DPP8 and DPP9 peptide inhibitors
09593148 ยท 2017-03-14
Assignee
Inventors
Cpc classification
International classification
C07K9/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to non-competitive allosteric peptide inhibitors of DPP9 and/or DPP8, competitive peptides binding to SUMO1, nucleic acid molecules and expression vectors coding said peptide inhibitors, host cells expressing said inhibitors, kits comprising said inhibitors, as well as methods of producing said inhibitors, and uses of said peptide inhibitors; as further defined in the Claims.
Claims
1. A peptide of 8 to 20 amino acids length, comprising the N-terminal sequence Xaa.sub.1-Leu-Arg-Phe-Leu-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8, wherein Xaa.sub.1 is selected from Ser and Thr, wherein Xaa.sub.6 is selected from Phe, Tyr, Trp, Val and Ile, wherein Xaa .sub.7 is selected from Ala and Glu, and wherein Xaa.sub.8 is selected from Gly and Ala (SEQ ID NO: 48); with the proviso that if Xaa.sub.6 is Phe, then Xaa.sub.7 is Ala or Xaa.sub.1 is Thr; wherein the peptide is a non-competitive allosteric inhibitor of a dipeptidyl peptidase selected from DPP9, DPP8, and a combination of DPP8 and DPP9.
2. The peptide of claim 1, having (i) a K.sub.i for DPP9 of 0.4-10 M; or (ii) a K.sub.i for DPP8 of 3.6 to 12 M; or (iii) a K.sub.i for DPP9 of 0.4-10 M and a K.sub.i for DPP8 of 3.6 to 12 M.
3. The peptide of claim 1, wherein the peptide is capable of inhibiting the activity by wild-type DPP9 (SEQ ID NO: 6) to a level of 35 to 5% of the activity if not inhibited, as determined in an assay using 25 nM DPP9, 0.2 mM GP-AMC, and 14 M of the peptide to be tested, in TB buffer (20 mM Hepes/KOH pH 7.3, 110 mM potassium acetate, 2 mM Mg acetate, 0.5 mM EGTA), supplemented with 0.2% polyoxyethylene (20) sorbitan monolaurate, 1 mM dithiothreitol, 380 nm excitation and 480 nm emission.
4. The peptide of claim 1, which has no inhibitory effect towards DPPIV (SEQ ID NO:8) as determined in an assay using 25 nM DPPIV, 0.2 mM GP-AMC, and 14 M of the peptide to be tested, in TB buffer (20 mM Hepes/KOH pH 7.3, 110 mM potassium acetate, 2 mM Mg acetate, 0.5 mM EGTA), supplemented with 0.2% polyoxyethylene (20) sorbitan monolaurate, 1 mM dithiothreitol, 380 nm excitation and 480 nm emission.
5. The peptide of claim 1, wherein positions 9 to 20 comprise a stretch of one, two or three Ala-residues followed by 5-9 Arg-residues for improving cell penetration of the peptide.
6. The peptide of claim 1, wherein positions 9 to 20 comprise 5-9 Arg-residues for improving cell penetration of the peptide.
7. The peptide of claim 1 having one of the amino acid sequences as shown in SEQ ID NO: 14, 38, 15, 39, 40, 28, 37, 29, 18, 1, 19, 27, or 41.
8. A polypeptide, comprising, fused to the N-terminus, a peptide of 8 to 20 amino acids length, comprising the N-terminal sequence Xaa.sub.1-Leu-Arg-Phe-Leu-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8, wherein Xaa.sub.1 is selected from Ser and Thr, wherein Xaa.sub.6 is selected from Phe, Tyr, Trp, Val and Ile, wherein Xaa.sub.7 is selected from Ala and Glu, and wherein Xaa.sub.8 is selected from Gly and Ala (SEQ ID NO: 48); with the proviso that if Xaa.sub.6 is Phe, then Xaa.sub.7 is Ala or Xaa.sub.1 is Thr; wherein the peptide is a non-competitive allosteric inhibitor of a dipeptidyl peptidase selected from DPP9, DPP8, and a combination of DPP8 and DPP9.
9. A peptide having the sequence Val-Glu-Xaa.sub.3-Ile-His-Val-Xaa.sub.7-Ser-Pro-Xaa.sub.10-Leu-Glu-Xaa.sub.13-Arg-Xaa.sub.15-Xaa.sub.16-Asp-Ser-Xaa.sub.19-Arg, wherein Xaa.sub.3 is any amino acid other than Val or Ile, wherein Xaa.sub.7 is selected from Pro and Thr, wherein Xaa.sub.10 is selected from Ala and Met, wherein Xaa.sub.13 is selected from Glu and Thr, wherein Xaa.sub.15 is selected from Lys and Arg, wherein Xaa.sub.16 is selected from Thr and Ala, and wherein Xaa.sub.19 is selected from Tyr and Phe, as represented by SEQ ID NO: 49, wherein the peptide competitively inhibits binding to the E67-interacting loop (EIL) of SUMO-1.
10. A polypeptide comprising a peptide having the sequence Val-Glu-Xaa.sub.3-Ile-His-Val-Xaa.sub.7-Ser-Pro-Xaa.sub.10-Leu-Glu-Xaa.sub.13-Arg-Xaa.sub.15-Xaa.sub.16-Asp-Ser-Xaa.sub.19-Arg, wherein Xaa.sub.3 is any amino acid other than Val or Ile, wherein Xaa.sub.7 is selected from Pro and Thr, wherein Xaa.sub.10 is selected from Ala and Met, wherein Xaa.sub.13 is selected from Glu and Thr, wherein Xaa.sub.15 is selected from Lys and Arg, wherein Xaa.sub.16 is selected from Thr and Ala, and wherein Xaa.sub.19 is selected from Tyr and Phe, as represented by SEQ ID NO: 49, wherein the peptide competitively inhibits binding to the E67-interacting loop (EIL) of SUMO-1; and wherein the polypeptide is not DPP8 or DPP9.
11. A method for the treatment of cancer, comprising the step of administering to a subject in need thereof a pharmaceutical composition comprising (i) a peptide of 8 to 20 amino acids length, comprising the N-terminal sequence Xaa.sub.1-Leu-Arg-Phe-Leu-Xaa.sub.6-Xaa.sub.7-Xaa.sub.8, wherein Xaa.sub.1 is selected from Ser and Thr, wherein Xaa.sub.6 is selected from Phe, Tyr, Trp, Val and Ile, wherein Xaa.sub.7 is selected from Ala and Glu, and wherein Xaa.sub.8 is selected from Gly and Ala (SEQ ID NO: 48), with the proviso that if Xaa.sub.6 is Phe, then Xaa.sub.7 is Ala or Xaa.sub.1 is Thr, wherein the peptide is a non-competitive allosteric inhibitor of a dipeptidyl peptidase selected from DPP9, DPP8, and a combination of DPP8 and DPP9; or (ii) a polypeptide, comprising, fused to the N-terminus, the peptide according to (i); and a pharmaceutically acceptable carrier, excipient or diluent.
12. The method of claim 11, wherein the cancer is selected from cervical cancer, melanoma, chronic myelogenous leukemia, colorectal adenocarcinoma, neuroblastoma, testicular tumors, breast cancer, ovarian cancer, and/or B cell chronic lymphocytic leukemia.
13. The method of claim 11, wherein the subject is a mammal.
14. The method of claim 11, wherein the subject is a horse, cow, pig, mouse, rat, guinea pig, cat, dog, goat, sheep, or non-human primate.
15. The method of claim 11, wherein the subject is a human.
16. The peptide of claim 1, wherein the amino acid residue at position 1 contains a free amino-terminus.
17. The polypeptide of claim 8, wherein wherein the amino acid residue at position 1 contains a free amino-terminus.
Description
DESCRIPTION OF THE FIGURES
(1)
(2) A. SUMO1 or SUMO2 were immobilized on sepharose beads and incubated with HeLa cell lysates. Interacting proteins were eluted with increasing salt concentrations, separated on SDS PAGE and analysed by mass spectrometry. Shown is a Coomassie staining of fractions eluted from bead-immobilized SUMO1 and SUMO2. B. SUMO-binding assay as in A, ovalbumin-coupled beads were used for control (Ov). Proteins were analysed by immunoblotting using specific antibodies, the input lane corresponds to 0.2% of the input. For detection of Uba2, DPP8, POP and PASP 10% of the eluate were loaded per lane. For DPP9 detection only 1% of the eluate were loaded per lane. C. HeLa cell extracts were incubated with immobilized ovalbumin, SUMO1 or SUMO2 beads. Bead-bound fractions (upper panel) were tested for hydrolysis of GP-AMC for 60 minutes. Fluorescence was measured using 380-nm (excitation) and 480-nm (emission) filters. The remaining unbound fractions (depleted lysates) were tested for release of AMC from GP-AMC (lower panel). These experiments were repeated four times, in triplicates, shown is one representative. D. Shown is a coomassie stained gel of recombinant DPP8 and DPP9 that were expressed and purified from insect Sf9 cells. E. Recombinant DPP8, DPP9 and Uba2 were incubated with immobilized SUMO1/SUMO2 or ovalbumin (Ov) coupled beads. Input (50 ng) and eluted proteins (50% of elutae) were detected by western blotting with DPP8 and Uba2-specific antibodies. For the detection of DPP9, 10% of eluted proteins were loaded. F. Results of an ELISA assay: binding of SUMO1 to immobilized DPP8, DPP9 or ovalbumin. Bound SUMO1 was quantified using SUMO1 specific antibodies. Experiments were repeated three times, in triplicates, error bars are shown.
(3)
(4) A. SUMO1 and SUMO1-SIG mutant proteins (SUMO1K37AV38A and SUMO1V38AK39A) were immobilized on Sepharose beads and incubated with HeLa cell extracts. Ovalbumin (Ov) coupled beads were used for control. Bound proteins were detected by western blotting using specific antibodies. B. Shown is a pull down assay as in A with recombinant DPP9, DPP8, Uba2 or GST-PIAS3. C. Recombinant DPP9 was incubated with 15 mer peptides of a peptide library covering SUMO1 amino acid sequence. The DPP9-peptide solutions were then incubated with SUMO1-immobilized beads. Following extensive washing steps, bound DPP9 was eluted with sample buffer and detected DPP9-specific antibodies. D. Recombinant DPP8 and DPP9 were incubated with SUMO1-immobilized beads. To elute bound proteins, beads were incubated with either SUMO1-peptide 5 (overlapping SIG) or with SUMO1-peptide 9. E. SUMO1 was mutated in amino acids covering the sequence of SUMO1 peptide 9. Recombinant SUMO proteins were immobilized on ELISA slips and incubated with recombinant DPP9. Interacting DPP9 was detected using a DPP9 specific antibody. Experiments were repeated three times in triplicates, shown is a representative, including error bars. F. Recombinant DPP9, Uba2 and GST-PIAS3 were incubated with bead-immobilized SUMO1 or SUMO1 mutants. Interacting proteins were eluted with sample buffer and detected using specific antibodies. G. Shown is a 3D structure of SUMO1 bound to a SIM peptide (Protein Data Bank accession code 2ASQ). The E67-interacting loop (EIL) is located on the opposite side (F66-G68) of the SIG.
(5)
(6) A. Alignment of SUMO1 (SEQ ID NO: 1), SUMO2 and SUMO3 (SEQ ID NO: 2) in the amino-acid sequence corresponding to the EIL in all homologs. B. Recombinant DPP9 or Uba2 (for control) were incubated with bead-immobilized SUMO1, SUMO2 or SUMO2 mutants (S2). Bead-Bound proteins were eluted and detected using specific antibodies.
(7)
(8) A. HA-tagged DPP9 mutants (DPP9V285A, DPP9V287A, DPP9I288A and DPP9V290) were expressed in HEK293T cells and purified using anti-HA beads agarose. Purified DPP9 proteins (input) were then incubated with SUMO1-immobilized beads. Bound DPP9 was eluted and analysed by western blotting using anti HA antibodies. B. Alignment of the amino-acid sequences corresponding to the arms of DPP9 (SEQ ID NO: 3), DPP8 (SEQ ID NO: 4) and DPPIV (SEQ ID NO: 5). C A homology model of DPP9 (published by (Park et al., 2008)), shown from the back side and the front (180 rotation) side, which also reveal the so-called side opening. Color code: the hydrolase domain is dark grey, the eight-bladed propeller is light grey and the extended SUMO-binding-arm is indicated. D. Results of an ELISA assay. Prior to the assay SUMO1 was incubated with varying concentrations of a peptide corresponding to the arm of DPP9 (or a control peptide). The SUMO1-peptide mix was then added to immobilized DPP9, and interacting SUMO1 was quantified using SUMO1-specific antibodies.
(9)
(10) A. DPP9 wild-type and V285 mutants form dimers in cells. HEK293T cells were transfected with either HA or Flag tagged DPP9, or both together. Alternatively, cells were transfected with HA or Flag tagged versions of DPP9V285A (Totals). Lysates were immunoprecipitated using anti-Flag beads, and tested for the presence of both HA and Flag DPP9. B. Elution profiles of 100 microgram recombinant DPP9 wild-type and DPP9V285A mutant on a gel exclusion chromatography, using analytical Superdex S200. C. Coomassie staining of recombinant wild-type DPP9 and DPP9 V285A mutant purified from insect cells. D. Michaelis-Menten analysis for GP-AMC hydrolysis by wild-type DPP9 or DPP9V285A mutant. Experiments were repeated 4 times, shown is an experiment performed in triplicates, including error bars. The hydrolysis of GP-AMC by recombinant DPP9 was measured using 380-nm (excitation) and 480-nm (emission) filters.
(11)
(12) A-F. Michaelis-Menten analysis: The hydrolysis of GP-AMC by recombinant DPP9 was measured using 380-nm (excitation) and 480-nm (emission) filters. The experiments were repeated at least 4 times, shown are examples, each performed in triplicates, including error bars. A. Activity of recombinant DPP9 alone, or in the presence of SUMO1. B. Activity of recombinant DPP9 was measured as in A, in the presence of SUMO2 or SUMO2R61L. Control samples contained DPP9 alone. C. Activity of DPP9 alone or in the presence of SUMO1, SUMO2 or SUMO1 E67A. D. Activity of DPP9 alone or in the presence of SUMO2 or SUMO2D71H. E. Activity of DPP9 V285A mutant in the presence of SUMO1 or SUMO2. F. Activity of DPP9 alone, in the presence of a model protein RanGAP, or in the presence of RanGAP modified with SUMO1 (S1-RG). G. HeLa cells were transfected with siRNA against SUMO1 (SUMO1-i, SUMO1-ii) and harvested after 72 h. Non-targeting siRNA was used for control. Shown is a Western blot of cytosol extracts from the siRNA treated cells (10 g of protein extract per lane) developed with antibodies against SUMO1 and actin as a loading control. H. Lysates from the silenced cells in were tested for release of AMC from GP-AMC. This assay was repeated three times.
(13)
(14) DPP9 (shown a monomer of a dimer) exists in two alternative conformations, which are found in equilibrium and differ in their activity. Protein X interacts weakly with DPP9. Sumoylation of protein X leads to a stronger association with DPP9 and stabilization of the more active form of the enzyme.
(15)
(16) HeLa cytosolic fractions were incubated with 0, 27.7 or 55.6 M peptide EIL, and immediately tested for hydrolysis of varying concentrations of A. GP-AMC or B. R-AMC. Fluorescence was measured using the Appliskan microplate fluorimeter (Thermo Scientific) with 380 nm (excitation) and 480 nm (emission) filters and the Skanit software, and analysed using the Prism software. Each experiment was performed at least three times, in replicates of four.
(17)
(18) A&B 25 nM purified recombinant (A) DPP9 or (B) DPP8 were incubated with varying concentrations of the EIL peptide (0, 3.3 M and 6.6 M) and tested for hydrolysis of GP-AMC (1000, 500, 250, 125, 0 M). C. 25 nM DPPIV was tested for the hydrolysis of GP-AMC (500, 250, 125, 0 M) in the presence of 0 or 13 M EIL peptide. Fluorescence was measured using the Appliskan microplate fluorimeter (Thermo Scientific) with 380 nm (excitation) and 480 nm (emission) filters and the Skanit software, and analysed using the Prism software. Each experiment was performed at least three times, in replicates of four.
(19)
(20) The figure shows an alignment of DPP9 (SEQ ID NO: 6), DPP8 (SEQ ID NO: 7), and DPPIV (SEQ ID NO: 8). The ARM-motif is boxed.
(21)
(22) Cells were incubated for 30 minutes with 0 M, 5 M or 10 M SLRFLYAG (SEQ ID NO: 40) and 100 M carrier pep-1. Cytosolic extracts of cells (5 g) were then tested for hydrolysis of 0.5 mM GP-AMC.
(23)
(24)
EXAMPLES
(25) Materials and Methods
(26) Cell culture and siRNA experiments: HEK293T and HeLa cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. siRNA oligos against SUMO1 were synthesized by Life Technologies: SUMO1-i (5-GGA UAG CAG UGA GAU UCA CUU CAA-3; SEQ ID NO: 9) SUMO1-ii (5-GGA AGA AGA UGU GAU UGA AGU UUA U-3; SEQ ID NO: 10). HeLa cells 30% confluent were transfected with 120 picomols of siRNA using Oligofectamin reagent (Life Technologies) in antibiotics-free medium. 48 h later cells were retransfected in the same conditions, and harvested after a total of 72 hours.
(27) Antibodies: Goat anti-Uba2 were previously described. Rabbit anti PIAS2 antibodies were purchased from Sigma, mouse-GST antibodies were purchased from Santa-Cruz. Mouse anti GMP1 antibodies were obtained from the Developmental Studies Hybridoma Bank. Rabbit anti DPP9 antibodies were purchased from Abcam. For ELISA assay we the inventors produced goat anti DPP9 specific antibodies, by injecting a goat with 500 mg full length DPP9 on days 0, 40 and 70. The goat anti-DPP9 antibodies were purified from the serum by incubation with DPP9-coupled beads followed by acetic acid elution.
(28) Plasmids: SUMO1 and SUMO2 were cloned into pET11. DPP8 and DPP9 were subcloned into a pFASTBacHT plasmid (Invitrogen), baculoviruses were generated according to the instructions of the Bac to Bac Baculovirus expression system, using SF9 cells. DPP9 was cloned into pcDNA3.1 vector for expression of C-terminal HA or Flagged tagged DPP9 (using the BamH1 and Not restriction sites). Single point mutations in SUMO1, SUMO2 or DPP9 were generated using primers for site directed mutagenesis.
(29) Recombinant Protein purification: Purification of RanGAP, SUMO1 and SUMO2 were described previously (Meulmeester et al., 2008; Pichler et al., 2002). For the expression of recombinant DPP8 and DPP9, SF9 insect cells were infected with a high titer DPP8 or DPP9 viral stock for 72 h. Cells were harvested by centrifugation and resuspended in 25 mM Hepes pH7.5, 400 mM NaCl, 5 mM imidazole, 5 mM beta-mercaptoethanol. Cells were then sonificated and centrifuged at 100,000 g for 1 hour at 4 C. The cleared lysate was then incubated with Ni-NTA beads for 2 hours at 4 C. (Qiagen). After extensive washing, proteins were eluted with 25 mM Hepes pH 7.5, 50 mM NaCl, 100 mM imidazole, 5 mM beta-mercaptoethanol. Eluted protein were then separated on a mono Q column (General electric), using a gradient of 0-0.5 M NaCl in 50 mM TRIS pH 8, 2 mM DTT. In a last step, proteins were loaded on a preparative Superdex 200 column (General electric) equilibrated with transport buffer (TB: 20 mM Hepes/KOH pH 7.3, 110 mM potassium acetate, 2 mM Mg acetate, 0.5 mM EGTA, 2 mM DTT).
(30) Peptides: All Peptides: DPP9 arm and the SUMO1-peptide library (>80% purity) were purchased from Genescript.
(31) SUMO pull down screen: SUMO proteins were coupled to CnBr Sepharose (Sigma-Aldrich) at a concentration of 1 mg protein per ml beads. To identify SUMO-interacting proteins, 25 g of frozen HeLa cell pellets were resuspended in 50 ml TB (20 mM Hepes/KOH pH 7.3, 110 mM potassium acetate, 2 mM Mg acetate, 0.5 mM EGTA), supplemented with protease inhibitors (1 g/ml leupeptine, pepstatine, aprotinin and 2 mM DTT) and homogenized by douncing. Cell lysates were centrifuged at 10,000 g for 15 minutes and again at 100,000 g for 1 hour. 10 ml supernatant was incubated at 4 C. with 200 ml SUMO1/SUMO2 or control beads that were inactivated with ethanolamine. After 2 hours of incubation, beads were washed and eluted in TB supplemented with protease inhibitors and increasing NaCl concentrations (0.25, 0.5 or 1 M). Eluted proteins were precipitated with methanol-chloroform, resuspended in LDS sample buffer, separated on a 4-12% NuPAGE gradient gel (Invitrogen), and stained with colloidal Coomassie. In-gel tryptic digestion, desalting, and LC-MSMS analysis were performed as reported previously (Sauer et al. 2005). All pkl files corresponding to one sample were merged into a single mascot generic data file and searched against the human IPI protein database (V3.26) using Mascot with Mascot Server (V2.2). The following settings were used: digestion with trypsin allowing one miss cleavage, carbamylation of cysteine as fixed modification, oxidation of methionine as variable, 150 ppm MS mass accuracy and 0.3 Da for fragmentation masses.
(32) SUMO pull downs with recombinant proteins and competition assays: 500 ng recombinant proteins were incubated at 4 C. with bead immobilized SUMO1, SUMO2 or ovalbumin in TB supplemented with 0.5% Tween-20 and 0.2 mg/ml ovalbumin. Following a 1 hour binding period, beads were washed in TB containing 0.5% Tween-20 and protease inhibitors. Proteins were eluted with sample buffer. For peptide competition assays: recombinant DPP9 (500 ng) was incubated with peptides from the SUMO1 peptide library (0.1 mg/ml) for 1 hour prior to incubation with the SUMO-immobilized beads.
(33) ELISA: A 96 well plate (Immuno 96 MicroWell Solid Plates, Nunc) was coated with 600 ng purified recombinant proteins per well in 100 l TB, overnight at 4 C. Next, wells were blocked with ELISA buffer (TB supplemented with 3% BSA) for 1 h at room temperature. After blocking, recombinant interacting proteins were added to the wells. Following 2 hours of incubation at 25 C., wells were washed four times. Interacting proteins were detected using specific antibodies diluted in ELISA buffer: goat anti DPP9 antibody or anti GMP antibody (SUMO1). After incubation with secondary antibodies, wells were washed with ELISA buffer and ddH20. Reactions were developed with TMB-substrate reagent set from BD (OptEIA substrate). Absorbance was measured at 420 nm on an Appliskan microplate fluorimeter (Thermo Scientific) and the Skanit software. Experiments were performed at least three times, in triplicates.
(34) Immunoprecipitations: HEK293T cells were transfected with pcDNA3 vectors using the calcium phosphate precipitation method. 48 hours later, cells were harvested and dounced in ice cold TB supplemented with 0.2% Tween-20. The homogenate was centrifuged at 4 C., 100,000 g for 20 min. Supernatant were precleared for 30 min with protein G beads, followed by immunoprecipitation for 2 hours at 4 C. with mouse anti-HA or mouse anti-FLAG beads (SIGMA). After extensive washing with TB containing 0.2% Tween-20, bound proteins were eluted with 0.5 mg/ml HA or FLAG peptide in the same buffer.
(35) Size exclusion chromatography: 100 microgram purified DPP9 wild-type or V285A mutant were diluted in 500 microliter TB supplemented with 0.2% Tween-20. Recombinant DPP9 wild-type or mutant were then loaded on an analytical Superdex 200 column (General electric) for size exclusion chromatography. DPP9 concentration in the loop equals 2 M.
(36) Kinetics assays: Purified recombinant DPP9 (25 nM) was incubated with varying concentrations of GP-AMC, in TB supplemented with 0.2% Tween-20, 1 mM ditiothreitol. For inhibition assays, inhibitory peptides were added directly to the reaction mixture. For activation by SUMO 0.2 mg/ml BSA, was added to the buffer to prevent non specific binding. DPP9 were incubated with 2.5 M of either SUMO1, SUMO1 E67A, SUMO2 for 2 hour on ice prior to the assay. Alternatively DPP9 was incubated with 1 M of RanGAP or RanGAP modified with either SUMO1 or SUMO2 for 2 hours prior to the assay. Fluorescence was measured using the Appliskan microplate fluorimeter (Thermo Scientific) with 380 nm (excitation) and 480 nm (emission) filters and the Skanit software, and analysed using the Prism software. Each experiment was performed at least three times, in replicates of four.
(37) Prolyl peptidase activity in SUMO1 silenced cells: HeLa cells were silenced for 72 hours with SUMO1-specific siRNA (SUMO1-1 and SUMO1-3). A non-targeting siRNA was used as control. After 72 h cells were washed in PBS, harvested and lysed in TB supplemented with 1 mM DTT and 400 nM SUMO2-VE (SENP inhibitor). Equal amounts of protein (6 M) were loaded on 15% SDS PAGE and analysed by western blot. 5 M of cell lysates from each silenced sample were tested for hydrolysis of 0.5 mM GP-AMC.
Example 1
Identification of a Novel Interaction Between SUMO1 and the Cytosolic Prolyl Peptidases DPP8 and DPP9
(38) The inventors identified DPP8 and DPP9 in a screen for proteins that interact preferentially with either SUMO1 or SUMO2 (
(39) Next, following the incubation of cell lysates with immobilized SUMO-beads, the SUMO-bound fractions were eluted and assayed for prolyl-peptidase activity by measuring the release of AMC from the model substrate Gly-Pro-AMC (GP-AMC). Prolyl-peptidase activity was recovered specifically in fractions eluted from the SUMO1, but not on SUMO2 or ovalbumin beads (bead-bound fraction
(40) To test whether these interactions are direct, the inventors expressed DPP8 and DPP9 in insect cells (
Example 2
DPP9 Interacts with a Novel Interaction Surface on SUMO1: E67-Interacting Loop (EIL)
(41) To study the preferential association of DPP8 and DPP9 with SUMO1 but not SUMO2, the inventors aimed to identify the surfaces on SUMO1 involved in these interactions. First the inventors analysed the interaction of DPP9 with SUMO1 mutants in residues which were previously shown via NMR and crystal structures to interact with SIM containing proteins (Song et al, 2004; Hecker et al, 2006; Reverter & Lima, 2005). The inventors constructed SUMO1K37AV38A and SUMO1V38AK39A mutants, and analysed the capacity of these SUMO proteins to pull-down endogenous DPP8 and DPP9 from cell lysates. For control, the inventors analysed the binding of known SIM-containing proteins, Uba2 and PIAS2, which did not interact with SUMO1K37AV38A and SUMO1V38AK39A. In a striking contrast, both endogenous and recombinant DPP8 and DPP9 still interacted with these SUMO1-SIG mutations (
(42) Next, single amino acids in SUMO1 between phenylalanine 64 and histidine 75 (corresponding to SUMO1 peptide 9) were mutated to alanines. SUMO1 mutants were immobilized on wells of a 96 well plates and their interaction with DPP9 was analysed in ELISA assays, and compared to their interaction with wild-type SUMO1 (
(43) These results verify that the sequence covering F66-H75, and specifically glutamic acid in position 67 of SUMO1 is important for the DPP9-SUMO1 interaction, but not the SIG (
(44) Taken together these results show for the first time that the EIL functions as a second and independent surface of SUMO for non-covalent interactions with downstream effectors.
Example 3
Gain-of Binding by Point Mutations in SUMO2
(45) To get more insight into the preferential SUMO1 binding of DPP9, the inventors analysed the homology between the SUMO paralogs in the newly identified EIL, and flanking regions. Sequence alignment shows that glutamic acid 67 in SUMO1 is replaced by aspartic acid in SUMO2; since both amino acids are negatively charged, the inventors assumed that this could not explain the strong preference in binding of DPP9 to SUMO1 (
(46) Strikingly, mutation of a single amino acid in SUMO2, aspartic acid 71 to the corresponding histidine residue in SUMO1, resulted in a full gain of binding to DPP9 (
(47) Taken together, the inventors conclude that the preferential interaction of DPP9 with SUMO1 but not with SUMO2, is due to a more negative charge of the SUMO2-EIL.
(48) Previously, crystal structures of the SUMO conjugating enzyme Ubc9 in complex with SUMO showed that non-covalent interaction between Ubc9 and SUMO also includes the surface covering the EIL (knipscheer et al., 2007). However, in contrast to DPP9, Ubc9 does not differentiate between the SUMO homologs.
Example 4
SUMO1 Binds to an Arm-like Structure in DPP9 Which Regulates Enzymatic Activity
(49) In parallel, the inventors aimed to identify the surface in DPP9 that interacts with SUMO1. For this the inventors constructed several DPP9 mutants, concentrating first on amino acids in the propeller domain of DPP9, assuming that it would play a role in protein-protein interactions. Mutants were expressed and purified from HEK293T cells and tested for binding to immobilized SUMO1 beads. Using this approach, the inventors identified a cluster of hydrophobic amino acids in DPP9 that is essential for binding to SUMO1. Replacement of valine 285, isoleucine 288 or valine 290 by alanines resulted in a strong loss of interaction with SUMO1 (
(50) To better understand where the SUMO1-binding surface is localized, the inventors turned to published homology models of DPP9, based on solved structures of other members of the DPPIV family: DPPIV, DPPX and FAP (Park et al, 2008)(Rummey & Metz, 2006). Crystal structures of members of the DPPIV family show that they all form homodimers, where each monomer is built of two domains, a barrel-like / hydrolase and an eight bladed propeller. An extended arm-like structure projects from blade 4 of the propeller, and is located next to a side opening in DPPIV, which is formed between the hydrolase and the propeller.
(51) Both published DPP9 homology models predict that valine 285, isoleucine 288 and valine 290 locate to the extended arm of DPP9 (Park et al, 2008; Rummey & Metz, 2006) (
(52) Taken together, the inventors conclude that the arm of DPP9 mediates its interaction with SUMO1. The arm is found in all the alternative transcripts for DPP8 and DPP9 described so far and is composed of the following minimal sequence (non-conserved residues are underlined):
(53) TABLE-US-00001 DPP9-ARM: (SEQIDNO:12) LYEEVDESEVEVIHVPSPALEERKTDSYRY DPP8-ARM: (SEQIDNO:13) LYEENDESEVEIIHVTSPMLETRRADSFRY
(54) The inventors define this surface as the SUMO-binding-arm (SUBA) of DPP9. The SUBA of DPP9 is part of the propeller domain, and is in close proximity to the side cavity leading to the active site.
(55) In DPPIV the arm-structure is localized to the dimer interface of DPPIV. Recently, Tang et al showed that DPPIV mutants deleted of the arm motif fail to dimerize and are less active. However, they found that the arm of DPP9 was not essential for dimerization, since a DPP9 construct lacking parts of the arm (amino acids corresponding to I288 to Y305) is correctly folded and forms dimers. Moreover, the deletion mutant was less active compared to the wild type protein (Tang et al, 2011). The reason for the reduced activity is currently not understood.
(56) The deletion mutant reported by Tang et al only partially covers the SUBA, since it does not include valine 285, which shows the most dramatic loss in interaction with SUMO1 if replaced by an alanine. Therefore, the inventors tested both the activity of the DPP9V285A mutant, and its ability to form dimers as the wild-type enzyme.
(57) First, the inventors transfected HEK293T cells with FLAG-DPP9, HA-DPP9, or both. The inventors then tested whether HA-tagged DPP9 co-purifies with FLAG tagged DPP9 in co-immunoprecipitation assays. As shown in
(58) Importantly, a single point mutation in the SUBA motif of DPP9, valine 285 to alanine, strongly reduces DPP9 activity, compared to the wild-type peptidase (
(59) Next, the inventors asked whether free SUMO1 has the ability to influence the enzymatic activity of DPP9. The inventors tested this question by measuring GP-AMC hydrolysis by DPP9 in the presence of recombinant SUMO1. For control, activity was compared to samples containing DPP9 alone, or samples in which DPP9 was incubated with equal concentrations of recombinant SUMO2, which does not interact with DPP9. Indeed the inventors found that the hydrolysis rate of GP-AMC by DPP9 was increased in the presence of SUMO1 in comparison to control reactions (
(60) Since SUMO1 is usually found in the cells in a conjugated form, the inventors tested whether a control protein, which otherwise does not interact with DPP9, could stimulate DPP9 activity, if it is modified by SUMO1. For this purpose the inventors used RanGAP as a model substrate. The inventors modified RanGAP, with SUMO1, in vitro, and tested for hydrolysis of GP-AMC by DPP9 in the presence of DPP9 alone, DPP9 with RanGAP, or DPP9 with sumoylated RanGAP. As shown in
(61) Taken together these results show that by binding to the arm of DPP9, SUMO1 but not SUMO2 can regulate DPP9 activity. This activation is dependent on the direct association between SUMO1 and the SUMO1-binding arm in DPP9.
(62) Finally, the inventors investigated whether SUMO1 is important for DPP9 activity in cells. To this end, the inventors transfected HeLa cells with two different siRNA oligos desgined to target SUMO-1, and tested for cleavage of GP-AMC in these lysates.
(63) How SUMO1 activates DPP9 is currently unclear. According to the DPP9 homology model, the SUBA is located next to a side opening, which is formed between the hydrolase and the propeller of DPP9. A crystal structure of DPPIV with a decapeptide suggests that substrates enter the hydrolase domain of DPPIV via this side opening, and not through the propeller funnel. The predicted proximity of the SUBA to the substrate entrance site raises the intriguing possibility that SUMO1 activates DPP9 by regulating the access of substrates into the hydrolase domain, or stabilizing a more active state of the peptidase (
Example 5
The SUMO1 EIL Peptide (SEQ ID NO: 1) Inhibits Post Prolyl Peptidase Activity in Cells
(64) Since the EIL peptide can displace SUMO1 from a DPP9-SUMO1 or a DPP8-SUMO1 complex (
Example 6
The SUMO1 EIL Peptide (SEQ ID NO: 1) Acts as a Selective, Non-competitive, Allosteric Inhibitor of DPP8 and DPP9
(65) The inventors found that pure recombinant DPP8 and DPP9 are inhibited by the EIL peptide, also in the absence of SUMO1 (
Example 7
Improved Inhibition: Variants of EIL Peptide
(66) To improve the inhibition of DPP9, the inventors tested several peptide libraries composed of truncated variants of the EIL peptide (>80% pure, ordered from Genescript). These peptides were incubated with recombinant DPP9 and analysed GP-AMC hydrolysis. AMC release was then compared to samples containing either DPP9 alone, or the EIL peptide. The results of this screen are summarized in the table below.
(67) TABLE-US-00002 TABLE1 InhibitionofDPP9byvariantsoftheEIL. PurifiedrecombinantDPP9(25nM)wasincubated with0.2mMGP-AMC,and14MoftheEILvariant peptides.Fluorescencewasmeasuredusingthe Appliskanmicroplatefluorimeter(Thermo Scientific)with380nm(excitation)and480nm (emission)filtersandtheSkanitsoftware. %DPP9 Inhibitorypeptide activity Std. (aasequence) (Mean) Deviation 1. Nopeptide 100 4.013 2. SLRFLFEGQRIADNH 27.45 1.92 (EILpeptide; SEQIDNO:1) 3. SLRFLFAGQRIADNH 5.093 0.6078 (SEQIDNO:14) 4. SLRFLYEGQRIADNH 13.06 0.5645 (SEQIDNO:15) 5. SLRFLFEGQRIADNR 56.38 2.135 (SEQIDNO:16) 6. SLRFLFDGQRIADNH 74.9 2.172 (SEQIDNO:17) 7. SLRFLWEGQRIADNH 26.93 1.991 (SEQIDNO:18) 8. SLRFLVEGQRIADNH 28.61 1.223 (SEQIDNO:19) 9. SLRFLAEGQRIADNH 72.87 4.704 (SEQIDNO:21) 10. SLRFLFEAQRIADNH 19.46 2.709 (SEQIDNO:22) 11. LRFLFEGQRIADNH 79.82 2.022 (SEQIDNO:23) 12. RFLFEGQRIADNH 42.7 2.16 (SEQIDNO:24) 13. LFEGQRIADNH 67.77 3.627 (SEQIDNO:25) 14. FEGQRIADNH 97.12 1.522 (SEQIDNO:26) 15. SLRFLFEGQRIAD 29.07 0.9588 (SEQIDNO:27) 16. SLRFLFEGQRI 18.36 0.8809 (SEQIDNO:28) 17. SLRFLFEGQR 24.27 0.5411 (SEQIDNO:29) 18. SLRFLFEGQ 16.43 0.5832 (SEQIDNO:30) 19. LRFLFEGQRIADN 66.42 5.493 (SEQIDNO:31) 20. RFLFEGQRIAD 38.41 1.362 (SEQIDNO:32) 21. FLFEGQRIA 55.93 5.38 (SEQIDNO:33) 22. FLFEGQRI 48.41 1.53 (SEQIDNO:34) 23. LFEGQR 51 6.869 (SEQIDNO:35) 24. FLIEGQRI 49.04 2.345 (SEQIDNO:36)
(68) The inventors selected three peptides form the library for a more detailed analysis to measure the Ki values of these peptides compared to the EIL peptide, as shown in Table 2 below.
(69) TABLE-US-00003 TABLE2 InhibitionofDPP9byvariantsoftheEIL. PurifiedrecombinantDPP9(25nM)wasincubated with0,5,or20Mpeptideandtestedforthe hydrolysisofGP-AMC(0,31,62.5,125,250and 500M).Fluorescencewasmeasuredusingthe Appliskanmicroplatefluorimeter(ThermoScientific) with380nm(excitation)and480nm(emission filtersandtheSkanitsoftware. Inhibitory Ki(M)for peptidesequence inhibitionofDPP9 1 SLRFLFEGQRIADNH 7.316 (EIL;SEQIDNO:1) 2 SLRFLYEGQRIADNH 2.557 (SEQIDNO:15) 3 SLRFLFAGQRIADNH 1.43 (SEQIDNO:14) 4 SLRFLFEG 2 (SEQIDNO:37)
(70) The analysis of the first library shows that (1) the serine residue is important for inhibition; (2) the minimal sequence includes SLRFLFEG (SEQ ID NO: 37); (3) position 6 shall contain a hydrophobic amino acid: Tyr, Phe, Val, Trp, (Ile), replacement by an Ala losses the inhibitory effect (cf. peptide #9 in Table 1; SEQ ID NO: 21); (4) position 7 may be Glu or an Ala, where Ala is more efficient; and (5) position 8 may be Gly or Ala.
(71) In a following library the inventors tested several variations of the SLRFLFEG (SEQ ID NO: 37) sequence, summarized in Table 3.
(72) TABLE-US-00004 TABLE3 InhibitionofDPP9byvariantsoftheSLRFLFEG (SEQIDNO:37). PurifiedrecombinantDPP9(25nM)wasincubated with0.2mMGP-AMC,and10MoftheSLRFLFEG (SEQIDNO:37)variantpeptides.Fluorescencewas measuredusingtheAppliskanmicroplatefluorimeter (ThermoScientific)with380nm(excitation)and480 nm(emission)filtersandtheSkanitsoftware. %DPP9 Inhibitorypeptide activity Std. (aasequence) (Mean) Deviation 1. DPP9 100.3 8.857 2. SLRFLFEG(SEQIDNO:37) 24.08 2.275 3. SLRFLYEG(SEQIDNO:38) 10.92 1.564 4. SLRFLFAG(SEQIDNO:39) 15.33 1.017 5. SLRFLYAG(SEQIDNO:40) 16.27 1.535 6. TLRFLFEG(SEQIDNO:41) 29.2 2.598 7. Acet-SLRFLFEG(SEQIDNO:42) 90.74 3.223 8. Acet-SLRFLYEG(SEQIDNO:43) 89.09 1.433 9. AAASLRFLYEG(SEQIDNO:44) 91.15 4.535 10. SLRFLYEGAAA(SEQIDNO:45) 23.86 0.1565 11. RRRRSLRFLFEG(SEQIDNO:46) 92.2 1.984 12. RRRRSLRFLYAG(SEQIDNO:47) 91.47 2.428
(73) The analysis of the second library shows that (1) strongest inhibition was observed for SLRFLYEG (SEQ ID NO: 38); (2) position 1 must contain a free NH3 group (no acetylation), and can be Ser or Thr; (3) position 6 shall contain a hydrophobic amino acid: Tyr, Phe, Val, Trp (Ile); (4) a stretch of Ala starting from position 9 Is tolerated (and may be used as a linker to introduce R residues for enhanced cell penetration).
(74) Peptide SLRFLYEG (SEQ ID NO: 38) was analysed further and was found to have the lowest k.sub.i and highest selectivity towards DPP9 over DPP8: The K.sub.i for DPP9 is 0.4 M, (18.5 fold increased efficiency compared to the EIL peptide), whereas the K.sub.i for DPP8 is 3.6 M (the affinity is 9 fold lower compared to DPP9). It has no inhibitory effect towards DPPIV, in the concentrations measured (up to 50 M).
Example 8
Cytosolic Targeting of Inhibitory Peptides
(75) The inventors tested whether the inhibitory peptides can be inserted into cells. For this purpose the inventors established a method using a well characterized carrier peptide: Peptide 1 (KETWWETWWTEWSQPKKKRKV; SEQ ID NO: 48), in combination with SLRFLYAG (SEQ ID NO: 40). To test for effective inhibition, the inventors measured for hydrolysis of an artificial DPP8 and DPP9 substrate: GP-AMC, specifically in cytosolic extracts. For this, 5 M or 10 M of SLRFLYAG (SEQ ID NO: 40) were incubated with 100 M Peptide 1, at 37 C. for 30 minutes, in 250 ml DMEM without FCS, supplemented with glutamine and antibiotics. Once a complex was formed between the SLRFLYAG (SEQ ID NO: 40) and peptide 1, it was added to HEK293 cells (human embryonic kidney cells), grown in 24 well plates (50% confluence), and incubated for 30 minutes at 37 C. Cells were then trypsinized and washed twice with PBS buffer. Cell pellet was shock frozen in liquid nitrogen. For preparation of cytosolic extracts: cell pellet were resuspended in TB buffer, dounced and centrifuged at 14,000 g. Cytosolic extracts (5 g) were then tested for the hydrolysis of 500 M GP-AMC. Fluorescence was measured using the Appliskan microplate fluorimeter (Thermo Scientific) with 380 nm (excitation) and 480 nm (emission) filters and the Skanit software, and analysed using the Prism software.
(76) Importantly, using this setup, the inventors show that the inhibitory peptide SLRFLYAG (SEQ ID NO: 40) can successfully enter the cell and inhibit the cytosolic prolyl peptidase activity by ca 30% using 5 M SLRFLYAG (SEQ ID NO: 40) peptide (
Example 9
SLRFLYEG (SEQ ID NO: 38) is Cell Permeable and Inhibits Cytosolic Prolyl Peptidase Activity
(77) To test whether the SLRFLYEG peptide (SEQ ID NO: 38) enters the cells, it was labeled with FITC. 5 M SLRFLYEG labelled with FITC was allowed to form a complex with the cell-penetrating peptide (Pep-1) in DMEM FCS-free at a molar ratio of 1:10 (cargo:carrier). As a control, a mixture with Peptide-1 alone was used. Complexes were formed for 30 minutes at 37 C. The mixtures were then overlaid onto HeLa cells grown on glass coverslips at a confluence of 60-70%, allowing the complexes to enter the cells for 30 minutes at 37 C. For observation, cells were fixed for 10 minutes with 4% formaldehyde, nuclei were stained with 10 g/ml Hoechst 33258 (Molecular Probes) and cells were mounted with Fluorescent mounting medium (DAKO). For analysis, cells were imaged using a LSM 510-Meta confocal microscope, oil immersion objective 63/1.3 (Carl Zeiss MicroImaging, Inc.). The obtained images were processed using the LSM image Browser (Carl Zeiss MicroImaging, Inc.) and Adobe Photoshop.
(78) The resulting images (not shown) demonstrated that HeLa cells treated with Pep-1 in complex with FITC-SLRFLYEG show cellular localization of the FITC-labelled inhibitor, together with Hoechst 33258 nuclear staining. Thus, it could be clearly shown that the peptide enters the cells.
(79) In another experiment, HeLa cells were treated for 30 minutes with either 5 M SLRFLYEG (SEQ ID NO: 38) alone or in complex with the carier peptide Pep-1. Cells were then typsinized and cytosolic extracts were prepared and tested for cleavage of the model substrate GP-AMC. The results are shown in
(80) The results in
Example 10
Cells Exposed to SLRFLYEG (SEQ ID NO: 38) Show Reduced Proliferation
(81) To test whether DPP8/9 inhibition effects cell viability, the inventors performed MTT (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) based assays, which are widely used to measure cell proliferation and growth (e.g. in clinical settings for testing tumor sensitivity to chemotherapeutic agents). This assay involves the conversion of the water soluble MTT to an insoluble formazan. The formazan is then solubilized in 0.1N HCl. Absorbance is then determined at 570 nm. For a prolonged exposure of cells to the allosteric DPP8/9 inhibitor SLRFLYEG (SEQ ID NO: 38), the inventors cloned the peptide into a mammalian expression vector pEYFP-N1 from Clontech. The peptide inhibitor contains a serine at its amino-termini. Thus, to avoid expression of the peptide with a methionine residue at its amino terminus, it was cloned in frame with ubiquitin. Once expressed in cells, the ubiquitin moiety is cleaved by ubiquitin isopeptidases to release the SLRFLYEG-YFP (SEQ ID NO: 38) construct. A control construct was included that lacked the SLRFLYEG (SEQ ID NO: 38) peptide (Ubiquitin-YFP). HeLa cells were plated in wells of a 96 well plate and transfected with Fugene HD transfection reagent (Promega) following the manufacturer protocols with the following constructs for 32 hours: Ubiquitin (YFP_UBI), Ubiquitin-SLRFLYEG (YFP_UBI_YEG) or mock treated. 32 hours after transfection, cells were analyzed via a standard MTT assay.
(82) Briefly, cell media was removed and replaced by 0.5 mg/ml of MTT reagent. Following 3 hours of incubation at 37 C., the MIT reagent was removed and replaced by 0.1 N HCl. Absorbance was measured with a microplate fluorimeter. Each experiment was performed in triplicates.
(83) Using this setup the inventors observe a drastic reduction in cell proliferation of at least 50% in Hela cells exposed to the allosteric DPP8/9 inhibitor SLRFLYEG (SEQ ID NO: 38) compared to the control cells transfected with the control plasmid or mock transfected. The results suggest that inhibition of DPP8 and/or DPP9 using the disclosed inhibitors can be used to control cell viability. The HeLa cell line used here is derived from cervical cancer for use in cancer research. Accordingly, it is very likely that the disclosed inhibitors have therapeutic potential for use in oncology.
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