PEPTIDES FOR TREATMENT OF SEPSIS AND CANCER
20230220047 · 2023-07-13
Inventors
Cpc classification
C07K14/70596
CHEMISTRY; METALLURGY
C07K2319/10
CHEMISTRY; METALLURGY
C07K14/4705
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention provides oligopeptidic compounds comprising a first oligopeptidic component derived from SLAMF1 and a second oligopeptidic component which is a cell-penetrating peptide. The oligopeptidic compounds provided have been found unexpectedly to block signalling from TLR4, TLR7, TLR8 and TLR9 in response to stimulus by their ligands, and also to display an anti-proliferative effect on cancer cells. The oligopeptidic compounds provided may be used in therapy for conditions associated with overactive immune responses, such as sepsis, and for treatment of cancer.
Claims
1. An oligopeptidic compound comprising: a) a first oligopeptidic component which: i) is capable of blocking signalling from TLR4, TLR7, TLR8 and/or TLR9; and/or ii) has an anti-proliferative and/or cytotoxic effect on cancer cells; and comprising the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 70% sequence identity thereto; and b) a second oligopeptidic component, which is a cytosol-targeting cell penetrating oligopeptidic component.
2. The oligopeptidic compound of claim 1, wherein the first oligopeptidic component has an anti-proliferative and/or cytotoxic effect on ANBL6 multiple myeloma cells and/or VK-MYC multiple myeloma cells.
3. The oligopeptidic compound of claim 1 or 2, where the first oligopeptidic component is 8-13 amino acids long, preferably 9-11 amino acids long.
4. The oligopeptidic compound of any one of claims 1 to 3, wherein the first oligopeptidic component (i) is not extended at its C-terminus relative to SEQ ID NO: 1 and/or (ii) comprises an N-terminal extension of up to 3 amino acids relative to SEQ ID NO: 1.
5. The oligopeptidic compound of any one of claim 1 to 3 or 4(i), wherein the first oligopeptidic component is not extended at its N-terminus relative to SEQ ID NO: 1.
6. The oligopeptidic compound of any one of claims 1 to 5, wherein the second oligopeptidic component is located C-terminal to the first oligopeptidic component.
7. The oligopeptidic compound of any one of claims 1 to 6, wherein the first oligopeptidic component comprises 1 to 3 amino acid substitutions relative to SEQ ID NO: 1, and said substitutions are located at positions 2, 4 and/or 10 of SEQ ID NO: 1.
8. The oligopeptidic compound of claim 7, wherein the threonine at position 2 of SEQ ID NO: 1 is substituted for asparagine, aspartic acid, histidine or lysine; the tyrosine at position 4 of SEQ ID NO: 1 is substituted for alanine, phenylalanine, asparagine, aspartic acid or serine; and/or the threonine at position 10 of SEQ ID NO: 1 is substituted for proline, asparagine or arginine.
9. The oligopeptidic compound of any one of claims 1 to 6, wherein the first oligopeptidic component consists of the amino acid sequence set forth in SEQ ID NO: 1.
10. The oligopeptidic compound of any one of claims 1 to 8, wherein the first oligopeptidic component consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2-6, 16-20, 104 or 118-121.
11. The oligopeptidic compound of any one of claims 1 to 10, wherein the second oligopeptidic component: (i) is a polyarginine peptide, wherein preferably the polyarginine peptide consists of the amino acid sequence set forth in SEQ ID NO: 7; (ii) is penetratin, consisting of the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 80% sequence identity thereto; or (iii) consists of the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 80% sequence identity thereto.
12. The oligopeptidic compound of any one of claims 1 to 11, wherein the first oligopeptidic component and the second oligopeptidic component are joined by a linker, optionally wherein the linker is a glycine residue.
13. The oligopeptidic compound of any one of claims 1 to 12, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 10-15, 107-113, 115 or 117.
14. A pharmaceutical composition comprising an oligopeptidic compound as defined in any one of claims 1 to 13, and one or more pharmaceutically-acceptable diluents, carriers or excipients.
15. An oligopeptidic compound as defined in any one of claims 1 to 13, or a composition as defined in claim 14, for use in therapy.
16. An oligopeptidic compound as defined in any one of claims 1 to 13, wherein the first oligopeptidic component of the oligopeptidic compound is capable of blocking signalling from TLR4, TLR7, TLR8 and/or TLR9, or a composition as defined in claim 14 comprising said oligopeptidic compound, for use in treatment or prevention of a disease associated with hypercytokinemia in a human subject, wherein preferably said hypercytokinemia is characterised by up-regulation of expression of interferon 13, IL-1β, IL-6 and/or TNF.
17. The oligopeptidic compound or composition for use according to claim 16, wherein the first oligopeptidic component consists of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, 16-20 or 104.
18. The oligopeptidic compound or composition for use according to claim 17, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 10-15, 108-110, 115 or 117.
19. The oligopeptidic compound or composition for use according to any one of claims 16 to 18, wherein said disease is sepsis.
20. The oligopeptidic compound or composition for use according to any one of claims 16 to 19, wherein said disease is associated with infection by a bacterium and/or virus.
21. The oligopeptidic compound or composition for use according to any one of claims 16 to 20, wherein said treatment or prevention further comprises administration to the subject of a second therapeutically active agent, preferably wherein the second therapeutically active agent is an antibiotic or an antiviral agent.
22. An oligopeptidic compound as defined in any one of claims 1 to 13, wherein the first oligopeptidic component of the oligopeptidic compound is capable of blocking signalling from TLR4, TLR7, TLR8 and/or TLR9, or a composition as defined in claim 14 comprising said oligopeptidic compound, for use in treatment or prevention of an inflammatory disease, an autoimmune disease or ischemia-reperfusion injury; preferably wherein the oligopeptidic compound or composition is as defined in claim 17 or 18.
23. The oligopeptidic compound or composition for use according to claim 22, wherein the inflammatory disease or autoimmune disease is selected from type II diabetes, a neurodegenerative disease, preferably Alzheimer's disease or Parkinson's disease, gout, non-alcoholic steatohepatitis, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, Schnitzler's syndrome, atherosclerosis, graft-versus-host disease, cryopyrin-associated periodic syndromes or a fibrotic disease.
24. An oligopeptidic compound as defined in any one of claims 1 to 13, wherein the first oligopeptidic component of the oligopeptidic compound has an anti-proliferative and/or cytotoxic effect on cancer cells, or a composition as defined in claim 14 comprising said oligopeptidic compound, for use in the treatment of cancer.
25. The oligopeptidic compound or composition for use according to claim 24, wherein the first oligopeptidic component consists of the amino acid sequence set forth in any one of SEQ ID NOs: 1-3 or 118-121.
26. The oligopeptidic compound or composition for use according to claim 25, comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 10-14, 107 and 111-113.
27. The oligopeptidic compound or composition for use according to any one of claims 24 to 26, wherein the cancer is breast cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, lung cancer, melanoma, endometrial cancer, a haematological cancer, cervical cancer, ovarian cancer, colorectal cancer or pancreatic cancer.
28. The oligopeptidic compound or composition for use according to claim 27, wherein the haematological cancer is multiple myeloma, acute myeloid leukaemia, T cell leukaemia, Hodgkin lymphoma or non-Hodgkin lymphoma.
29. The oligopeptidic compound or composition for use according to any one of claims 24 to 28, wherein said treatment further comprises administration to the subject of a second therapeutically active agent, preferably wherein the second therapeutically active agent is a chemotherapy, radiotherapy, hormonal therapy and/or immunotherapy agent, and/or a small molecule inhibitor or biologic.
30. The oligopeptidic compound or composition for use according to claim 29, wherein said second therapeutically active agent is melphalan, preferably wherein said treatment is for multiple myeloma.
31. A method of treating or preventing a disease in a human subject, comprising administering to the subject an oligopeptidic compound as defined in any one of claims 1 to 13 or a composition as defined in claim 14; wherein the disease and oligopeptidic compound are as defined in any one of claims 16 to 20 or 22 to 28, and optionally wherein the treatment or prevention is as defined in claim 21, 29 or 30.
32. Use of an oligopeptidic compound as defined in any one of claims 1 to 13 in the manufacture of a medicament for use in the treatment or prevention of a disease in a human subject, wherein the disease and oligopeptidic compound are as defined in any one of claims 16 to 20 or 22 to 28, and optionally wherein the treatment or prevention is as defined in claim 21, 29 or 30.
33. A kit comprising an oligopeptidic compound as defined in any one of claims 1 to 13 or a pharmaceutical composition as defined in claim 14, and a second therapeutically active agent; preferably wherein the oligopeptidic compound is as defined in any one of claims 16 to 18, and the second therapeutically active agent is an antibiotic or an antiviral agent; or the oligopeptidic compound is as defined in any one of claims 24 to 26, and the second therapeutically active agent is a chemotherapy, radiotherapy, hormonal therapy and/or immunotherapy agent, and/or a small molecule inhibitor or biologic.
34. A product comprising an oligopeptidic compound as defined in any one of claims 1 to 13 or a pharmaceutical composition as defined in claim 14, and a second therapeutically active agent, as a combined preparation for separate, simultaneous or sequential use in the treatment or prevention of a disease in a human subject, wherein: (i) the oligopeptidic compound is as defined in any one of claims 16 to 18 and the disease is as defined in any one of claim 16, 19, 20, 22 or 23, and the second therapeutically active agent is preferably an antibiotic or an antiviral agent; or (ii) the oligopeptidic compound is as defined in any one of claims 24 to 26 and the disease is as defined in any one of claim 24, 27 or 28, and the second therapeutically active agent is preferably a chemotherapy, radiotherapy, hormonal therapy and/or immunotherapy agent, and/or a small molecule inhibitor or biologic.
35. A nucleic acid molecule comprising a nucleotide sequence which encodes an oligopeptidic compound as defined in any one of claims 1 to 13.
36. A construct comprising the nucleic acid molecule of claim 35, or a vector comprising said construct or nucleic acid molecule.
37. A method of downregulating expression of interferon β by a human cell, comprising contacting the cell with an oligopeptidic compound as defined in any one of claims 1 to 13, wherein the first oligopeptidic component of the oligopeptidic compound is capable of blocking signalling from TLR4, TLR7, TLR8 and/or TLR9; wherein optionally expression of IL-1β, TNF, IL-6 and/or CXCL10 is also downregulated.
Description
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EXAMPLES
[0242] Peptides were designed based on the information about interaction domains for SLAMF1 and TRAM proteins described above and published in Yurchenko et al. (supra). The amino acid sequence of the SLAMF1 protein was selected as basis to design peptides to target and inhibit the interaction of TRAM with SLAMF1 (such that the peptides would compete with endogenous SLAMF1 protein for the SLAMF1 binding region of TRAM), in order to inhibit synthesis of pro-inflammatory cytokines.
[0243] Materials & Methods
[0244] Primary Human Monocytes
[0245] Use of human monocytes from blood donors was approved by the Regional Committees for Medical and Health Research Ethics at the Norwegian University of Science and Technology (NTNU). Human monocytes were isolated from buffy coats by adherence as previously described (Husebye et al., Immunity 33: 583-596, 2010). Briefly, freshly prepared buffy coat (The Blood Bank, St. Olav's Hospital, Trondheim, Norway) was diluted in 100 ml PBS, and PBMCs isolated by density gradient centrifugation using Lymphoprep (Axis-Shield) according to the manufacturer's instructions. PBMCs were collected and washed with Hank's Balanced Salt Solution (Sigma-Aldrich, USA) four times by low speed centrifugation (150-200× g). Cells were counted using a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter) on program B, re-suspended in RPM11640 (Sigma) supplemented with 5% pooled human serum at a concentration of 8×10.sup.6 per ml and seeded into 6-well (1 ml per well) or 24-well (0.5 ml per well) cell culture dishes. Following a 45 min incubation, allowing surface adherence of monocytes, the dishes were washed 3 times with Hank's Balanced Salt Solution to remove non-adherent cells. Monocytes were maintained in RPM11640 supplemented with 10% pooled human serum (The Blood Bank, St Olav's Hospital) and used within 24 h after isolation.
[0246] Cell Lines
[0247] SW480 (ATCC CCL-228) and Jurkat (ATCC Clone E6-1; TIB-152) cells were cultured in RPMI 1640 (Sigma-Aldrich, Schnelldorf, Germany) supplemented with 10% heat-inactivated FCS, penicillin/streptomycin (Life Technologies) and 20 mM L-Glutamine. THP-1 cells (ATCC TIB-202) were cultured in RPMI-1640 (high-glucose, ATCC) supplemented with 10% heat inactivated foetal calf serum (FCS), 100 nM penicillin/streptomycin and 5 μM β-mercaptoethanol (Sigma). THP-1 cells were differentiated with 60 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) for 72 h, followed by 24 or 48 h in medium without PMA.
[0248] Multiple myeloma cells JJN-3 (DSMZ (Germany) no. ACC 541), ANBL-6 (Cellosaurus accession number CVCL_5425, obtained from Dr Diane Jelinek, Mayo Clinic, Rochester, Minn.), INA-6 (Cellosaurus accession number CVCL_5209, obtained from Dr M. Gramatzki, University of Erlangen-Nurnberg, Erlangen, Germany), RPMI-8226 (ATCC CCL-155), and IH-1 (Cellosaurus accession number CVCL_WZ44, established from the pleural effusion of a myeloma patient by Myeloma research group, IKOM, NTNU, Trondheim, Norway) were grown in RPMI-1640 (Sigma-Aldrich Norway, Oslo, Norway) supplemented with 100 nM penicillin/streptomycin and 10% FCS, and for ANBL-6, IH-1 and INA-6 cells 2 ng/ml recombinant IL-6 (Gibco, Thermo Fisher Scientific, Waltham, Mass., USA). Murine multiple myeloma Vk*MYC cells were obtained from M.D. Leif Bergsagel (Mayo Clinic, Richester, Minn., USA) and cultured in RPMI-1640 supplemented with 2% human serum (B.sup.+, The blood bank, St Olav's Hospital, Trondheim, Norway), 100 nM penicillin/streptomycin, L-Glutamine (2 mM) and recombinant IL-6 (1 ng/ml). HEK293T (ATCC CRL-3216) were cultured in DMEM with 10% FCS. THP-1 TRAM knockout (KO) and TLR4 KO cell lines, and matching lentivirally transduced control cell lines, were differentiated in the same way as wild type (WT) THP-1. TRAM KO THP-1 cell line is described in Yurchenko et al. (supra).
[0249] To generate the TLR4 KO cell line, the LentiCRISPRv2 plasmid (Sanjana et al., Nature Methods 11: 783-784, 2014; Addgene #52961) was ligated with 5′-CACCGCCAGCTTTCTGGTCTCACGC-3′ (SEQ ID NO: 100) and 5′-AAACGCGTGAGACCAGAAAGCTGGC-3′ (SEQ ID NO: 101) to target TLR4. For the control cell line, the LentiCRISPRv2 plasmid was ligated with 5′-CACCGTTTGTAATCGTCGATACCC-3′ (SEQ ID NO: 102) and 5′-AAACGGGTATCGACGATTACAAAC-3′ (SEQ ID NO: 103) (which have no specific targets in the human genome). Packaging plasmids pMD2.G and psPAX2 were used for producing lentivirus (Addgene plasmids #12260 and #12259). HEK293T cells were co-transfected with the packaging and lentiCRISPRv2 plasmids and washed after 16 h. The lentivirus-containing supernatants were collected after 48 h and used for transduction of THP-1 cells along with 8 μg/ml protamine sulphate. Transduced THP-1 cells were then selected with Puromycin (1 μg/ml) for 1 month. The control cell line was tested along with the THP-1 cell line in LPS stimulations and did not demonstrate differences in cytokine expression compared to THP-1 WT cells. The TLR4 KO cell line was, as expected, not sensitive to LPS-treatment, but has unaffected responses to the TLR2/6 ligand FSL-1 (as detailed below). All cell lines were regularly checked for mycoplasma contamination.
[0250] Immortalized bone-derived-macrophages (iBMDM) from wild type, Tram−/− or TIr4−/− C57BL/6 mice were maintained at 37° C. and 5% CO.sub.2 in DMEM supplemented with 10% FCS.
[0251] Peripheral Blood Mononuclear Cells (PBMCs)
[0252] The use of PBMCs was approved by the Regional Committees from Medical and Health Research Ethics at the Norwegian University of Science and Technology (REK no. 2009/2245). Cells were isolated from buffy coats using Lymphoprep separation medium (Axis-Shield, Oslo, Norway) and washed with Hanks' Balanced Salt solution (Sigma Aldrich, Schnelldorf, Germany). Cells were resuspended in RPMI-1640 supplemented with 10% FCS, 2% human serum (A.sup.+, The Blood bank, St Olav's Hospital, Trondheim, Norway), 100 nM penicillin/streptomycin and L-Glutamine (2 mM). 50,000 cells/well of PBMCs were seeded in white-walled flat-bottomed 96-well plates in 200 μl of media and treated with media with solvent (water) or 20 μM peptides. PBMCs were kept in a total volume of 250 μl/well and incubated with treatment for 48 hr.
[0253] Whole Blood Assay
[0254] To evaluate the effect of peptides in an ex vivo system, a human whole blood model was used. Peripheral venous blood was drawn from 6 healthy donors into sterile polypropylene tubes (Thermo Scientific™ Nunc™ Biobanking and Cell Culture Cryogenic Tubes) containing the anti-coagulant Lepirudin (Refludan®; Celgene). Peptide solutions were made by diluting peptide stocks in DPBS (Dulbecco's Phosphate Buffered Saline with MgCl.sub.2 and CaCl.sub.2; Sigma-Aldrich) to obtain a final concentration of 140 μM. Peptide solution or DPBS was distributed in equal volume (50 μL) to 1.8 mL sterile polypropylene tubes (Thermo Scientific™Nunc™ Biobanking and Cell Culture Cryogenic Tubes). 250 μL whole blood was then added to the 1.8 mL tubes containing peptide solution or DPBS, and incubated at 37° C. under constant rotation for 1 h (concentration of peptides for pre-incubation: 23.3 μM). Stimulation solutions were made by diluting K12 LPS or CL075 in DPBS to obtain a final concentration in whole blood of 100 ng/mL (LPS) or 1 μg/mL (CL075) after addition of 50 μL stimulation solution or DPBS. Concentration of peptides in whole blood samples was reduced by this to 20 μM. Tubes were incubated at 37° C. under constant rotation for 4 h. Finally, plasma was isolated from the whole blood specimens by centrifugation, collected in 96 well plates (Corning® 96 Well CellBIND® Microplates), and stored at −20/−80° C. until analysis by multiplex/27-plex cytokine assay or IFNβ ELISA (VeriKine-HS Human Interferon Beta Serum ELISA Kit; pbl Assay Science).
[0255] Reagents and Cell Stimulation
[0256] pHrodo Red E. coli were purchased from Thermo Fisher Scientific. Ultrapure E. coli K12 LPS, polyinosinic-polycytidylic acid [poly(I:C)], and the thiazoloquinoline compound CL075, were obtained from InvivoGen. Ultrapure K12 LPS was used at a concentration of 100 ng/ml. E. coli bioparticles were reconstituted in 2 ml PBS, and 50 μl/well (1.5×10.sup.7 particles) in 1 ml of media was used for cells in 6-well plates (NUNC) or 35-mm glass bottom tissue cell dishes (MatTek Corp.), or 15 μl/well (0.45×10.sup.7 particles) in 0.5 ml of media for 24-well plates (NUNC).
[0257] Peptides
[0258] Peptides were synthesised to custom order as TFA-free (with TFA substitution to acetate ions), with N-terminal acetylation and C-terminal amidation, by GenScript. Peptides were dissolved in the suggested optimal solvent (DMSO or sterile milliQ water) to concentrations from 0.5 to 5 mM, aliquoted and stored at −80° C., avoiding freezing-unfreezing cycles. Peptides were dissolved to the working concentration in cell culture media (cell-based assays) or PBS (whole blood assays) just before use.
[0259] Antibodies
[0260] The following primary antibodies were used: rabbit-anti-TICAM-2/TRAM (GTX112785) from Genetex; rabbit anti-human SLAMF1/SLAMF1 (#10837-R008-50) from Sino Biological Inc.; anti-tubulin antibodies from abcam; IkB-α (44D4) #4812, phospho-IkB-α (14D4) #2859, phospho-p38 MAPK (Thr180/Tyr182) (D3F9) #4511, phospho-TBK1/NAK (Ser172) (D52C2) #5483, phospho-TAK1 (T184/187) (9007) #4508, TAK1 #5206, anti-DYKDDDDK Tag (D6W5B)/Flag-tag #14793, anti-MyD88 (D80F5) #4283, anti-IRAK1 (Human Specific) #4359, anti-IRAK1 (D51G7) #4504, anti-IRAK4 #4363, anti-TRAF6 (D21G3) #8028, phospho-STAT1 (Tyr701) (D4A7) #7649 from Cell Signaling; Living Colors rabbit anti-full-length GFP polyclonal Abs (#632592) from Clontech; 4G10® Platinum anti-phosphotyrosine antibody biotin conjugated (#16-452) from Merck-Millipore (Merck Life Science AS); mouse anti-Glutathione-S-Transferase (SAB4200237), monoclonal mouse ANTI-FLAG M2 antibodies (#F1804-200UG) from Sigma; rabbit anti-caspase-3 (#9662) from Cell Signaling Technology. Secondary antibodies (HRP linked) for Western blotting were swine anti-rabbit (P039901-2) and goat anti-mouse (P044701-2) from DAKO/Agilent.
[0261] Live Cell Imaging
[0262] Human primary monocytes were seeded into 24-well glass-bottomed plates (MatTek Corporation) in RPMI media containing 25% pooled human serum. The day after isolation, cells were washed once more with RPMI and media changed to RPMI supplemented with 10% human serum, followed by addition of peptides (Arg11 or Pep7-Arg11) labelled with N-terminal TAMRA (GenScript) in a sterile hood. Cells were immediately transferred to a microscope and peptide accumulation was followed using a ZEISS AiryScan microscope. Images were taken at different time points following accumulation of peptides inside the cells.
[0263] Confocal Microscopy Analysis
[0264] THP-1 cells, THP-1.sup.cherry cells or human PBMC were seeded in 24-well glass-bottom plates (MatTek Corporation), 125,000 cells/well. The THP-1 cells were differentiated into macrophages with 60 ng/ml PMA for 72 h and thereafter rested for 48 h, whereas the PBMC were used the day after isolation. The P7-penatratin peptide or control peptide was added to the cells 30 min before stimulation at 15 μM concentration. If used, 3 μM cytochalasin D (CytoD) was added simultaneously. The cells were stimulated with Alexa Fluor 488-conjugated E. coli K-12 BioParticles or Alexa Fluor 488-conjugated S. aureus BioParticles (12 particles/cell). Prior to stimulation bacterial particles were sonicated and opsonised in medium containing 10% human A.sup.+ serum for 5 min at 37° C. After incubation for 15, 30 or 45 minutes the cells were washed with PBS and fixed with 2% PFA for 10 min on ice, and thereafter stained with rhodamine phalloidin or SiR-actin. Confocal images were captured using an apochromat 63×/1.4 CS2 oil-immersion objective on a Leica TCS SP8 (Leica Microsystems). The 488 nm, 561 nm and 633 nm white laser lines were used for detection and three-dimensional data was acquired from 12-bit raw imaging data used to the build Z-stacks for the individual channels by LAS X software. A spot detection mode in the Bitplan Imaris software was used to define individual phagosomes and cells were counted manually. The data is presented as the average number of phagocytosed bacteria per cell. Fluorescence voxel intensity of TRAM.sup.cherry at the phagosome is also presented. Statistical significance was calculated in GraphPad Prism by One-way ANOVA Kruskal-Wallis multiple comparison test.
[0265] LDH Cytotoxicity Assay
[0266] To address the potential cytotoxicity of different peptides, supernatants from the cell-based assays were collected and analysed using the Thermo Scientific™ Pierce™ LDH Cytotoxicity Assay Kit as suggested by the manufacturer. For every assay, separate wells were seeded for the required controls (Spontaneous LDH Release Controls and Maximum LDH Release Control) with the same amount of cells as used for peptide treatments. All supernatants were analysed in triplicate with the average value of technical replicates taken into calculations.
[0267] qPCR
[0268] Total RNA was isolated from the cells using Qiazol reagent #79306 from QIAGEN, and chloroform extraction followed by purification on RNeasy Mini columns with DNAse digestion (Qiagen). cDNA was prepared using the Maxima First Strand cDNA Synthesis Kit for quantitative real-time polymerase chain reaction (RT-qPCR) (Thermo Fisher Scientific) according to the manufacturer's protocol. qPCR was performed using the PerfeCTa qPCR FastMix (Quanta Biosciences) in replicates and cycled in a StepOnePlus™ Real-Time PCR cycler. The following TaqMan® Gene Expression Assays (Applied Biosystems®) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), CXCL10 (Hs01124251_g1), IL-6 (Hs00174131_m1) and IL-1β (Hs01555410_m1), for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Tbp (Mm01277042_m1) and II-1β (Mm00434228_m1), for mouse cells. No RT controls were negative. The level of TBP mRNA was used for normalisation and results presented as relative expression compared to the control untreated sample. Relative expression was calculated using Pfaffl's mathematical model (Pfaffl, Nucleic Acids Research 29(9): e45, 2001). Results are presented as the mean and SD expression fold change for biological replicates relative to non-stimulated cells. Statistical significance was evaluated in GraphPad Prizm 5.03. Data distribution was assumed to be normal but this was not formally tested. The difference between the two groups was determined by two-tailed t test.
[0269] Expression Vectors and DNA Transfection
[0270] SLAMF1 and mutants thereof in a C-terminal Flag-tag vector and TRAM.sup.FLAG construct are described in Yurchenko et al. (supra), and SLAMF1 mutants not disclosed in Yurchenko et al. were synthesised equivalently to those previously detailed; Human TRAM.sup.YFP, Mal and Mal.sup.FLAG were obtained from K. Fitzgerald (University of Massachusetts Medical School, Worcester, Mass., USA). IRAK1 and IRAK4.sup.EGFP were obtained from MRC PPU Reagents and Services, University of Dundee, UK. HEK293T cells in 6-well plates were transfected with 0.2-0.4 μg vectors/well using Genejuice transfection reagent (Millipore). The total amount of DNA per well was always normalised to empty vectors. Media was changed 24 h after transfection and lysates were prepared 48 h after transfection.
[0271] Immunoprecipitations (IPs)
[0272] HEK293T cells expressing Flag-tagged proteins were lysed using 1×lysis buffer (150 mM NaCl, 50 mM TrisHCl (pH 8.0), 1 mM EDTA, 1% NP40), supplemented with EDTA-free Complete Mini protease Inhibitor Cocktail Tablets and PhosSTOP phosphatase inhibitor cocktail from Roche, 50 mM NaF and 2 mM Na.sub.3VO.sub.3 (Sigma). IPs were carried out by rotation of cell lysates at 4° C. for 2 h with anti-flag (M2) agarose (Sigma). Beads were washed 5 times with lysis buffer and heated for 5 min with 1×NuPAGE® LDS Sample Buffer (Thermo Fisher Scientific) before analysis of precipitates by Western blotting. For testing peptides as CFP-tagged constructs, primers coding for the described amino acid sequences were cloned into the ECFP-C1 vector yielding N-terminal CFP-tagged peptides.
[0273] Immunoprecipitations with the biotinylated peptides Penetratin-biotin and Pep7-Penetratin-biotin (synthesised by custom order by GenScript) were performed by coating NeutrAvidin® Agarose Resin (Thermo Scientific) with peptide by incubating 40 μl 50% agarose slurry with 10 μl 2 mM peptides in 300 μl lysis buffer (for one precipitation reaction) (300 mM NaCl, 50 mM TrisHCl (pH 8.0), 1 mM EDTA, 1% Triton X100). Beads were washed 3 times with lysis buffer to remove unbound peptides before applying cellular extracts. Cell lysis was performed in the same lysis buffer, both for HEK293T cells and primary human macrophages. Precipitations were carried out by rotation at 4° C. for 30′-1 h, followed by four washes with lysis buffer. Agarose resin with precipitated protein complexes was heated in loading buffer 1×NuPAGE® LDS Sample Buffer (Thermo Fisher Scientific) containing 20 mM DTT and analysed by Western blotting.
[0274] Western Blotting
[0275] Cell lysates, other than when used as controls in immunoprecipitations, were prepared by simultaneous extraction of proteins and total RNA using Qiazol reagent (Qiagen) as suggested by the manufacturer. Protein pellets were dissolved by heating protein pellets for 10 min at 95° C. in buffer containing 4 M urea, 1 SDS (Sigma) and NuPAGE® LDS Sample Buffer (4×) (Thermo Fisher Scientific). Otherwise, lysates were made using 1× RIPA lysis buffer (150 mM NaCl, 50 mM TrisHCl (pH 7.5), 1% Triton X100, 5 mM EDTA, protease inhibitors, phosphatase inhibitors). For Western blot analysis we used pre-cast protein gels NuPAGE™ Novex™ and iBlot Transfer Stacks iBlot Gel Transfer Device (Thermo Fisher Scientific). Proteins were transferred to the membrane by dry-blotting using iBlot® 2 NC Regular/Mini Stacks (Life Technologies) in the iBlot 2 Dry Blotting System (Life Technologies). After secondary antibody incubation, membranes were washed and incubated with HRP substrate solution (SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific)). Images were taken using Odyssey® Fc (LI-CORE). Quantification graphs were generated by the analysis of signal intensities for protein bands (protein of interest and endogenous loading control—β-tubulin from the same membrane) using Odyssey software (LI-CORE). Data were normalised to the expression levels of β-tubulin and presented as a relative fold change to the control sample.
[0276] ELISA and Multiplex Cytokine Assay
[0277] TNF in supernatants was detected using a human TNF-alpha DuoSet ELISA (DY210) (R&D Systems); CXCL10 using a DuoSet ELISA (DY266) (R&D Systems); IL-1β using a BD OptEiA human IL-1β ELISA SetII (#557953, BD Biosciences): IFNβ using a VeriKine-HS™ Human Interferon-Beta Serum ELISA Kit (#41415) from PBL Assay Science. Some supernatants were also analysed by multiplex cytokine assay (Bio-Plex; Bio-Rad Laboratories Inc.) for IL-1β, IL-6, IL-8, CXCL10/IP-10. Results are presented as mean and SD for biological replicates for a representative donor (primary human macrophages), or at least three independent experiments for the model cell line THP-1. Statistical significance was evaluated in GraphPad Prizm 5.03. Data distribution was assumed to be normal but this was not formally tested. The difference between the two groups was determined by two-tailed t test.
Example 1—Effect of Peptide Size on Efficacy
[0278] As detailed in Yurchenko et al., murine SLAMF1 and TRAM do not interact with each other. The C-termini of human and murine SLAMF1 are largely conserved, with the exception of four amino acids at positions 329-332 of murine SLAMF1, which correspond to positions 321-324 of human SLAMF1. These amino acids have the sequence Thr-Asn-Ser-Ile in human SLAMF1 and Pro-Asn-Pro-Thr in murine SLAMF1. It was hypothesised that this sequence could be important in binding to TRAM, though it was also considered possible that this sequence could be important for the interaction in the context of the whole protein secondary structures (due to the positioning of the β-sheet formed by this sequence), but not critical for designing of small free peptides. The two proline residues in murine SLAMF1ct will change the positioning of the C-terminal β-sheet (because prolines introduce turns to the protein backbone structure), but may not have an essential role when the interaction region is freely exposed in a short peptide sequence. Peptides were designed which encompass positions 321-324 of human SLAMF1 and the surrounding regions.
[0279] Prior to designing the peptides, we performed an immunoprecipitation screen for some of the SLAMF1 c-terminus (SLAMF1ct) variants with single or double aa substitutions to identify amino acid residues important in binding of SLAMF1 to TRAM (
[0280] Inhibition of the TRAM-SLAMF1 interaction using SLAMF1-derived peptides was tested. For the first line of experiments, SLAMF1-derived peptides were co-expressed as N-terminal ECFP-tagged proteins together with SLAMF1 and TRAM.sup.FLAG constructs in HEK-293T cells. Cell lysates were used for anti-Flag immunoprecipitations (IPs) as shown in
[0281] Modelling of peptide secondary structures with an attached Arg11 CPP (SEQ ID NO: 48) was performed using the PEP FOLD server (http://bioservspbs.univ-paris-diderotfr/services/PEP-FOLD/). This showed more potential intramolecular interactions between P6 and the Arg11 CPP than between P7 and Arg11 (not shown). Such internal contacts could interfere with cell penetration or the affinity of the peptide for the target protein. Modelling of a longer peptide (P3, SEQ ID NO: 49) attached to the Arg11 CPP showed an even higher probability of formation of a closed structure with electrostatic contacts between P3 and the Arg11 CPP (not shown). Accordingly a longer peptide may not penetrate effectively, or form a secondary structure with incorrect positioning of the TRAM-interacting motif interfering with the affinity of the peptide for TRAM. Accordingly, peptide P7 was selected as lead candidate of the tested candidate sequences.
Example 2—Intracellular Localization of P7-Arg11
[0282] Before proceeding with functional analysis, we tested the penetrating ability of CPP-tagged P7, and its resulting intracellular distribution in human primary macrophages. P7 peptide tagged with Arg11 CPP was labeled with TAMRA fluorescent dye (TAMRA-P7-Arg11) along with Arg11 alone (TAMRA-Arg11) and applied to live human macrophages differentiated from PBMCs to follow intracellular distribution of the peptides. P7-Arg11 (SEQ ID NO: 51) was distributed both to vesicles and cytoplasm in the cells (not shown) showing potential for co-localization with cytoplasmic targets.
Example 3—Primary Functional Testing of P7 Peptide
[0283] We tested whether pre-treatment of THP-1 macrophages with the P7-Arg11 peptide would have an inhibitory effect on LPS-mediated IFN/3, TNF and CXCL10 mRNA expression.
[0284] THP-1 cells were pre-treated with increasing concentrations of P7-Arg11 and control peptide Arg11 for 1 h (
[0285] In further experiments peptides were removed from media after pre-treatment of macrophages but before LPS stimulation. No changes in LPS-induced cytokine expression levels (IFNβ, TNF and CXCL10 mRNA expression) were observed for P7-Arg11 pre-treated cells, which demonstrates a reversible effect of the P7-Arg11 peptides (data not shown).
[0286] Due to the larger molecular weight of P7-Arg11 than of the Arg11 CPP alone, we required another more suitable control peptide with a similar molecular weight and amino acid composition to P7. We tested the control/decoy peptide P7C3 (SEQ ID NO: 50), which contains 4 amino acid substitutions relative to Pep7 but has a similar molecular weight to P7-Arg11, the same length as P7-Arg11 and an almost identical overall amino acid composition to P7-Arg11. Pre-treatment of THP-1 cells with P703-Arg11 was found to have no effect on LPS- or E. coli particle-induced expression of IFNβ, CXCL10 and TNF (data not shown).
Example 4—CPP Screening
[0287] P7/Pep7 linked to two different CPPs—Arg11 and penetratin (Pen, SEQ ID NO: 8) with or without a TAMRA label was equally effective in down-regulating LPS-mediated expression of IFNβ (
[0288] In light of these results in the THP-1 model system, we proceeded with testing P7 peptides linked to two different CPPs (and at two different concentrations) in PBMC-derived human primary monocytes stimulated by LPS or E. coli particles (
[0289] We then set up a CPP screen in THP-1 cells to identify the best CPPs for P7 delivery (
TABLE-US-00002 TABLE 2 SEQ ID Name Sequence NO P7- ITVYASVTLTGRRRRRRRRRRR 51 Arg11 Arg11- RRRRRRRRRRRGITVYASVTLT 52 P7 P7C3- IATYASTALTGRRRRRRRRRRR 53 Arg11 P7-Arg9 ITVYASVTLTGRRRRRRRRR 13 P7C3- IATYASTALTGRRRRRRRRR 54 Arg9 P7-Arg7 ITVYASVTLTGRRRRRRR 55 P7-Pen ITVYASVTLTGRQIKIWFQNR 10 RMKWKK Pen-P7 RQIKIWFQNRRMKWKKGITVY 56 ASVTLT P7C3- IATYASTALTGRQIKIWFQNR 57 Pen RMKWKK P7- ITVYASVTLTGRGGRLSYSRR 58 Pegelin RFSTSTGR P7-TAT ITVYASVTLTGGRKKRRQRRR 59 PPQ P7-KLA ITVYASVTLTGKLALKLALKA 12 LKAALKLA KLA-P7 KLALKLALKALKAALKLAGIT 60 VYASVTLT P7C3-KLA IATYASTALTGKLALKLALKA 61 ALLKAKLA P7- ITVYASVTLTGVKRGLKLRHV 62 Vectocell RPRVTRMDV P7-Pen_sh ITVYASVTLTGNRRMKWKK 63
[0290] Several CPPs were tested in parallel—protegrin class CPPs (TAT, SEQ ID NO: 36; pegelin, SEQ ID NO: 36), amphipathic CPPs (vectocell, SEQ ID NO:43; KLA/MAP, SEQ ID NO: 9), shorter versions of Arg sequences (Arg9, SEQ ID NO: 7; Arg7, SEQ ID NO: 47), short penetratin CPP (Pen_sh, SEQ ID NO: 22) along with previously tested Pen, Arg11, some control peptides and respective solvents. Also we performed some tests to address the effect of CPP positioning (C-terminal vs. N-terminal to P7) on the inhibitory activity of P7 peptide towards expression of several LPS-mediated cytokines (CXCL10, TNF, IL-13) (
[0291] Data is presented for cytokine secretion levels in supernatants of treated cells (representative experiment,
Example 5—Screening Longer SLAMF1-Derived Peptides
[0292] In the next screens we tested if the extension of peptides at the N- or C-terminus could alter their efficacy, and peptides used for the screens are listed in Table 3.
TABLE-US-00003 TABLE 3 SEQ ID Name Sequence NO P7-Pen ITVYASVTLTGRQIKIWFQNRRMKWKK 11 P6-Pen ITVYASVTLPGRQIKIWFQNRRMKWKK 12 P3-Arg11 TNSITVYASVTLTESGRRRRRRRRRRR 64 P3sh1-Pen TNSITVYASVTLTGRQIKIWFQNRRMK 65 WKK P3sh2-Pen NSITVYASVTLTGRQIKIWFQNRRMKW 66 KK P10-Pen ITVYASVTLPEGRQIKIWFQNRRMKWKK 67 P10-Ala11- ITVYASVTLPAGRQIKIWFQNRRMKWKK 68 Pen P11-Pen ITVYASVTLTEGRQIKIWFQNRRMKWKK 69 P11-Ala11- ITVYASVTLTAGRQIKIWFQNRRMKWKK 70 Pen
[0293] As we expected from the peptide modelling (see above), the P3-Arg11 peptide (SEQ ID NO: 64) was not effective in reducing LPS-mediated cytokine mRNA expression (
[0294] To test which additional amino acids are responsible for the loss of the peptide's inhibitory effect, THP-1 cells were pre-treated with several variants of SLAMF1-derived peptides, which corresponded to P7 elongated at either the N-terminus or the C-terminus (
Example 6—Screen of Substitutions within P7
[0295] The sequences of peptides synthesised for the screen of single amino acid substitutions in P7 are listed in Table 4.
TABLE-US-00004 TABLE 4 SEQ ID Name Sequence NO P7-Pen ITVYASVTLTGRQIKIWFQNRRMKWKK 10 P7-A1-Pen ATVYASVTLTGRQIKIWFQNRRMKWKK 75 P7-A2-Pen IAVYASVTLTGRQIKIWFQNRRMKWKK 76 P7-S2-Pen ISVYASVTLTGRQIKIWFQNRRMKWKK 77 P7-N2-Pen INVYASVTLTGRQIKIWFQNRRMKWKK 15 P7-A3-Pen ITAYASVTLTGRQIKIWFQNRRMKWKK 78 P7-T3-Pen ITTYASVTLTGRQIKIWFQNRRMKWKK 79 P7-L3-Pen ITLYASVTLTGRQIKIWFQNRRMKWKK 80 P7-A4-Pen ITVAASVTLTGRQIKIWFQNRRMKWKK 14 P7-V4-Pen ITVVASVTLTGRQIKIWFQNRRMKWKK 81 P7-T4-Pen ITVTASVTLTGRQIKIWFQNRRMKWKK 82 P7-L5-Pen ITVYLSVTLTGRQIKIWFQNRRMKWKK 83 P7-A6-Pen ITVYAAVTLTGRQIKIWFQNRRMKWKK 84 P7-A7-Pen ITVYASATLTGRQIKIWFQNRRMKWKK 85 P7-A8-Pen ITVYASVALTGRQIKIWFQNRRMKWKK 86 P7-A9-Pen ITVYASVTATGRQIKIWFQNRRMKWKK 87 P7-I9-Pen ITVYASVTITGRQIKIWFQNRRMKWKK 88 P7-G9-Pen ITVYASVTGTGRQIKIWFQNRRMKWKK 89 P7-A10-Pen ITVYASVTAAGRQIKIWFQNRRMKWKK 90 P7-S10-Pen ITVYASVTLSGRQIKIWFQNRRMKWKK 91 P7-V10-Pen ITVYASVTLVGRQIKIWFQNRRMKWKK 92 P6-Pen ITVYASVTLPGRQIKIWFQNRRMKWKK 11 P7C3A-Pen IATYASVTLTGRQIKIWFQNRRMKWKK 93
[0296] The screen was performed in THP-1 cells pre-treated with 15 μM peptides for 30 min and stimulated with LPS for 5 h. We found that the amino acids at positions 2, 4 and 10 of the P7 peptide could be substituted to other amino acids without an increase in peptide toxicity (
Example 7—Timing of Cell Treatment with Peptide
[0297] Next, we examined if the inhibitory effect of the peptide on the TLR4-IFNβ signaling axis would be preserved if the peptide was added 30 min before addition of LPS, simultaneously with LPS or 1 h after LPS treatment. We found that the peptide remained very effective in inhibition of CXCL10 secretion when applied to cells 30 min before LPS addition, and when applied simultaneously with LPS (
Example 8—Effect of P7 on Murine Cells
[0298] Murine disease models are the most accessible and widely used pre-clinical model for testing new drugs. We decided to test if the P7-Pen peptide would interact with murine TRAM protein, which would suggest a similar effect of the P7 peptide on TLR4-mediated signaling in murine cells. To test the potential interaction of the P7 peptide with murine TRAM vs. human TRAM protein, we performed precipitations of C-terminally biotinylated Pen or P7-Pen peptides. Human or murine TRAM coding constructs were overexpressed in HEK 293T cells, and lysates used for precipitations on Neutravidin agarose beads. We found that P7-Pen peptide could effectively precipitate not only human TRAM, but also murine TRAM protein (data not shown).
[0299] We proceeded with cellular assays to address the effect of peptide pre-treatment on TLR4-mediated cytokine expression in B6 immortalised murine macrophages (
Example 9—Effect of P7 Peptide on Primary Human Monocytes and Whole Blood
[0300] To clarify the inhibitory potential of the P7 peptide on TLR-mediated signaling in primary human cells, we examined the peptide's effect on peripheral blood (PB) monocytes stimulated by TLR4 and TLR8 ligands. As seen before in THP-1 macrophages (
[0301] Next, we proceeded with a pilot whole blood assay to determine whether the P7 peptide could downregulate TLR4-mediated IFNβ secretion induced by LPS or E. coli particles. Due to the detected inhibitory activity of the P7 peptide on TLR8-mediated IFNβ expression by primary human monocytes, a TLR8 ligand was also included in the pilot whole blood assay (
[0302] Therefore, CL075, LPS and Gram-negative and Gram-positive bacterial particles were included in a wider setup for whole blood assays, in which the secretion of IFNβ and a range of pro-inflammatory cytokines was monitored (
[0303] While significantly inhibiting E. coli-mediated IFNβ secretion, P7-Pen did not have so clear an effect on all pro-inflammatory cytokines. Significant inhibition of TNF production was seen, a degree of inhibition of IL-6 (though not to a statistically significant degree) and no significant effect on IL-1β secretion (
[0304] As seen in monocytes and the pilot whole blood assay, P7-Pen inhibited TLR8-mediated IFNβ secretion but had no significant effect on pro-inflammatory cytokine secretion (
Example 10—Effect of Peptides on Signalling Via Other PRRs or the IL-1β Receptor
[0305] P7-Pen was then screened for effects on signaling from several other pattern recognition receptors (PRRs). LPS-stimulation was performed in parallel as a positive control for P7-Pen-mediated inhibition of cytokine expression.
[0306] The effect of P7-Pen on TLR2-mediated pro-inflammatory cytokine expression was investigated. Pre-treatment of primary human macrophages with P7-Pen totally abrogated TLR4-mediated expression of TNF, IL-6 and IL-1β mRNA, but had no effect on expression of pro-inflammatory cytokines induced by a TLR2/6 agonist—the synthetic lipopeptide FSL-1 (Pam.sub.2CGDPKHPKSF, SEQ ID NO: 94) (data not shown).
[0307] As previously shown, P7-Pen inhibits TLR8-mediated IFNβ expression in human monocytes, without significant effect on the expression of pro-inflammatory cytokines. Human cells express one more endosomal RNA sensor in addition to TLR8: TLR7, which uses similar signaling pathways to induce cytokine expression as the TLR8 receptor. To investigate the effect of P7-Pen on TLR7-mediated signaling, human monocytes were stimulated with imiquimod (R837), an imidazoquinoline amine analog to guanosine, which activates only TLR7, not TLR8 (Lee et al., PNAS 100(11): 6646-6651 (2003)). As shown in
[0308] We also tested the effect of P7-Pen on cytokine expression mediated by TLR7 in murine macrophages (using the murine TLR7 agonist resiquimod (R848) to activate TLR7), and found that the peptide had no effect on TLR7-mediated cytokine expression (data not shown). Thus, P7-Pen interferes with signaling initiated by human TLR7 and TLR8, but has no effect on signaling via murine TLR7 (murine TLR8 is believed to be non-functional). As previously shown, P7-Pen efficiently inhibits TLR4-mediated cytokine expression in both human and murine macrophages.
[0309] We then tested whether P7-Pen could alter signaling via another endosomal PRR: TLR9, which recognises specific unmethylated CpG motifs prevalent in microbial DNA. In humans, TLR9 is almost exclusively expressed in B cells and plasmacytoid dendritic cells (pDC), while in mice TLR9 is expressed more widely, including in myeloid immune cells. To test the effect of P7-Pen on signaling via human TLR9 we used a model system of THP-1 cells expressing an inducible TLR9.sup.CHERRY construct. 48 hr after induction of TLR9 expression by doxycycline, undifferentiated THP-1 TLR9.sup.CHERRY cells were pre-treated with control or P7-Pen peptide for 1 hr, then stimulated with the Class B CpG oligonucleotide CpG 2006 ODN (a human-specific TLR9 agonist). P7-Pen did not alter cell viability during stimulation of cells with CpG oligonucleotide (
[0310] TLR2 and 4, and the endosomal TLRs 7-9, use the Myddosome signaling complex to activate transcription of pro-inflammatory cytokines, and endosomal TLRs also activate expression of type I IFNs via the same signaling complex. There are also several IL-1-like receptors including the IL-1 receptor (IL-1R) itself, which is characterized by extracellular immunoglobulin-like domains and intracellular Toll/Interleukin-1R (TIR) domains. These receptors also use the Myddosome complex to initiate downstream signaling. Therefore, we decided to test if signaling downstream of human IL-1R could be altered by the P7-Pen peptide. We used HEK-Blu IL-1R cells (Invivogen) that endogenously express the human IL-1 receptor. HEK-Blue IL-1R cells express a SEAP reporter gene under the control of the IFNβ minimal promoter fused to five NF-κB and five AP-1 binding sites. Binding of IL-1β to its receptor IL-1R on the surface of HEK-Blue IL-1R cells triggers a signaling cascade leading to the activation NF-κB and the subsequent production of SEAP.
[0311] In the assay, HEK-Blue IL-1R cells were pre-treated with control peptide or P7-Pen and stimulated with human recombinant II-1β (hII-1β) for up to 3 hr or for 24 hr. Stimulation of cells by TLR2 and 4 as well as by IL-1β would result in fast posttranslational modifications of IRAK1 in the Myddosome, and in phosphorylation of p38 MAPK. Pre-treatment of HEK-Blue IL-1R cells with P7-Pen had no effect on IL-1β-induced IRAK1 posttranslational modifications or on p-p38 MAPK levels. Moreover, IL-1β-mediated secretion of SEAP in the supernatants of cells pre-treated with the control peptide Pen was comparable with cells pre-treated with the P7-Pen peptide (data not shown). This showed that P7-Pen does not entirely block the Myddosome signaling complex, but has a specific target in this complex that is crucial for the TLR4, and one of the TLR7-9, signaling axes, but is not required for TLR2 and IL-1R-mediated signaling.
[0312] The inhibitory effect of the SLAMF1-derived peptide P7-Pen on signaling via endosomal TLRs could not be explained by its targeting of the TRAM adaptor protein, since TRAM is not involved in signal transduction from endosomal TLRs. Moreover, application of P7-Pen almost completely abrogated pro-inflammatory cytokine expression and secretion in response to LPS, a TLR4 agonist, which could not be fully explained by targeting of TRAM, since the TLR4-TRIF-TRAM signaling complex enhances only the late phase of pro-inflammatory cytokine expression.
Example 11—Bacterial Uptake and TRAM Recruitment to Bacterial Phagosome
[0313] Due to the strong effect of the P7 peptide on TLR4-mediated cytokine expression and secretion, and the important role of TRAM in the regulation of bacteria phagocytosis (Skjesol et al. PLoS Pathogens 15(3): e1007684, 2019), we addressed the effect of the P7 peptide on bacteria uptake in the THP-1 model system and in primary human monocytes, and analysed the effect on TRAM recruitment to endocytosed bacteria particles in a TRAM overexpression system expressing TRAM.sup.CHERRY (
[0314] Cell were pre-incubated with Pen or P7-Pen peptides for 30 min, followed by the addition of Alexa Fluor 488-labeled E. coli or S. aureus bioparticles for the times indicated (
[0315] The striking effect of the P7 peptide on bacterial uptake could explain the blockade of TLR4-mediated IFNβ secretion by this peptide, because activation of IFNβ expression can only be initiated from the endosomal compartment. This effect could also be mediated by peptide interaction with Rab11 FIP2 (data not shown), which is a crucial regulator of TRAM trafficking and the uptake of both Gram-negative and Gram-positive bacteria by macrophages (Skjesol, supra). Interestingly the peptide could also co-precipitate SLAMF1 itself (data not shown). The PHYRE2-predicted SLAMF1 cytoplasmic tail secondary structure demonstrates close intermolecular positioning of C-terminal and N-terminal β-sheets (not shown). P7 is derived from a C-terminal β-sheet, and so could complex with the SLAMF1 N-terminal beta sheet. Given the effect of P7-Pen on bacterial uptake, it could be used to block bacterial uptake by monocytes, thus making bacteria more exposed to antibiotic treatment.
Example 12—Mechanism of Inhibition of TLR4-Mediated Cytokine Secretion by P7
[0316] It is known that regulation of cytokine expression in response to TLR ligands could differ between species, like humans and mice. In murine cells, expression of II-1β is highly dependent on the TLR4-TRAM-TRIF signaling complex (data not shown). Both Ifnβ and II-1β mRNA expression mediated by LPS or E. coli particles were totally abrogated in TIr4.sup.−/− and Tram.sup.−/− cells. However, Tram.sup.−/− cells had intact Tnf expression in response to LPS, with a partial reduction of expression in response to bacterial stimulation (data not shown). Thus, P7-Pen's inhibitory effect on both Tnf and II-1β mRNA expression in murine cells (
[0317] In human cells (THP-1 cell line) TRAM expression was not required for TLR4-mediated IL-1β or TNF expression in response to LPS or E. coli particles (
[0318] To clarify the mechanisms behind the P7-Pen inhibitory effect on the expression of TRAM-independent cytokines, we examined the effect of the peptide on posttranslational modification/phosphorylation of molecules involved in signalling through the Myddosome complex. We proceeded with precipitations of biotinylated peptides and endogenous immunoprecipitations of key signaling components of the Myddosome upon treatment of cells with control or P7 peptide.
[0319] It was found that in murine macrophages the P7 peptide interfered with phosphorylation of the TAK1 kinase and IRAK1 ubiquitination/phosphorylation (modified IRAK1 is not detected with the antibody for murine IRAK1) upstream of TAK1 (
[0320] Further, we tested the effect of P7-Pen on phosphorylation of p38 MAPK, IκBα degradation and modification of IRAK1 in human macrophages (THP-1 cells) and primary human monocytes. P7-Pen inhibited LPS-mediated phosphorylation of p38 MAPK (
[0321] A correlation was also seen between the inhibitory effect of the P7 peptide on pro-inflammatory cytokine expression and phosphorylation/activation of p38 MAP kinase in primary human macrophages (
[0322] To look for interactions between P7 and proteins in the Myddosome complex, biotinylated P7 peptides were pre-incubated with lysates of primary human macrophages (unstimulated and stimulated by LPS or CL075) and NeutrAvidin beads, followed by Western blot analysis of precipitates (
[0323] Like TRAM, Mal comprises a TIR domain. As detailed above, the SLAM F1 C-terminus (and also thus peptide P7) interacts with a region of TRAM at the start of its TIR domain. Mal also comprises a TIR domain, the sequence at the start of which was compared to the corresponding SLAMF1-binding sequence of TRAM. The two protein regions displayed low sequence homology (not shown), but are predicted by PHYRE2 modelling to display structural homology (not shown). MyD88 does not co-precipitate with SLAM F1 (Yurchenko et al., supra), but Mal/TIRAP was not previously tested. Thus, we examined if P7-Pen could precipitate overexpressed Mal protein from HEK293T cells, and this was found to be the case (
[0324] At the same time, pre-incubation of macrophages with P7-Pen resulted in the disruption of Mal recruitment to the MyD88 complex and IRAK1 (
[0325] Pre-treatment of monocytes with P7-Pen abrogated recruitment of all tested IRAKs (1,2 and 4) to MyD88 (
[0326] Indeed, a potential P7-Pen interaction motif is located within the protein kinase domain of IRAK4. Alignment of IRAKs with the TRAM-derived sequence that is targeted by the peptide identified sequences in IRAK1 and IRAK4 that are highly similar to the P7-interacting motif in TRAM/TICAM2 (not shown). In endogenous IPs, SLAMF1 was found to interact with modified IRAK1, but not with IRAK2, upon LPS stimulation (
[0327] When we tested the effect of the P7 peptide on endogenous IPs of IRAKs after ligation of TLR2 or TLR8, we found that the P7 peptide does not inhibit phosphorylation of p38 MAPK, posttranslational modifications of IRAK1 in TLR2-stimulated cells, or TLR2-mediated recruitment of IRAK1 and 4 to MyD88 (
[0328] IRAKs 1 and 4 could be responsible for phosphorylation of Mal after LPS signalling. In an overexpression system this interaction can target Mal for degradation (Dunne et al., Journal of Biological Chemistry 285(24): 18276-18282 (2010)). Although this has not been demonstrated with endogenous proteins, phosphorylation of Mal by IRAK1 and/or 4 could be an important step in Myddosome formation. Inhibition of IRAK 1 and 4 kinase activity results in the stabilisation of Mal protein in HEK293 cells (Dunne et al., supra), and so does ECFP-Pep7 when co-expressed with Mal in HEK293 cells (
[0329] It is possible that P7 interacts with the Mal-IRAK1 and/or IRAK4 complex, and blocks the protein kinase activities of IRAKs 1 and/or 4. This would result in the inhibition of all signalling pathways initiated via TLR4, and some signalling from other TLRs.
Example 13—Effect of P7 on Cancer Cells
[0330] To evaluate the potential impact of the P7 peptide on proliferation of tumour cells of lymphoid origin, we performed a CellTiter-Glo Luminescent Cell Viability Assay to address proliferation of several multiple myeloma (MM) cell lines. For the assay, cells were incubated for 48 h in respective media containing either solvent (water), 10 μM control peptide (Pen) or the SLAMF1-derived peptide P7-A4-Pen for 48 h (
Example 14—Testing of Further Peptides
[0331] A number of additional peptides were synthesised, as set out in Table 5, and their effect on THP-1 cell responses to LPS stimulation tested as previously (
TABLE-US-00005 TABLE 5 SEQ ID Name Sequence NO P3sh3-Pen SITVYASVTLTGRQIKIWFQNRRMKWKK 105 P7-Q2-Pen IQVYASVTLTGRQIKIWFQNRRMKWKK 106 P7-K2-Pen IKVYASVTLTGRQIKIWFQNRRMKWKK 107 P7-H2-Pen IHVYASVTLTGRQIKIWFQNRRMKWKK 108 P7-D2-Pen IDVYASVTLTGRQIKIWFQNRRMKWKK 109 P7-F4-Pen ITVFASVTLTGRQIKIWFQNRRMKWKK 110 P7-N4-Pen ITVNASVTLTGRQIKIWFQNRRMKWKK 111 P7-D4-Pen ITVDASVTLTGRQIKIWFQNRRMKWKK 112 P7-S4-Pen ITVSASVTLTGRQIKIWFQNRRMKWKK 113 P7-G10- ITVYASVTLGGRQIKIWFQNRRMKWKK 114 Pen P7-N10- ITVYASVTLNGRQIKIWFQNRRMKWKK 115 Pen P7-D10- ITVYASVTLDGRQIKIWFQNRRMKWKK 116 Pen P7-R10- ITVYASVTLRGRQIKIWFQNRRMKWKK 117 Pen
[0332] As shown in
[0333] The further peptides, and others described previously, were also tested for their effect on ANBL-6 multiple myeloma cells, in the same manner as described above in Example 13, except the peptides were applied at the higher concentration of 15 μM. The results are shown in
Example 15—Concentration Effect on Anti-Proliferative Activity
[0334] The peptides P7-Pen, C3A-Pen, P6-Pen, P-P6-Pen (which corresponds to P6-Pen in which the tyrosine at position 4 of P6 is modified to phosphorylated tyrosine) P7-A4-Pen, P7-N4-Pen, P7-D4-Pen and P7-K2-Pen were applied to ANBL-6 cells as described above in Example 13, at a series of concentrations (5, 10, 12.5, 15, 20 and 30 μM; or 5, 10 and 20 μM) to determine the impact of peptide concentration on their anti-proliferative activity. As shown in
Example 16—Combination of Anti-Proliferative Peptides and Melphalan
[0335] The effect of the anti-proliferative peptide P7-A4-Pen on multiple myeloma ANBL-6 cells, and the effect of the anti-proliferative peptide P7-N4-Pen on multiple myeloma JJN-3 cells, were tested in combination with the licensed multiple myeloma drug melphalan, and the results compared to those obtained with melphalan alone or the peptide alone. As shown in
Example 17—Effect of Anti-Proliferative Peptides on Other Cancer Cell Lines
[0336] To investigate whether the anti-proliferative peptides of the invention have a broader anti-proliferative effect against other types of cancer cells (beyond multiple myeloma), P7-Pen, P7-A4-Pen and P7-N4-Pen were tested against the acute myeloid leukaemia cell line THP-1 (and compared to the control peptide C3A-Pen), and P7-Pen and P7-N4-Pen were tested against the T cell leukaemia Jurkat cell line and the SW480 colon cancer cell line with the Penetratin peptide as control, using the methods as set out above. The peptides were tested against the SW480 cell line in the presence and absence of LPS.
[0337] As shown in
[0338] While the anti-proliferative peptides had a lesser effect on some cell lines (e.g. SW480) than others, the results nonetheless indicate a potential utility in treatment of these cancers, particularly when used within a combination therapy.
Example 18—Effect of Peptides on Primary Multiple Myeloma Cells
[0339] Primary multiple myeloma cells were isolated from Vk*Myc mice and provided by the Multiple Myeloma Research Group, IKOM, NTNU. The peptides P7-Pen, P7-N4-Pen and P7-A4-Pen were applied to the cultured primary cells and effect of the peptides on cell growth analysed as previously. All three peptides displayed a strong cytotoxic effect on primary multiple myeloma cells (
Example 19—Toxicity of Anti-Proliferative Peptides
[0340] The toxicities of the anti-proliferative peptides P7-Pen and P7-A4-Pen were tested against PBMCs from three healthy donors (PBMCs isolated as described above). P7-Pen and P7-A4-Pen were applied to the PBMCs for 48 hours at a range of concentrations up to 20 μM. No toxicity was seen against the PBMCs from any of the three donors (
Example 20—Caspase Activation by Anti-Proliferative Peptides
[0341] To investigate the mechanism of cytotoxicity mediated by the anti-proliferative peptides of the invention, the effect of the peptide P7-A4-Pen on caspase-3 activation in JJN-3 multiple myeloma cells was investigated. Caspase-3 is a pro-apoptotic caspase which is activated by cleavage by upstream caspases 8, 9 and 10 in response to cell death stimuli. Activated, cleaved caspase-3 cleaves and activates caspases 6 and 7, which induce apoptosis. JJN-3 multiple myeloma cells were incubated with 10 μM P7-A4-Pen or Penetratin control for 24 or 48 hours, lysed and analysed by Western blot to investigate caspase-3 activation (by blotting cleaved caspase-3).
[0342] As shown in