units as anti-microbial agents may be used, in particular, negatively charged amphiphathic helical peptides as Nef inhibitors.
  • Anti-sense Peptide Nucleic Acid (PNA) Inhibitors Anti-sense PNAs directed to essential regions of the Nef gene are synthesised and the anti-HIV effects of such compounds determined.
  • Rl may be an acetamide cap or a solubilizing group as stated above, R2 is Phe or its 2-naphthyl counterpart, R3 is Cys (free acid) or Abu (free acid), R4 is Asp or Asn, R5 is Cys or (S-4-nitrobenzyl)Cys and may be terminally amidated or not .
  • Nef inhibitors All compounds designed as Nef inhibitors were assembled by peptide or amide bond formation using conventional peptide chemistry coupling techniques. This involved activation of a carboxylic acid group, through generation of an acid chloride, an anhydride or an active ester, with subsequent reaction with an amine. Constituent units were natural and unnatural amino acids, N-alkylated amino acids ("peptoids") and lipophilic carboxylic acids. Some units were synthesised because they were not commercially available. Where required, amino acids were fully protected. In all cases, incremental assembly was diagnostically monitored by IH NMR. As a final step, full deprotection yielded the target molecules.
  • the HBTU coupling method was predominantly used with solution-phase t-BOC chemistry, but any peptide coupling method and strategy may be used.
  • Carboxylic acids were protected as esters and removed by saponification, acidolysis or hydrogenolysis, but any other sensible protecting groups could also be used. Dissolution of carboxylic esters prior to hydrogenolysis required solvents such as hexafluoropropanol, trifluoroethanol, trifluoroacetie acid or DMF and were often unprecedentedly slow, requiring many days reaction for completion. This was a function of the substrate and not the solvent .
  • the thiol of cysteine was protected as a nitrobenzyl or disulfide (removal by reduction) , an acetamidomethyl (removal by oxidation) , or a trityl (removal by acidolysis) , but any other suitable protecting groups could also be used.
  • Amines were protected as t-butyloxycarbamates (BOC; removal by acidolysis or heat), fluorenylmethoxycarbonyl (FMOC; removal by secondary amines) or benzyloxycarbamates (CBZ; removal by acidolysis or hydrogenolysis), but any other suitable protecting groups may be used.
  • HBTU 0-benzotriazole-N,N,N' ,N' -tetramethyl-uronium hexafluorophosphate
  • DIPEA diisopropylethylamme
  • the reaction mixture was dumped into water. If the precipitated product was solid, this was filtered off and sucked to dryness, with final trituration with ether. If the precipitated product was a gum, this was taken up in ethyl acetate and the organic layer was subjected to the standard washing protocol, which will be outlined once as follows:
  • TFA.D (Bn) Abu (Bn) was dissolved in 1:1 TFA:DCM, stirred for 30 mins, and concentrated rigorously under vacuum to give the trifluoroacetate salt, TFA.D (Bn) Abu (Bn) as a gum.
  • this dipeptide was added to an HBTU-activated (as above) solution of BOC-Phe (32 mmol) in DMF (120ml) and treated in the usual way to furnish BOC- FD(Bn) Abu(Bn) (91 % overall yield) as a gum which solidified on standing. This was treated with TFA as above to give the title compound as an oil.
  • BOC-IVI(Bn) was made by the above HBTU coupling/TFA deprotection protocol.
  • AcIVI could be readily made by two methods.
  • the BOC group was removed and the resulting TFA.
  • IVI (Bn) was acylated with excess acetyl chloride or acetic anhydride in DMF in the presence of excess triethylamine to give Ac- IVI(Bn) as a solid after precipitation with water, filtering, and drying.
  • BOC-IVI(Bn) was debenzylated by catalytic hydrogenation transfer using ammonium formate in methanol to give, after concentrating and precipitating with water, BOC-IVI as a gum which hardened to a solid on standing. After removal of the BOC group, the resulting TFA. IVI could be acylated and treated by the above protocol to give Ac-IVI as the triethylamine salt .
  • succinimidyl carboxymethyl polyethyleneglycol monomethyl ether (MW 5000) [MPEG ( 5000 )SCM] (17.5mg) in dry DMF (2ml) was added to a solution of Ile-Val-Ile-Phe-Asn-Abu-NH2 (2.3mg) in DMF (2ml). The solution was stirred for 30 minutes, then triethylamine (2 drops) added and the mixture stirred overnight at room temperature. Diethyl ether (30ml) was then added and the precipitated white solid collected by filtration, washed with diethyl ether and dried to give a white powder (15mg) .
  • the hydroxyl group of monoether-protected polyglycols was conventionally functionalized to a carboxylic acid or activated carbonate.
  • the hydroxyl proton was removed with strong base and alkylated with an alkyl bromoacetate followed by saponification to give the oxyacetate.
  • the hydroxyl group was reacted with succimidyl carbonate to give the active carbonate.
  • hexa (1, 2-butylene glycol) monobutyl ether in dry acetonitrile over molecular sieves was treated with disuccinimidyl carbonate (1.5 equivalents) and triethylamine (1.5 equivalents) and stirred overnight under dry nitrogen.
  • Cysteine ethyl ester hydrochloride (20g) in water (50ml) was treated with l.leq. H202 (13ml of 30% aq. solution) slowly and external cooling was applied during a 30 min period. The reaction mixture was stirred for another hour, basified (HC03-) and extracted with ethyl acetate (4 X 100ml) . The organic layer was dried and evaporated and the residue dissolved in dioxan (200 ml) and acidified with cone. HCl (10 ml). After cooling, the product, cystine diethyl ester, was filtered off as colourless crystals in the form of the dihydrochloride salt.
  • BOC-D(Bn) and BOC-F were then sequentally used in the standard HBTU coupling/TFA deprotection procedure to finally give TFA.FD(Bn)C(OEt) as the disulfide dimer which could be recrystallized from acetonitrile.
  • HBTU coupling with cholic acid gave the title compound.
  • BRI6209 was obtained after ester saponification and disulfide cleavage with 2- mercaptoethanol .
  • a useful system for column chromatography with silica gel used n-butanol :28%aq. NH3 : water: ethanol in a ratio of 28:8:4:3 and product visualisation was aided by vanillin spray.
  • Nef peptide-induced membrane damage in human cells was developed for the screening of putative Nef inhibitory compounds. Based on the release of the fluorochrome calcein from pre-loaded cells, the method was developed from a cell-mediated cytotoxicity assay described by Lichtenfels et al (1994) . Unlike the LDH assay, this assay doest not rely on the measurement of an enzymic activity, and so is not susceptible to the presence of similar activity, or inhibitors of activity, in the samples to be tested. CEM cells or PBMC were washed twice in PBS containing 5% FBS and resuspended at a concentration of 2xl06/ml in PBS/5% FBS. Calcein AM (Molecular Probes,
  • % cytotoxicity 100 x (Flpeptide - Flspontaneous) / (Flmaximum - Flspontaneous) Putative inhibitors of Nef peptide cytotoxicity were tested by mixing equal volumes (50 ⁇ l) of Myr-Nef2-22 peptide and synthetic compound, diluted in Nef assay buffer as required, in a U-bottom microplate (Disoposable
  • Results were expressed as the percent inhibition of the cytotoxicity in the presence of the Nef N-terminal peptide alone. Compounds were also tested in the absence of Nef peptide to assess cytotoxicity
  • Tables 3 and 4 list the compounds synthesised and the diagnostic protein NMR signals for some representative compounds respectively.
  • Nb para-Nitrobenzyl (on S atom)
  • -Hexapeptides containing a variant C-terminus residue such as L-2-aminobutyric acid (Abu; dlAbu for the racemate) and/or modified end-caps to enhance solubility/activity -Truncated sequences
  • Ch-FNC(Nb) -NH2 Ch-FD (t- Phe and Tft ArH (20; 7.2-7.5) Bu)C(Trt) (Pip) Pip ArH (3; 6.7-6.9)
  • Ch-FDC Loss of protecting group signals [from, for example, Ch-FD (Bn) C (Et ) ) 2 or Ch-FD ( Bn) C (Nb) (OEt) or Ch-FD (t- Bu)C(Trt) (Pip) ] iBu (RLeu) FN-Abu- ⁇ -CH (1; 3.95; triplet) NH2 "N- ⁇ -CH2" (2; 3.0-3.4; pair of (CDC13) doublets of doublets) isobutyryl CH (1; 2.8-2.9; multiplet) isobutyl CHs (2; 1.9-2.1; multiplet) ⁇ -CH2 (2; 1.5-1.8; multiplet) isobutyryl CH3s (6; 1.15; doublet) isobutyl CH3s (12; 0.9-1.0; multiplets) iBu (RGly) 3FD-Abu ⁇ -CH2s (6; 3.9-4.3) CDC13 "N- ⁇ -CH2s" (6; 3.1-3.3) is
  • AP13, DER45 and DER-AP2 also showed at least 50% Nef inhibition and gave less than 25% cytotoxicity. Titration of these compounds revealed one, BRI6199, with which caused 50% inhibition of Nef peptide-induced calcein release at a molar ratio of approximately 1:1 (compound: Nef peptide) and 90% inhibition at a molar ratio of 2:1 (compound: Nef peptide) as shown in Table 6.
  • the next best compounds, DER-AP2, DER-AP3, AP13, DER45, BRI6209 showed 50% inhibition of Nef peptide cytotoxicity at 1 to 5:1 compound :Nef peptide molar ratios and 90% inhibition at 4-14:1 compound:Nef peptide molar ratios.
  • Example 8 Structural Requirements for the Cytotoxicity of the Nef N-Terminal .
  • the effects of modifications to the sequence and secondary structure of the Nef N-terminus on the cytotoxicity of the peptide were investigated.
  • Far ultraviolet circular dichroism spectra of the peptides in methanol were measured over the range 250 nm to 200 nm using an AVIV spectropolarimeter at 309K using 0.01 or 0.05 cm pathlength rectangular quartz cells. Peptide concentration in the sample solution was measured by quantitative amino acid analysis. Thirty six spectra were averaged, baseline-corrected and analysed for % a-helix, b- strand and disordered structures using the K2d Kohonen neural network program (Andrade et al . 1993; Merelo et al. 1994) obtainable by ftp anonymous to swift. e bl- heidelberg.de directory /group/andrade.
  • the fusion assay was carried out in a darkened room by adding 100 mg of the labelled vesicles to 1 mg of unlabelled SUV in 2 ml of 50 mM Tris-HCl buffer (pH 7.0) and adding the fusogenic agent at the desired molar ratio to this mixture.
  • the fluorescence of the mixture was followed at 43oC using excitation and emission wavelengths of 460 and 680 nm, respectively in a Perkin Elmer-Hitachi M35 spectrofluorimeter .
  • the fluorescence intensity of the mixture in the absence of fusogen was taken as baseline.
  • DPPC phosphatidylcholine
  • hexane:propanol 9:1 v/v
  • the DPPC solution was added dropwise to the surface of a subphase (170 ml) contained in a Teflon® Langmuir trough.
  • the trough was mounted in a thermostatted cabinet with high humidity and the temperature was controlled to 30.2° C.
  • the film was compressed at a rate of 38 mm2 s-1. Isotherms of DPPC were measured on subphases of both Milli-Q® (Millipore) water (pH 5.9) and 25 mM HEPES (Merck) adjusted to a pH 7.2 with sodium hydroxide (AR) .
  • Nef 2-22a and Nef 2 -22c Small differences were in structure observed between peptides Nef 2 -22a and Nef 2 -22c.
  • Nef 2-22b Nef 2-22e and Nef 31-50 showed a marked increase in a-helix and a corresponding reduction in disorder.
  • Increasing the chain length from 21 to 25 residues (Nef 2-26) had little effect on the CD spectrum.
  • Theoretical calculation of the secondary- structures showed that most of the increase m a-helix m Nef 2-22b,c &e occurred over residues 2-10 while Nef 31- 50, in contrast to its CD spectrum showed a equal probability of a-helix and loop over its entire length, as shown in Figure 22.
  • Increasing the positive charge of this sequence resulted in a modest increase in a- helicity .
  • the overall CD-derived value for a-helix for Nef 2-22 and Nef 2-26 and the distribution of the probability for a-helicity for Nef 2-22 and Nef 2-26 agreed well with the structure obtained from NMR3 for Nef 2-26.
  • Fluorescence fusion assays were carried out on the non-Myristylated Nef 2-22 and Nef 9-22 peptides to find out whether the fusion of SUV by non-Myristylated Nef N- terminal peptides that we had observed previously required the presence of the relatively unstructured seven N- terminal residues. These assays rely on the fact that the fluorochrome R18 is heavily self quenched when inserted in a lipid bilayer at high lipid/fluorochrome ( ⁇ 1/100) . In the absence of lipid mixing the dye only very slowly transfers from the bilayers of the labelled to those of the unlabelled vesicles.
  • Figure 25 shows dose-response curves for the lysis of sheep red blood cells by all the Nef peptides. Broadly, reduction of the positive charge in the 2-7 region of the peptide (Nef 2-22b) reduced the lytic activity of the peptide as did truncation of the peptide at the C- terminal end (Nef 2-10). An increase in positive charge in the C-terminal region (Nef 2-22c) led to a slight increase in lytic activity. Secondary structure at the N-terminus seemed to take second place to positive charge as a determining lytic activity. Nef 2-26 had a much higher a- helical content than Nef 2-22c or Nef 2-22, yet had the same order of haemolytic activity.
  • Nef 31-50 which has a very high a-helical content is poorly lytic.
  • the addition of two positively-charged lysine residues to its N-terminal region increased its lytic activity fourfold.
  • the kinetics of haemolysis as shown in Figure 26 show a similar trend with the peptides with positive charges at the N-terminal end reaching almost 100% haemolysis in the first five minutes after addition. It should be noted that the other peptides, with the exception of Nef 31-50, attained more than 50% haemolysis after 160 minutes.
  • the secondary structure at the N-terminus appeared to be of lesser importance than the positive charge in determining Nef peptide cytotoxic activity.
  • Myristylated Nef31-50 which had a high alpha- helical content, caused only moderate LDH release from CEM cells, but this is increased to the level of the N-terminal peptide (Myr-Nef2 -22 ) following the addition of two lysine residues (positively charged) at positions 32 and 36.
  • Nef N-terminal 7 residue sequence lacks hydrophobic residues with the exception of Trp4. Adding the myristyl chain would increase the hydrophobicity of this region, possibly making Nef 2-2n peptides behave more like melittin.
  • Nef 2-22d is still quite haemolytic although the a-helix apparently does not commence until around residue 14. It should be noted, however, that sequence changes that even slightly reduce the a-helicity of melittin render it non-toxic, although simply shortening the peptide at the C-terminal end by 3-5 residues has little effect (cf the very slight activity differences between Nef 2-22 and Nef 2-26)
  • Nef 9-22 Supporting an internal toxicity role for Nef 9-22 are the data showing that Nef 31-50 is significantly haemolytic after 160 min., whereas it is of low toxicity towards CEM cells .
  • the film balance data show that the ability to interact with membrane lipid is not a sufficient condition for lytic activity. If anything, the poorly-lytic Myr-Nef 31-50 reacts more strongly with DPPC monolayers than the lytic Myr-Nef 2-22. On the other hand, it should be noted that non-Myristylated Nef 2-22 does not penetrate the monolayer to any extent showing that myristoylation is essential for lipid interaction, although it should be noted that sufficient interaction does occur with the SUV membranes to bring about fusion. There is a remarkable correspondence between the proximate N-terminus of Nef and that of a number of Myristylated proteins of the src family (Silverman and Resch, 1992) (Table 9) .
  • Nef interacts with a number of kinases kinases (Sawai et al 1995) , and phosphorylated serine residues have been found at the N-terminus (Bodeus et al 1995) , it is possible that a similar mechanism operates to control the interaction of Nef with cell membranes and so modulate its interactions with other membrane-associated systems. Interference with such a switch mechanism could be an attractive therapeutic objective.
  • Nef GGKWSKSS src GSSKSKPK hck GCMKSKFL lyn GCIKSKGK Example 9 : Further Studies of the Cytotoxicity of Nef N-terminus in Leukocytes and Lymphoid Tissue. The effects of the Nef N-terminus on membrane integrity were investigated in a range of cultured eukocytic cell lines and in freshly-isolated PBMC and lymphoid tissue cells. The human cell lines CEM, Jurkat, RPMI 8226, U266, THP-1 and U-937, PBMC and tonsil lymphoid cells used in these studies were obtained as described above under "General Methods". The peptides used and their cytotoxicity were respectively synthesised and measured in the LDH release assay as described above under "General Methods” .
  • a range of leukocytic cell lines were examined for susceptibility to peptide-induced cytotoxicity, using the LDH release assay, to determine whether the cytotoxic activity of the Nef N-terminus is restricted to CD4+ T cells.
  • Myristylated N-terminal Nef peptide was highly cytotoxic for both the Jurkat and CEM T cell lines, the U266 B cell line and the RC2a monocyte cell line, as shown in Figure 28A.
  • near-maximal LDH release was observed in all four cell lines.
  • a lower concentration of peptide (5 ⁇ M) although all the cell lines tested were susceptible to Nef N- terminal cytotoxicity, some differences in the percentage of LDH release were apparent, as shown in Figure 28B.
  • Lymphoid tissues are the major site of viral replication and are progressively destroyed in HIV-infected individuals.
  • the effect of Nef peptides on lymphoid tissue cells was assessed using cells freshly isolated from human tonsils. Myristylated N-terminal Nef peptide was cytotoxic for uncultured tonsil lymphoid cells, causing near-maximal LDH release after 30 min. While some LDH release occurred following exposure of cells to the Myristylated non-N- terminal control peptide, this was only apparent after 2 hr and levels were less than 25% of maximum. The non- Myristylated N-terminal peptide caused virtually no LDH release, even after 2 hrs. Results are shown in Figure 30.
  • the deletion of residues 16-22 abolished serine kinase binding and reduced infectivity
  • the lymphoid tissue site is the site of a majority of viral replication and that the tissue progressively destroyed
  • the extracellular Nef detected in culture and serum has N-terminal protease sites and is present in virions mainly as a 20kD cleaved molecule (cleaved by the HIV PR) .
  • N-terminus of Nef The importance of the N-terminus of Nef is highlighted by the fact that the artifically myristylated sequence comprising amino acid residues 31-50 of Nef does not have cytotoxic activities comparable to that of the N-terminus myristylated peptide.
  • Nef belongs to a suite of membrane active proteins associated with HIV. Others include the accessory protein Vpr which has regions capable of creating pores in membranes (Macreadie et al 1996, Piller et all996) , and the gp41 transmembrane protein which has a number of membrane-active regions associated with lysis and fusion (Gallaher et al 19887, Gawrisch et al 1993). For example, the N-terminal gp41 peptide (519-541) is also lytic for red cells and toxic to cultured lymphocytes (Mobley et al 1992). It seems possible that during infection there may be a synergism between these membrane active proteins which would enhance cell membrane disruption and death.
  • Nef is biologically relevant in AIDS pathogenesis, it is an advantage to reproduce the effect in a microbial system. Apart from allowing the mechanism of cell killing to be investigated, it enables methods for the convenient, rapid screening of inhibitors of such an activity to be developed.
  • Nef2-26 peptide In aqueous solution no stable structure was observed for the Nef2-26 peptide. Addition of up to 300 mM phosphate buffer at pH 5.3 produced no change in the ID spectrum. The Nef peptide, seems less prone to aggregation than melittin, presumably as a reflection of its smaller hydrophobic surface compared with melittin.
  • Nef is a multifunctional protein, and it has been shown that the N-terminus is of vital importance to some of these functions. In aqueous solution the N-terminus is unstructured, but is likely that the functions of this region of the protein involve interactions with cell membranes, in which environment the structure is probably significantly different from that in aqueous solution.
  • the structure of Nef2-26 in methanol may be expected to resemble more closely the structure adopted by the N-terminus of Nef when it interacts with cell membranes .
  • Bodeus, M . Cardine, A.-M., Bougeret, C, Ramos-Morales, F, and Benarous, R.
  • Kestler, H.W. III. Ringler, D.J., Mori, K., Panicali, D.L., Sehgal, P.K., Daniel, M.D. and Desrosiers R.C.
  • Macreadie I.G., Arunagiri, C.K., Hewish, D.R., White, J.F. and Azad, A.A.
  • Macreadie I.G., Castelli, L.A., Lucantoni, A. and
  • Piller, S . C . Ewart, G.D., Premku ar, A., Cos, G.B. and Gage, P.W. Proc. Natl. Acad. Sci. USA, 1996 93 111-115.
  • Ranee, M. Sorenson, O.W. , Bodenhausen, G., Wagner, G.,

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Abstract

La présente invention concerne des peptides cytotoxiques myristilés dérivés de la terminaison N de la protéine Nef. Ces peptides comprennent de préférence un domaine comportant une charge positive nette et une région d'hélice α. L'invention, qui concerne également des composés inhibiteurs de la cytotoxicité par la protéine Nef, des techniques de prévention de la toxicité par la Nef, de prévention de l'infection par le VIH, concerne enfin des compositions pharmaceutiques comprenant de tels composés.
EP97941730A 1996-09-27 1997-09-26 Peptides cytotoxiques Withdrawn EP0935608A4 (fr)

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PCT/AU1997/000640 WO1998013377A1 (fr) 1996-03-06 1997-09-26 Peptides cytotoxiques

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CURTAIN C C ET AL: "FUSOGENIC ACTIVITY OF AMINO-TERMINAL REGION OF HIV TYPE 1 NEF PROTEIN" AIDS RESEARCH AND HUMAN RETROVIRUSES, NEW YORK, NY, US, vol. 10, no. 10, October 1994 (1994-10), pages 1231-1240, XP009005690 ISSN: 0889-2229 *
CURTAIN CYRIL C ET AL: "Cytotoxic activity of the amino-terminal region of HIV type 1 Nef protein" AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 13, no. 14, 20 September 1997 (1997-09-20), pages 1213-1220, XP008029579 ISSN: 0889-2229 *
CURTAIN CYRIL C ET AL: "Structural requirements for the cytotoxicity of the N-terminal region of HIV type 1 Nef" AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 14, no. 17, 20 November 1998 (1998-11-20), pages 1543-1551, XP008029578 ISSN: 0889-2229 *
See also references of WO9813377A1 *

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