MACROMOLECULAR CONJUGATES FOR ISOLATION, IMMOBILIZATION AND VISUALIZATION OF PROTEINS

Abstract

Synthetic macromolecular conjugate for selective interaction with proteins has a synthetic copolymer, and at least one binding group and at least one further group selected from an affinity tag and an imaging probe, and at least one binding group and at least one further group being bound via covalent bond to the synthetic copolymer. The macromolecular conjugate is suitable in particular for identification, visualization, quantification or isolation of proteins and/or cells.

Claims

1: Synthetic macromolecular conjugate for selective interaction with proteins, characterized in that it comprises a synthetic copolymer, and at least one binding group and at least one further group selected from an affinity tag and an imaging probe, said at least one binding group and at least one further group being bound via covalent bond to said synthetic copolymer.

2: The synthetic macromolecular conjugate according to claim 1, characterized in that the synthetic copolymer is a copolymer obtainable by copolymerization of at least one monomer of Formula 1: ##STR00006## wherein: R.sup.1 is selected from H, CH.sub.3; and R.sup.2 is selected from NH.sub.2, NH—CH.sub.2—CH(OH)—CH.sub.3, NH—CH.sub.3, NH—CH.sub.2CH.sub.3, NH—CH.sub.2CH.sub.2—OH, NH—CH.sub.2CH.sub.2CH.sub.2—OH, NHC(CH.sub.2OH).sub.3, NH—CH.sub.2CH.sub.2—N.sup.+(CH.sub.3).sub.3Cl.sup.−, O—CH.sub.2CH.sub.2—OH, O—(CH.sub.2CH.sub.2O).sub.2—H O—(CH.sub.2CH.sub.2O).sub.3—H, O—CH.sub.2CH.sub.2—N.sup.+(CH.sub.3).sub.3Cl.sup.−, NH—(CH.sub.2).sub.3N.sup.+(CH.sub.3).sub.2—(CH.sub.2).sub.2—COO.sup.−; and at least one monomer of Formula 2: ##STR00007## wherein: R.sup.1 is selected from H, CH.sub.3; and X is selected from NH—(CH.sub.2).sub.2—CO, NH—(CH.sub.2).sub.3—CO, NH—(CH.sub.2).sub.4—CO, NH—(CH.sub.2).sub.5—CO, Gly, GlyGly, GlyPheLeuGly; and R.sup.3 is selected from: ##STR00008## whereas in the copolymer at least one reactive group R.sup.3 is replaced by the binding group, at least one reactive group R.sup.3 is replaced by the affinity tag and/or at least one reactive group R.sup.3 is replaced by the imaging probe.

3: The macromolecular conjugate according to claim 1, characterized in that the molecular weight of the conjugate is in the range of 1000 to 500000 g/mol, preferably in the range from 20000 to 150000 g/mol.

4: The macromolecular conjugate according to claim 1, characterized in that the binding group is selected from moieties binding His-tag and glutathione, preferably the binding group is selected from glutathione and complexes of ions and chelating agents including iminodiacetic acid, nitrilotriacetic acid, carboxylmethylaspartate and tris(nitriloacetic) acid.

5: The macromolecular conjugate according to claim 1, characterized in that the binding group is bound to the synthetic copolymer via a flexible linker, preferably via a linker based on polyethylene glycol, peptide, preferably a peptide having a molecular weight in the range of 100 to 5000 g/mol, or nucleic acid, preferably a nucleic acid containing 1 to 40 nucleotides, or oligosaccharide, preferably an oligosaccharide containing 1 to 40 monosaccharides.

6: The macromolecular conjugate according to claim 1, characterized in that the affinity tag is present and it is selected from the group comprising biotin, FLAG tag, His-tag, HA tag, Strep-tag, Avi-tag, GST, c-myc-tag, V5-tag, E-tag, S-tag, SBP-tag, poly(Glu)-tag, calmodulin tag.

7: The macromolecular conjugate according to claim 1, characterized in that the imaging probe is present and is selected from the group comprising fluorescent moieties, radionuclides and metal complexes.

8: The macromolecular conjugate according to claim 7, characterized in that the imaging probe is selected from the group comprising fluorophores with an excitation maximum in the range of 350 to 850 nm, preferably ATTO488 or DY676; lanthanide complexes, preferably of Gd, Mn, Dy, Eu; radionuclide complexes .sup.64Cu, .sup.68Ga, .sup.18F, .sup.99mTc, .sup.123I, .sup.125I, .sup.131I, .sup.57Co, .sup.51Cr, .sup.67Ga, .sup.64Cu, .sup.111In, .sup.90Y.

9: Use of the macromolecular conjugate according to claim 1 for identification, visualization, quantification or isolation of proteins and/or cells in vitro.

10: Use of the macromolecular conjugate according to claim 1 in an immunochemical method, preferably selected from ELISA, Wester blotting and modifications thereof, flow cytometry, immunoprecipitation, immunocytochemistry.

11: Use of the macromolecular conjugate according to claim 1 for screening of inhibitors, ligands, and compounds and substrates capable of binding to the target protein.

12: Use of the macromolecular conjugate according to claim 4 for identification, visualization, quantification or isolation of proteins containing the affinity tag His-tag or GST-tag.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 shows a schematic structure of the polymeric conjugates.

[0039] FIG. 2 shows the structure of Compound A intended for targeting of the protein His-tag.

[0040] FIG. 3 shows the structure of Conjugate 1 intended for targeting of proteins with a His-tag.

[0041] FIG. 4 shows the structure of Conjugate 2 intended for targeting proteins with a His-tag.

[0042] FIG. 5 shows the structure of Conjugate 3 without a binding group (negative control).

[0043] FIG. 6 shows the structure of Conjugate 4 without a binding group (negative control).

[0044] FIG. 7 shows a Western blot with recombinant purified M1 protein from influenza virus labeled with His-tag (M1-HisTag), which was visualized using a commercial antibody against His-tag and Conjugate 1. Lane 1: M1-HisTag (10 ng); 2: M1-HisTag (5 ng); 3: M1-HisTag (1 ng); 4: M1-HisTag (0.5 ng); 5: All blue marker (2 μl); 6: M1-HisTag (0.1 ng); 7: M1-HisTag (0.5 ng); 8: M1-HisTag (1 ng); 9: M1-HisTag (5 ng); 10: M1-HisTag (10 ng).

[0045] FIG. 8 shows a Western blot with bacterial lysate containing an E. coli poly(A)-polymerase labeled with HisTag (PAP-HisTag) which was visualized both with commercial antibody against His-tag, and with Conjugate 1 and Conjugate 2. Lane 1: bacterial lysate (10 μl); 2: bacterial lysate (5 μl); 3: bacterial lysate (1 μl); 4: All blue marker (2 μl); 5: bacterial lysate (10 μl); 6: bacterial lysate (5 μl); 7: bacterial lysate (1 μl); 8: All blue marker (2 μl); 9: bacterial lysate (10 μl); 10: bacterial lysate (5 μl); 11: bacterial lysate (1 μl).

[0046] FIG. 9A shows affinity isolation (“pull-down”) of a protein containing His-tag (NEDD8-HisTag) using Conjugate 1 under native conditions. Proteins were separated by SDS-PAGE electrophoresis and the gel was stained with silver. Lane 1: NEDD8-His Tag (500 ng); 2: Conjugate 1 (25 μg); 3: All Blue Marker (2 μl); 4: Load; 5: FT: Conjugate 1; 6: FT: Conjugate 3; 7: FT: Conjugate 4; 8: FT: control without polymer; 9: Elution: Conjugate 1; 10: Elution: Conjugate 3; 11: Elution: Conjugate 4; 12: Elution: control without polymer. All lanes of each fraction were loaded with 5 μl sample.

[0047] FIG. 9B shows affinity isolation (“pull-down”) of a protein containing His-tag (NEDD8-HisTag) using Conjugate 1 under denaturing conditions. Proteins were separated by SDS-PAGE electrophoresis and the gel was stained with silver. Lane 1: NEDD8-His Tag (500 ng); 2: Load; 3: FT: Conjugate 3; 4: FT: Conjugate 4; 5: FT: Conjugate 1; 6: FT: control without polymer; 7: Elution: Conjugate 3; 8: Elution: Conjugate 4; 9: Elution: Conjugate 1; 10: Elution: control without polymer. All lanes of each fraction were loaded with 5 μl sample.

[0048] FIG. 10 shows affinity isolation (“pull-down”) of a protein containing His-tagg (Ddi1-HisTag) from bacterial lysate using Conjugate 1. Proteins were separated by SDS-PAGE electrophoresis and the gel was stained with silver. Track 1: All blue marker (2 μl); 2: free lane; 3: Load; 4: free lane; 5: The elution: Conjugate 1; 6: Elution: Conjugate 3; 7: Elution: control without polymer. All lanes of each fraction were loaded with 5 μl sample.

[0049] FIG. 11 shows the structure of Compound B designed for the targeting of protein GST-tag.

[0050] FIG. 12 shows the structure of Conjugate 5 designed for the targeting of proteins with GST-tag.

[0051] FIG. 13 shows the structure of Conjugate 6 designed for the targeting of proteins with GST-tag.

EXAMPLES OF CARRYING OUT THE INVENTION

[0052] All chemicals used were from Sigma-Aldrich unless stated otherwise. All compounds tested in biological assays were purified using Waters Delta 600 preparative HPLC system (flow rate 7 ml/min; gradient shown for each compound, including retention times), with Waters SunFire C18 OBD Prep Column, 5 μm, 19×150 mm. Purity of compounds was checked on an analytical Jasco PU-1580 HPLC system (flow rate 1 ml/min with a constant gradient of 2-100% acetonitrile in 30 minutes; retention time is shown for each compound) with Watrex C18 Analytical Column, 5 μm, 250×5 mm. Final compounds were at least of 99% purity and their structure was further confirmed using HR-MS on LTQ Orbitrap XL (Thermo Fisher Scientific) and NMR (Bruker Avance I™ 500 MHz equipped with a cryo-probe). All interaction constants are given in Hz.

Example 1: Preparation of Compound A

[0053] Compound A was prepared according to the following scheme:

##STR00004##

(1) Compound A.SUB.0

[0054] Compound A.sub.0, NH.sub.2-triNTA(o-tBu).sub.9: Synthesis of Compound A.sub.0 was performed according to published procedure [7], with a small deviation in one step: in a reaction between a derivative of a tricarboxylic acid (derived from lysine), and three monomers of nitrilotriacetic acid, N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (TSTU) was used as the activating agent instead of NHS/EDC, as TSTU provided significantly higher yields.

(2) Compound A

[0055] Compound A, NH.sub.2—PEG.sub.5-triNTA: Compound A.sub.0 (52 mg, 38 μmol, 1.0 eq, purified using HPLC prior to this step) was dissolved in 1 ml of DMF to which 15 mg (38 μmol, 1.0 eq) of Boc-O2Oc-O2Oc-OH linker (Iris Biotech, #BAA1485) was subsequently added in one step. 16 mg (76 μmol, 2.0 eq) of DCC was further added to the reaction mixture and the reaction was left to react for 24 hours at room temperature. The solvent was evaporated and the raw mixture was mixed with 1 ml of pure TFA; the reaction mixture was alternately stirred and sonicated in a water bath for 3 hours. The TFA was removed with nitrogen gas and the final product was purified by preparative HPLC (gradient 2-30% ACN in 50 min, R.sub.T=35 min). The weight of the obtained pure product was 4 mg (yield=32%).

[0056] Analytical HPLC (gradient 2-100%, 30 min) RT=12.0 min. HR-MS (ESI+): counted for C.sub.43H.sub.71O.sub.27N.sub.10 [M].sup.+ 1159.44846. Found 1159.44849.

Example 2: Preparation of Conjugate 1

(1) Preparation of the Polymeric Precursor Poly(HPMA-Co-Ma-β-Ala-TT)

[0057] Monomeric compounds N-(2-hydroxypropyl) methacrylamide (HPMA) and 3-(3-methacrylamido propanoyl) thiazolidine-2-thione (Ma-β-Ala-TT) were prepared according to a published procedure [1, 5]. The polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) was prepared using RAFT-copolymerization (reversible addition-fragmentation chain-transfer). 1.0 g of HPMA (85% mol) was dissolved in 7.3 ml of tert-butanol; 318 mg of Ma-β-Ala-TT (15% mol) dissolved in 1.9 ml of DMSO, 2.42 mg of 2-cyano-2-propylbenzodithioate and 0.90 mg of 2,2′-azobis (2-methylpropionitrile) was added to the solution and the solution was transferred into a polymerization vial. The mixture was purged with argon for 10 min and then the vial was sealed. The polymerization reaction was performed at 70° C. (16 h). The polymeric precursor was isolated by precipitation into acetone:diethyl ether mixture (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum. Dithiobenzoate end groups were removed according to a previously published procedure [8].

[0058] This procedure resulted in the polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) with molecular weight of M.sub.w=81600 g/mol, polydispersity D=1.18 and containing 14.6 mol % of reactive thiazolidin-2-thione (TT) groups.

(2) Preparation of Conjugate 1

[0059] The polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) (0.040 g, M.sub.w=81600 g/mol, 14.6 mol % TT), Compound A (6.0 mg) and N-(2-aminoethyl) biotinamido hydrobromide (biotin-NH.sub.2) (5 mg) was dissolved in 0.2 ml of DMSO. ATTO488-NH.sub.2 (2.5 mg) was dissolved in 0.1 ml of DMSO and added to a solution of the polymeric precursor. N,N-diisopropylethylamine (DIPEA) (8.0 μl) was added and the reaction mixture was stirred for 4 hours at room temperature. Subsequently, 1-amino-propan-2-ol (5 μl) was added to the solution and the reaction mixture was stirred for 10 min. Then, the polymeric conjugate 1 poly(HPMA-co-Ma-β-Ala-CompoundA-co-Ma-β-Ala-ATTO488-co-Ma-β-Ala-NH-biotin) was isolated by precipitation in acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum. Polymeric conjugate was purified from low-molecular impurities by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum. The yield of Conjugate 1 was 22 mg. Content of ATTO488 4.17% was determined spectrophotometrically (ε.sub.502nm=90000 l.Math.mol.sup.−1.Math.cm.sup.−1, distilled water) and the inhibitor content 11.28% was determined in the sample hydrolysate (6N—HCl, 115° C., 16 hr) by HPLC with fluorescence detector (Ex. 229 nm, Em. 450 nm), column: Chromolith C18, precolumn derivatisation method with o-phthaldialdehyde.

(3) Preparation of Conjugate 3 (Comparative Conjugate Serving as a Negative Control)

[0060] The polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) for the preparation of Conjugate 3 was prepared by RAFT-copolymerization as described in Example 2 (see above), using the following composition of the polymerization mixture: 500 mg of HPMA (85 mol %), 159 mg of Ma-β-Ala-TT (15 mol %) dissolved in 0.8 ml of DMSO, 1.21 mg of 2-cyano-2-propylbenzodithioate and 0.45 mg 2,2′-azobis(2-methylpropionitrile) were dissolved in 3.8 ml of tert-butanol. This procedure resulted in the polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) with a molecular weight M.sub.w=85900 g/mol and a polydispersity of D=1.22 and containing 13.4 mol % of the reactive thiazolidine-2-thione groups.

[0061] The polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) (0.045 g, M.sub.w=85900 g/mol, 13.4 mol % TT) and 5 mg of biotin-NH.sub.2 was dissolved in 0.2 ml of DMSO. ATTO488-NH.sub.2 (2.5 mg) was dissolved in 0.1 ml of DMSO and added to the solution of polymeric precursor. N,N-diisopropylethylamine (DIPEA) (2.5 μl) was added and the reaction mixture was stirred for 4 hours at room temperature, then 1-amino-propan-2-ol (5 μl) was added to the solution and the reaction mixture was stirred for 10 min. Then, the polymeric conjugate 3 poly(HPMA-co-Ma-β-Ala-ATTO488-co-Ma-β-Ala-NH-biotin) was isolated by precipitation into acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum. Polymeric conjugate was purified by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum. The yield of Conjugate was 3 was 32 mg, content of ATTO488 was 5.1%, and the content of biotin 10.8%.

Example 3: Preparation of Conjugate 2

(1) Preparation of Conjugate 2

[0062] The polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) (0.030 mg, M.sub.w=81600 g/mol, 14.6 mol % TT; see Preparation of Conjugate 1), Compound A (3.5 mg) and N-(2-aminoethyl)biotinamido hydrobromide (biotin-NH.sub.2) (4 mg) were dissolved in 0.3 ml of DMSO. N,N-diisopropylethylamine (DIPEA) (4.0 μl) was added and the reaction mixture was stirred for 4 hours at room temperature; then, 1-amino-propan-2-ol (2 μl) was added to the solution and the reaction mixture was stirred for 10 min. Then, the polymeric conjugate 2 poly(HPMA-co-Ma-β-Ala-CompoundA-co-Ma-β-Ala-NH-biotin) was isolated by precipitation into acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum. Polymeric conjugate was purified from low-molecular impurities by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum. The yield of Conjugate 2 was 21 mg. Biotin content 5.53% was determined using the HABA/Avidin kit (Sigma) and the inhibitor content of 10.43% was determined in the sample hydrolysate (6N—HCl, 115° C., 16 hours) by HPLC with fluorescence detector (Ex. 229 nm, Em. 450 nm), column: Chromolith C18, precolumn derivatisation method with o-phthaldialdehyde.

(2) Preparation of Conjugate 4 (Comparative Conjugate Serving as a Negative Control)

[0063] The polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) for the preparation of Conjugate 4 was prepared by RAFT-copolymerization as described in Example 2 (see above), using the following composition of the polymerization mixture: 500 mg of HPMA (90% mol), 100 mg of Ma-β-Ala-TT (10% mol), 4.29 mg of 2-cyano-2-propylbenzodithioate, 1.59 mg of 2,2′-azobis(2-methylpropionitrile) and 4.5 ml of tert-butanol. This procedure resulted in polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) with a molecular weight of M.sub.w=26600 g/mol and a polydispersity of D=1.07 and containing 10.4 mol % of the reactive thiazolidine-2-thione groups.

[0064] The polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) (0.04 g, M.sub.w=26600 g/mol, 10.4 mol % TT) was dissolved in 0.25 ml of DMSO, 5 mg of biotin-NH.sub.2 was added to the solution. N,N-diisopropylethylamine (DIPEA) (3.0 μl) was then added. The compounds reacted together for 4 hours at room temperature and then 1-amino-propan-2-ol (5 μl) was added to the solution. Polymeric conjugate 4 poly(HPMA-co-Ma-β-Ala-NH-biotin) was isolated by precipitation into acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum. Polymeric conjugate was purified by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum. The yield of Conjugate 4 was 28 mg and biotin content was 6.4%.

Example 4: Visualization of Proteins with His-Tag by Western Blot Using Conjugates 1 and 2

[0065] Before use, Conjugate 1 and Conjugate 2 (30 μM, 100 μl) were first incubated for 1 hour at room temperature in the presence of 94 mM nickel chloride (i.e. in a 100 fold molar excess of nickel cations to NTA groups) for filling the binding groups with nickel cations. The unbound cations were then removed by dialysis using Slide-A-Lyzer Mini Dialysis Devices (10 kDa MWCO). Dialysis was carried out at room temperature first for 3 hours, against 5 l of distilled water and then for 12 hours against 3 l of TBS.

[0066] Various amounts (0.1-10 ng) of purified recombinant M1 protein from influenza virus labeled with His-tag (M1-HisTag) was applied on SDS-PAGE electrophoresis; after electrophoresis, the gel was blotted on a membrane (wet blot: 100 V/60 min). The surface of the membrane was then blocked using 1.1% (w/v) solution of casein in PBS (Casein Buffer 20×-4× Concentrate, SDT) at room temperature for 1 hour. To visualize the M1-HisTag protein, membrane was incubated with 5 nM Conjugate 1 in PBS containing 0.05% Tween 20 (PBST), or with antibody against His-tag conjugated with horseradish peroxidase (Sigma, #A7058-1VL; 1:2000 in PBST) at room temperature for 1 hour. The blots (incubated in a solution of a Conjugate 1) were then washed three times with PBST and incubated for 1 h with NeutrAvidin conjugated to horseradish peroxidase (Thermo Scientific, #31001, 1:2500 in PBST). Finally, the blots were washed three times with PBST, and SuperSignal West Classic/Dura/Femto Chemiluminescent Substrate (Thermo Scientific) was applied to the membrane. Chemiluminescence was recorded using ChemiDoc It™-600 Imaging System (UVP).

[0067] When comparing the detection sensitivity of the recombinant purified M1-HisTag protein using 5 nM Conjugate 1 and a commercial antibody (FIG. 7), it is clear that the detection method using Conjugate 1 is more sensitive than with the antibody (approximately 5 times) and thus has a lower limit of detection (100 pg vs. 500 pg).

[0068] Nonspecific reactivity of Conjugate 1 and Conjugate B on the Western blot was tested using a bacterial lysate containing a poly(A)-polymerase from E. coli labeled with His-tag (PAP-HisTag) (FIG. 8).

Example 5: Affinity Isolation (“Pull-Down”) of NEDD8 Protein Labeled with His Tag (NEDD8-HisTag) Using Conjugate 1

[0069] Affinity isolation of the NEDD8-HisTag protein was performed both under native conditions in 20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.4 (TBST) and under denaturing conditions in 8 M urea, 20 mM Tris-HCl, 300 mM NaCl, pH 7.4.

[0070] Conjugate 1, Conjugate 3 and Conjugate 4 (i.e. negative controls showing nonspecific binding; only pure resin without any added conjugate was used as the fourth sample) were pre-bound to 20 μl of Streptavidin Sepharose (200 nM solution in 1000 μl of TBST containing 1 mM NiCl.sub.2, 1 hour, 6° C.). After washing with 3×1000 μl TBST, the resin was mixed with 1000 μl of a solution of NEDD8-HisTag (5 ng/μl, either under native or denaturing conditions) and incubated at 6° C. for 3 hours. The resin was then washed with 3×1000 μl TBST and subsequently, proteins were eluted by addition of 50 μl of sample buffer for SDS-PAGE and heating to 98° C. for 10 min.

[0071] NEDD8-HisTag protein was successfully isolated using Conjugate 1 both under native conditions (FIG. 9A) and under denaturing conditions (FIG. 9B). Each of the negative controls (conjugate without inhibitor; conjugate without inhibitor and ATTO488; empty resin Streptavidin Sepharose) showed that binding NEDD8-HisTag takes place specifically via Compound A with the NTA groups.

Example 6: Affinity Isolation (“Pull-Down”) of the Protein Marker Ddi1 with His-Tag (Ddi1-HisTag) from the Bacterial Lysate Using Conjugate 1

[0072] Affinity isolation of Ddi1 protein (DNA-damage inducible protein 1), labeled with His tag (Ddi1-HisTag) was performed using Conjugate 1 form bacterial lysate containing Ddi1-HisTag protein (FIG. 10).

[0073] Conjugate 1, Conjugate 3 (i.e. negative control showing nonspecific binding; only pure resin without any added conjugate was used as the third sample) were pre-bound to 30 μl of Streptavidin Agarose (200 nM solution in 1000 μl of TBST for 1 h, containing 1 mM NiCl.sub.2). After washing with 3×1000 μl TBST, the resin was mixed with 1000 μl of Ddi1-HisTag solution and incubated at room temperature for 1 hour. The resin was then washed with 3×1000 μl TBST and proteins were then eluted by addition of 30 μl of 250 mM imidazole and incubating for 30 min.

[0074] Ddi1-HisTag protein was successfully isolated by Conjugate 1 from bacterial lysate (FIG. 10). Besides the Ddi1-HisTag protein, the elution fraction contained two other proteins: the presence of one (˜15 kDa) was given by the non-specificity of the triNTA group, the second (˜22 kDa) by binding of this protein to the Streptavidin Agarose resin.

Example 7: Immobilization of Recombinant Human GCPII Labeled with His-Tag (his-rhGCPII) and the Subsequent Testing of the Inhibitory Potency of GCPII Inhibitors

[0075] 10 μl solution of streptavidin (10 μg/μl) in 100 mM borate buffer, pH 9.5, was applied to the bottom of wells in a 96 well FrameStar 480/96 plate and incubated at room temperature for 1 hour.

[0076] The contents of wells was then tapped out and wells were washed three times with 200 μl of TBS. Unoccupied surface of the wells was blocked with 0.55% (w/v) solution of casein in TBS (Casein Buffer 20×-4× Concentrate, SDT, 24 h). After further washing with 3×200 μl TBST, Conjugate 2 or 4 (100 nM in TBST containing 1 mM NiCl.sub.2, 2 h) was bound to streptavidin. Unbound conjugates were washed away by washing with 3×200 μl of TBST and a solution of recombinant His-rhGCPII in TBST (10 ng/well, 1 hr, prepared according to [9]) was subsequently added to the wells. After washing with 3×200 μl of TBST, either detection probe ssPSMA alone (1 nM in TBST) binding to the active site of His-rhGCPII, or a mixture of this probe and a selected test substance in a selected concentration (typically 100 μM in TBST) were added. After incubation for 1 hr at room temperature, the wells were washed 5×200 μl of TBST and the amount of bound detection probe was then determined by qPCR. From the changes in the amount of bound probe in wells incubated with test compound compared to wells incubated with the probe alone, the fraction of active sites of the His-rhGCPII occupied by a given test substance was calculated, and consequently the inhibition constant of the substance (a detailed description of the ssPSMA detection probe and a method to calculate the inhibition constants are given in Czech patent application PV 2014-527).

[0077] With this method, it was possible to determine the inhibition constant of the tested inhibitor by measuring the sample in a single well; this method was used to measure twenty inhibitors and K.sub.i values obtained corresponded to the K.sub.i values acquired by measuring His-rhGCPII enzyme kinetics.

[0078] In this patent application, conjugates have been described containing nitrilotrisacetic acid (NTA) based compounds binding polyhistidine sequence (His-tag), which is used as a purification and visualization tag for a large part of recombinantly prepared proteins. Thanks to NTA His-tag binding is therefore possible to use these conjugates universally for all proteins carrying this affinity tag. Dissociation constant for binding of the conjugate to His-tag was determined by SPR. Further, conjugates were used for isolation and purification of proteins labeled with His-tag, both under native and denaturing conditions. When using the conjugate in the Western blot method, it was possible to detect very small quantities of proteins transferred to the membrane after SDS-PAGE electrophoresis. The detection limit on Western blot using a 5 nM solution of Conjugate 1 was about 100 pg. Conjugate was further used for immobilization of proteins with His-tag in assays derived from ELISA (sandwich arrangement), where proteins after immobilization via a His-tag were incubated in the presence of a test substance and their known ligand/inhibitor (or generally a substance binding to the protein), thereby to determine the bond strength of the tested substances with the protein and thus their inhibitory potency.

Example 8: Preparation of Compound B

[0079] Compound B was prepared according to the following scheme:

##STR00005##

[0080] (1) Compound B.sub.0

[0081] (2S,2′S)-5,5′-(((2R,2′R)-disulfanediylbis(1-((carboxymethyl)amino)-1-oxopropan-3,2-diyl))bis(azanediyl))bis(2-amino-5-oxopentanoic) acid, Compound B.sub.0: 150 mg of oxidized glutathione (0.24 mmol, 1.0 eq) was dissolved in 3 ml of water and 111 mg of Boc-anhydride (0.50 mmol, 2.05 eq) dissolved in 3 ml of methanol wad added to the solution. To provide basic pH, DIEA was added to the reaction mixture. After 12 hours, all substances were evaporated and the crude product was used without further purification in the next step. Analytical HPLC showed 99% purity (R.sub.T=16.5 min). HRMS (ESI−) m/z for C.sub.30H.sub.48O.sub.16N.sub.6S.sub.2[M-H].sup.−: calculated: 811, 24954, found 811, 24991.

[0082] (2) Compound B.sub.1

[0083] (6S,6′S,11R,11′R)-11,11′-(disulfanediylbis(methylen))bis(6-carboxy-2,2-dimethyl-4,9,12-trioxo-3-oxa-5,10,13-triazapentadecane-15-oic) acid, Compound B.sub.1: 90 mg of Compound B.sub.0 (0.11 mmol, 1.0 eq) was dissolved in 1 ml of water and the solution was purged with nitrogen under stirring for 10 min. 35 mg of tris(2-carboxyethyl) phosphine (TCEP; 0.12 mmol, 1.1 eq) was added and the reaction was stirred for 2 h under inert atmosphere. HPLC analysis proved complete disappearance of Compound B.sub.0. Then, 114 mg of mal-dPEG.sub.4-NHS (0.22 mmol, 2.0 eq, #PEG1575.0001, IRIS Biotech GmbH) dissolved in 1 ml of methanol was added. After 3 hours the volatiles were evaporated and the crude product was purified by preparative HPLC (gradient of 15-50% ACN in 50 min, R.sub.T=32 min). After lyophilization, 95 mg of Compound B.sub.1 was isolated (yield 47%). Analytical HPLC: R.sub.T=16.8. HRMS (ESI−) m/z for C.sub.37H.sub.56O.sub.19N.sub.6S [M-H].sup.− calculated 919.32482. found 919.32444.

[0084] (3) Compound B

[0085] 46 mg of Compound B.sub.1 (50 μmol, 1.0 eq) was dissolved in 1 ml of methanol, and 20 μl of freshly redistilled ethylenediamine (300 μmol, 6.0 eq) was added to the reaction mixture. The reaction mixture was allowed to react under stirring for 3 hours. The volatiles were evaporated and the product was purified by preparative HPLC (gradient of 15-40% ACN in 50 min, R.sub.T=29 min). After lyophilization, 43 mg of Compound B was isolated (yield 46%). Analytical HPLC: R.sub.T=14.7. HRMS (ESI−) m/z for C.sub.45H.sub.58O.sub.16N.sub.7S [M-H].sup.−: calculated 866.38143. found 866.38118.

Example 9: Preparation of Conjugate 5

[0086] The polymeric precursor poly (HPMA-co-Ma-β-Ala-TT) (0.040 g, M.sub.w=81600 g/mol, 14.6 mol % TT; see Preparation of Conjugate 1), Compound B (5.5 mg) and N-(2-aminoethyl) biotinamid hydrobromide (biotin-NH.sub.2) (5 mg) were dissolved in 0.2 ml of DMSO. ATTO488-NH.sub.2 (2.5 mg) was dissolved in 0.1 ml of DMSO and added to a solution of the polymeric precursor. Then, N,N-diisopropylethylamine (DIPEA) (8.0 μl) was added and the reaction mixture was stirred for 4 hours at room temperature. Subsequently, 1-amino-propan-2-ol (5 μl) was added to the solution and the reaction mixture was stirred for 10 min. Then, the polymeric conjugate 5 poly(HPMA-co-Ma-β-Ala-CompoundB-co-Ma-β-Ala-ATTO488-co-Ma-β-Ala-NH-biotin) was isolated by precipitation in acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum. Polymeric conjugate was purified from low-molecular impurities by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum. The yield of the Conjugate 5 was 20 mg. Content of ATTO488 4.78% was determined spectrophotometrically (ε.sub.502nm=90000 l.Math.mol.sup.−1.Math.cm.sup.−1, distilled water) and the glutathione content 10.87% was determined in sample hydrolyzate (6N—HCl, 115° C., 16 hr) by HPLC with fluorescence detector (Ex. 229 nm, Em. 450 nm), column: Chromolith C18, precolumn derivatisation method with o-phthaldialdehyde.

Example 10: Preparation of Conjugate 6

[0087] The polymeric precursor poly(HPMA-co-Ma-β-Ala-TT) (0.030 mg, M.sub.w=81600 g/mol, 14.6 mol % TT; see Preparation of Conjugate 1), Compound B (3.8 mg) and N-(2-aminoethyl) biotinamid hydrobromide (biotin-NH.sub.2) (4 mg) were dissolved in 0.3 ml of DMSO. N,N-diisopropylethylamine (DIPEA) (4.0 μl) was added, the reaction mixture was stirred for 4 hours at room temperature and then 1-amino-propan-2-ol (2 μl) was added to the solution and the reaction mixture was stirred for 10 min. Then, the polymeric conjugate 6 poly(HPMA-co-Ma-β-Ala-CompoundB-co-Ma-β-Ala-NH-biotin) was isolated by precipitation into acetone:diethyl ether (3:1), filtered, washed with acetone and diethyl ether and dried in vacuum. Polymeric conjugate was purified from low-molecular impurities by column chromatography on Sephadex LH-20 in methanol, precipitated in diethyl ether, filtered and dried in vacuum. The yield of Conjugate 6 was 18 mg. Biotin content 5.23% was determined using the HABA/Avidin kit (Sigma) and glutathione content 10.85% was determined in the sample hydrolysate (6N—HCl, 115° C., 16 hours) by HPLC with fluorescence detector (Ex. 229 nm, Em. 450 nm), column: Chromolith C18, precolumn derivatisation method with o-phthaldialdehyde.

Example 11: Affinity Isolation (“Pull-Down”) of Recombinant Human Protein GCPII with GST Affinity Tag (GST-rhGCPII) Using Conjugate 5 and Conjugate 6

[0088] Affinity isolation of GST-rhGCPII was conducted analogously as the isolation of the protein labeled the His-tag (see Example 5).

[0089] Conjugate 5, Conjugate 6, Conjugate 3 and Conjugate 4 were pre-bound to 20 μl of Streptavidin Sepharose (200 nM solution in 1000 μl of TBST, 1 hr, 6° C.). After washing with 3×1000 μl TBST, the resin was mixed with 1000 μl of a solution of GST-rhGCPII (5 ng/μl in TBST or lysate of LNCaP cells) and incubated at 6° C. for 3 hours. The resin was then washed with 3×1000 μl TBST and subsequently, proteins were eluted by addition of 50 μl of sample buffer for SDS-PAGE and by heating to 98° C. for 10 min.

[0090] GST-rhGCPII protein was successfully isolated with Conjugate 5 and Conjugate 6 from both samples (both from pure buffer, and from the lysate of LNCaP cells). Each of the negative controls (conjugate without inhibitor and conjugate without inhibitor and ATTO488) showed that the binding of GST-rhGCPII happens specifically via a binding group present on the conjugate.

Example 12: Quantification of the Interactions of Polymeric Conjugates with the GST-rhGCPII Using Surface Plasmon Resonance (SPR)

[0091] Measuring the interaction of GST-rhGCPII with Conjugates 5 and 6 using surface plasmon resonance (SPR) was performed on a four-channel SPR sensor developed at the Institute of Photonics and Electronics AS CR in Prague [10-11]. In a typical experiment, the SPR chip (supplied by IPE ASCR) immersed in ethanol solution (7:3) of alkanethiols HS—(CH.sub.2).sub.11-PEG.sub.4-OH and HS—(CH.sub.2).sub.11-PEG.sub.6-O—CH.sub.2—COOH (Prochimia) at a final concentration of 0.2 mM for 1 h at 37° C. The chip was subsequently rinsed with ethanol for UV spectroscopy, with deionized water and dried with nitrogen. Finally, the chip is attached to a SPR chip prism; all measurements were performed at 25° C.

[0092] Activation of the terminal carboxyl groups on the sensor surface was carried out in situ by addition of a mixture (1:1) 11.51 mg/ml N-hydroxysuccinimide (NHS, Biacore), and 76.68 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC, Biacore) in deionized water for 5 min at 20 μl/min. Following steps of the experiment were then conducted at a flow rate of 30 μl/min. Subsequently, neutravidin solution (20 ng/μl) in 10 mM sodium acetate, pH 5.0 was applied for 8 min. To remove non-specifically bound molecules of neutravidin, buffer of high ionic strength (PBS with 0.5 M NaCl) was used, and then for deactivation of the remaining activated carboxyl groups, 1 M ethanolamine (Biacore) was applied. Conjugate of 5 or 6 (1 μM in TBS, for 10 min) was then bound to immobilized neutravidin. Finally, a solution of recombinant protein GST-rhGCPII in TBS in varying concentrations was injected on this prepared layer (the concentrations of GST-rhGCPII were 100, 200, 400 and 800 nM) and subsequently only TBS (dissociation phase). Curves describing the bond were exported and analyzed in TraceDrawer v.1.5 (Ridgeview Instruments AB) to obtain the parameters k.sub.on a k.sub.off.

[0093] The value of the dissociation constant between the GST-rhGCPII and Conjugate 5 was determined K.sub.D=12 nM; between GST-rhGCPII and Conjugate 6 K.sub.D=9 nM.

Example 13: Immobilization of GST-rhGCPII and Subsequent Testing of the Inhibitory Potency of GCPII Inhibitors

[0094] The experiment was performed analogously to the experiment with immobilization of GCPII via His-tag (Example 7).

[0095] 10 μl solution of streptavidin (10 ug/μl) in 100 mM borate buffer, pH 9.5, was applied to the bottom of wells in a 96 well FrameStar 480/96 plate and incubated at room temperature for 1 hour. The contents of wells was then tapped out and wells were washed three times with 200 μl of TBS. Unoccupied surface of the wells was blocked with 0.55% (w/v) solution of casein in TBS (Casein Buffer 20×-4× Concentrate, SDT, 24 h). After further washing with 3×200 μl TBST, Conjugate 6 or 4 (100 nM in TBST, 2 hrs) was bound to streptavidin. Unbound conjugates were washed away by washing with 3×200 μl of TBST and a solution of recombinant GST-rhGCPII in TBST (10 ng/well, 1 hr, prepared according to [9]) was subsequently added to the wells. After washing with 3×200 μl of TBST, either detection probe ssPSMA alone (1 nM in TBST) binding to the active site of GST-rhGCPII, or a mixture of this probe and a selected test substance in a selected concentration (typically 100 μM in TBST) were added. After incubation for 1 hr at room temperature, the wells were washed 5×200 μl of TBST and the amount of bound detection probe was then determined by qPCR. From the changes in the amount of bound probe in wells incubated with test compound compared to wells incubated with the probe alone, the fraction of active sites of the GST-rhGCPII occupied by a given test substance was calculated, and consequently the inhibition constant of the substance (a detailed description of the ssPSMA detection probe and a method to calculate the inhibition constants are given in Czech patent application PV 2014-527).

[0096] With this method, it was possible to determine the inhibition constant of the tested inhibitor by measuring the sample in a single well; this method was used to measure twenty inhibitors and K.sub.i values obtained corresponded to the K.sub.i values acquired by measuring GST-rhGCPII enzyme kinetics.

[0097] Conjugates 5 and 6 containing a binding group for the GST-tag were used for the affinity isolation and purification of proteins with GST-tag from various samples. Bond between these polymers and the protein carrying the GST-tag was analyzed by SPR; dissociation constant of the binding was in the nanomolar range (about 10 nM). Further, in an analogous way as for the conjugate binding His-tag, the conjugate was used for the immobilization of proteins with GST-tag and subsequent testing of substances competing for binding with a known ligand of the protein.

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