METHOD OF DETECTION OF ANALYTE ACTIVE FORMS AND DETERMINATION OF THE ABILITY OF SUBSTANCES TO BIND INTO ANALYTE ACTIVE SITES
20170219583 · 2017-08-03
Assignee
Inventors
- Vaclav NAVRATIL (Praha 5, CZ)
- Pavel SACHA (Praha 6, CZ)
- Jiri SCHIMER (Praha 8, CZ)
- Jan KONVALINKA (Prague 6, CZ)
- Pavel MAJER (Praha 4, CZ)
Cpc classification
C12Y402/01001
CHEMISTRY; METALLURGY
C12Y302/01018
CHEMISTRY; METALLURGY
G01N2333/988
PHYSICS
C12Y304/17021
CHEMISTRY; METALLURGY
G01N33/54353
PHYSICS
International classification
Abstract
A method for detection of active form of analytes in a sample and/or for determination of ability of tested substances to bind to the active site of these analytes has the following steps: a) analyte or group of analytes from the sample is immobilized on the surface of a solid carrier; b) analyte or group of analytes is incubated with a detection probe; c) then the solid carrier is washed to remove unbound detection probe; and subsequently, the amount of bound detection probe is determined.
Claims
1. A method for detection of active form of analytes in a sample and/or for determination of ability of tested substances to bind to the active site of these analytes, characterized in that it comprises the following steps: a) analyte or group of analytes from the sample is immobilized on the surface of a solid carrier either by non-specific non-covalent adsorption or by covalent binding of surface functional groups of the analyte and corresponding functional groups of the solid carrier, or preferably via a binding molecule which is bound to the surface of the solid carrier before immobilization of the analyte or group of analytes and is capable of selectively binding the analyte or group of analytes contained in the sample during incubation of the solid carrier with the sample; b) analyte or group of analytes is incubated with a detection probe which binds selectively to the analyte or group of analytes via a compound for selective binding to the analyte active site; whereas the probe consists of a low molecular compound having molecular weight of up to 2500 Da for selective binding to the analyte active site; an oligonucleotide tag, optionally with a covalently attached fluorophore, biotin or a chemical group, and a chemical linker covalently linking the compound for selective binding to the analyte active site and the oligonucleotide tag; and optionally, the incubation is carried out in the presence of a tested substance whose ability to bind to the active site is to be tested, or a mixture of such substances; c) then the solid carrier is washed to remove unbound detection probe; d) subsequently, the amount of bound detection probe is determined, preferably directly on the solid carrier or after releasing, whereas this amount is proportional to the amount of the analyte or group of analytes in the sample.
2. The method according to claim 1, wherein in step b) incubating the detection probe with a solid carrier, or in step a) incubating the sample with a solid carrier, at least one additive selected from the group consisting of ionic detergents, nonionic detergents, casein and therefrom prepared casein blocking agents, serum albumin, DNA, and immunoglobulins, is added to the incubated solution.
3. The method according to claim 1, wherein the sample containing the analyte or group of analytes is first incubated with the detection probe according to step b), then the immobilization according to step a) is performed, and subsequently steps c) and d) are performed
4. The method according to claim 1, wherein: the analyte is selected from the group consisting of enzyme or group of enzymes, wherein the compound for selective binding to the active site is a selective inhibitor of the enzyme or group of enzymes; receptor or group of receptors, wherein the compound for selective binding to the active site is a selective agonist or antagonist of the receptor or group of receptors; transporter or group of transporters, wherein the compound for selective binding to the active site is a substance capable of selective binding of the transporter or group of transporters in the binding site of transported molecules; the oligonucleotide tag is single stranded or double stranded DNA, optionally with one or more modifying groups selected from the group consisting of a fluorophore, biotin, and a reactive chemical group, covalently attached via a chemical linker to one or both strands of the oligonucleotide tag.
5. The method according to claim 1, wherein the detection probe contains two or more molecules of the same compound for selective binding to the analyte active site, individually covalently linked via a chemical linker into different positions of the oligonucleotide tag.
6. The method according to claim 1, wherein detection is carried out using a conjugate of a detection probe consisting of four probe molecules with attached biotin, and of avidin, neutravidin or streptavidin to which fluorophores or enzymes are optionally covalently attached.
7. The method according to claim 1, wherein the amount of bound detection probe is determined by quantitative polymerase chain reaction, by fluorescence or through coupled enzyme reactions spectrophotometrically or chemiluminescently.
8. The method according to claim 1, wherein the binding molecule capable of selectively binding the analyte from the sample is selected from the group consisting of antibodies or fragments thereof, protein molecules mimicking antibodies such as affibodies, anticalins, or DARPins, and lectins, avidin, neutravidin, streptavidin, oligopeptides, and chelating agents.
9. The method according to claim 1, wherein in step a), selective binding of the analyte or group of analytes to a binding molecule immobilized on the solid carrier takes place via hapten, biotin, a universal epitope, affinity or purification tag, which is covalently attached to the analyte or group of analytes.
10. The method according to claim 1, wherein the sample is a complex biological matrix, optionally containing interfering antibodies, selected from the group consisting of blood, blood plasma, blood serum, cerebrospinal fluid, urine, bacterial, yeast, tissue or cell lysate, conditioned bacterial, yeast or cell culture medium, synovial fluid, amniotic fluid, ascites, pleural fluid, pericardial fluid, stool extract, saliva, sweat and seminal plasma.
11. The method according to claim 1, wherein the ability of the tested substance or mixture of such substances to bind to the active site of the analyte is determined from the difference in the amount of bound detection probe after incubation without tested substance and after incubation with the tested substance.
12. The method according to claim 11, wherein the value of binding constant of binding of the tested substance into the active site of analyte is determined from the difference of the amounts of bound detection probe after incubation without tested substance and after incubation with a single concentration of the tested substance.
13. The method according to claim 1, wherein the analyte is a human prostate specific membrane antigen, known as glutamate carboxypeptidase II, and compound for selective binding is inhibitor of human prostate specific membrane antigen; or the analyte is human glutamate carboxypeptidase III and the compound for selective binding is inhibitor of human glutamate carboxypeptidase III.
14. The method according to claim 1, wherein the analyte is human prostate specific antigen and the compound for binding is selective inhibitor of human prostate specific antigen.
15. The method according to claim 1, wherein the analyte is human carbonic anhydrase IX and the compound for selective binding is inhibitor of human carbonic anhydrase IX; or the analyte is human carbonic anhydrase XII and the compound for selective binding is inhibitor of human carbonic anhydrase XII.
16. The method according to claim 1, wherein the analyte is influenza neuraminidase and the compound for selective binding is the inhibitor of influenza neuraminidase.
17. The method according to claim 1, wherein the analyte is human fibroblast-activating protein and the compound for selective binding is inhibitor of human fibroblast-activating protein; or the analyte is human dipeptidyl peptidase 4, known as CD26 and the compound for selective binding is inhibitor of the human dipeptidyl peptidase 4.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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A) A detection probe with a compound for selective binding of HIV protease (ssHIV1)
B) A detection probe with a compound for selective binding of carbonic anhydrases (ssCA)
C) A detection probe with a compound for selective binding of aspartic proteases (ssAP)
D) A detection probe with a compound for selective binding of influenza neuraminidases (ssAD_NA).
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EXAMPLES
Composition of Solutions
[0147] Modification buffer 100 mmol.Math.l.sup.−1 phosphate buffer; 150 mmol.Math.l.sup.−1 NaCl; pH=7.8 [0148] TBS 20 mmol.Math.l.sup.−1 Tris; 150 mmol.Math.l.sup.−1 NaCl; pH=7.4 [0149] TBST 20 mmol.Math.l.sup.−1 Tris; 150 mmol.Math.l.sup.−1 NaCl; pH=7.4; 0.05% Tween20 (vol./vol.) [0150] TBST200 20 mmol.Math.l.sup.−1 Tris; 200 mmol.Math.l.sup.−1 NaCl; pH=7.4; 0.05% Tween20 (vol./vol.) [0151] TSBT′ 20 mmol.Math.l.sup.−1 Tris; 150 mmol.Math.l.sup.−1 NaCl; pH=7.4; 0.1% Tween20 (vol./vol.) [0152] CaSDS 20 mmol.Math.l.sup.−1 Tris; 150 mmol.Math.l.sup.−1 NaCl; pH=7.4; 0.1% Tween20 (vol./vol.); 0.005% SDS (hm./obj.); 500-fold diluted casein blocker (SDT; cat. no. CBC1) [0153] TBSE 20 mmol.Math.l.sup.−1 Tris; 150 mmol.Math.l.sup.−1 NaCl; pH=7.4; 5 mmol.Math.l.sup.−1 EDTA [0154] MEST 20 mmol.Math.l.sup.−1 MES; 750 mmol.Math.l.sup.−1 NaCl; pH 6.0; 0.05% Tween20 (vol./vol.) [0155] CLP 50 mmol.Math.l.sup.−1 Tris; 100 mmol.Math.l.sup.−1 NaCl; pH 7.4 [0156] HEPESTC 100 mmol.Math.l.sup.−1 HEPES; 400 mmol.Math.l.sup.−1 NaCl; pH=7.5; 0.01% Tween20 (vol./vol.); 2000-fold diluted casein blocker (SDT; cat. no. CBC1) [0157] HEPESTC′ 100 mmol.Math.l.sup.−1 HEPES; 400 mmol.Math.l.sup.−1 NaCl; pH=7.5; 0.1% Tween20 (vol./vol.); 500-fold diluted casein blocker (SDT; cat. no. CBC1)
Explanation of Terms and Abbreviations
[0158] GCPII glutamate carboxypeptidase II [0159] PSMA prostate specific membrane antigen [0160] Avi-PSMA protein consisting of extracellular part of prostate specific membrane antigen with N-terminally attached Avi-tag [0161] rhPSMA recombinant human prostate specific membrane antigen [0162] GCPIII glutamate carboxypeptidase III [0163] Avi-GCPIII protein consisting of extracellular part of glutamate carboxypeptidase III with N-terminally attached Avi-tag [0164] ssPSMA designation of detection probe for the detection of PSMA (containing single stranded oligonucleotide tag) [0165] dsPSMA designation of detection probe for the detection of PSMA (containing double stranded oligonucleotide tag) [0166] dsA3PSMA designation of detection probe for the detection of PSMA (containing double stranded oligonucleotide tag) [0167] dsbiotPSMA designation of detection probe for the detection of PSMA (containing biotinylated double stranded oligonucleotide tag) [0168] Neu_dsbiotPSMA designation of detection probe for the detection of PSMA (containing biotinylated double stranded oligonucleotide tag bound to neutravidin) [0169] NeuHRP_dsbiotPSMA designation of detection probe for the detection of PSMA (containing biotinylated double stranded oligonucleotide tag bound to neutravidin conjugated with peroxidase) [0170] ssHIV designation of detection probe for the detection of HIV protease (containing single stranded oligonucleotide tag) [0171] CA-II carbonic anhydrase II [0172] CA-IX carbonic anhydrase IX [0173] ssCA designation of detection probe for the detection of carbonic anhydrases (containing single stranded oligonucleotide tag) [0174] ssCAbis designation of bivalent detection probe for the detection of carbonic anhydrases [0175] Neu_dsbiotCA designation of detection probe for the detection of carbonic anhydrases (containing biotinylated double stranded oligonucleotide tag bound to neutravidin) [0176] ssAP designation of detection probe for the detection of aspartic proteases (containing single stranded oligonucleotide tag) [0177] ssAD designation of oligonucleotide with bound DBCO [0178] ssAD_NA designation of detection probe for the detection of influenza neuraminidases [0179] eq equivalent [0180] RT retention time [0181] Tween 20 polyoxyethylene (20) sorbitanmonolaurate (USB, cat. no. 20605) [0182] DIAD diisopropyl azodicarboxylate [0183] DBCO dibenzyl cyclooctyne [0184] HEPES N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid [0185] SDS sodium dodecyl sulfate [0186] SDS-PAGE polyacrylamide gel with sodium dodecyl sulfate for electrophoresis [0187] LC-MS liquid chromatography-mass spectrometry [0188] ESI electrospray ionisation [0189] FBS fetal bovine serum [0190] THF tetrahydrofuran [0191] DMF dimethylformamide [0192] DIEA diisopropylethylamine [0193] ACN acetonitrile [0194] TFA trifluoroacetic acid [0195] DPPA diphenylphosphorylazide [0196] TEA triethylamine [0197] PEG5 5 linked ethylene glycol units [0198] HOBT/DIC hydroxybenzotriazole/diisopropylcarbodiimide [0199] AAZ acetazolamide [0200] DCC dicyclohexylcarbodiimide [0201] DCU dicyclohexylurea [0202] TBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate [0203] HRMS high resolution mass spectrometry
Example 1: Quantification of PSMA, Testing of PSMA Inhibitors Potency
1a: Preparation of PSMA Inhibitor with a Linker and an Activated NHS Ester
[0204] The detection probe for PSMA was prepared by linking of an urea based PSMA inhibitor S,S-2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid to the DNA. Following derivatives of the inhibitor were prepared: inhibitor with linker with terminal NHS-ester (Compound 3) for linking to the amino group of the DNA oligonucleotide and this compound reacted with ethanolamine (Compound 4) for determination of the impact of linking of the DNA oligonucleotide on the inhibition potency.
[0205] All chemicals were purchased from Sigma-Aldrich, unless stated otherwise. The purity of compounds was tested on analytical Jasco PU-1580 HPLC (flow rate 1 ml/min, invariable gradient 2-100% (vol./vol.) ACN in 30 minutes, RT shown for each compound) with column Watrex C18 Analytical Column, 5 μm, 250×5 mm. All final products were purified using preparative scale HPLC Waters Delta 600 (flow rate 7 ml/min, gradient and RT shown for each compound) with column Waters SunFire C18 OBD Prep Column, 5 μm, 19×150 mm. All final products were of at least 99% purity. Structure of the final products was further confirmed by HRMS at LTQ Orbitrap XL (Thermo Fisher Scientific) and by NMR (Bruker Avance ITM 500 MHz equipped with Cryoprobe or Bruker Avance ITM 400 MHz).
[0206] Preparation of 3,3′-oxydipropanoic acid (Compound 1): 2.38 ml (20 mmol) of 3,3′-oxydipropanenitrile was dissolved in 7 ml of concentrated HCl and was heated to 50° C. for 24 hours. The reaction mixture was then left to cool down overnight and the hydrochloric acid was removed by flow of nitrogen. The resulting slurry was dissolved in water and lyophilized; 2.25 g of white product was obtained (yield=70%). The spectral analysis of this product was identical to that described in (White et al. 2003, Tetrahedron-Asymmetry, p. 3633).
[0207] Preparation of bis(2,5-dioxopyrrolidin-1-yl) 3,3′-oxydipropanoate (Compound 2): To a solution of Compound 1 (260 mg, 1.6 mmol, 1 eq) and N-hydroxy succinimide (660 mg, 3.2 mmol, 2 eq) in 10 ml of THF, solid DCC (368 mg, 3.2 mmol, 2 eq) was added in one portion. The reaction was left overnight, after which the DCU was filtered of and the volatiles rotary evaporated. The crude product was further purified by chromatography (He:EtOAc 1:2); 338 mg of pure product obtained (isolated yield=60%). Analytical HPLC RT=16.2 min.
[0208] Result of analysis by .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.85 (t, J=6.4 Hz, 4H), 2.90 (t, J=6.4 Hz, 4H), 2.83 (bs, 8H).
[0209] Result of analysis by .sup.13C NMR (101 MHz, CDCl.sub.3): δ 169.07, 166.77, 65.78, 32.20, 25.73.
[0210] Result of analysis by HRMS (ESI+): calculated mass of C.sub.14H.sub.16O.sub.9N.sub.2 [MNa].sup.+ 379.07480, detected mass 379.07469.
[0211] Preparation of 19-((2,5-dioxopyrrolidin-1-yl)oxy)-5,13,19-trioxo-16-oxa-4,6,12-triazanonadecane-1,3,7-tricarboxylic acid (Compound 3): To a stirring solution of Compound 2 (69 mg, 193 μmol, 1.2 eq) dissolved in 1 ml of DMF, a solution of di-tert-butyl 2-(3-(6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)ureido)pentanedioate (100 mg, 161 μmol, 1.0 eq, prepared as described in (Murelli et al. 2009, Journal of the American Chemical Society, str. 17090)) and DIEA (34 μl, 193 μmol, 1.2 eq) in 1 ml of DMF was added dropwise during 1 hour. The reaction mixture was left stirring for 2 hours after which an HPLC analysis proved total disappearance of reactants. The solvents were then removed by rotary vacuo and the compound was fully dried. 1 ml of TFA was then added into the crude mixture to yield title compound, after 1 hour incubation at room temperature the trifluoracetic acid was removed by flow of nitrogen. The crude product was purified using preparative HPLC (gradient 5-50% (vol./vol.) ACN in 40 minutes, RT 18 minutes); 20 mg of the product was obtained (isolated yield=22%). Analytical HPLC RT=13.7 min.
[0212] Result of analysis by .sup.1H NMR (500 MHz, DMSO-d6): δ 7.80 (t, J=5.6, 1H, NH-Lys-6), 6.32 (d, J=8.3, 1H, NH-Glu-2), 6.29 (d, J=8.2, 1H, NH-Lys-2), 4.09 (m, 1H, Glu-2), 4.03 (m, 1H, Lys-2), 3.55 (m, 4H, O—CH.sub.2—CH.sub.2—COO, Lys-CO—CH.sub.2—CH.sub.2—O), 3.00 (m, 2H, Lys-6), 2.80 (bs, 4H, CO—CH.sub.2—CH.sub.2—CO), 2.41 (t, J=6.3, 2H, O—CH.sub.2—CH.sub.2—COO), 2.31-2.20 (m, 4H, Lys-CO—CH.sub.2—CH.sub.2, Glu-4), 1.91 (m, 1H, Glu-3b), 1.71 (m, 1H, Glu-3a), 1.63 (m, 1H, Lys-3b), 1.51 (m, 1H, Lys-3a), 1.37 (m, 2H, Lys-5), 1.26 (m, 2H, Lys-4).
[0213] Result of analysis by .sup.13C NMR (125.7 MHz, DMSO-d6): δ 174.77 (Lys-1), 174.40 (GLu-1), 173.95 (Glu-5), 172.89 (O—CH.sub.2—CH.sub.2—COO), 170.39 (CO—NH—CO), 170.00 (lys-CO—CH.sub.2—CH.sub.2—O), 157.52 (NH—CO—NH), 66.87 (lys-CO—CH.sub.2—CH.sub.2—O), 66.07 (O—CH.sub.2—CH.sub.2-000), 52.46 (Lys-2), 51.85 (Glu-2), 38.54 (Lys-6), 36.25 (Lys-CO—CH.sub.2—CH.sub.2—O), 34.84 (O—CH.sub.2—CH.sub.2-000), 31.97 (Lys-3), 30.09 (Glu-4), 29.00 (Lys-5), 27.71 (Glu-3), 25.66 (CO—CH.sub.2—CH.sub.2-00), 22.82 (Lys-4).
[0214] Result of analysis by HRMS (ESI+): calculated mass of C.sub.22H.sub.32O.sub.13N.sub.4 [MNa].sup.+ 583.18581; detected mass 583.18596.
[0215] Preparation of 1-hydroxy-4,10,18-trioxo-7-oxa-3,11,17,19-tetraazadocosane-16,20,22-tricarboxylic acid, Compound 4: 5 mg (9.87 μmol, 1 eq) of Compound 3 were dissolved in 200 μl of DMF and 6 μl (99 μmol, 10 eq) of ethanolamine was added into the mixture along with 14 μl (80.4 μmol, 8 eq) of DIEA and the mixture was left stirring overnight. The solvent was rotary evaporated and the mixture was dissolved in ACN/water and lyophilized three times (to evaporate the remaining ethanolamine). The compound was used in biochemical studies without further purification (the only contaminant is NHS, otherwise purity was higher than 95%). Analytical HPLC RT=11.3 min. Result of analysis by HRMS (ESI+): calculated mass of C.sub.20H.sub.33O.sub.11N.sub.4 [M].sup.+505.21513, detected mass 505.21515.
1b: Preparation of a Detection Probe for Selective Binding to PSMA
[0216] Detection probe for selective binding to PSMA was prepared by reacting of Compound 3 and single-stranded DNA with the 3′-terminal 6-amino-2-(hydroxymethyl)hexyl phosphate modification and the sequence CCT GCC AGT TGA GCA TTT TTA TCT GCC ACC TTC TCC ACC AGA CAA AAG CTG GAA A (custom synthesis Generi-Biotech, OPC purification).
[0217] Oligonucleotide (hereinafter referred to as iqPCR_amino) was dissolved in double distilled water at a concentration of 1 mmol.Math.l.sup.−1, and subsequently, for part of the solution, water was replaced with 100 mmol.Math.l.sup.−1 phosphate buffer solution with 150 mmol.Math.l.sup.−1 NaCl (p.a.; Penta), at pH 7.8 (hereinafter “modification buffer”) by repeated ultrafiltration on Amicon Ultra 0.5 ml 3K column (Millipore, cat. no. UFC500396). The rest of the solution was treated the same, but after each step of ultrafiltration, it was diluted with solution of double distilled water. In both cases, the total dilution of the original solvent was 10.sup.5 fold. The concentration of oligonucleotide in the resulting solution was calculated from the measured absorbance at 260 nm (Nanodrop ND-1000, Thermo Scientific) and the estimated optical density of oligonucleotide solution 1 OD=1744 pmol.
[0218] To verify the identity and purity, the solution of iqPCR_amino in distilled water was analyzed with LC/ESI-MS method in the Agilent 6230 TOF LC/MS device (Agilent Technologies) equipped with dual AJS ESI source in the settings for detecting negative ions (4 GHz, HiRes). Separation was carried out at room temperature on Agilent Zorbax Extend-C18 1.8 μm (2.1×50 mm) column by gradient elution in changing ratio of HFIP solution (200 mmol.Math.l.sup.−1 aqueous solution of 1,1,1,3,3,3-hexafluoro-2-propanol, pH adjusted to 7.0 by addition of triethylamine) and acetonitrile, at a flow rate of 0.3 ml.Math.min.sup.−1 (2-45% (vol./vol.) ACN in 6 minutes). The result of the analysis of 5 pmol iqPCR_amino was a single absorption peak at 260 nm and a retention time of 4.84 min and the measured mass (calculated from the most intense peaks for each of the charge z) 16981.87; the most abundant mass predicted with ChemBioDraw Ultra 13.0.0.3015 program (CambridgeSoft) is 16979.91 for the molecular weight of 16983.09.
[0219] Preparation of a conjugate of the oligonucleotide (iqPCR_amino) with PSMA inhibitor (Compound 3): 6.9 μl of 1 mol.Math.l.sup.−1 HEPES buffer, pH=8.0, was added to 10 μl of the oligonucleotide in the modification buffer (10.2 nmol, 1 eq) and after stirring, 3.1 μl of a solution of compound 3 at a concentration of 100 mmol.Math.l.sup.−1 in anhydrous DMSO (307 nmol, 30 eq) was added. Anhydrous DMSO was prepared by several hours incubating of DMSO (Sigma, A.C.S. spectrophotometric grade) with activated molecular sieves (Sigma, cat. No. 688363) at continuous shaking. The molecular sieves were then removed by a brief centrifugation at 16000 g.
[0220] The resulting mixture was incubated at room temperature for 24 hours, diluted to 500 μl with distilled water and then applied to Amicon Ultra 0.5 ml 10K column (Millipore, cat. No. UFC501096) for purification from the hydrolysis products of Compound 3. Through repeated concentrating by ultrafiltration on the column and repeated dilutions, the original solvent (together with the hydrolysis reaction products) was diluted 10.sup.10-fold with double distilled water. This way we obtained 43 μl of solution with oligonucleotide concentration of 215 pmol.Math.μl.sup.−1 determined by absorbance at 260 nm (9.2 nmol, 90% yield); the resulting product is hereinafter called ssPSMA, and its predicted structure is shown in
[0221] To verify the efficiency of conjugation, ssPSMA was analyzed by LC-MS, the procedure was identical to the original iqPCR_amino (described above in this section), and the result was a single peak with absorbance at 260 nm, retention time 4.85 min (i.e. the same retention time as the original iqPCR_amino) and the measured mass (calculated from the most intense peaks for each of the charges z) was 17426.84 (
[0222] The ssPSMA solution was also analyzed for the presence of residual products of hydrolysis of Compound 3, in particular 6,14-dioxo-3-oxa-7,13,15-triazaoctadecan-1,12,16,18-tetracarboxylic acid. This compound obviously binds to the active site of PSMA and if it occurred in the sample in a similar or greater amount than the actual probe, it might compete with the probe for binding and thus reduce the sensitivity of the PSMA assay. The analysis itself was performed by LC-MS in the same manner as described above, with the use of Waters Acquity C18 BEH column 1.8 μm (100×2.1 mm), mobile phase 0.1% (vol./vol.) formic acid and acetonitrile. Elution gradient was 2-100% (vol./vol.) ACN in 6 minutes, and during the whole elution, no signal was detected corresponding at least approximately (±0.2) to the estimated mass 463.18 (m/z=462.18).
[0223] The ssPSMA conjugate, as well as the original oligonucleotide iqPCR_amino, was diluted prior to use in bioassays to a final concentration of 5 nmol.Math.l.sup.−1 in water and 10× concentrated TBS buffer (final concentration 1×TBS: 20 mmol.Math.l.sup.−1 Tris, 150 mmol.Math.l.sup.−1 NaCl, pH=7.4). In a volume of 50 μl in a thin-wall polypropylene eppendorf PCR tube the (Biotix, cat. No. 3423.AS) it was exposed to to the following temperature cycle (hereinafter thermal annealing) in a Tgradient Biometra thermocycler (Labrepco): rapid heating to 98° C., followed by repeated cooling by increments of 1° C. (0.2° C./s) and remaining for 5 min in each step; after reaching a temperature of 60° C., repeated cooling by increments of 5° C. (0.2° C./s) followed and remaining for 5 min in each step until reaching 20° C.; the temperature of the lid was set to 99° C. during the entire procedure.
[0224] Detection probes with double-stranded oligonucleotide tags were prepared in a similar manner: before thermal annealing, the ssPSMA conjugate was mixed in approximately equimolar ratio successively with several different complementary oligonucleotides. The dsPSMA detection probe was formed by mixing ssPSMA with single-stranded DNA sequence TTT CCA GCT TTT GTC TGG TGG AGA AGG TGG CAG ATA AAA ATG CTC AAC TGG CAG G (optical density of the complementary strand solution used for calculation of its concentration: 1 OD=1649 pmol); the dsA3PSMA detection probe was formed by mixing ssPSMA with single-stranded DNA sequence CCA GCT TTT GTC TGG TGG AGA AGG TGG CAG ATA AAA ATG CTC AAC TGG CAG G (1 OD=1721 pmol); whereas the detection probe dsbiotPSMA probe was formed by mixing ssPSMA with single-stranded DNA (hereinafter iqPCR_biotin) sequence CCA GCT TTT GTC TGG TGG AGA AGG TGG CAG ATA AAA ATG CTC AAC TGG CAG GTA (1 OD=1639 pmol), 3′-terminally biotinylated; the structure of this modification is shown in
1c: Determination of the Inhibition Constants of the Compounds Prepared and the Detection Probes
[0225] Biotinylated extracellular portion of PSMA (hereinafter called Avi-PSMA) used in the enzyme assay was prepared and purified according to (Tykvart et al. 2012, Protein Expression and Purification, p. 106); the concentration of pure recombinant protein was determined by amino acid analysis on the Biochrom30 device (Biochrom) according to the manufacturer's instructions. The enzyme was then stored frozen in aliquots at −80° C. The concentration of oligonucleotides was determined spectrophotometrically (see above), the concentration of Compound 4 was derived from the weight on an analytical balance (dissolved in distilled water).
[0226] IC.sub.50 values of all compounds were determined using a method based on HPLC. In a 96 well plate, 0.2 ng Avi-PSMA in 25 mmol.Math.l.sup.−1 Bis-Tris propane, 150 mmol.Math.l.sup.−1 NaCl, 0.001% (wt./vol.) octaethyleneglycol monododecyl ether (C.sub.12E.sub.8), pH 7.4 (hereinafter referred to as a reaction buffer) were mixed together with the tested inhibitor in a total volume of 180 μl. Ten different inhibitor concentrations in a quad dilution series were used to determine the inhibition curve. Reactions were preincubated at 37° C. for 5 min and then started by adding 20 μl 4 μmol.Math.l.sup.−1 pteroyl-bis-L-glutamate (Schircks Laboratories) and incubated at 37° C. for an additional 20 min. The enzymatic reaction was stopped by adding 20 μl of 25 μmol.Math.l.sup.−1 inhibitor 2-PMPA (2-(phosphonomethyl) pentanedioic acid (Jackson et al., 1996, Journal of Medicinal Chemistry, p. 619)).
[0227] Subsequently, 100 μl of the reaction mixture was analyzed by HPLC on an Agilent 1200 Series device equipped with UPLC HSS T3 column 1.8 μm (2.1×100 mm, Waters). Elution itself was isocratic in 2.7% (vol./vol.) acetonitrile and 97.3% (vol./vol.) 20 mmol.Math.l.sup.−1 phosphate, pH=6.0 at a flow rate of 0.4 ml.Math.min′. Substrate and product absorbances were detected at 281 nm, their amounts were determined by automatic integration. The obtained data were evaluated in GraFit v.5.0.11 program (Erithacus Software) and thus the IC.sub.50 values were obtained.
[0228] The kinetic parameters (K.sub.M and k.sub.cat) of pteroyl-bis(L-glutamate) Avi-PSMA cleavage in the reaction buffer were determined according to the procedure described above without the added inhibitor, with various substrate concentrations ranging from 15 nmol.Math.l.sup.−1 to 400 nmol.Math.l.sup.−1, wherein the conversion of all reactions was 13±2%. These parameters were then used, assuming competitive inhibition, to convert values of measured IC.sub.50 to values of the inhibition constants (K.sub.i) according to the Cheng-Prusoff equation (Cheng et al., 1973, Biochemical Pharmacology, p. 3099).
[0229] The resulting K.sub.i value of the Compound 4 was 3.3 nmol.Math.l.sup.−1, K.sub.i of the original iqPCR_amino oligonucleotide was not determined (even at the highest concentration used, 0.5 μmol.Math.l.sup.−1, there was no inhibition observed), K.sub.i of the ssPSMA detection probe was 0.14 nmol.Math.l.sup.−1, K.sub.i of the dsbiotPSMA probe was 0.16 nmol.Math.l.sup.−1 and K.sub.i of the dsPSMA probe was 0.28 nmol.Math.l.sup.−1. From these data it is clear that the connection of single-stranded oligonucleotide tag not only doesn't worsen the inhibition constant, but actually it improved it by more than an order of magnitude. Furthermore, it was determined that addition of the second strand didn't influence the inhibitory constant and thereby the ability of the detection probe to bind to the active site of PSMA, which means that various modifications can be deliberately added to the original single-stranded detection probe ssPSMA by pairing with a modified complementary strand.
1d: Determination of Detection Probes Using qPCR
[0230] The designed sequence of single-stranded DNA contained in ssPSMA was optimized in the Vector NTI 10.3 (Invitrogen) so that it should not form strong secondary structure. At the margins were used sequences complementary to primers, for which it was previously verified that they allow the amplification of template DNA with high efficiency and that they don not form primer dimers at a given PCR conditions, thus ensuring the sensitivity of determination of the oligonucleotide tag in the ssPSMA probe in the order of single molecules. The sequence of primers used was CCA GCT TTT GTC TGG TGG AG and CCT GCA GCC AGT TGA TTT TT (Generi-Biotech; desalting purification); and a hydrolysis probe #87 from Roche “Universal Probe Library” (LNA octamer sequence CTG CCA CC, cat. no. 04689127001) was chosen to detect amplified template DNA during qPCR.
[0231] To test the effectiveness of the determination, we prepared a decimal dilution series of the ssPSMA detection probe in double-distilled water in a concentration range of 10 nmol.Math.l.sup.−1 to 10 mol.Math.l.sup.−1, corresponding to a concentration of 6 to 6×10.sup.9 copies per μl of solution. The dilution series was then used for qPCR calibration: 10 μl of a reaction mixture consisted of LC 480 Probes Master (Roche, cat. no. 04707494001; diluted to the final concentration recommended by the manufacturer), both primers (final conc. of each of them 1 nmol.Math.l.sup.−1), fluorescent hydrolysis probe#87 (final conc. 50 nmol.Math.l.sup.−1) and 1 μl of template DNA or 1 μl of distilled water in the no template control; each concentration and no template control was measured in triplicates. 96 well plates FrameStar 480/96 (4titude, cat. no. 4ti-0951) were used and after pipetting the reaction mixture into the wells they were sealed with adhesive optical films (Roche, cat. no. 4729692001). The time course of the PCR included successively 3 min at 95° C.; then 45 repetitions consisting of three steps: 10 s at 95° C., 30 sec at 66° C. and 30 sec at 72° C.; and finally 2 min at 37° C. We used the Light Cycler 480 II (Roche) with the excitation and emission filter adjusted to the FAM fluorophore. Threshold cycles (C.sub.q) were obtained from the measured fluorescence curves using the method of maxima of the second derivative in the Light Cycler 480 II Software (Roche).
[0232] The obtained C.sub.q values plotted against the decimal logarithm of the concentration of template showed that the linear range of the assay was in the range of 6 to 6×10.sup.8 copies at over 90% efficiency of amplification, and 6 copies (C.sub.q 37 cycles) differed significantly from the no template control, which had no measurable signal. The data are listed in Table 1.
TABLE-US-00001 TABLE 1 C.sub.q values measured depending on the number of copies of the ssPSMA detection probe number of ssPSMA copies C.sub.q 6000000000 9.22 600000000 10.13 60000000 12.72 6000000 15.87 600000 19.10 60000 22.44 6000 26.17 600 29.96 60 33.47 6 37.39 0 >45.00
1e: Determination of the Optimal Working Concentration and Optimal Diluent for the Detection Probe for Determining the Amount of PSMA
[0233] In individual tests to determine the dissociation constant of the detection probe towards PSMA, 10 μl of a solution of various antibodies recognizing the native form of PSMA (2G7, J415, J591, D2B, 107-1A4; described in Tykvart et al. 2014, Prostate, p. 1674) at a concentration of 10 ng.Math.μl.sup.−1 in TBS buffer were loaded to the bottom of wells of a 96 well plate FrameStar 480/96 (4titude, cat. No. 4ti-0951) and incubated at room temperature for 30-120 minutes. The content of wells was then tapped out and the wells were washed three times with 200 μl of TBS. Then, 100 μl of casein blocking agent five times diluted in TBS (casein blocker biotin free 5.5% w/v″; SDT; cat. no. CBC1) was applied to the bottom of the wells and incubated for 1-15 hours at room temperature. Then content of the wells was tapped out again and the wells were washed three times with 200 μl of TBST (TBS with 0.05% (vol./vol.) Tween 20). Thereafter, either 10 μl of pure TBST′ buffer (TBS with 0.1% (vol./vol.) Tween 20) or 10 μl of TBST′ solution with purified recombinantly prepared extracellular portion of human PSMA (hereinafter rhPSMA) at a concentration of 1 μg.Math.μl.sup.−1 i.e. approximately 10 pmol.Math.l.sup.−1 was applied to the bottom of the wells. rhPSMA was prepared and purified as described in (Barinka et al., 2002, Journal of Neurochemistry, p. 477), purity was checked by SDS-PAGE and the concentration determined by amino acid analysis on Biochrom30 (Biochrom) according to the manufacturer's instructions; aliquots of protein stock solution were stored at −80° C. After 60 to 120 minutes incubation at room temperature, the content of the wells was tapped out and the wells were washed five times with 200 μl TBST. Finally, 10 μl of TBST′ solution with the ssPSMA detection probe of several different concentrations in tenfold dilution series from 0.1 pmol.Math.l.sup.−1 to 10 nmol.Math.l.sup.−1 was added to the bottom of wells and incubated for 15-75 minutes at room temperature. Then content of the wells was tapped out again and the wells were washed ten times with 200 μl of TBST. Subsequently, 10 μl of a qPCR mixture of the same composition as in the case of no template control in the previous example 1d was added to the bottom of the wells and the amount of bound detection probe was than determined using qPCR as described in the example 1 d.
[0234] By the described procedure, the amount of the non-selectively adsorbed probe depending on its used concentration (dilution series of detection probe in wells with no added rhPSMA) was measured as well as the amount of the probe selectively bound to the active site of PSMA depending on its used concentration (dilution series of detection probe in wells with added rhPSMA). Dependence of selectively bound probe on its concentration was fitted by the function described by equation (3) using the “Solver” in Microsoft Office Excel 2003, where the variables solved were E.sub.tot (the maximum amount of selectively bound probe) and K.sub.d (dissociation constant of the probe), with minimizing the sum of squared relative deviations between the measured values and values calculated from the fitted function.
[0235] The whole assay was successively repeated with all the above mentioned antibodies and it was shown that the value of K.sub.d of the ssPSMA probe binding into the active site of the immobilized enzyme was always in the range of 100-200 pmol.Math.l.sup.−1, which corresponds very well to the inhibition constant of 140 pmol.Math.l.sup.−1, measured in the enzyme assay Immobilization of the enzyme on any of these antibodies therefore does not affect the binding affinity of the detection probe to the active site of the enzyme. The maximum amount of selectively bound probe for each antibody provided the C, read from qPCR of between 15 and 16; thus, there was not any significant difference among the antibodies in the efficiency of immobilization of the enzyme from the solution. The amount of non-selectively adsorbed detection probe was also similar for all antibodies and it was directly proportional to the concentration of the probe in the entire concentration range used. Subtracting the measured C.sub.q in the well without the enzyme (corresponding to a non-selective binding of the probe) from C.sub.q in a well with the enzyme (corresponding to selective binding of the probe) with the same antibody used and the same concentration of the probe, the signal/background ratio was determined. This was the highest for the probe concentration less than or equal to K.sub.d of its binding to the active site of PSMA and depending on the antibody ranged from 8 to 12 qPCR cycles, which corresponds to a hundred-fold to thousand-fold difference for the used amount of 10 pg rhPSMA.
[0236] The whole experiment was repeated with the 2G7 antibody wherein different concentrations of the ssPSMA detection probe were applied after dilution in TBST′ or in TBST′ with the addition of SDS in a concentration range of 0.005% to 0.02% (wt./vol.) or with the addition of a casein blocker diluted in the range of hundred to thousand fold, or in TBST′ with both additives within the same concentration range. TBST′ with 0.005% (wt./vol.) SDS and 500-fold diluted casein blocking agent (hereinafter “buffer CaSDS”) was determined as the optimal; in which dissociation constant for the selective binding of the detection probe only slightly increased and selective binding thus remained almost unchanged and at the same time, non-selective adsorption was reduced, which showed as an increase of the measured C.sub.q by 5-6 cycles and thus increasing the signal/background ratio by a corresponding extent. Further experiments showed that the same effect is also achieved when using the other antibodies recognizing native form of PSMA.
[0237] Using the 2G7 antibody, the dissociation constant of not only the ssPSMA probe, but also dsPSMA, dsA3PSMA and dsbiotPSMA, was measured more accurately by the same procedure in further experiments. Unlike previous procedures, the applied concentration of rhPSMA was 0.1 pg.Math.μl.sup.−1 and each probe was applied usually in twelve different concentrations ranging from 3 to 1600 pmol.Math.l.sup.−1. For some probes, the determination of the dissociation constant was repeated several times, and the resulting K.sub.d values determined from individual measurements were almost identical to each other. It was found that K.sub.d of the ssPSMA, dsA3PSMA a dsbiotPSMA probes in TBST′ is approximately 60 pmol.Math.l.sup.−1, whereas K.sub.d of the dsPSMA probe was about 100 pmol.Math.l.sup.−1. In the CaSDS buffer, the dissociation constant of all these probes was very similar, approximately 100 pmol.Math.l.sup.−1. Given that for each concentration of each probe, control wells without added antigen were also included, it was found that non-selective binding of single-stranded and double-stranded probes differ from each other. While the concentration of 1000 pmol.Math.l.sup.−1 of the ssPSMA probe in TBST′ results in non-selective binding of the probe amount corresponding to C.sub.q equal to 24 and the same concentration of the same probe in CaSDS results in C.sub.q equal to 30, the concentration of 1000 pmol.Math.l.sup.−1 of the dsbiotPSMA probe (or other double-stranded probes) in TBST′ results in non-selective binding of the probe amount corresponding to C.sub.q equal to 28 and the same concentration of the same probe in CaSDS results in k C.sub.q equal to 33. Dissociation constant for the ssPSMA probe and other forms of PSMA was determined in both buffers by the same procedure; this time, either lysate of human cell line expressing PSMA containing PSMA at a concentration of approximately 0.1 pg.Math.μl.sup.−1 (1 ng of total protein; the HEK line 1-750 is described in (Mlcochova et al. 2009, Prostate, p. 471), or tenfold diluted human citrate blood plasma containing endogenous PSMA at concentration of approximately 0.1 pg.Math.μl.sup.−1 was applied into the wells. The determined dissociation constant of the probe towards PSMA present in the cell lysate was approximately 140 pmol.Math.l.sup.−1 in TBST′, while approximately 250 pmol.Math.l.sup.−1 in CaSDS; dissociation constant of the probe towards the endogenous PSMA contained in plasma was approximately 280 pmol.Math.l.sup.−1 in TBST′, whereas 450 pmol.Math.l.sup.−1 in CaSDS. C, values obtained for the dilution series of the ssPSMA probe with various antigens are summarized in Table 2.
TABLE-US-00002 TABLE 2 C.sub.q values obtained for the dilution series of the ssPSMA probe with various antigens TBST′ buffer CaSDS buffer concentration of 2 pg 1 ng 1 μl 2 pg 1 ng 1 μl ssPSMA, pmol .Math. l.sup.−1 rhPSMA HEK1-750 plasma rhPSMA HEK1-750 plasma 1600 15.86 16.36 16.96 15.81 16.43 17.03 800 15.95 16.48 17.19 15.85 16.64 17.39 400 16.04 16.66 16.86 16.29 17.02 17.98 200 16.26 17.07 18.01 16.47 17.63 18.57 150 16.49 17.30 18.03 16.84 17.72 18.95 100 16.60 17.63 18.66 17.04 18.39 19.60 75 16.85 18.22 19.22 18.19 18.70 19.93 50 16.85 18.46 19.63 17.59 18.98 20.29 25 17.67 19.13 20.50 18.15 19.91 21.13 12.5 18.67 19.98 21.21 19.17 20.78 22.06 6.25 19.40 20.96 22.28 20.16 21.85 22.99 3.125 20.35 22.02 23.55 21.14 22.79 24.24
[0238] Dissociation constant of the ssPSMA probe towards purified recombinantly prepared biotinylated Avi-tagged proteins (Avi-PSMA and Avi-GCPIII) was determined in a similar manner. Purified Avi-GCPIII was prepared according to the procedure described in (Tykvart et al. 2014, Prostate, in press), the concentration of purified protein was determined by amino acid analysis on Biochrom30 (Biochrom) according to the manufacturer's instructions, aliquots of protein stock solution were stored at −80° C. GCPIII is a close human homologue of PSMA having very similar enzyme activity and is therefore another suitable target for quantification. The procedure was identical to the procedure described at the beginning of this paragraph; only in the first step, a solution of neutravidin (Pierce, cat. no. 31000) in TBS at concentration of 10 ng.Math.μl.sup.−1 was applied to the wells instead of the antibody. Following steps were the same, only instead of rhPSMA, aforementioned Avi-PSMA diluted in TBST′ to a concentration of 0.24 pg.Math.μl.sup.−1 or Avi-GCPIII diluted in TBST′ to a concentration of 100 pg.Math.μl.sup.−1 were applied. The determined dissociation constant of the ssPSMA probe was 160 pmol.Math.l.sup.−1 towards Avi-PSMA (140 pmol.Math.l.sup.−1 by enzyme kinetics) and 1700 pmol.Math.l.sup.−1 towards Avi-GCPIII, meaning that the probe effectively binds also into the active site of Avi-GCPIII.
1f: Determination of Concentrations of PSMA and its GCPIII Homologue in a Solution
[0239] In individual tests to determine PSMA concentrations, either 10 μl of the 2G7 antibody solution or 10 μl of neutravidin solution, both at the concentration of 10 ng.Math.μl.sup.−1 in TBS, were applied to the bottom of wells in a 96-well plate FrameStar 480/96 and incubated at room temperature for 30 to 120 minutes. Content of the wells was then tapped out and the wells were washed three times with 200 μl of TBS. 100 μl of casein blocking agent five times diluted in TBS was then applied to the bottom of the wells and incubated 1-15 hours at room temperature. Content of the wells was tapped out again and the wells were washed three times with 200 μl of TBST. 10 μl of TBST′ solution with variously concentrated proteins to be determined was then added to the bottom of the wells. After 60 to 120 minutes of incubation at room temperature, the content of the wells was tapped out and the wells were washed five times with 200 μl of TBST. Finally, 10 μl of TBST′ solution of the detection probe was added to the bottom of wells and incubated for 15-75 minutes at room temperature. Content of the wells was tapped out and the wells were washed ten times with 200 μl of TBST. 10 μl of a qPCR mixture of the same composition as in the case of no template control in Example 1d was then added to the bottom of the wells and subsequently the amount of bound detection probe was determined by qPCR the same way as described in Example 1d.
[0240] In the first embodiment, the 2G7 antibody was applied to the wells, and after blocking the surface, 10 μl of rhPSMA solution in a concentration range of 1 ng.Math.μl.sup.−1 to 0.1 fg.Math.μl.sup.−1 (prepared by a dilution series of the purified rhPSMA of known concentration in TBST′ buffer) was added. For the detection, 100 pmol.Math.l.sup.−1 solution of ssPSMA in CaSDS, 1000 pmol.Math.l.sup.−1 solution of dsbiotPSMA in CaSDS and 60 pmol.Math.l.sup.−1 solution of Neu_dsbiotPSMA in CaSDS were successively tested. The Neu_dsbiotPSMA detection probe was prepared by mixing 3 μl of a neutravidin solution at a concentration of 1 mg.Math.ml.sup.−1 with 20 μl of a solution of biotinylated dsbiotPSMA detection probe at a concentration of 10 μmol.Math.l.sup.−1 (corresponding to a fourfold molar excess compared to neutravidin) in TBS buffer. After overnight incubation on ice, the resulting complex was purified from any excess free dsbiotPSMA probe by ultrafiltration on Amicon Ultra 0.5 ml 100K; the retentate volume was diluted tenfold in TBS, twice consecutively. The final concentration of the detection probe in the complex was determined by qPCR by comparison with a standard dilution series of ssPSMA as described in Example 1d.
[0241] It was found that using the solution of dsbiotPSMA detection probe in CaSDS at a concentration of 1000 pmol.Math.l.sup.−1, concentration of rhPSMA can be determined throughout the test range, i.e. from 10 ng to 1 fg with linear range of the determination being approximately six orders of magnitude (from 1 ng to 1 fg; the value of R.sup.2 of reliability of logarithmic fitting of the results from 7 concentrations in this range was 1.00, as calculated in Microsoft Office Excel 2003, see
TABLE-US-00003 TABLE 3 C.sub.q Values measured with single- and double-stranded probes (ssPSMA and dsbiotPSMA) depending on the amount of rhPSMA ssPSMA in CaSDS, dsbiotPSMA in CaSDS, amount of rhPSMA, pg 100 pmol .Math. l.sup.−1 1000 pmol .Math. l.sup.−1 10000 N/A 8.20 1000 N/A 9.22 316 11.55 N/A 100 12.47 11.23 32 13.97 N/A 10 15.40 14.66 3.2 17.15 N/A 1.0 18.58 18.18 0.32 20.34 N/A 0.10 22.14 21.21 0.032 23.87 N/A 0.010 25.26 24.78 0.0032 26.92 N/A 0.0010 27.88 27.47 0 29.91 29.84
[0242] As demonstrated in the previous example 1e, the detection probe for PSMA binds also into the active site of its close homologue GCPIII. Selectivity of the determination was therefore tested by the same procedure as above; besides the 10 μl of rhPSMA solution, 10 μl of a solution of purified tagged and biotinylated Avi-GCPIII, or 10 μl of solution of purified extracellular portion of the human GCPIII prepared by recombinant expression (hereinafter rhGCPIII, prepared according to the procedure in (Hlouchová et al. 2007, Journal of Neurochemistry, p. 682)), were applied to the other wells, both at various concentrations ranging from 1 ng.Math.μl.sup.−1 to 0.1 fg.Math.μl.sup.−1. A solution of ssPSMA probe in CaSDS at a concentration of 1000 pmol.Math.l.sup.−1 was used for the detection. While rhPSMA was detectable even at the lowest application quantity of 1 fg, with all applied quantities of Avi-GCPIII or rhGCPIII except the two highest (1 and 10 ng), there was no detectable difference from the wells without any analyte. The amount of bound detection probe in the wells with 10 ng of said proteins approximately corresponded to the amount of bound probe in the well with 10 fg of PSMA; that means that the determination of PSMA would lead to false positive results only in case that the concentration of GCPIII in the analysed sample would be at least about six orders of magnitude higher than the concentration of PSMA. Moreover, further order increase in the selectivity can be achieved with a tenfold reduction in the concentration of detection probe down to the K.sub.d of the probe towards PSMA, as the K.sub.d of the probe towards GCPIII is nearly twenty times higher. This example shows that in combination with a selective antibody, extraordinary high selectivity can be achieved even with a probe binding to more analytes.
[0243] In another embodiment, neutravidin solution was applied into the wells in the first step and after blocking the surface, 10 μl solution of purified tagged and biotinylated Avi-PSMA, or purified tagged and biotinylated Avi-GCPIII diluted in TBST′ was applied into the wells in various concentrations. The ssPSMA probe solution at a concentration of 1000 pmol.Math.l.sup.−1 in CaSDS was used for detection of Avi-PSMA, and at a concentration of 10000 pmol.Math.l.sup.−1 in CaSDS for detection of Avi-GCPIII. Linear range of Avi-PSMA determination was in the range of 10 ng to 100 fg, while linear range of Avi-GCPIII determination was in the range of 10 ng to 10 pg. Measured values of C, depending on the amount of Avi-PSMA or Avi-GCPIII are summarized in Table 4.
TABLE-US-00004 TABLE 4 Measured values of C.sub.q depending on the amount of Avi-PSMA or Avi-GCPIII: Quantity, pg Avi-PSMA Avi-GCPIII 10000 9.65 11.71 1000 13.02 15.37 100 16.72 19.11 10 19.88 21.77 1 23.26 23.59 0.1 26.20 23.95 0.01 25.81 23.98 0.001 28.04 24.00 0 27.24 22.13
[0244] Controls confirming the selectivity of the analyte determination were included in all assays; in the control wells, no antibody or no neutravidin for the selective immobilization of the test protein were applied to the wells and the surface was only blocked, in which cases the amount of bound probe corresponding to wells without the analyte was observed. Alternatively, detection probe was added to the wells in a solution containing a known competitive inhibitor of PSMA or GCPIII at concentrations significantly higher than their inhibition constants, leading to a strong decline in bound probe compared to wells without the inhibitor.
[0245] In another embodiment, elution of probe selectively bound to the active site of PSMA was tested; procedure was the same as described at the beginning of the example. 2G7 antibody was first immobilized on the surface of the wells, and after blocking the surface, besides TBST′ buffer alone, decimal dilution series of rhPSMA in TBST′ from 2 pg to 2 fg of rhPSMA was applied to the wells. Solution of ssPSMA in CaSDS at a concentration of 100 pmol.Math.l.sup.−1 was added to the wells for the detection. Finally, after washing the unbound probe away, 10 μl of TBST′ buffer was added in some wells. After 1 hour at room temperature, the solution was collected from these wells (hereinafter “elution”) and wells were washed again ten times with 200 μl of TBST. Only then, 10 μl of a qPCR mixture of the same composition as in the case of no template control in Example 1d were added to the wells, and subsequently the amount of bound probe was determined by qPCR by the same procedure as described in Example 1d. In addition, the amount of probe in 1 μl of collected elution was determined in the same manner (10 μl of the qPCR mixture of the same composition as above was applied to a clean well, 1 μl of the elution was added and the amount of detection probe was determined by qPCR as above). Comparing the amount of bound detection probe in the wells eluted with TBST′ and in the non-eluted wells showed that approximately 50% of the detection probe was released from the surface within one hour, which was in accordance with the measured concentration of the probe in the elution. C, measured depending on the PSMA concentration and the determination process are summarized in Table 5.
TABLE-US-00005 TABLE 5 C.sub.q measured depending on the PSMA concentration and the determination process Quantity of rhPSMA, fg non-eluted eluted elution 2000 18.88 19.60 23.88 200 22.35 23.50 27.45 20 25.73 26.68 30.61 2 28.85 29.76 32.85 0 29.96 31.91 34.79
[0246] It is clear that the linear range and detection limit are the same when determining the eluted probe as when determining the probe bound to a solid carrier. This procedure can thus be used to release the bound probe to a solution allowing determination in other types of solid carriers than the one to which the immobilization was done.
1g: Determination of the Limit of Detection of PSMA in a Solution
[0247] 10 μl of 2G7 antibody solution at a concentration of 2.5 ng.Math.μl.sup.−1 in TBSE buffer (TBS with EDTA at a concentration of 5 mmol.Math.l.sup.−1) was applied to the bottom of the wells of a 96-well plate FrameStar 480/96 and incubated at room temperature for 6 hours. Content of the wells was then tapped out and the wells were washed three times with 200 μl of TBS. 200 μl of casein blocking agent five times diluted in TBSE was then applied to the bottom of the wells and incubated for 18 hours at room temperature. Content of the wells was tapped out and the wells were washed five times with 200 μl of TBST. Thereafter, either 10 μl of TBST′ buffer or 10 μl of rhPSMA solution in TBST′ of known concentration of rhPSMA in the range of 0.1 fg.Math.μl.sup.−1 to 0.5 ag.Math.μl.sup.−1, i.e. in a total amount of rhPSMA between 1 fg to 5 ag, were applied to the bottom of wells wherein each concentration was applied at least in triplicate. After 3 hours incubation at room temperature the content of the wells was tapped out and the wells were washed three times with 200 μl TBST. Finally, 10 μl of solution of detection probe dsA3PSMA(1:1.2) in CaSDS buffer at the probe concentration of 75 pmol.Math.l.sup.−1 was applied to the bottom of wells and incubated for 1 hour at room temperature. The dsA3PSMA(1:1.2) probe was prepared by the same procedure as the dsA3PSMA probe with the difference that the ssPSMA probe was paired with a 1.2 molar excess of the complementary strand, at a concentration of ssPSMA 1 nmol.Math.l.sup.−1. Contents of the wells was subsequently tapped out and the wells were washed ten times with 200 μl TBST. 10 μl of a qPCR mixture of the same composition as in the case of no template control in Example 1d was then applied to the bottom of the well and the quantity of bound detection probe was subsequently determined by qPCR in the same way as described in Example 1d. Measured C.sub.q values depending on the amount of applied rhPSMA are summarized in Table 6 and
TABLE-US-00006 TABLE 6 Measured C.sub.q values depending on the amount of applied rhPSMA quantity of rhPSMA, fg C.sub.q (1) C.sub.q (2) C.sub.q (3) 0 35.74 36.44 35.60 0 36.10 35.32 34.65 0 34.64 35.03 35.36 0.005 34.52 34.68 35.01 0.005 35.41 34.92 35.06 0.010 34.93 34.86 34.58 0.025 34.17 34.19 33.41 0.050 33.45 34.30 34.83 0.10 33.55 33.25 32.94 0.25 32.44 32.02 32.38 0.50 31.72 31.44 31.01 1.00 30.51 30.68 29.15
[0248] Each tested sample was measured in triplicate, 5 ag sample in six copies and zero sample in nine copies, so these samples have more rows in the table.
1h: Determination of PSMA Concentration in Complex Biological Matrices
[0249] 10 μl of 2G7 antibody solution at 5 ng.Math.μl.sup.−1 in TBS was loaded to the bottom of the wells of a 96-well plate FrameStar 480/96 and incubated at room temperature for 1 to 1.5 hours. Content of the wells was then tapped out and the wells were washed three times with 200 μl of TBS. then 100 μl of casein blocking agent five times diluted in TBS was applied to the bottom of the wells and incubated for 24 hours at room temperature. Content of the wells was tapped out and the wells were washed three times with 200 μl TBST. Thereafter, either 10 μl of rhPSMA standard solution at 12 different concentrations in TBST′ (range 32 pg.Math.μl.sup.−1 to 0.1 fg.Math.μl.sup.−1), or analysed samples of cell lysate, urine and blood plasma in various dilutions in TBST′ buffer, were added to the bottom of the wells. After 1.5 hours incubation at determining PSMA in urine and cell lysate or after 18 hours incubation at the determining PSMA in blood plasma, always at room temperature, the content of the wells was tapped out and the wells were washed five times with 200 μl TBST. Finally, 10 μl solution of ssPSMA detection probe in CaSDS buffer at the probe concentration of 1000 pmol.Math.l.sup.−1 was added to the bottom of wells and incubated for 1 hour at room temperature. Content of the wells was subsequently tapped out and the wells were washed ten times with 200 μl of TBST. 10 μl of a qPCR mixture of the same composition as in the case of no template control in Example 1d was then added to the bottom of the wells and subsequently the amount of bound detection probe was determined using qPCR as described in Example 1d.
[0250] The linear range of the detection was according to the standard dilution series of the rhPSMA the same as in the previous example. The concentration of PSMA in the biological samples was then calculated from the obtained calibration curves (dependence of C.sub.q on the logarithm of rhPSMA concentration fitted with linear function) and from knowledge of dilution of the analysed biological samples. First, the PSMA concentrations in 15 samples of citrate plasma of healthy donors was determined; wherein the final value was the average of concentrations determined in a ten-, hundred- a thousand-fold diluted plasma samples of individual donors. PSMA concentrations determined from various dilutions were identical to each other (except for minor variations); moreover, the determined amount of PSMA were always well above the limit of detection (C.sub.q difference of at least three cycles compared to null controls), even for the most diluted samples with the lowest concentrations of PSMA. This means that plasma volume of no more than 10 nl is sufficient for the described determination of PSMA. Furthermore, the selectivity of binding of the detection probe via its ligand portion was verified both by a suppressed binding to the surface of the wells with analysed samples when a competitive inhibitor of PSMA was added to the applied probe solution; and further by the fact that binding of oligonucleotide iqPCR_amino without the ligand portion to the surface of the wells with analysed samples did not exceed non-selective binding to the surface without a sample. The measured concentrations were then compared with the concentrations measured in the same samples using radio-enzymatic assay (procedure of collecting and processing citrate plasmas and determination of the PSMA concentration in these samples by means of a radio-enzymatic assay are described in (Knedlík et al. 2014, Prostate, p. 768). Although the absolute values determined by the method disclosed herein were approximately ten times smaller compared to the radio-enzymatic determination (Table 7), values from the two methods are very well correlated. This can be seen from the graphical plot comparing the results of both methods in
TABLE-US-00007 TABLE 7 Comparison of PSMA concentration measured by a reference method and by our method concentrations of PSMA (ng .Math. ml.sup.−1) measured by: a method according to the invention radio-enzymatically sample 1 0.43 5.7 sample 2 0.40 3.7 sample 3 0.14 1.4 sample 4 0.11 1.3 sample 5 0.26 3.0 sample 6 0.23 3.2 sample 7 0.94 9.9 sample 8 0.37 4.6 sample 9 0.41 4.0 sample 10 1.37 17 sample 11 0.25 3.4 sample 12 0.15 1.4 sample 13 0.18 2.3 sample 14 0.15 1.5 sample 15 0.20 2.4
[0251] The concentration of PSMA in urine samples ten- and a hundred-fold diluted in TBST′ was also determined with the described method; variation between measured concentrations were very small, typically ten percent. In two samples of urine from patients suffering from prostate cancer, the measured concentrations of PSMA were 33 and 192 pg.Math.ml.sup.−1; in urine of a healthy male, the concentration was 15 pg.Math.ml.sup.−1, while in the urine of healthy female, it was just 8 pg.Math.ml.sup.−1. Although 1 μl of urine was sufficient for the determination of PSMA with our method, there is currently no available reference method sensitive enough, with which the measured concentrations could be compared. However, as well as in the blood plasma, binding of the detection probe was suppressed by addition a competitive inhibitor of PSMA, which demonstrates selective binding of the detection probe to the active site of PSMA via its ligand portion.
[0252] Described method was also used to determine concentrations in lysates of cultures of cell lines derived from prostate cancer, particularly from metastatic cells LNCaP, DU-145 and PC-3. Cells grown at 37° C. in an atmosphere of 5% (vol./vol.) CO.sub.2 in RPMI medium (Sigma, cell line LNCaP) or IMDM medium (Invitrogen) supplemented with 10% (vol./vol.) FBS (Sigma) in Petri dishes of 100 mm diameter, designed for tissue cultures (SPL Life Sciences) were after reaching approximately 90% confluence resuspended in this medium, transferred to a microtube and centrifuged for 5 min at 250 g at room temperature. The medium was then removed and the cells were washed with 50 mmol.Math.l.sup.−1 Tris with 100 mmol.Math.l.sup.−1 NaCl at pH 7.4 (hereinafter referred to as CLP). Approximately 20 million cells were then suspended in 300 μl of CLP and transferred into 2 ml microtube with round bottom; and a steel ball with a diameter of approximately 3 mm was added to them. Cells were subsequently lysed and homogenized in a Tissue Lyzer (Qiagen; three minutes at maximum power). Solution was then transferred to a new tube, 1/10 volume of 10% (wt./vol.) octaethylenglycolmonododecyl ether (Affymetrix, cat. No. 0330; C.sub.12E.sub.8) was added and after mixing, the solution was sonicated for 1 min in ice-cold sonication bath Elmasonic S30. The resulting solution was centrifuged for 15 min/600 g/4° C. and supernatant was collected, which represents the lysate. The total protein concentration in the lysate was determined using the Biorad Protein Assay reagent. PSMA concentration was determined at various lysate dilutions in TBST′; applied amount of total protein ranged from 100 ng to 100 pg. Measured concentration of PSMA in LNCaP line was 0.27 ng/μg total protein, whereas in lines DU-145 and PC-3, PSMA was not detectable, which represented a concentration of less than 0.1 pg/ng of total protein under the given conditions of determination. Binding of the detection probe was also suppressed by adding a competitive inhibitor of PSMA to the solution of the detection probe, and the measured concentrations were also in accordance with the determination by Western blotting.
1i: Testing the Inhibitory Potency of Compounds Towards Various Forms of PSMA and Towards its Homologue GCPIII
[0253] 10 μl of a neutravidin solution at 10 ng.Math.μl.sup.−1 in TBS buffer was applied to the bottom of the wells of a 96-well plate FrameStar 480/96 and incubated at room temperature for 1.5 hours. Contents of the wells was then tapped out and the wells were washed three times with 200 μl of TBS. 200 μl of casein blocking agent five times diluted in TBS was then applied to the bottom of the wells and incubated for 24 hours at room temperature. Content of the wells was tapped out and the wells were washed three times with 200 μl TBST. Thereafter, 10 μl of the standard solutions with known concentrations of Avi-PSMA (50 pg.Math.μl.sup.−1) or Avi GCPPIII (2 ng.Math.μl.sup.−1) in TBST′ were added to the bottom of wells; zero controls with buffer alone were also included. After 2 hours incubation at room temperature the contents of the wells was tapped out and the wells were washed five times with 200 μl TBST. Then, 10 μl of ssPSMA detection probe solution in TBST′ buffer was applied to the bottom of the wells, at the concentration of the probe 200 pmol.Math.l.sup.−1 for Avi-PSMA wells, or the concentration of 1000 pmol.Math.l.sup.−1 to the Avi-GCPIII wells. Subsequent incubation was carried out for 1 hour at room temperature. Contents of the wells was subsequently tapped out and the wells were washed ten times with 200 μl of TBST. 10 μl of a qPCR mixture of the same composition as in the case of no template control in Example 1d was then added to bottom of the wells and the quantity of bound detection probe was determined by qPCR as described in Example 1d.
[0254] Adding a certain concentration of a tested substance to the detection probe allowed deriving the inhibition constant of the substance from the decrease in bound detection probe amount. The tested substance was always added in one concentration only, and the assay was performed in duplicate. 15 substances have been selected for testing; their inhibitory constant towards Avi-PSMA was also measured by enzymatic assay described in Example 1c. Their inhibition constants were in the range of tens pmol.Math.l.sup.−1 to hundreds μmol.Math.l.sup.−1. Subtracting C.sub.q measured in the wells with enzyme and pure detection probe from C.sub.q measured in the wells with the same enzyme and the detection probe, accompanied by the tested substance, ΔCq was calculated from which the percentage of the active sites of enzyme occupied with the tested substance was derived, using the formulas (9) and (14); qPCR efficiency has been replaced by the value of one. ΔCq obtained by subtracting C.sub.q measured in the wells with enzyme from C.sub.q measured in zero controls (wells without enzyme) was used to determine the maximum extent of determining the percentage quantity of occupied active sites of the enzyme.
[0255] By substituting the concentration of tested substance, ΔC.sub.q, K.sub.d of the detection probe identified in Example 1e (160 pmol.Math.l.sup.−1 for ssPSMA and Avi-PSMA in TBST′; 1700 pmol.Math.l.sup.−1 for ssPSMA and Avi-GCPIII in TBST′) and the concentration of the detection probe into the equation (15), the value of the inhibition constant of the substance was determined. Table 8 summarizes the concentrations of the tested substances used in the determination, the percentage of active sites of the enzyme occupied by them, measured inhibition constants towards the given enzyme and reference values of inhibition constants measured with enzyme kinetics described in Example 1c. Reference values for the Avi-GCPIII enzyme are not known because greater amount of the enzyme would be necessary for measuring K.sub.i of several substances, than was available. The table shows that for Avi-PSMA, deviation between the two methods is at most tens of percent for the highly-binding inhibitors; and for the six weakest-binding inhibitors, the constants determined by the method disclosed herein are two to four times higher. Excellent agreement between the two methods is evident from a graphical comparison of the results of both methods shown in
[0256] Minor differences between the two methods can be due to inaccuracy in our determination or inaccuracies in the determination of enzyme kinetics. Our determination could be refined by measuring multiple replicates, either with the same or different concentrations of the tested substances, and averaging the resulting values. Errors in the determination by enzyme kinetics arise mainly from inaccurate determination of substrate K.sub.M (systematic shift in all K.sub.i) and incorrect fitting of the reaction rate dependence on the inhibitor concentration. At this point it should be stressed that even a properly measured K.sub.i value cannot be considered as a proper physical constant, since the properties of enzymes, in our context particularly the affinity of the enzyme to the substrate and the rate of the enzyme catalysed reaction, is fundamentally dependent on the composition of the solution. Measured K.sub.i thus generally strongly depends on the particular pH, temperature, buffer substance used, the ionic strength of the solution, the nature of the ions in the solution, various additives (e.g. detergents), and other influences. Even if K.sub.i is determined by enzyme kinetics at very similar conditions, results are quite often different, for example as seen in the K.sub.i determination of a known inhibitor of 2-PMPA. In the work described in (Jackson et al. 1996, Journal of Medicinal Chemistry, p. 619) K.sub.i of 2-PMPA was determined as 0.3 nmol.Math.l.sup.−1, whereas in (Kozikowski et al. 2004, Journal of Medicinal Chemistry, p. 1729) it was determined as 1.4 nmol.Math.l.sup.−1. In our assay, another buffering agent and another detergent were used compared to the enzyme kinetics, and they were also in higher concentrations, which might contribute to the observed small differences in the results.
[0257] In a similar manner, with very similar results, inhibitory potency of substances was tested for unpurified recombinant PSMA with N-terminally attached His-tag. The procedure was identical to the procedure described above for Avi-PSMA, but after the immobilization of neutravidin, there was one additional step of one hour incubation of the wells with 10 μl tris-nitrilotriacetic acid with covalently attached biotin (biotin-tris-NTA) at a concentration of 10 nmol.Math.l.sup.−1 in the presence of NiCl.sub.2 at a concentration of 1 mmol.Math.l.sup.−1 in TBST′ buffer, and only after washing, the step of incubation with His-tagged PSMA followed.
TABLE-US-00008 TABLE 8 Comparison of K.sub.i values and the percentage of occupied active sites identified by the method according to the invention and a reference method measuring enzyme kinetics Avi-PSMA Concentration K.sub.i Avi-GCPIII of the Percentage K.sub.i (enzyme Percentage K.sub.i substance, of occupied determined, kinetics), of occupied determined, Designation μmol .Math. l.sup.−1 active sites nmol .Math. l.sup.−1 nmol .Math. l.sup.−1 active sites nmol.l.sup.−1 Substance 1 1 99.86 0.7 0.4 96.4 24 Substance 2 1 99.964 0.2 0.2 99.18 5 Substance 3 1 89 56 40 54 570 Substance 4 1 99.982 0.09 0.06 86 110 Substance 5 1 99.991 0.04 0.09 95.0 35 Substance 6 1 92.9 36 50 40 1 000 Substance 7 1 99.915 0.4 0.3 95.5 31 Substance 8 1 99.63 2 2 87 96 Substance 9 1 99.81 1 1 96.3 26 Substance 10 113 99.0 550 260 88 10 000 Substance 11 85 75 13 000 4 200 85 9 900 Substance 12 1 000 60 310 000 230 000 22 ~2 400 000 Substance 13 1 000 86 76 000 31 000 60 440 000 Substance 14 1 000 94.9 25 000 12 000 38 ~1 100 000 Substance 15 1 000 82 100 000 23 000 64 380 000
[0258] The different number of digits for the percentage of occupied active sites by the tested substance corresponds to different measurement accuracies at different occupancy percentage. K.sub.i derived from occupancy percentage less than 50 percent are considered as less reliable. Important is also that by means of said testing, first inhibitors selective for Avi-PSMA compared to Avi-GCPIII were found, wherein the inhibitory potency of tested substance 4 towards Avi-GCPIII was verified by enzyme kinetics (measured K.sub.i=140 nmol.Math.l.sup.−1).
[0259] The fact that the tested substances are usually dissolved in various organic solvents, has led to testing the reliability of our method in the presence of acetonitrile, methanol, dimethyl sulfoxide (DMSO) and the detergent Tween 20. The procedure was identical to those described above, only at the beginning, instead of the solution of neutravidin, 2G7 antibody solution at a concentration of 5 ng.Math.μl.sup.−1 in TBS was applied to the wells. Instead Avi-tagged proteins, rhPSMA solution of known concentration 2 pg.Math.μl.sup.−1 in TBST′ was then applied. The ssPSMA probe solution at a concentration of 60 pmol.Math.l.sup.−1 in TBST′, or in TBST′ with variously concentrated organic solvent, or in TBS with varying concentrations of Tween 20 was used for the detection. Zero controls without antigen were included for each detection probe solution used, and everything was measured in duplicates. Possible influence of the composition of probe diluent on the assay was determined by comparing the measured C, in wells with and without antigen. It was found that DMSO, acetonitrile or methanol did not influence the measured results at concentrations of 0.1%, 1% or 10% (vol./vol.). Similarly, various concentrations of Tween 20 in the range of 0% to 1% (vol./vol.) in the diluent had no effect on the determination. At said concentrations of DMSO, Tween 20, optionally with addition of 500-fold to 2000-fold diluted casein blocker, a set of inhibitors with respective K.sub.i values ranging from 100 pmol.Math.l.sup.−1 to 100 nmol.Math.l.sup.−1 was tested and it was found, that the additives had no effect on accuracy of the determination of respective K.sub.i values. Addition of three inhibiting substances in various concentrations was also tested; with inhibition constants of hundreds pmol.Math.l.sup.−1, tens nmol.Math.l.sup.−1 and tens nmol.Math.l.sup.−1, and it was found that within the linear range of the determination, very similar K.sub.i values are obtained, irrespective of the concentration of the tested substances.
[0260] The procedure described in the preceding paragraph allows testing of the inhibitory potency of substances also against an endogenous enzyme; and therefore inhibitory constants of set of 36 substances against not only rhPSMA but also endogenous PSMA contained in human blood plasma were determined with the same method as in the preceding paragraph. To determine the inhibitory potency towards rhPSMA, solution of rhPSMA at a concentration of 2 pg.Math.μl.sup.−1 in TBST′ was applied to the wells and the ssPSMA detection probe was used at a concentration of 60 pmol.Math.l.sup.−1 in TBST′, while for determination of inhibitory potency towards endogenous PSMA, citrate blood plasma tenfold diluted in TBST′ was applied to the wells and the ssPSMA detection probe was used at a concentration of 300 pmol.Math.l.sup.−1 in TBST′. Decrease in amount of bound detection probe was measured again at a single concentration for each tested substance only. From the measured data, we calculated ΔC.sub.q, the percentage of active site of the enzyme occupied with tested substances and the K.sub.i of the tested substances by the same procedure as previously described (K.sub.d=60 pmol.Math.l.sup.−1 for ssPSMA and rhPSMA in TBST; K.sub.d=300 pmol.Math.l.sup.−1 for ssPSMA and endogenous PSMA in TBST′, both determined in Example 1e). Concentration of the tested substance, the percentage of active sites of the enzymes occupied with them and their measured inhibition constants towards rhPSMA or endogenous PSMA are summarized in Table 9; comparison of the inhibition constants measured for rhPSMA and endogenous PSMA is plotted graphically in
[0261] Even wider range of inhibition constants of tested substances was quantitatively determined from their single tested concentration by a procedure, wherein 250 pg rhPSMA was immobilized via antibody 2G7 onto the bottom of wells in a multiwell plate and washed afterwards; the particular wells were then incubated with the mixture of particular tested substance at concentration of 100 μmol.Math.l.sup.−1 and of detection probe dsA3PSMA at concentration of 125 pmol.Math.l.sup.−1 in TBST′ buffer with addition of 500-fold diluted casein blocker. After subsequent wash, the amount of bound detection probe was determined via qPCR and respective K.sub.i values of the substances were calculated from the difference of bound probe in wells incubated with the detection probe alone and of bound probe in wells incubated with the mixture of the detection probe and particular tested substance according to formula 15 described in the description of the invention. In this manner, inhibition constants of 40 substances were determined. As determined by enzyme kinetics described in example 1c, the inhibitory potencies of the substances were approximately evenly distributed in the range of K.sub.i values ranging from 19 pmol.Math.l.sup.−1 to 250 μmol.Math.l.sup.−1 and it was found that K.sub.i values of all substances were determined very accurately by the procedure described here: the determined values corresponded on the average to 85% of the values from enzyme kinetics and they did not differ in any case more than twofold from the values from enzyme kinetics (R.sup.2=0.991). These results show, that it is possible to accurately determine the K.sub.i value of the tested substances in the range of seven logs (range of 19 pmol.Math.l.sup.−1 to 250 μmol.Math.l.sup.−1) from single tested concentration of the substances (100 μmol.Math.l.sup.−1) by the here described procedure.
TABLE-US-00009 TABLE 9 Inhibition constants of substances measured towards rhPSMA or endogenous PSMA Concentration rhPSMA endogenous PSMA of the Percentage of K.sub.i Percentage of K.sub.i substance, occupied determined, occupied determined, Designation nmol .Math. l.sup.−1 active sites nmol .Math. l.sup.−1 active sites nmol .Math. l.sup.−1 Substance 1 1000 99.959 0.21 99.65 1.8 Substance 2 1000 99.966 0.17 99.73 1.3 Substance 3 1000 95.9 22 88 70 Substance 4 1000 99.968 0.16 99.70 1.5 Substance 5 1000 99.932 0.34 99.36 3.2 Substance 6 1000 96.9 16 94.0 32 Substance 7 1000 99.915 0.43 98.5 7.9 Substance 8 1000 99.84 0.81 N/A N/A Substance 9 1000 99.928 0.36 99.55 2.3 Substance 10 100000 99.57 220 98.7 650 Substance 11 100000 89 6100 78 14000 Substance 12 1000000 57 380000 55 410000 Substance 13 1000000 73 180000 83 100000 Substance 14 1000000 86 80000 94.8 28000 Substance 15 1000000 91.7 45000 87 78000 Substance 16 1000 99.961 0.19 99.48 2.6 Substance 17 1000 99.49 2.5 98.3 8.7 Substance 18 1000 98.7 6.4 96.3 19 Substance 19 1000 98.6 7.0 95.9 21 Substance 20 1000 99.56 2.2 98.3 8.4 Substance 21 1000 99.938 0.31 99.52 2.4 Substance 22 1000 99.962 0.19 99.60 2.0 Substance 23 1000 99.86 0.72 99.31 3.5 Substance 24 1000 98.1 9.4 94.1 31 Substance 25 1000 98.9 5.5 97.1 15 Substance 26 1000 99.903 0.48 99.45 2.8 Substance 27 1000 99.930 0.35 99.35 3.3 Substance 28 1000 99.73 1.37 98.8 5.9 Substance 29 1000 99.967 0.16 99.67 1.7 Substance 30 1000 99.85 0.75 99.69 1.6 Substance 31 1000 99.60 2.0 98.9 5.7 Substance 32 1000 99.80 1.0 98.7 6.7 Substance 33 1000 99.941 0.30 99.60 2.0 Substance 34 1000000 94.9 27000 91.7 45000 Substance 35 100000 0 does not 0 does not inhibit inhibit Substance 36 100000 0 does not 0 does not inhibit inhibit
[0262] Designation of the substances meets the description in the preceding table. The different number of digits for the percentage of occupied active sites (with the tested substance) corresponds to different determination accuracies at different percentage of occupancy.
1j: Determination of PSMA in Solution Using Chemiluminescent Detection
[0263] 100 μl of the 2G7 antibody solution at a concentration of 2.5 ng.Math.μl.sup.−1 in TBS was applied to the wells of 96-well Nunc Maxisorb microplates (cat. no. 437111) and incubated at room temperature for 1 hour. Content of the wells was then tapped out and the wells were washed three times with 200 μl of TBS. 200 μl of casein blocking agent five times diluted in TBS was then applied to the wells and incubated for 18 hours and 30 minutes at room temperature. Content of the wells was then tapped out and the wells were washed three times with 200 μl TBST. 100 μl of rhPSMA standard solution of various known concentrations in TBST′, the resulting applied amount in the range of 1 ng to 1 pg, was then added to the wells. Zero controls without rhPSMA were also included, all in two replicates. After 2 hours and 45 minutes incubation at room temperature, the content of the wells was tapped out and the wells were washed three times with 200 μl TBST. Finally, 100 μl of a solution of the NeuHRP_dsbiotPSMA detection probe at a concentration of 600 pmol.Math.l.sup.−1 in CaSDS was added to the wells. NeuHRP_dsbiotPSMA detection probe was prepared by mixing 6.1 μl of neutravidin-HRP conjugate solution (Pierce, cat. no. 31001) at a concentration of 1 mg.Math.ml.sup.−1 with 10 μl solution of biotinylated detection probe dsbiotPSMA at 10 nmol.Math.l.sup.−1 (corresponding fourfold molar excess compared to neutravidin-HRP conjugate) in TBS buffer. After overnight incubation on ice, the resulting complex was purified from the remaining free dsbiotPSMA probe by ultrafiltration on a membrane with a permeability cutoff of 100 kDa; the original solution was diluted hundredfold in sum. The final concentration of the detection probe in the complex was determined by qPCR by comparison with a standard dilution series of ssPSMA as described in Example 1d. After incubation for 1 hour at room temperature, the content of the wells was tapped out and the wells were washed ten times with 200 μl of TBST. 160 μl of chemiluminescent substrate was then added to the wells (aqueous solution of 4-iodophenol (Acros Organics, cat. no. 122390100) at a concentration of 2 mmol.Math.l.sup.−1, luminol (5-amino-2,3-dihydro-1,4-phthalazinedion, Sigma Aldrich, cat. no. A8511) at a concentration of 2.5 mmol.Math.l.sup.−1; 3.2% DMSO (vol./vol.), 0.02% (wt./vol.) of hydrogen peroxide and 0.1 mol.Math.l.sup.−1 Tris-HCl, pH 8.0) and the luminescence was measured in each well using a Tecan reader Infinite M1000.
[0264] The dynamic range of detection was observed in the range of the applied amount of rhPSMA 1 ng to 1 pg. Detection limit of 1 pg indicates that it is a more sensitive determination than nowadays the most sensitive available determination of PSMA by ELISA (Sokoloff et al. 2000, Prostate, p. 150). Measured values of the luminescence duplicate measurements are summarized in Table 10.
TABLE-US-00010 TABLE 10 Determination of rhPSMA in a using chemiluminescent detection amount of luminescence (1), luminescence (2), rhPSMA, pg relative units relative units 0 2071 2011 1 2455 2477 10 17055 16840 100 308210 320440 1000 4059300 4351800
1k: Determination of PSMA Catalytic Activity for Substrate Hydrolysis
[0265] Following the procedure described in section 1i, 20 pg of Avi-PSMA was immobilized to the bottom of the wells via immobilized neutravidin and subsequently incubated with the dsA3PSMA detection probe at a concentration of 35 pmol.Math.l.sup.−1 and simultaneously with various concentrations of the folyl-γ-L-glutamate substrate in the range of 10 nmol.Math.l.sup.−1 to 100 μmol.Math.l.sup.−1 for 40 minutes, and after washing, the amount of bound probe was determined with qPCR. K.sub.M of the substrate (corresponding to IQ was calculated from the ΔC.sub.q difference between wells with the highest concentration of the substrate and the wells with only the detection probe according to the equation (15). Based on the this calculated K.sub.i and the ΔC.sub.q difference measured for each initial concentration of the substrate, final substrate concentrations at the end of incubation, S.sub.t, were then calculated according to the same formula, and according to equation (17) described above in the description of the invention, catalytic efficiency k.sub.cat was calculated. At an initial concentration of folyl-γ-L-glutamate 107 nmol.Math.l.sup.−1, 80% is cleaved during incubation, which corresponds to a k.sub.cat of 1.2 s.sup.−1. At a concentration of 336 nmol.Math.l.sup.−1, 43% was cleaved, corresponding to k.sub.cat of 2.3 s.sup.−1 and at a concentration of 1049 nmol.Math.l.sup.−1, 16% was cleaved, corresponding to k.sub.cat of 2.6 s.sup.−1. Obtained k.sub.cat values are in conformity with the k.sub.cat value of 5 s.sup.−1 obtained from enzyme kinetics as described in Section 1c.
Example 2: Detection of HIV-1 Protease, Testing Potency of HIV-1 Protease Inhibitors
2a: Preparation of an HIV-1 Protease Inhibitor with a Linker and an Activated NHS Ester
[0266] The detection probe for HIV-1 protease was prepared by linking of a HIV-1 protease inhibitor with linker with terminal NHS-ester (Compound 7) with the amino group of the DNA oligonucleotide. Compound 8, prepared by reaction of Compound 7 with ethanolamine was used for determination of the impact of linking of the DNA oligonucleotide on the inhibition potency. All compounds were purified and characterized as described in example 1a.
[0267] Ritonavir (RTV, available under the brand name Norvir from Abbott Laboratories) was isolated from commercially available capsules in which RTV is suspended in an oily mixture of rather nonpolar compounds. 50 tablets (100 mg RTV each) were cut open and the oily substance was squeezed out into a bottom round shaped 21 flask. 200 ml of hexane was added along with 500 ml of diethylether. The resulting suspension was triturated and sonicated for 3 hours until all oil turned into a white precipitate or was dissolved in solvents. This precipitate was filtered and again triturated/sonicated in pure diethylether, after which the pure RTV was filtered. 3.6 g of RTV was obtained (yield 72%). The purity of RTV was determined by HPLC and was well above 99%.
[0268] Preparation of thiazol-5-ylmethyl ((2S,3S,5S)-5-amino-3-hydroxy-1,6-diphenylhexan-2-yl)carbamate (Compound 5) by partial hydrolysis of ritonavir (RTV): 1.00 g of RTV was dissolved in 50 ml of dioxane in a bottom round flask. 50 ml of concentrated hydrochloric acid was added and the resulting mixture was stirred at 65° C. for 20 hours (note that different temperature and/or time lead to different cleavage products). After 20 hours the mixture was let to cool down to RT. The mixture was neutralized by addition of K.sub.2CO.sub.3 until the resulting mixture showed basic pH. The solvents were concentrated using rotary evaporater to roughly 50 ml and the slurry was diluted by 150 ml of water and washed 3 times by 100 ml of EtOAc. The water phase was discarded and organic phase was dried and evaporated. 885 mg of crude mixture was obtained and was used in the next reaction without further purification (purity approx. 80% as determined by analytical HPLC).
[0269] For spectral determination, 50 mg were purified using preparative HPLC (gradient: 20-50% (vol./vol.) ACN in 40 minutes, RT 15 minutes). Analytical HPLC RT=17.3 min.
[0270] Result of analysis by NMR (500 MHz, DMSO-d6): δ 9.06 (d, .sup.4J=0.8, 1H, N—CH—S), 7.84 (q, .sup.4J=0.8, 1H, S—C—CH—N), 7.81 (bs, 3H, NH.sub.3+), 7.32-7.15 (m, 10H, 2×Ph-), 7.20 (bs, 1H, NH), 5.50 (bs, 1H, OH), 5.15 (dd, J.sub.gem=13.2, .sup.4J=0.8, O—CH.sub.2), 5.11 (dd, 1H, J.sub.gem=13.2, .sup.4J=0.8, COO—CH.sub.2), 3.69 (m, 1H, HO—CH), 3.67 (m, 1H, HO—CH—CH—NH), 3.50 (bm, 1H, NH.sub.3.sup.+—CH), 2.87 (dd, 1H, J.sub.gem=14.0, J=6.4, NH.sub.3.sup.+—CH—CH.sub.2-Ph), 2.80 (dd, 1H, J.sub.gem=14.0, J=7.3, 1H, NH.sub.3.sup.+—CH—CH.sub.2-Ph), 2.79 (dd, 1H, J.sub.gem=13.7, J=3.7, 1H, NH—CH—CH.sub.2-Ph), 2.79 (dd, J.sub.gem=13.7, J=10.5, 1H, NH—CH—CH.sub.2-Ph), 1.58 (bs, 2H, OH—CH—CH.sub.2—CH).
[0271] Result of analysis by .sup.13C NMR (125.7 MHz, DMSO-d6): δ 155.39 (O—C—N), 155.77 (N—CH—S), 143.23 (S—C—CH—N), 139.52 (Ph), 136.37 (Ph), 134.14 (S—C—CH—N), 129.61 (Ph), 129.18 (Ph), 128.81 (Ph), 128.23 (Ph), 127.07 (Ph), 126.12 (Ph), 69.81 (HO—CH), 57.49 (COO—CH.sub.2), 56.94 (HO—CH—CH—NH), 50.87 (NH.sub.3.sup.+—CH), 38.71 (NH.sub.3.sup.+—CH—CH.sub.2-Ph), 35.69 (NH—CH—CH.sub.2-Ph), 34.66 (CH—CH.sub.2—CH).
[0272] Result of analysis by HRMS (ESI+): calculated mass of C.sub.23H.sub.28O.sub.3N.sub.3S [M].sup.+426.18459; detected mass 426.18454.
[0273] Preparation of (S)-1-(((2S,4S,5S)-4-hydroxy-1,6-diphenyl-5-(((thiazol-5-ylmethoxy)carbonyl)amino) hexan-2-yl)amino)-3-methyl-1-oxobutan-2-aminium 2,2,2-trifluoroacetate (compound 6): 526 mg (1.64 mmol, 1 eq) of TBTU was added to 356 mg (1.64 mmol, 1 eq) BOC-Val, dissolved in 1.5 ml of DMF along with 690 μl of DIEA (3.94 mmol, 2.4 eq). The crude hydrolysate of RTV (700 mg, 1.64 mmol, 1 eq), dissolved in 1 ml of DMF, was added after 5 minutes of stirring in one portion. The reaction was left overnight and the DMF was rotary evaporated. The reaction mixture was dissolved in 50 ml of EtOAc and washed two times by saturated NaHCO.sub.3, two times by 10% KHSO.sub.4 (wt./vol.) and once with brine. The organic mixture was dried, evaporated and the product was purified using Flash chromatography (TLC analysis: EtOAc, Rf=0.65). Product was further dissolved in 5 ml of hot EtOAc and 5 ml of diethyl ether were added. The resulting gel was filtrated and dried to give very pure (>99%, HPLC) 250 mg of product. The BOC protected compound was dissolved in pure TFA and sonicated for 10 minutes. The TFA was removed by flow of nitrogen and the resulting oil was dissolved in water/ACN and lyophilized to remove residual TFA. Overall yield: 25% (the low yield was due to discarded fractions with impurities from TLC).
[0274] Analytical HPLC RT=17.4 min.
[0275] Result of analysis by .sup.1H NMR (500 MHz, DMSO-d6): δ 9.06 (d, .sup.4J=0.8, 1H, N—CH—S), 8.24 (d, J=8.2, 1H, —NH—CO), 8.00 (bd, J=5.2, 3H, —NH.sub.3.sup.+), 7.85 (q, .sup.4J=0.8, 1H, S—C—CH—N), 7.28-7.13 (m, 10H, 2×Ph-), 6.94 (d, J=9.4, 1H, NH—CO—O), 5.12 (d, .sup.4J=0.8, 2H, O—CH.sub.2), 4.16 (m, 1H, CH—NH—CO), 3.78 (m, 1H, CH—NH.sub.3.sup.+, partial overlap with water residual peak), 3.58 (td, J=6.8, J=2.0, 1H, CH—OH), 3.48 (m, 1H, Ph-CH.sub.2—CH—NH), 2.72-2.67 (m, 4H, 2×CH—CH.sub.2-Ph), 2.00 (m, 1H, CH—(CH.sub.3).sub.2), 1.50 (m, 1H, OH—CH—CH.sub.2), 1.43 (m, 1H, OH—CH—CH.sub.2), 0.89 (d, J=6.8, 3H, —CH.sub.3), 0.84 (d, J=6.8, 3H, —CH.sub.3).
[0276] Result of analysis by .sup.13C NMR (125.7 MHz, DMSO-d6): δ 167.33 (CO Val), 158.33 (q, J.sub.C,F=34.4, CF.sub.3COO—), 155.79 (O—C—N), 155.71 (N—CH—S), 143.23 (S—C—CH—N), 139.50 (Ph), 138.55 (Ph), 134.23 (S—C—CH—N), 129.56 (Ph), 129.17 (Ph), 128.30 (Ph), 128.25 (Ph), 126.26 (Ph), 126.09 (Ph), 116.44 (q, J.sub.C,F=294.8, CF.sub.3—COO.sup.−), 68.90 (HO—CH), 57.56 (CO—CH—NH.sub.3), 57.44 (COO—CH.sub.2), 55.74 (HO—CH—CH—NH), 47.98 (CONH—CH), 39.75 (NH—CH—CH.sub.2-Ph), 37.77 (—CH.sub.2—CH—CH—), 37.33 (Ph-CH.sub.2—CH—NH), 30.04 (CH(CH.sub.3).sub.2), 17.26 and 18.69 (2×CH.sub.3).
[0277] Result of analysis by HRMS (ESI+): calculated mass of C.sub.28H.sub.37O.sub.4N.sub.4S [M].sup.+525.25300, detected mass 525.25292.
[0278] Preparation of (5 S,6S,8S,11S)-2,5-dioxopyrrolidin-1-yl 5,8-dibenzyl-6-hydroxy-11-isopropyl-3,10,13-trioxo-1-(thiazol-5-yl)-2,16,19,22,25,28-hexaoxa-4,9,12-triazahentriacontan-31-oate (compound 7): 50 mg (78.3 nmol, 1 eq) of NHS-PEG5-NHS (Broadpharm) was dissolved in 0.5 ml of DMF along with 30 μl (172 nmol, 2.2 eq) of DIEA and 46 mg (86.1 nmol, 1 eq) of compound 6 dissolved in 0.5 ml of DMF was added dropwise during 30 minutes. The reaction was left to react overnight, the reaction mixture was then rotary evaporated and the crude product was purified using preparative HPLC (gradient: 20-50% (vol./vol.) ACN in 40 minutes, RT 32 minutes). 30 mg were isolated after lyophilization with purity well above 99% as determined by analytical HPLC (yield 40%). Analytical HPLC RT=21.2 min.
[0279] Result of analysis by HRMS (ESI+): calculated mass of C.sub.46H.sub.63O.sub.14N.sub.5S [MNa].sup.+964.39844, detected mass 964.39922.
[0280] Preparation of thiazol-5-ylmethyl ((24S,27S,29S,30S)-27-benzyl-1,29-dihydroxy-24-isopropyl-4,22,25-trioxo-31-phenyl-7,10,13,16,19-pentaoxa-3,23,26-triazahentriacontan-30-yl)carbamate (compound 8): 4 mg (4.25 μmol, 1 eq) of compound 7 were dissolved in 200 μl of DMF and 3 μl (49.7 μmol, 12 eq) of ethanolamine were added into the mixture along with 7 μl (42.5 μmol, 10 eq) of DIEA and the whole reaction mixture was left stirring overnight. The solvent was rotary evaporated and the mixture was dissolved in ACN/water and lyophilized 3 times (to remove ethanolamine). The compound was used in biochemical studies without further purification (the only contaminant is NHS, otherwise purity was higher than 98%). Analytical HPLC RT=19.0 min.
[0281] Result of analysis by HRMS (ESI+): calculated mass of C.sub.44H.sub.65O.sub.12N.sub.5S [MNa].sup.+910.42426, detected mass 910.42479.
2b: Preparation of a Detection Probe for Selective Binding of the HIV-1 Protease
[0282] Detection probe for quantification of HIV-1 protease was prepared by reacting the iqPCR_amino oligonucleotide with Compound 7 in the modification buffer: 8 μl of DMSO was added to 10 μl of the oligonucleotide in the modification buffer (10.2 nmol; 1 eq), and after mixing, the resulting solution was added to 2 μl of a solution of Compound 7 (205 nmol; 20 eq) in anhydrous DMSO. This mixture was incubated for 4.5 hours at room temperature, then 480 μl of TBS was added and incubation continued at the same temperature overnight.
[0283] The resulting detection probe (shown in
2c: Determination of Inhibition Constants of the Prepared Compounds and the Detection Probe
[0284] HIV-1 protease enzyme used in the assay was expressed, refolded and purified as described in (Kozisek et al. 2008, Journal of Virology, p. 5869; Weber et al. 2002, Journal of Molecular Biology, p. 739). The concentration of HIV-1 protease in the final composition was determined by titration of the active site with brecanavir inhibitor; the enzyme was stored frozen in aliquots at −20° C. before use. Concentration of ssHIV1/TBS was determined spectrophotometrically, the concentration of compound 8 was derived from its weight on an analytical balance.
[0285] Inhibition analyses were performed using a chromogenic peptide substrate KARVNle*NphEANle-NH.sub.2 as described in (Weber et al. 2002, Journal of Molecular Biology, p. 739). Reactions were carried out in 100 mmol.Math.l.sup.−1 sodium acetate and 300 mmol.Math.l.sup.−1 NaCl at pH 4.7 in a total volume of 1 ml. The final substrate concentration was maintained near K.sub.M (i.e. 16 nmol.Math.l.sup.−1), the total amount of the protease in the reaction was from 6 to 8 pmol. Various concentrations of Compound 8 (dissolved in DMSO) or ssHIV1/TBS were added to the mixture. Final DMSO concentration was always lower than 2.5% (vol./vol.). Substrate hydrolysis was monitored by the decrease in absorbance at 305 nm in a UV-Vis spectrometer UNICAM UV500 (Thermo Scientific). Data were subsequently analysed using the equation for competitive inhibition by Williams and Morrison in the GraFit program. K.sub.i=2.3±0.1 nmol.Math.l.sup.−1 was thus measured for compound 8, while K.sub.i was 0.23±0.03 nmol.Math.l.sup.−1 for ssHIV1/TBS.
2d: Detection of HIV-1 Protease and Testing of HIV-1 Protease Inhibitors by Direct Adsorption on a Solid Carrier
[0286] In this embodiment, the stock solution of purified HIV-1 protease at a concentration of 244 ng.Math.μl.sup.−1 in 10% glycerol (vol./vol.) was diluted to 10 ng.Math.μl.sup.−1 of the protease in TBS and 10 μl was applied to the bottom of the wells of 96-well plates FrameStar 480/96; the same volume of only pure TBS was applied to wells of the zero controls. After 15 minutes incubation at room temperature, the content of the wells was tapped out and the wells were washed three times with 200 μl of TBS. 200 μl of casein blocking agent five times diluted in TBS was then applied to the bottom of the wells and incubated for 1 hour at room temperature. The content of the wells was tapped out and the wells were washed three times with 200 μl of TBS. 10 μl of ssHIV1 detection probe was added in an aqueous solution of 20 mmol.Math.l.sup.−1 MES with 750 mmol.Math.l.sup.−1 NaCl and 0.05% Tween 20 (vol./vol.) at pH 6.0 (hereinafter “MEST”). After 45 minutes incubation at room temperature, the content of the wells was tapped out and the wells were washed eight times with 200 μl of TBST. 10 μl of qPCR mixture of the same composition as in the case of no template control in Example 1d was then added to the bottom of the wells and the amount of bound detection probe was determined by qPCR as described in Example 1d.
[0287] With the method described, the amount of bound detection probe was measured depending on the concentration in which it was applied, and it was found that the dissociation constant of the probe for the immobilized HIV protease is higher than the highest used concentration of the probe, i.e. 32 nmol.Math.l.sup.−1. At the detection probe concentration of 3.2 nmol.Math.l.sup.−1, the difference in the measured C, in the wells of zero control and in the wells with 100 ng of sorbed HIV protease was approximately eight cycles, which corresponds to two orders of magnitude difference in the amount of bound probe. Under the same conditions, it was verified that the addition of DMSO at 0.1%, 1% or 10% (vol./vol.) to the solution of the probe did not affect selective or non-selective binding of the probe.
[0288] Finally, various concentrations of 12 different HIV-1 protease inhibitors were added to the detection probe solutions applied into the wells and their inhibition constants were determined by the procedure described in Example 1i. For calculations, only the values of occupancy of active sites in the range from 50 to 99% were used and the corresponding concentrations of the inhibitor. Only a single high concentration of the substance was sufficient for qualitative information about the ability of the tested substances to inhibit HIV protease, however, for a quantitative information, due to the dynamic range of two orders of magnitude, it was necessary to test a series of tenfold diluted concentrations. Obtained K.sub.i values were then compared with reference values K.sub.iref obtained from enzyme kinetics method described in example 2c, whereby it was found that the values of both methods correlate very well with each other, as seen from a graphical comparison of the results of both methods in
TABLE-US-00011 TABLE 11 Comparison of K.sub.i values of inhibitors, measured in two independent experiments, and the respective K.sub.iref K.sub.i (1), K.sub.i (2), K.sub.iref, Inhibitors of HIV PR nmol .Math. l.sup.−1 nmol .Math. l.sup.−1 nmol .Math. l.sup.−1 saquinavir 0.61 1.7 0.04 ritonavir 0.16 0.11 0.015 indinavir 1.3 1.5 0.12 amprenavir 0.80 0.44 0.184 lopinavir 0.18 0.41 0.018 atazanavir 0.11 0.18 0.024 tipranavir 1.3 2.9 0.14 darunavir 0.03 0.11 0.0053 brecanavir 0.04 0.05 0.001 Substance 37 600 380 116 Substance 38 N/A 110 10 Substance 39 40 N/A 0.3 K.sub.i (1) and K.sub.i (2) indicate inhibitory constants determined sequentially in two independent experiments (pH 6.0), K.sub.iref is the reference value obtained by enzyme kinetics (pH 4.7).
2e: Detection of HIV-1 Protease and Testing its Inhibitors by Binding to an Immobilized Antibody
[0289] In another embodiment, 10 μl of a solution of a polyclonal antibody binding the HIV-1 protease (MyBiosource, MBS536030) at 5 ng.Math.μl.sup.−1 in TBS was applied to the bottom of wells of a 96 well plate FrameStar 480/96 and incubated at room temperature for 45 minutes. Content of the wells was then tapped out and the wells were washed three times with 200 μl of TBS. 200 μl of casein blocking agent five times diluted in TBS was then applied to the bottom of the wells and incubated for 3 hours at room temperature, then the content of the wells was tapped out and the wells were washed three times with 200 μl TBST. Subsequently, 10 μl of purified HIV-1 protease solution at different concentrations in TBST was applied to the bottom of wells. After incubation for 1 hour at room temperature, the content of the wells was tapped out and the wells were washed three times with 200 μl TBST. Then, 10 μl of ssHIV1 solution detection probe in TBST was added. Further procedure was the same as in Example 2d.
[0290] This procedure showed that the range of detection reached from 100 ng to 1 ng of HIV-1 protease, when detection probe was applied at a concentration of 10 nmol.Math.l.sup.−1, and ΔC.sub.q between wells with 100 ng of the protease and without it was nine cycles. It was also found that the addition of casein blocking agent to the detection probe solution in a final twothousand-fold dilution increased the ΔC.sub.q between wells with 100 ng of the protease and without it to twelve cycles, corresponding to thousand-fold difference in binding of the probe; therefore the probe was diluted in TBST buffer containing casein blocking agent for further determinations. In the case that instead of the detection probe solution, a solution of the original oligonucleotide without the iqPCR_amino ligand portion was used, no binding was observed, even when applying an amount of 100 ng of HIV-1 protease, confirming the selectivity of the detection probes binding via the ligand portion. The procedure was also tested for the influence of solvents added to the solution of the detection probe; at final concentrations of 0.1%, 1% and 10% (vol./vol.) of DMSO, acetonitrile or methanol had no effect on binding of detection probe, either selective to the protease or the non-selective to the surface without the protease. Finally, various concentrations of 12 different inhibitors of HIV-1 protease were added to the detection probe solutions applied to the wells and their inhibition constants were determined by the procedure described in Example 1i, only the values of occupancy of active sites in the range from 50 to 99% were used and the corresponding concentrations of the inhibitor. A number of substances that do not inhibit the HIV-1 protease were gradually added at high concentration (1 mmol.Math.l.sup.−1) to check the correctness of the determination (substances), and none of them lead to decrease in the amount of bound detection probe, i.e. no false positive results were observed. For qualitative information about the ability of tested substances to inhibit HIV protease, a single high concentration of the substance is sufficient; however, for a quantitative information, due to the dynamic range of two to three orders of magnitude, it was necessary to test a series of tenfold diluted concentrations. K.sub.i values obtained were compared to reference K.sub.iref values obtained by enzyme kinetic method described in example 2c, and it was found that the values from both methods correlate very well, as is apparent from the graphical comparison of the results of both methods in
TABLE-US-00012 TABLE 12 Comparison of the measured values of inhibitors' K.sub.i and the respective K.sub.iref K.sub.i, K.sub.iref, Inhibitors of HIV-1 PR nmol .Math. l.sup.−1 nmol .Math. l.sup.−1 saquinavir 1.1 0.04 ritonavir 3.0 0.015 indinavir 8.7 0.12 amprenavir 3.5 0.184 lopinavir 1.3 0.018 atazanavir 1.4 0.024 tipranavir 14 0.14 darunavir 0.44 0.0053 brecanavir 0.41 0.001 Substance 37 N/A 116 Substance 38 830 10 Substance 39 19 0.3 K.sub.i refers to determined inhibition constants (pH 7.4), K.sub.iref to the reference value obtained from enzyme kinetics (pH 4.7).
Example 3: Detection of Carbonic Anhydrases II and IX and Testing of their Inhibitors
3a: Preparation of a Common Inhibitor of Carbonic Anhydrases II and IX, and its NHS Ester
[0291] All compounds were purified and characterized as described in example 1a.
[0292] Preparation of methyl 4-(4-((tert-butoxycarbonyl)amino)butoxy)benzoate (compound 9): To a solution of 161 mg (1 eq, 1.06 mmol) of methyl 4-hydroxybenzoate, 300 mg (1.5 eq, 1.59 mmol) of tert-butyl (4-hydroxybutyl)carbamate and 400 mg (1.5 eq, 1.59 mmol) of triphenylphosphine in 10 ml of THF was added 312 μl (1.5 eq, 1.59 mmol) of DIAD in one portion and the reaction was left stirring overnight. The reaction mixture was then evaporated and the crude product was purified by column chromatography (He:EtOAc 4:1, RF=0.25; note: the methyl 4-hydroxybenzoate has identical RF with the product, therefore 1.5 eq of other reactants was used) 260 mg of white powder was obtained (yield 75%).
[0293] Result of analysis by .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.95 (d, J=8.9 Hz, 2H), 6.87 (d, J=8.9 Hz, 2H), 4.71 (s, 1H), 3.99 (t, J=6.2 Hz, 2H), 3.85 (s, 3H), 3.17 (dd, J=12.8, 6.3 Hz, 2H), 1.86-1.75 (m, 2H), 1.69-1.61 (m, 2H), 1.42 (s, 9H).
[0294] Result of analysis by .sup.13C NMR (101 MHz, CDCl.sub.3) δ 166.92 (s), 162.78 (s), 156.10 (s), 131.64 (s), 122.57 (s), 114.12 (s), 79.20 (s), 67.73 (s), 51.89 (s), 40.29 (s), 28.49 (s), 26.86 (s), 26.49 (s). Result of analysis by MS (ESI+): calculated mass of C.sub.17H.sub.25O.sub.5N [MNa].sup.+346.17; detected mass 346.2.
[0295] Preparation of 4-(4-((tert-butoxycarbonyl)amino)butoxy)benzoic acid (compound 10): 270 mg of compound 9 were dissolved in 5 ml of methanol and 5 ml of 5 mol.Math.l.sup.−1 NaOH was added. The mixture was refluxed until TLC analysis showed complete disappearance of compound 9 (6 hours). The reaction mixture was diluted by EtOAc (20 ml), the water phase was acidified by 10% KHSO.sub.4 (wt./vol.) to acidic pH and extracted 2 more times by 20 ml of EtOAc. 240 mg of oily product which turned to crystalline white after removal of solvent traces was obtained (yield 95%).
[0296] Result of analysis by .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.03 (d, J=8.9 Hz, 2H), 6.91 (d, J=9.0 Hz, 2H), 4.65 (s, 1H), 4.04 (t, J=6.2 Hz, 2H), 3.27-3.20 (m, 2H), 1.91-1.78 (m, 2H), 1.69 (dd, J=14.8, 7.2 Hz, 2H), 1.44 (s, 9H).
[0297] Result of analysis by .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.51 (s), 163.46 (s), 156.20 (s), 132.42 (s), 121.92 (s), 114.28 (s), 79.42 (s), 67.86 (s), 40.36 (s), 28.56 (s), 26.89 (s), 26.53 (s).
[0298] Result of analysis by MS (ESI−): calculated mass of C.sub.16H.sub.22O.sub.5N [M].sup.− 308.16; detected mass 308.2.
[0299] Preparation of tert-butyl (4-(4-(3-(4-sulfamoylphenyl)ureido)phenoxy)butyl)carbamate (compound 11): 720 mg (1 eq, 2.33 mmol) of compound 10 was dissolved in 15 ml of dry toluene and 810 μl (2 eq, 4.65 mmol) of DIEA was added. DPPA (552 μl, 1.1 eq, 2.56 mmol) was added to the reaction mixture in one portion and the reaction mixture's temperature was raised to 90° C. for 2 hours. The reaction mixture was then evaporated and dissolved in dry ACN; 601 mg (1.5 eq, 3.49 mmol) of sulfanilamide was added in one portion and reaction mixture was heated up to 60° C. overnight while stirring. All volatiles were evaporated after 12 hours and the crude product was purified by column chromatography on silica (He: EtOAc, 2:5, RF=0.25). 340 mg of product was obtained (isolated yield 30%).
[0300] Result of analysis by .sup.1H NMR (400 MHz, DMSO) δ 8.98 (s, 1H), 8.59 (s, 1H), 7.71 (d, J=8.8 Hz, 2H), 7.59 (d, J=8.9 Hz, 2H), 7.34 (d, J=9.0 Hz, 2H), 7.20 (s, 2H), 6.91-6.81 (m, 3H), 3.91 (t, J=6.4 Hz, 2H), 2.96 (dd, J=12.9, 6.7 Hz, 2H), 1.71-1.61 (m, 2H), 1.51 (dt, J=13.1, 6.5 Hz, 2H), 1.37 (s, 9H).
[0301] Result of analysis by .sup.13C NMR (101 MHz, DMSO) δ 155.37 (s), 154.02 (s), 152.16 (s), 142.99 (s), 136.40 (s), 132.04 (s), 126.61 (s), 120.14 (s), 117.12 (s), 114.50 (s), 77.06 (s), 67.05 (s), 40.35 (overlap with solvent peak) 27.77 (s), 26.85 (s), 25.73 (s).
[0302] Result of analysis by MS (ESI+): calculated mass of C.sub.22H.sub.3O.sub.6N.sub.4S [MNa].sup.+501.17; detected mass 501.2.
[0303] Preparation of 4-(4-(3-(4-sulfamoylphenyl)ureido)phenoxy)butan-1-aminium 2,2,2-trifluoroacetate, (compound 12): 500 mg of compound 11 was dissolved in 1 ml of TFA and was sonicated and stirred alternately for 15 minutes. TFA was then removed by flow of nitrogen and the product was used in further steps without any characterization or purification.
[0304] Preparation of 2,5-dioxopyrrolidin-1-yl 19-oxo-24-(4-(3-(4-sulfamoylphenyl)ureido)phenoxy)-4,7,10,13,16-pentaoxa-20-azatetracosan-1-oate (compound 13): 33 mg (1 eq, 67 μmot) of compound 12 was added slowly (during 1 hour) into a solution of bisNHS-PEG5 (36 mg, 1 eq, 67 μmob Broadpharm) and DIEA (22 μl, 2.5 eq, 168 μmot) in DMF (1 ml). The reaction mixture was left for 3 hours stirring and then the volatiles were evaporated. The final product was purified by preparative HPLC (gradient: 15-50% (vol./vol.) ACN in 40 minutes, RT 30 minutes). 15 mg of product were isolated with purity well above 99% (yield 28%). Analytical HPLC RT=18.7 min.
[0305] Result of analysis by HRMS (ESI+): calculated mass of C.sub.35H.sub.50O.sub.14N.sub.5S [MH]+795.30695, detected mass 796.30678.
[0306] Preparation of 18-oxo-23-(4-(3-(4-sulfamoylphenyl)ureido)phenoxy)-3,6,9,12,15-pentaoxa-19-azatri-cosan-1-aminium 2,2,2-trifluoroacetate (compound 14): 46 mg (1 eq, 112 μmot) of Boc-PEG5-COOH was dissolved in 0.5 ml of DMF along with 36 mg (1 eq, 112 μmot) of TBTU and 49 μl (2.5 eq, 279 μmot) of DIEA. To this solution 55 mg (1 eq, 112 μmot) of compound 12 was added and the mixture was stirred overnight. The solvent was then evaporated and the crude product dissolved in 10 ml of EtOAc. The organic phase was washed two times by saturated bicarbonate, two times by 10% (wt./vol.) KHSO.sub.4, dried and evaporated; 53 mg of product were isolated. 1 ml of TFA was added and the mixture was alternately sonicated and stirred for 15 minutes. The TFA was then removed by flow of nitrogen and the product was purified by preparative HPLC (gradient: 10-50% ACN in 40 minutes, RT=22 minutes). 17 mg of product were isolated (yield 31%). Analytical HPLC RT=16.5 min. Result of analysis by HRMS (ESI+): calculated mass of C.sub.30H.sub.48O.sub.10N.sub.5S [MH].sup.+670.31164; detected mass 670.31164.
3b: Preparation of Detection Probe for Selective Binding of Carbonic Anhydrases
[0307] Detection probe for selectively binding of carbonic anhydrases was prepared by reacting the iqPCR_amino oligonucleotide and Compound 13 in the modification buffer: 2 μl of 1 mol.Math.l.sup.−1 HEPES aqueous solution at pH 8.0 were first added to 10 μl of the oligonucleotide in the modification buffer (8.2 nmol, 1 eq.). After stirring, 8.2 μl of a solution of Compound 13 at a concentration of 50 mmol.Math.l.sup.−1 in anhydrous DMSO (410 mmol, 50 eq.) was added and stirred again. Finally, 5 μl of anhydrous DMSO was added to the mixture and after stirring incubated overnight at room temperature. The mixture was then diluted in 900 μl of an aqueous solution of 0.1 mol.Math.l.sup.−1 HEPES, pH 8.0, and incubated another day at room temperature. The resulting detection probe (hereinafter ssCA,
[0308] The ssCA sample was analysed with LC/ESI-MS on Agilent 6230 TOF LC/MS in the same manner as described in Example 1b, only 0.05% (wt./vol.) aqueous ammonium acetate solution was used as the mobile phase instead of HFIP with TEA. The result of the analysis was a major absorption peak at 260 nm with retention time 5.14 min and the corresponding weight of 17663.28, whereas the predicted molecular weight was 17663.86. The difference between measured masses of ssCA and the original iqPCR_amino is 681.40 compared to the expected difference 680.30. The product purity was about 80%.
[0309] A complex of neutravidin with a detection probe, Neu_dsbiotCA, was prepared for use in detection of carbonic anhydrases and testing their inhibitors. First, 750 pmol of ssCA probe together with 500 pmol iqPCR_biotin was diluted in 50 μl TBS and thermally paired by the procedure described in Example 1b; 10 μl of the resulting solution was mixed with 3 μl neutravidin at a concentration of 1 mg.Math.ml.sup.−1 and, after mixing, incubated first for 3 hours at room temperature and then overnight on ice. Resulting Neu_dsbiotCA complex was purified by ultrafiltration on Amicon Ultra 0.5 ml 100K, the volume of the retentate containing the complex was twice consecutively tenfold diluted in TBS. The final concentration of the detection probe in the complex was determined by qPCR by comparison with a dilution series of ssPSMA standard as described in Example 1d.
3c: Detection of CA-II and Testing Inhibitors of CA-II
[0310] The purified standard of human carbonic anhydrase II was ordered from Sigma-Aldrich (cat. no. C6165). After dissolving the lyophilized protein in double-distilled water, the protein was diluted in TBS to a final concentrations of 10 ng.Math.μl.sup.−1 to 10 pg.Math.μl.sup.−1 and 10 μl of these solutions were applied to the bottom wells of a 96-well plate FrameStar 480/96; 10 μl of pure TBS was applied for controls. After incubation for 40 minutes at room temperature, the content of the wells was tapped out and the wells were washed three times with 200 μl of TBS. Then, 100 μl of casein blocking agent five times diluted in TBS was applied to the bottom of the wells and incubated for 2 hours at room temperature. Content of the wells was subsequently tapped out and the wells were washed three times with 200 μl TBST. 10 μl of a solution of the detection Neu_dsbiotCA probe at a concentration 1 nmol.Math.l.sup.−1 of was added in a solution of 20 mmol.Math.l.sup.−1 Tris, 200 mmol.Math.l.sup.−1 NaCl and 0.05% Tween 20 (vol./vol.) pH=7.4 (hereinafter TBST200 buffer) with the addition of casein blocker diluted thousand fold in sum. Further procedure was the same as in Example 2d.
[0311] The procedure described could detect both 100 ng of protein CA-II (AC, compared to zero control=9 cycles) and 10 ng (ΔC.sub.q compared to zero control=5 cycles). It was also found that the addition of DMSO to a final concentration of 1% (vol./vol.) in the solution of the applied detection probe did not alter the selective binding of the probe to the immobilized protein CA-II, or non-selective binding to the surface in the zero control. Finally, 12 different known inhibitors of CA-II at a final concentration of 100 nmol.Math.l.sup.−1 were individually added to the solution of detection probe applied to the wells. This qualitatively verified that all 12 substances inhibit CA-II, i.e. that for all inhibitors tested, there was an observable decrease in bound detection probe.
3d: Detection of CA-IX and Testing CA-IX Inhibitors
[0312] 10 μl of a solution of purified antibody M75 (Zavada et al. 2000, British Journal of Cancer, p. 1808) was applied to the bottom of the wells of a 96-well plate FrameStar 480/96 at a concentration of 10 ng.Math.μl.sup.−1 in TBS and incubated at room temperature for 75 minutes. Content of the wells was then tapped out and the wells were washed three times with 200 μl of TBS. 100 μl of casein blocking agent five times diluted in TBS was then applied to the bottom of the wells and incubated for 2 hours at room temperature, then the content of the wells was tapped out and the wells were washed three times with 200 μl of TBST. Subsequently, 10 μl of a solution of purified carbonic anhydrase IX in various concentrations in TBST200 was added to the bottom of the wells. The construct containing the catalytic domain and the PG domain of carbonic anhydrase IX (amino acids 55 to 390, further referred to as CA-IX PG) was prepared by recombinant expression in insect S2 cells and purified as described in (Mader, 2010 Doctoral Thesis, Charles University Prague). After two hours incubation at room temperature, the content of the wells was tapped out and the wells were washed three times with 200 μl TBST. Then 10 μl of a solution of Neu_dsbiotCA detection probe at various concentrations in TBST200 was added with casein blocker at a resulting two thousand fold dilution. Further procedure was the same as in Example 2d.
[0313] With the described procedure, after application of 1 ng CA-IX PG into the well, the amount of bound detection probe depending on its concentration was measured, and it was found that the dissociation constant of the probe is significantly higher than the highest used concentration of the probe, i.e. 50 nmol.Math.l.sup.−1. Further, at the concentrations of the detection probe 5 nmol.Math.l.sup.−1, the difference in the measured C, in wells with zero control and in the wells with 1 ng CA IX PG was approximately ten cycles, which corresponds to a difference of more than two orders of magnitude in the amount of bound probe. Dynamic range of the determination of CA-IX PG was 50 pg to 1 ng under the same conditions, see Table 13. In the same manner, it was verified that the addition of DMSO to the solution of detection probe at concentrations of 0.1% to 10% (vol./vol.) does not affect the selective or non-selective binding of the probe.
TABLE-US-00013 TABLE 13 Dynamic range of CA-IX PG determination Amount in ng C.sub.q (1) C.sub.q (2) 10 10.32 10.21 3.2 9.95 10.03 1.0 10.61 10.33 0.50 11.40 11.25 0.25 12.31 12.26 0.10 15.69 15.51 0.05 18.71 18.24 0.025 18.10 19.74 0.010 20.35 19.86 0.005 20.55 20.36 0 20.92 20.70
[0314] Finally, various concentrations of a total of 12 different CA-IX inhibitors (the same substances as tested for CA-II above) were added to the solution of the detection probe applied to the wells and with the procedure described in Example 1i, their inhibition constants were determined. Only values of active sites occupancy in the range of 40 to 99.5% and the corresponding concentrations of inhibitor were used for calculations. For qualitative information about the ability of the tested substances to inhibit CA-IX, testing a single high concentration of the substance (100 nmol.Math.l.sup.−1) was sufficient. For quantitative information, due to the dynamic range of the setting (two to three orders of magnitude), it was necessary to test a tenfold dilution series of concentrations, whereby the measured K.sub.i value was practically identical always in two to three consecutive dilutions of the inhibitor. Obtained K.sub.i values were then compared with reference K.sub.iref values obtained from enzyme kinetics. The values obtained by the two methods are summarized in Table 14; enzyme kinetics also determined the K.sub.iref compound 13, i.e. the ligand part of the detection probe alone, to be approximately 400 nmol.Math.l.sup.−1. As evident from the table and a graphic comparison of the results of both methods in
TABLE-US-00014 TABLE 14 Comparison of K.sub.i of CA-IX inhibitors with the appropriate values of K.sub.iref K.sub.i determined, K.sub.iref, Designation μmol .Math. l.sup.−1 μmol .Math. l.sup.−1 CB4 0.50 0.06 CB5 0.12 0.026 CB7 130 161 CB8 3.6 0.43 CB10 50 90.7 CB12 8.1 32.7 CB19 0.22 1.2 CB20 0.077 0.23 CB21 6.8 6.5 CB31 0.0022 0.0016
3e: Detection of CA-IX and Testing of CA-IX Inhibitors Using Tight Binding Bivalent Probes
[0315] Bivalent probe for CA-IX detection was prepared by reacting Compound 13 with an oligonucleotide with the sequence AAA CCT GCC AGT TGA GCA TTT TTA TCT GCC ACC TTC TCC ACC AGA CAA AAG CTG GAA A containing the 3′-terminal 6-amino-2-(hydroxymethyl) hexyl phosphate modification and the 5′-terminal 6-aminohexyl phosphate modification (custom synthesis Generi-Biotech, OPC purification). The preparation, purification and LC/MS analysis was identical to the procedure described in Example 3b. The measured weight of the original oligonucleotide was 18100.85, while the weight of the oligonucleotide after reaction with Compound 13 (hereinafter ssCAbis) was 19462.36, corresponding weight difference of 1361.50 which corresponds to twice the mass of the attached Compound 13 (680.30).
[0316] Following the procedure described in the preceding examples, approximately 80 pg of CA-IX contained in a cell lysate of line HT-29 (used amount of total protein determined by Bradford assay was 1 μg) was immobilized using the M75 antibody. The immobilized CA-IX was subsequently incubated with various concentrations of ssCAbis detection probe diluted in HEPESTC′ buffer (concentration range 10 pmol.Math.l.sup.−1 to 100 nmol.Math.l.sup.−1) and K.sub.d of the probe was determined as 2.1 nmol.Math.l.sup.−1 (+−0.3 nmol.Math.l.sup.−1) which is more than twenty times improvement compared to the affinity of monovalent probe (a probe containing only one molecule of Compound 13). The same procedure was repeated with the CA-IX immobilized from 1 μl of blood serum, and the K.sub.d measured was almost identical (2.2 nmol.Math.l.sup.−1+−0.3 nmol.Math.l.sup.−1).
[0317] To determine the detection limit and dynamic range of the assay, a standard was prepared (cell lysate of line HT-29) in which the concentration of CA-IX was determined using a commercial ELISA kit from RnD Systems. The dilution series of this standard was then incubated for 3 hours in wells with immobilized M75 antibody and after washing, ssCAbis detection probe diluted to a concentration of 200 pmol.Math.l.sup.−1 in HEPESTC′ buffer was added to the wells for 1 hour, and after subsequent washing, the quantity of bound probe was determined using qPCR. As shown in
[0318] The concentration of CA-IX in blood serum samples taken from 36 subjects: 12 healthy males; 10 males and 2 females with histologically confirmed renal clear cell carcinoma; and 12 males with histologically confirmed prostate cancer, was determined with the same procedure. Amount of CA-IX was determined in 10 μl of undiluted serum, which was incubated in wells with immobilized M75 antibody for 21 hours. The concentrations of CA-IX in the samples were determined by comparing the amount of bound probe with a dilution series of the standard as described in the previous paragraph, and ranged from 0.1 to 1.5 ng.Math.ml.sup.−1. To verify the obtained data, all samples were incubated with the probe also in the presence of a competitive CA-IX inhibitor acetazolamide (AAZ); it was confirmed that the binding of the probe is suppressed by adding AAZ, and the strength of inhibition of the probe binding corresponded to the K.sub.i of acetazolamide. Adding acetazolamide suppressed probe binding to an amount corresponding to the concentration of CA-IX less than 1 pg.Math.ml.sup.−1 showing that the limit of detection is approximately 1 pg.Math.ml.sup.−1 with a consumption of 10 μl of serum (10 fg in total). For further validation of the values obtained, the concentration of CA-IX in all samples was measured also with commercially available ELISA kit from RnD Systems, and as shown in
[0319]
TABLE-US-00015 TABLE 15 Comparison of K.sub.i of CA-IX inhibitors with the corresponding K.sub.iref values K.sub.i determined K.sub.iref, K.sub.i determined in in blood serum, Designation μmol .Math. l.sup.−1 cell lysate, μmol .Math. l.sup.−1 μmol .Math. l.sup.−1 AAZ 0.025 0.052 0.018 CB1 0.38 0.40 0.43 CB7 160 14 8.2 CB8 0.43 1.2 2.4 CB19 1.1 0.18 0.10 CB20 0.23 0.19 0.13 CB2 5.1 2.1 0.94 CB6 2.3 0.93 1.9 CB18 26 11 6.2 Substance 40 0.41 0.12 0.052
Example 4: Universal Detection Probe for Selective Binding to the Active Sites of Aspartic Proteases
4a: Preparation of Pepstatin NHS Ester
[0320] All compounds were purified and characterized as described in example 1a.
[0321] Preparation of (21S,24S,27S,28S,32S,35S,36S)-1-((2,5-dioxopyrrolidin-1-yl)oxy)-28,36-dihydroxy-27,35-diisobutyl-21,24-diisopropyl-32-methyl-1,19,22,25,30,33-hexaoxo-4,7,10,13,16-pentaoxa-20,23,26,31,34-pentaazaoctatriacontan-38-oic acid, NHS-PEG.sub.5-pepstatin (compound 15): Pepstatin was synthesized by standart amino-Fmoc synthesis on solid phase, using 2-Chlortrityl chloride resin (Iris-Biotech). The first amino acid (Fmoc-Sta-OH) was attached to the solid phase according to the manufacturer's instructions. The resin was left to react with Fmoc-Sta-OH (0.6 eq to resin substitution) in presence of 4 equivalents of DIEA for 2 hours in dichlormethane (DCM). The remaining reactive residues were quenched with mixture of DCM/MeOH/DIEA (17:2:1) for 15 minutes. All other amino acids were added using HOBUDIC method. The peptide was then cleaved from the solid phase using 95% (vol./vol.) TFA (2.5% (vol./vol.) water; 2.5% (vol./vol.) triisopropyl silane) and the crude product was used in further step without further purification (the purity after cleavage was above 95%). 18 mg (1.1 eq, 33 μmot) of bis-PEG5-NHS ester (Broadpharm) was dissolved in 0.25 ml of DMF along with 25 μl (5 eq, 165 μmot) of DIEA. 20 mg (1 eq, 30 μmot) of peptide was then added dropwise slowly to the stirring solution (during 1 hour) and the reaction was left for 3 hours. The volatiles were then evaporated and the final product was purified by preparative scale HPLC (gradient: 15-50% (vol./vol.) ACN in 40 minutes, RT=31 minutes). 10 mg of product were isolated with purity well above 99% (yield 33%). Analytical HPLC RT=19.5 min.
[0322] Result of analysis by HRMS (ESI−): calculated mass of C.sub.47H.sub.81O.sub.18N.sub.6 [M].sup.− 1017.56128; detected mass 1017.56079.
4b: Preparation of Detection Probe for Selective Binding of Aspartic Proteases
[0323] Detection probe for selective binding of aspartate proteases was prepared by reacting the iqPCR_amino oligonucleotide with compound 15 in the modification buffer: First, 4 μl of aqueous solution 1 mol.Math.l.sup.−1 HEPES pH 8.0 was added to 20 μl of the oligonucleotide in the modification buffer (16.3 nmol, 1 eq.). After stirring, 16.3 μl of compound 15 solution at a concentration of 20 mmol.Math.l.sup.−1 in anhydrous DMSO (326 nmol, 20 eq.) was added and stirred again. Finally, 15 μl of DMSO was added to the mixture and, after stirring, the mixture was incubated overnight at room temperature. The resulting detection probe (hereinafter ssAP,
4c: Determination of the Inhibitory Potency of the Prepared Detection Probe for Human Cathepsin D
[0324] Inhibitory potency of the ssAP detection probe to human cathepsin D was determined by enzyme kinetics. The procedure was similar to that described in (Masa et al. 2006, Biochemistry, p. 15474), cathepsin D was prepared according to the procedure in (Fusek et al. 1992, Journal of Molecular Biology, p. 555). Wells of a white 96-well plate with a conical bottom (NUNC V96) were successively loaded with 93.5 μl of acetate buffer pH 4.0 (100 mmol.Math.l.sup.−1 CH.sub.3COONa and 300 mmol.Math.l.sup.−1 NaCl), 0.5 μl of cathepsin D solution and 1 μl of detection probe solution of known concentration. Just before the measurement, 5 μl of a solution of the fluorogenic substrate (Abz-Lys-Pro-Ala-Glu-Phe-Nph-Ala-Leu; Abz, aminobenzoic acid; Nph, 4-nitrophenylalanine) at a concentration of 40 nmol.Math.l.sup.−1 in 2% (vol./vol.) DMSO was added and cleavage rate of the substrate was then observed using a Tecan infinite M1000 reader (excitation at 330 nm and emission 410 nm). The IC.sub.50 value was determined to be around 1 nmol.Math.l.sup.−1 from the dependence of ν.sub.i/ν.sub.0 ratio on the concentration of the detection probe. Such affinity of the probe is sufficient for very sensitive detection of cathepsin D.
Example 5: Detection Probe for Selective Binding to the Active Sites of Influenza Neuraminidases
5a: Preparation of a Selective Inhibitor of Influenza Neuraminidases with Attached Azide Group
[0325] Compound 17; 1-(6-Azidohexyl)-1-methyl-(3R,4R,5 S)-4-acetylamino-5-N-tert-butoxycarbonyl-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-phosphonate; was prepared analogously to the procedure described in (Carbain 2010, Doctoral thesis, University of Sussex).
[0326] Preparation of Compound 18; 1-(6-Azidohexyl)-(3R,4R,5S)-4-acetylamino-5-N-tert-butoxycarbonyl-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-phosphonate: a mixture of diastereomers of Compound 17 (0.068 g, 0.12 mmol) was dissolved in 4 ml of dry THF and thiophenol (0.05 ml, 0.66 mmol) and triethylamine (0.15 ml; 1.08 mmol) were added to this solution. The reaction mixture was stirred at room temperature for two days, then thiophenol (0.05 ml, 0.66 mmol) and triethylamine (0.15 ml; 1.08 mmol) were again added. The next day, the reaction mixture was concentrated on a rotary evaporator and separated by column chromatography (silica gel; eluent ethyl acetate:methanol/3:1 to 1:2). Yield: 0.042 g of the demethylated product.
[0327] Result of analysis by HRMS (ESI−): calculated mass of C.sub.24H.sub.43O.sub.7N.sub.5P 544.2906; detected 544.2902. Preparation of Compound 19; 1-(6-Azidohexyl)-(3R,4R,5 S)-4-acetylamino-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-phosphonate: Compound 18 (0.04 g; 0.073 mmol) was dissolved in 3 ml of trifluoroacetic acid and after stirring for two hours at room temperature, the reaction mixture was evaporated and the residue was purified by preparative HPLC on a reverse column (stationary phase C-18 modified silica gel; mobile phase: acetonitrile/water with 0.1% (vol./vol.) trifluoroacetic acid). Yield: 0.02 g of the final product.
[0328] Result of analysis by HRMS (ESI+): calculated mass of C.sub.19H.sub.37O.sub.5N.sub.5P 446.2527; detected 446.2527.
5b: Preparation of Detection Probe for Influenza Neuraminidases
[0329] Preparation of oligonucleotide with dibenzylcyclooctyne group (hereinafter ssAD): 50 μl of 2× concentrated modification buffer was first added to the iqPCR_amino oligonucleotide (20.2 nmol; 1 eq) dissolved in 48 μl of double distilled water. 50.4 μl of dibenzylcyclooctyne NHS-ester (Sigma, cat. no. 761524) at a concentration of 20 mmol.Math.l.sup.−1 (1.008 μmol, 50 eq) in anhydrous DMSO was added and stirred. As precipitation was observed, an additional 70 μl of DMSO was subsequently added and stirred. After incubation for two days at room temperature, the resulting modified oligonucleotide (ssAD) was purified by ultrafiltration on Amicon Ultra 0.5 ml 10K column; the reaction mixture was diluted to 1 ml in double distilled water prior to application onto the column and then the retentate volume was ten times successively tenfold diluted in double distilled water during the ultrafiltration. Oligonucleotide concentration in the retentate was determined spectrophotometrically (OD 1=1744 pmol). A sample of the product was analysed by LC/ESI-MS on Agilent 6230 TOF LC/MS as described in Example 4b. Analysis resulted in an absorption peak at 260 nm with retention time 4.47 min corresponding to weight 17269.75. The weight of original iqPCR_amino was not found, suggesting complete conversion of the reaction. The difference between the measured weight of ssAD and the original iqPCR_amino was 278.88 compared to the expected difference of 287.40.
[0330] Preparation of detection probe for influenza neuraminidases (hereinafter ssAD_NA;
5c: Determination of Inhibition Constant of the Prepared Detection Probe
[0331] The tested neuraminidase type N1 came from the pandemic virus A/California/07/2009 (GenBank CY121682). The coding sequence of the catalytic domain (amino acids 82-469) was synthesized by Genscript company, cloned into pMT BiP vector, the resulting construct contained (in addition to the signal peptide) an N-terminal Avi-tag and thrombin cleavage site for cleavage of the tag. Neuraminidase was expressed in insect S2 cells, the secreted protein was purified from SF900 medium (commercially available media from Invitrogen, USA) by precipitation with ammonium sulphate. The soluble fraction resulting from 50 percent sulphate saturation was dialyzed into 50 mmol.Math.l.sup.−1 Tris, 150 mmol.Math.l.sup.−1 NaCl pH 8.0; concentrated and separated by gel permeation chromatography on a Superdex 200 column. Fractions with neuraminidase catalytic activity were combined, a solution of calcium chloride to a final concentration of 10 mmol.Math.l.sup.−1 and Thrombin-Agarose (Sigma-Aldrich, USA) were added and incubated for 12 hours at 4° C. This mixture was then divided by gel permeation chromatography on Superdex 75 column. Fractions containing the active neuraminidase without Avi-tag (determined by SDS polyacrylamide electrophoresis and Western blotting) were frozen for kinetic experiments.
[0332] Enzyme activity of neuraminidase was measured with a fluorimetric assay using a fluorescent substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA) in 100 mmol.Math.l.sup.−1 MES, 150 mmol.Math.l.sup.−1 NaCl, 10 mmol.Math.l.sup.−1 CaCl.sub.2 at pH 6.15. Final concentrations in the reaction mixture were: 16 nmol.Math.l.sup.−1 neuraminidase, 500 nmol.Math.l.sup.−1 4-MUNANA substrate (at K.sub.M=1.1 mmol.Math.l.sup.−1) and 2% DMSO (vol./vol.). Cleavage of substrate was monitored at excitation and emission wavelengths λ.sub.exc=365 nm and lea, =450 nm on a Tecan Infinite M1000 reader, the reaction was incubated for 20 minutes at 37° C. and then quenched by addition of sodium carbonate solution to a final concentration of 0.5 mol.Math.l.sup.−1. Values of apparent K.sub.i′ and K.sub.i were obtained by evaluating the ratio of the inhibited and uninhibited reaction rates ν.sub.i/ν.sub.0 according to Williams and Morrison. Measured K.sub.i of clinically used Oseltamivir was 24 nmol.Math.l.sup.−1 (±4 nmol.Math.l.sup.−1), K.sub.i of Compound 19 was 24 nmol.Math.l.sup.−1 (±5 nmol.Math.l.sup.−1) and K.sub.i of the ssAD_NA detection probe was 0.79 nmol.Math.l.sup.−1 (±0.09 nmol.Math.l.sup.−1). Apparent K.sub.i for the Compound 19 and the detection probe ssAD_NA was also determined at physiological pH 7.4 in 100 mmol.Math.l.sup.−1 Tris, 150 mmol.Math.l.sup.−1 NaCl, 10 mmol.Math.l.sup.−1 CaCl.sub.2; K.sub.i of Compound 19 was approximately 40 nmol.Math.l.sup.−1, while of the ssAD_NA probe approximately 2 nmol.Math.l.sup.−1. Connecting the oligonucleotide to the compound 19 thus resulted in a substance having at least twenty-fold higher affinity for neuraminidase compared to both Compound 19 and Oseltamivir.
5d: Testing the Inhibitory Potency of Substances Against Influenza Neuraminidase N1
[0333] Following the procedure described in the previous examples, 1 ng of purified recombinant neuraminidase N1, containing an N-terminal strep-tag, diluted in TBST′ buffer with thousand-fold diluted casein blocker and CaCl.sub.2 at a concentration of 5 mmol.Math.l.sup.−1 was immobilized to the bottom of wells of a PCR plate using an antibody recognizing the strep-tag. The immobilized neuraminidase was subsequently incubated in the same buffer with the ssAD_NA detection probe at a concentration of 250 pmol.Math.l.sup.−1 and also with various concentrations of several different tested substances in the range of 100 nmol.Math.l.sup.−1 to 1 mmol.Math.l.sup.−1 and after washing, the amount of bound probe was determined by qPCR. From the ΔC.sub.q difference between wells with tested substances and wells with the detection probe itself, inhibitory constants K.sub.i of the tested substances were calculated based on equation (15) and the used concentrations of the tested substances. K.sub.i was determined 48 nmol.Math.l.sup.−1 for oseltamivir (reference value by enzyme kinetics as described in the previous section was 24 nmol.Math.l.sup.−1), for the Compound 19 it was 138 nmol.Math.l.sup.−1 (24 nmol.Math.l.sup.−1) and for other substances: for Substance 41, 85 nmol.Math.l.sup.−1 (26 nmol.Math.l.sup.−1); for Substance 42, 159 nmol.Math.l.sup.−1 (39 nmol.Math.l.sup.−1); for Substance 43, 6100 nmol.Math.l.sup.−1 (2100 nmol.Math.l.sup.−1); and for Substance 44, 38100 nmol.Math.l.sup.−1 (12700 nmol.Math.l.sup.−1). Measured values correlate very well with those obtained by enzyme kinetics reported in brackets (R.sup.2=1).
INDUSTRIAL APPLICABILITY
[0334] The described method has broad application in medicine. Given the exceptional sensitivity in the order of only several tens of molecules, it offers the possibility of determining the protein markers in blood in concentrations unmeasurable so far (e.g., PSA after prostate surgery).
[0335] Furthermore, due to the probe binding to the active site of the analyte, it is possible to measure bond strength of other tested substances to the same active site. In combination with the large dynamic range, our assay allows to determine the value of inhibition constants of the tested substances from only one measurement and using a single concentration of tested substance. Due to the high sensitivity and selectivity of the method, a minimum amount of an analyte contained in a biological matrix, e.g. blood or a cell or tissue lysate, is sufficient.