CZ33391U1 - Equipment for determining the specific effect of adenylate cyclase isoforms using HEK293 cell membrane fractions - Google Patents

Description

Technical field

The present invention relates to a kit for determining a specific effect on the activity of various forskolin-sensitive adenylate cyclase (FSK) isoforms or other chemical compounds expressed by HEK293 cells using their membrane fractions.

BACKGROUND OF THE INVENTION

Adenylate cyclase (AC) is an enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to the cyclic adenosine monophosphate (cAMP) signaling molecule. cAMP plays a key role in regulating cell functions and mediates signal transduction upon activation of a significant group of receptors called G-protein coupled receptors. A large number of available drugs currently used in clinical practice (eg beta-blockers, antihistamines, antiasthmatics) act as ligands for G-protein coupled receptors and modulate AC activity. Over 750 G-protein coupled receptor subtypes are currently described. Thus, it is a very complex family of receptors and their specific modulation is problematic. In addition, modulation of these receptors not only affects their activation, but also indirectly modulates AC activity. However, the indirect effect of these drugs on AC is not well studied today.

To date, 10 different AC isoforms have been identified that are distributed across tissues and can be regulated differently. Research into new isoform-specific modulators suggests that ACs can become an interesting direct therapeutic target for next-generation drugs. Targeted testing of substances across individual isoforms will thus allow the selection and directed synthesis of substances that will be highly specific to individual AC isoforms.

The known non-selective activator forskolin, diterpene isolated from the roots of Coleus forskohlii, is used to investigate the activity of various AC isoforms. Forskolin is able to stimulate the activity of 8 membrane-bound ACs (AC1-8).

Currently, there are no commercially available technologies allowing testing of selective modulation of the activity of these forskolin-sensitive membrane-bound ACs.

The essence of the technical solution

SUMMARY OF THE INVENTION The object of the present invention is to provide a kit by which the influence of various compounds, for example pharmaceuticals, on the activity of adenylate cyclase isoforms can be determined. Such pharmaceuticals may include compounds affecting the activity of adenylate cyclase isoforms, for example forskolin or derivatives thereof.

The present invention fulfills the above-mentioned disadvantages and drawbacks, which comprises at least one human embryonic kidney 293 cell clone which selectively expresses at least one adenylate cyclase isoform and contains a plasmid selected from the group consisting of plasmid no. SEQ. ID. NO. 1, plasmid No. 2 with SEQ ID NO.2, plasmid No. 3 with SEQ ID NO.3, plasmid No. 4 with SEQ ID NO.4, plasmid No. 5 with SEQ ID NO.5, plasmid # 6 of SEQ ID NO: 6, plasmid # 7 of SEQ ID NO, or plasmid # 8 of SEQ ID NO, further separately comprising a lysis buffer containing hydroxyethyl piperazineethanesulfonic acid (HEPES), ethylenediaminetraacetic acid ( EDTA), MgCl2PFO, dithiotreitol (DTT), sucrose and a protease inhibitor mixture containing

- 1 (4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride) (AEBSF), aprotinin (bovine pancreatic trypsin inhibitor (BPTI)), V - [(2 S, 3 R) - 3-amino-2] -liydixy-4-phenylbutyryl] -L-lucine (Bestatin), (trans-epoxysuccinyl-1-leucylamido- (4-guanidino) butane) (E-64), N-acetyl-L-leucyl-L-leucyl-L -argininal (Leupeptin), isovaleryl-Val-Val-Sta-Ala-Sta hexapaptide (Iva-Val-Val-Sta-Ala-Sta) (Pepstatin A), separately containing a stimulation buffer containing Hanks Balanced Salt Solution (HBSS), 3-isobutyl-1-methylxanthine, bovine serum albumin (BSA stabilizer), hydroxyethyl piperazinethanesulfonic acid, 3-isobutyl-1-methylxanthine, MgCF.EFO, ATP (adenosine triphosphate) and further separately contain a detection kit that includes an aqueous solution containing europium labeled cAMP, a cAMP-specific monoclonal antibody labeled with a fluorescent dye having an emission in the range of 600 to 635 nm.

According to a preferred embodiment of the invention, the HEK293 cells are in the form of a cell suspension.

Preferably, in the lysis buffer, the concentration of hydroxyethyl piperazineethanesulfonic acid is in the range of 1 to 10 mmol / l, the EDTA is in the range of 0.5 to 5 mmol / l, the MgCl 2 · FFO is in the range of 0.5 to 5 mmoFl. mmol / l, sucrose in the range 100 to 400 mmol / l, hydrochloride (4- (2-aminoethyl) benzenesulfonyl fluoride) in the range 50 to 100 mmol / l, aprotinin in the range 40 to 80 pmol / l, N - [( 25 ', 3R-3-amino-2-liydroxy-4-phenylbutyryl] -L-1-succin in the range of 1 to 4 mmol / l, (trans-epoxysuccinyl-1-leucylamido- (4-guanidino) butane) in range 0.5 to 1.5 mmol / l, N-acetyl-L-leucyl-L-leucyl-L-argininal in the range 0.5 to 2 mmoFl, isovaleryl-Val-Val-Sta-Ala-Sta in the range 0 5 to 1.5 mmol / l.

Preferably, in the stimulation buffer, the concentration of hydroxyethyl piperazineethanesulfonic acid is in the range of 1 to 10 mmol / l, 3-isobutyl-1-methylxanthine is in the range of 0.1 to 1 mmol / l, bovine serum albumin is in the range of 0.05 to 0.5 mmoFl, MgCl 2 -FO in the range of 5 to 20 mmoFl, ATP in the range of 0.05 to 0.5 mmol / L.

Preferably, the concentration of europium-labeled cAMP in the aqueous solution is in the range of 1 to 5 nmol / L and the cAMP-specific monoclonal antibody labeled with the fluorescent dye is in the range of 0.1 to 1 µg / ml.

Preferably, the detection kit comprises a cAMP calibration standard.

Eight HEK293 cell clones were developed, each clone containing a different specific plasmid 1-8. HEK293 cells containing plasmid # 1 are capable of expressing the AC1 isoform. The same principle applies to HEK293 cells containing plasmids Nos. 2 to 8 individually, thus expressing the corresponding isoforms AC2 to AC8 individually.

Each of the above plasmids contains the same portion - the vector pCMV6-Entry see SEQ. 9 and differ in the sequence of a given isoform inserted into the multi-cloning site of the vector. In particular, the plasmids below having the sequences of plasmids SEQ NO.1 to 8, as listed in the Sequence Listing, were used.

HEK293 cell clones comprising at least one plasmid having SEQ. 1 to 8 are not reproducible and therefore cannot be considered as a biologically reproducible material.

Further, the kit according to the invention comprises components for detecting the enzymatic activity of AC, which comprises europium-labeled cAMP in an aqueous solution at a concentration ranging from 1 to 5 nmol / L and a cAMP-specific monoclonal antibody labeled with a fluorescent dye having an emission range of 600-635 nm. suitable as FRET acceptor. Examples include Seta640-di-NHS or sulfo-Cyanine7, in an aqueous solution at a concentration ranging from 0.1 to 1 µg / ml, cAMP calibration standard, and stimulation buffer (see Table I for composition).

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Table I - Stimulation buffer composition used for HEK293 cell suspension

Concentration range in buffer HBSS (Hanks Balanced Salt Solution) Ix HEPES (hydroxyethyl piperazinethanesulfonic acid) 1-10 mmol / l IB MX (3-isobutyl-1-methylxanthine) 0.1-1 mmol / l Bovine serum albumin 0.05-0.5 mmol / l MgCl ₂ .H ₂ O 5-20 mmol / l ATP 0.05-0.5 mmol / l

Hanks Balanced Salt Solution (HBSS) has a composition of 1.26 mM CaCl ₂ , 0.4 mM MgSO ₄ -7H ₂ O, 0.49 mM MgCl ₂ .6H ₂ O, 5.33 mM KCl, 137.99 mM NaCl, 4.16 mM NaHCO ₃ , 0.44 mM KH ₂ PO ₄ , 0.34 mM Na ₂ HPO ₄ , 5.56 mM glucose, pH 7.4, MilliQ water.

The calibration standard is an aqueous solution of cAMP (monohydrate, adenosine sodium 3 ', 5'-cyclic monophosphate) at a precise concentration, which is used to generate a calibration curve.

The structural formula of the fluorescent dye sulfo-Cyanine7 is:

Table II - Lysis buffer composition used for HEK293 cell suspension

Range of concentrations in buffer HEPES ((hydroxyethyl piperazinethanesulfonic acid)) 1-10 mmol / l EDTA 0.5-5 mmol / l MgCl ₂ .H ₂ O 0.5-5 mmol / l dithiotreitol 0.5-5 mmol / l sucrose 100-400 mmol / l hydrochloride (4- (2-aminoethyl) benzenesulfonyl fluoride) 50-100 mmol / l aprotinin 40-80 pmol / l N- | (2S, 3R) -3-Amino-2-hydroxy-4-phenylbutyryl] -L-leucine 1-4 mmol / l (trans-epoxysuccinyl-1-leucylamido- (4-guanidino) butane) 0.5-1.5 mmol / l N-acetyl-L-leucyl-L-leucyl-L-argininal 0.5-2 mmol / l isovaleryl-Val-Sta-Ala-Sta 0.5-1.5 mmol / l

Aprotinin is a small protein - bovine pancreatic trypsin inhibitor (BPTI), or the basic inhibitor of bovine pancreatic trypsin, which is an anti-cybrinol molecule that inhibits trypsin and its related proteolytic enzymes. Leupeptin is N-acetyl-L-leucyl-L-leucyl-L-argininal.

It is a naturally occurring protease inhibitor. Pepstatin A is a potent inhibitor of aspartyl proteases. It is a hexapeptide containing the unusual amino acid statin (Sta, (3S, 4S) -4-amino-3-hydroxy-6-methylheptanoic acid) having the sequence isovaleryl-Val-Val-Sta-Ala-Sta (Iva-Val- Val-Sta-Ala-Sta).

Such a type of baptism according to the present invention, using membrane fractions of HEK293 cells stably overexpressing selected human AC isoforms to determine the degree of modulation and cross-compare between individual AC isoforms, is not commercially available. Baptism is involved not only in the development of new drugs targeted to modulate cAMP production, but also in pharmacology and toxicology in testing the effects of substances on cAMP production.

The utility model was created within the project TG020I0048 “Support of verification and commercialization of the results of research and development in the University Hospital of St. George. Anny v Brně “, Technology Agency of the Czech Republic, Program title: GAMA Applied Research, Experimental Development and Innovation Program.

Clarification of drawings

Figure 1: Scheme of plasmid # 1

Figure 2: Scheme of plasmid # 2

Figure 3: Scheme of plasmid # 3

Figure 4: Scheme of plasmid # 4

Figure 5: Scheme of plasmid # 5

Figure 6: Scheme of plasmid # 6

Figure 7: Scheme of plasmid # 7

Figure 8: Scheme of plasmid # 8

Examples of technical solutions

A kit for analyzing the activity of specific human membrane-bound isoforms AC18. Membrane fractions isolated from unique HEK293 cell clones are utilized. Analysis of affecting the activity of individual membrane-bound AC isoforms is detected by measuring cAMP production by the homogeneous fluorescence time-resolution (TR-FRET) method.

Example 1

Methodology of preparation of unique HEK293 cell clones with high expression of individual AC isoforms (1-8)

Plasmid DNA amplification and isolation

In order to create unique clones, eight plasmids made of the same vector (pCMV6Entry, Origene) containing the gene sequence for a single AC isoform (acl-8) were used. The plasmid contains a sequence encoding kanamycin resistance genes that serve as a selectable marker

In the transformation of prokaryotic cells and neomycin resistance for selection of eukaryotic cell transfection (use of the antibiotic G418 in mammalian cells, see below).

Transformation of Escherichia coli chemocompetent cells (E. coli, strain DH5-a, NEB) was performed by the heat shock method. The plasmid DNA solution (40 ng) was mixed with 100 μΐ of competent cells. The mixture was left on ice for 10 min, then incubated for 2 min at 42 ° C. The mixture was then left on ice for 2 min and incubated by shaking in 0.9 ml LuriaBertani medium (LB-medium, Duchefa Biochemie) for 1 hour at 37 ° C. After centrifugation (3500 rpm, 1 min at room temperature), the pellet was resuspended in 0.1 ml LB-medium (Duchefa Biochemistry). Cells were plated on selective LB-agar plates (1% agar in Luria-Bertani medium, Oxoid) containing the selective antibiotic kanamycin (25 μΙ / ml, Serva) and left overnight in an incubator at 37 ° C.

The amplified plasmid was isolated from transformed E. coli cells using the Plasmid MaxiPrep Kit (JetStar) according to the attached instructions. DNA purity and concentration was measured on a NanoDrop® ND-1000 (Thermo Scientific) spectrophotometer according to the ratio of absorbance values at 260 and 280 nm.

Transfection of HEK293 tissue culture

Cultured HEK293 cells (Human Embryonic Kidney 293 cells) to 70-80% confluency were transfected sequentially with individual 8 plasmids pCMV6-Entry (15 pg, Origene) carrying individual AC (1-8) genes using an electroporation method. The Gene Pulser® II electroporator (Bio-Rad) was set at 1050 pF, 270 V and pulsed twice (25-40 ms) with a 10-second pause. The resulting transfected clones were selected for 3-4 weeks using the antibiotic G418 (2 , 2 mg / ml, Biotech).

Isolation of membrane fractions

For the isolation of membrane fractions, uniquely engineered HEK293 cell clones with high expression of individual AC isoforms, as described above, were used. Three 150 mm Petri dishes with a 90% confluent HEK293 cell line were lysed (lysis buffer, Tab. Ill) and homogenized using a Dounce homogenizer. The homogenized cell lysate thus prepared was centrifuged (1800 g, 5 min, 4 ° C) to remove the nuclei, and the supernatant was subsequently cetrifuged in an XL-80 Ultracentrifuge (Beckman) at 23,000 rpm, 20 min, 4 ° C. The resulting membrane pellet was resuspended in 100 µl lysis buffer. The membrane fraction concentration was determined by total protein determination by the Bradford method (Pierce Detergent Compatible Bradford Assay Kit, Thermo Scientific).

Table III - Lysis Buffer Composition for Isolation of Membrane Fractions from HEK293 Cells.

Final concentration h ₂ o HEPES (hydroxyethyl piperazinethanesulfonic acid) 20 mmoFl EDTA (ethylenediaminetetraacetic acid) 1 mmoFl MgCh. 6 H ₂ O 2 mmoFl DTT (dithiotreitol) 1 mmoFl sucrose 250 mmol / l aprotinin 60 μ1 / 1 N - [(2S, 3R) -3-Amino-2-hydroxy-4-phenylbutyryl] -L-leucine 2 mmoFl

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(trans-epoxysuccinyl-1-leucylamido- (4-guanidino) butane) 1 mmoFl N-acetyl-L-leucyl-L-leucyl-L-argininal 1 mmoFl isovaleryl-Val-Sta-Ala-Sta 1 mmoFl

Analysis of influence of AC isoforms activity

In this technology, the time-resolved homogeneous fluorescence (TR-FRET) method is used to measure cAMP production after regulation of AC activity. The procedure combines two components - europium-labeled cAMP (3 nmoFl) and cAMP-specific monoclonal antibody labeled with fluorescent dye Seta-640-di-NHS (0.5 µg / ml) and cAMP calibration standard (50 µ (). Stimulation buffer (Table IV) was used to dilute the cAMP calibration standard and forskolin activator or test substances. The membranes were diluted to a stock concentration of 1 mg / ml in lysis buffer (Table III). Determination of the optimal amount of membranes per well was determined based on the dynamic range of the calibration curve.

Table IV - Composition of stimulation buffer used for membrane fractions of HEK293 cells

Final concentration HBSS (Hanks Balanced Salt Solution) Ix BSA Stab (7.5%, bovine serum albumin) 0.1 mmol / l HEPES (hydroxyethyl piperazinethanesulfonic acid) 5.0 mmol / l IB MX (3-isobutyl-1-methylxanthine) 0.5 mmol / l MgCh. 6H2O 10.0 mmol / l ATP 0.1 mmol / l

In a black 384-well plate (ThermoFisher Scientific), 5 μΐ of the single dilution of the calibration standard, respectively, was pipetted in duplicate. of test substance and 5 µl of stimulation buffer and 5 µl of each well was added to each well. membrane suspensions. The mixture was incubated for 30 min at room temperature, the plate was covered with foil (SealPlate film, Sigma-Aldrich). Components were pipetted into each well, first with Europium-labeled cAMP (3 nmol / L) and then with cAMP-specific monoclonal antibody labeled with fluorescent dye Seta-640-di-NHS (0.5 µg / ml). The plate was incubated for 1 hour at room temperature, without exposure to light. After incubation, samples were measured in a Spark 10M multifunctional fluorescence spectrophotometer (Tecan), with optimized parameter settings (see Table V).

Table V - Parameters for TR-FRET measurement

Parameter Value Excitation wavelength 320 nm Emission wavelength 620/665 nm Excitation bandwidth 25 nm Emission bandwidth 10 nm Number of measurements 30 Manual signal amplification 165 nm Integration time 500 ps lag time 150 ps Settle time 0 ms

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The resulting fluorescence value was normalized as the ratio of the experimental (at 665 nm) and the reference (at 620 nm) measured values. Normalized fluorescence at the highest concentration of forskolin (Y) was subtracted from the fluorescence so calculated. Thus, 100% activation is expressed as the difference between spontaneous fluorescence and fluorescence of the highest concentration of forskolin (zbc). The percentage of activation relative to the maximum possible forskolin stimulation was calculated according to the formula:

Rχ r% affinity - —---- x 100

Validation of membrane enzyme assay

(a) Intra-assay variability:

This test was performed by measuring two negative (N1 and N2 - ΟμΜ FSK) and two positive (PI and P2 - 10μΜ FSK) samples in 20 replicates of each sample according to the standard procedure above. The average relative fluorescence unit (RFU, 665/620 nm), sample standard deviation (SD) and variation coefficient (CV) were calculated for each sample (Table VI a-h). As a result, it tests the coefficient of variation for the entire measurement range. All 8 membrane-bound AC clones sensitive to forskolin stimulation (AC1-8) were included in the assay.

Table VI - Results of intra-assay variability measurements, a) AC1, b) AC2, c) AC3, d) AC4, e) AC5, f) AC6, g) AC7, h) AC8

a) AC1 Sample PI P2 RFU diameter SD CV NI N2 RFU diameter SD CV 1 0,0769 0.0794 0.0781 0.0017 2.2 0.1485 0.1438 0.1461 0,0033 2.3 2 0,0814 0,0799 0.0806 0.0011 1.3 0.1460 0.1535 0.1497 0.0053 3.5 3 0.0730 0.0745 0.0737 0.0011 1.4 0.1586 0.1518 0.1552 0,0049 3.1 4 0.0713 0.0739 0.0726 0.0019 2.6 0.1544 0.1491 0.1518 0.0037 2.5 5 0,0787 0.0780 0.0783 0.0005 0.6 0.1446 0.1488 0.1467 0,0029 2.0 6 0,0755 0,0789 0.0772 0,0023 3.0 0.1485 0.1433 0.1459 0.0036 2.5 7 0.0796 0,0798 0,0797 0.0002 0.2 0.1569 0.1524 0.1547 0.0032 2.1 8 0,0787 0,0813 0,0800 0.0019 2.3 0.1435 0.1433 0.1434 0.0002 0.1 9 0,0778 0.0748 0.0763 0,0021 2.8 0.1490 0.1457 0.1473 0,0023 1.6 10 0.0806 0,0801 0.0803 0.0003 0.4 0.1462 0.1521 0.1491 0,0042 2.8 11 0.0792 0.0775 0.0783 0,0012 1.5 0.1441 0.1494 0.1468 0.0037 2.5 12 0.0763 0,0863 0,0813 0.0070 8.6 0.1529 0.1444 0.1487 0.0060 4.0 13 0.0732 0.0776 0.0754 0.0032 4.2 0.1502 0.1466 0.1484 0.0025 1.7 14 0.0772 0,0892 0,0832 0,0084 10.1 0.1423 0.1436 0.1429 0.0009 0.6 15 Dec 0.0763 0,0813 0.0788 0.0035 4,5 0.1471 0.1492 0.1482 0.0015 1.0 16 0,0823 0.0760 0.0792 0.0044 5.6 0.1510 0.1447 0.1479 0.0045 3.0 17 0,0747 0.0766 0.0757 0,0013 1,8 0.1546 0.1480 0.1513 0.0047 3.1 18 0.0786 0.0794 0,0790 0.0005 0.7 0.1486 0.1508 0.1497 0.0015 1.0 19 Dec 0,0857 0,0814 0,0836 0,0030 3.6 0.1485 0.1523 0.1504 0,0027 1,8 20 May 0,0762 0,0672 0.0717 0.0064 8.9 0.1504 0.1579 0.1541 0.0053 3.4 Intra-assay (n = 20) mean CV (%) 3.3 2.2

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b) AC2 Sample Pl P2 RFU diameter SD CV N1 N2 RFU diameter SD CV 1 0,0939 0,0971 0,0955 0,0023 2.4 0.1273 0.1241 0.1257 0,0023 1,8 2 0.1023 0,0936 0,0980 0.0062 6.3 0.1253 0.1279 0.1266 0.0018 1.4 3 0,0912 0,0930 0.0921 0,0012 1.3 0.1212 0.1239 0.1226 0.0019 1.6 4 0.1006 0,0966 0,0986 0.0028 2.9 0.1264 0.1270 0.1267 0.0005 0.4 5 0,0885 0,0850 0,0868 0.0025 2.9 0.1247 0.1255 0.1251 0.0005 0.4 6 0.0927 0.0923 0,0925 0.0003 0.4 0.1293 0.1218 0.1255 0.0053 4.2 7 0,0919 0.0920 0.0920 0.0001 0.1 0.1348 0.1268 0.1308 0.0057 4.4 8 0,0908 0,0934 0.0921 0.0018 2.0 0.1264 0.1235 0.1250 0,0021 1.7 9 0,0769 0,0767 0,0768 0.0002 0.2 0.1214 0.1234 0.1224 0,0014 1,2 10 0,0974 0,0952 0,0963 0.0015 1.6 0.1251 0,1195 0.1223 0.0039 3.2 11 0,0859 0,0878 0,0869 0,0014 1.6 0.1347 0.1315 0.1331 0,0023 1.7 12 0,0912 0,0885 0,0899 0.0019 2.1 0.1279 0.1241 0.1260 0,0027 2.1 13 0,0846 0.0884 0,0865 0,0027 3.1 0,1193 0.1241 0.1217 0.0034 2.8 14 0,0939 0,0904 0.0921 0.0025 2.7 0.1207 0.1220 0.1213 0.0009 0.7 15 Dec 0,0867 0,0877 0,0872 0.0007 0.9 0.1140 0.1129 0.1135 0.0008 0.7 16 0.0920 0,0910 0,0915 0.0007 0.7 0,1193 0.1241 0.1217 0.0034 2.8 17 0.0713 0,0799 0.0756 0,0061 8.0 0.1218 0.1261 0.1240 0,0030 2.4 18 0,0812 0,0863 0,0838 0.0036 4.3 0.1216 0.1243 0.1230 0.0020 1.6 19 Dec 0,0952 0,0916 0,0934 0,0026 2.7 0.1252 0.1218 0.1235 0,0024 2.0 20 May 0.0776 0,0690 0.0733 0,0061 8.4 0.1252 0.1311 0.1282 0,0042 3.3 Intra-assay (n = 20) mean CV (%) 2.7 2.0

c) AC3 Sample Pl P2 RFU diameter SD CV N1 N2 RFU diameter SD CV 1 0,0400 0.0384 0,0392 0,0012 3.0 0.1213 0.1129 0,1171 0.0060 5.1 2 0,0351 0,0341 0,0346 0.0007 2.0 0.1066 0.1098 0.1082 0,0023 2.1 3 0.0310 0,0333 0.0322 0,0016 5.1 0,1192 0,1181 0,1186 0.0008 0.7 4 0,0359 0,0342 0.0350 0,0012 3.5 0.1199 0.1180 0,1189 0,0013 1.1 5 0,0378 0.0344 0,0361 0,0024 6.6 0.1121 0.1150 0.1136 0,0021 1,8 6 0,0300 0,0295 0,0297 0.0003 1,2 0,1162 0.1124 0,1143 0,0027 2.3 7 0.0310 0.0323 0,0316 0.0009 2.7 0,1183 0,1190 0,1187 0.0005 0.4 8 0,0372 0,0343 0,0357 0.0020 5.7 0,1154 0,1153 0,1154 0.0001 0.0 9 0,0317 0,0335 0.0326 0,0013 3.8 0.1224 0.1138 0,1181 0.0060 5.1 10 0.0302 0.0328 0,0315 0.0018 5.8 0,1196 0.1126 0,1161 0.0050 4.3 11 0.0306 0.0340 0.0323 0,0024 7.5 0.1101 0,1141 0.1121 0.0028 2.5 12 0.0324 0.0325 0.0325 0.0000 0.1 0.1283 0.1226 0.1254 0.0040 3.2 13 0,0333 0,0343 0,0338 0.0007 2.2 0,1181 0.1128 0,1154 0.0038 3.3 14 0,0311 0,0367 0,0339 0.0040 11.8 0,1146 0.1128 0,1137 0,0013 1.1 15 Dec 0,0367 0,0371 0,0369 0.0003 0.9 0,1188 0.1208 0,1198 0,0014 1,2 16 0.0327 0,0314 0.0321 0.0009 2.9 0,1155 0.1160 0,1158 0.0004 0.3 17 0.0302 0.0326 0,0314 0.0017 5.4 0.1204 0.1296 0.1250 0,0065 5.2 18 0.0321 0.0350 0,0336 0,0021 6.2 0.1211 0.1257 0.1234 0,0033 2.7 19 Dec 0,0334 0,0332 0,0333 0.0002 0.5 0.1120 0.1095 0,1108 0.0018 1.6 20 May 0,0336 0,0349 0,0342 0.0009 2.7 0,1190 0.1127 0,1158 0.0044 3.8 Intra-assay (n = 20) mean CV (%) 4.0 2.4

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d) AC4

Model to Pl P2 Avg rRFU SD CV N1 N2 Avg rRFU SD CV 1 0.0745 0.0751 0.0748 0.0004 0.6 0.1283 0.1311 0.1297 0.0020 1.5 2 0.0887 0,0890 0.0888 0.0002 0.3 0.1290 0.1273 0.1282 0,0012 0.9 3 0,0871 0,0892 0.0881 0.0015 1.7 0.1278 0.1280 0.1279 0.0001 0.1 4 0.0807 0,0836 0,0821 0,0021 2.5 0.1268 0.1335 0.1301 0.0048 3.7 5 0,0843 0,0891 0,0867 0.0034 3.9 0.1309 0.1229 0.1269 0.0057 4,5 6 0,0768 0.0889 0,0828 0.0085 10.3 0.1310 0.1271 0.1291 0,0027 2.1 7 0,0874 0,0870 0,0872 0.0003 0.3 0.1264 0.1257 0.1261 0.0005 0.4 8 0,0782 0,0862 0,0822 0.0057 6.9 0.1339 0.1288 0.1313 0.0036 2.7 9 0,0812 0,0861 0,0837 0.0034 4.1 0.1273 0.1296 0.1285 0.0017 1.3 10 0,0872 0,0795 0,0833 0.0055 6.6 0.1314 0.1232 0.1273 0,0058 4.6 11 0,0857 0,0777 0,0817 0.0056 6.9 0.1340 0.1208 0.1274 0.0093 7.3 12 0,0795 0.0786 0,0791 0.0006 0.8 0.1297 0.1236 0.1267 0,0043 3.4 13 0,0850 0,0840 0,0845 0.0007 0.8 0.1299 0.1293 0.1296 0.0005 0.4 14 0,0860 0,0877 0,0868 0,0012 1.4 0.1234 0.1287 0.1260 0.0037 2.9 15 Dec 0,0854 0,0839 0,0847 0.0011 1,2 0.1230 0.1326 0.1278 0.0068 5.3 16 0,0830 0,0851 0,0840 0.0015 1,8 0.1201 0.1144 0,1172 0.0040 3.4 17 0,0762 0,0652 0,0707 0.0078 11.0 0,1192 0.1229 0.1210 0,0026 2.2 18 0,0893 0,0857 0,0875 0.0025 2.9 0.1180 0.1274 0.1227 0.0066 5.4 19 Dec 0,0834 0,0932 0.0883 0.0069 7.9 0.1301 0,1185 0.1243 0.0082 6.6 20 May 0,0943 0,0965 0,0954 0.0015 1.6 0.1290 0,1170 0.1230 0.0085 6.9 Intra-assay (n = 20) mean CV (%) 3.7 3.3

e) AC5 Sample Pl P2 RFU diameter SD CV N1 N2 RFU diameter SD CV 1 0.0550 0.0533 0.0541 0,0012 2.2 0.1385 0.1438 0.1412 0.0037 2.7 2 0,0645 0.0572 0.0609 0,0051 8.4 0.1459 0.1412 0.1435 0,0033 2.3 3 0,0594 0.0575 0,0584 0,0013 2.3 0.1346 0.1517 0.1431 0.0121 8.5 4 0,0487 0.0542 0.0514 0.0039 7.6 0.1492 0.1451 0.1471 0,0029 2.0 5 0,0640 0,0614 0,0627 0.0018 2.8 0.1474 0.1417 0.1446 0.0040 2.7 6 0,0636 0,0687 0,0662 0.0036 5.4 0.1550 0.1502 0.1526 0.0034 2.2 7 0,0614 0,0565 0.0590 0.0034 5.8 0.1430 0.1400 0.1415 0,0021 1.5 8 0,0678 0.0571 0,0624 0.0076 12.1 0.1523 0.1519 0.1521 0.0003 0.2 9 0.0537 0,0585 0,0561 0.0034 6.1 0.1483 0.1450 0.1466 0,0023 1.6 10 0,0557 0,0615 0,0586 0,0041 7.0 0.1532 0.1518 0.1525 0.0009 0.6 11 0.0605 0,0679 0,0642 0.0052 8.2 0.1490 0.1446 0.1468 0.0031 2.1 12 0.0547 0.0534 0.0540 0.0009 1.7 0.1460 0.1450 0.1455 0.0008 0.5 13 0.0539 0.0560 0.0550 0.0015 2.7 0.1467 0.1459 0.1463 0.0006 0.4 14 0.0537 0.0598 0,0567 0,0043 7.5 0.1546 0.1590 0.1568 0.0031 2.0 15 Dec 0.0502 0,0583 0.0542 0.0057 10.5 0.1482 0.1443 0.1462 0.0028 1.9 16 0.0508 0.0596 0.0555 0.0062 11.3 0.1483 0.1489 0.1486 0.0004 0.3 17 0.0506 0.0520 0.0513 0,0010 1.9 0.1537 0.1551 0.1544 0,0010 0.7 18 0,0611 0.0602 0.0607 0.0007 1.1 0.1459 0.1440 0.1450 0,0013 0.9

-9cz 33391 U1

19 Dec 0.0632 0.0657 0,0644 0.0018 2.7 0.1404 0.1426 0.1415 0,0016 1.1 20 May 0.0648 0.0632 0,0640 0,0012 1,8 0.1486 0.1444 0.1465 0,0030 2.0 Intra-assay (n = 20) mean CV (%) 5.5 1,8

f) AC6 Model to Pl P2 RFU diameter SD CV N1 N2 RFU diameter SD CV 1 0,0862 0,0916 0.0889 0.0039 4.3 0.1412 0.1490 0.1451 0.0056 3.8 2 0,0885 0,0861 0,0873 0.0017 1.9 0.1445 0.1531 0.1488 0,0061 4.1 3 0.0776 0,0811 0,0793 0.0025 3.1 0.1565 0.1498 0.1531 0.0048 3.1 4 0,0815 0.0806 0,0811 0.0006 0.8 0.1561 0.1493 0.1527 0.0048 3.1 5 0,0916 0.0796 0,0856 0.0085 10.0 0.1494 0.1488 0.1491 0.0005 0.3 6 0.0756 0,0857 0.0806 0.0071 8.8 0.1429 0.1490 0.1459 0,0043 2.9 7 0.0737 0.0717 0.0727 0,0014 1.9 0.1463 0.1497 0.1480 0,0023 1.6 8 0,0871 0.0775 0,0823 0.0067 8.2 0.1472 0.1470 0.1471 0.0001 0.1 9 0,0820 0,0840 0,0830 0,0014 1.7 0.1513 0.1524 0.1518 0.0007 0.5 10 0.0772 0,0867 0,0819 0.0067 8.2 0.1598 0.1502 0.1550 0.0068 4.4 11 0,0873 0,0795 0,0834 0.0055 6.6 0.1293 0.1414 0.1354 0.0085 6.3 12 0,0865 0,0871 0,0868 0.0004 0.5 0.1521 0.1475 0.1498 0,0033 2.2 13 0,0866 0.0735 0,0801 0.0093 11.6 0.1448 0.1473 0.1461 0.0017 1,2 14 0,0891 0.0764 0,0827 0.0090 10.9 0.1451 0.1497 0.1474 0,0033 2.2 15 Dec 0,0778 0,0839 0.0809 0,0043 5.3 0.1471 0.1572 0.1521 0.0071 4.7 16 0,0801 0,0811 0.0806 0.0007 0.8 0.1419 0.1434 0.1427 0.0011 0.8 17 0,0818 0,0859 0,0838 0,0029 3.5 0.1401 0.1323 0.1362 0.0055 4.0 18 0,0824 0,0897 0,0861 0,0051 5.9 0.1532 0.1477 0.1505 0.0039 2.6 19 Dec 0.0760 0.0881 0,0820 0.0086 10.5 0.1514 0.1475 0.1494 0.0028 1.9 20 May 0,0859 0,0836 0,0847 0,0016 1.9 0.1522 0.1516 0.1519 0.0005 0.3 [ntra-assay (n = 20) mean CV (%) 5.3 2.5

(g) AC7 Model to Pl P2 Avg rRFU SD CV N1 N2 RFU diameter SD CV 1 0,0618 0,0650 0,0634 0,0022 3.5 0.1388 0.1313 0.1351 0.0053 4.0 2 0,0624 0,0636 0,0630 0.0009 1.4 0.1381 0.1367 0.1374 0,0010 0.7 3 0,0656 0,0667 0,0662 0.0008 1.3 0.1321 0.1295 0.1308 0.0019 1.4 4 0,0631 0,0673 0,0652 0,0029 4,5 0.1376 0.1251 0.1314 0.0088 6.7 5 0,0629 0,0667 0,0648 0,0026 4.1 0.1322 0.1319 0.1321 0.0003 0.2 6 0,0613 0,0641 0,0627 0.0020 3.2 0.1369 0.1360 0.1364 0.0006 0.5 7 0,0634 0,0565 0,0600 0,0049 8.1 0.1400 0.1424 0.1412 0.0018 1,2 8 0,0667 0,0649 0,0658 0,0013 1.9 0.1345 0.1438 0.1392 0.0066 4.7 9 0,0672 0,0585 0,0628 0.0062 9.9 0.1370 0.1392 0.1381 0,0016 1.1 10 0,0682 0,0589 0,0635 0.0066 10.3 0.1416 0.1321 0.1368 0.0068 4.9 11 0,0585 0,0653 0,0619 0.0048 7.7 0.1394 0.1413 0.1403 0,0013 0.9

- 10GB 33391 U1

12 0.0660 0.0628 0,0644 0,0022 3.4 0.1316 0.1394 0.1355 0.0055 4.1 13 0.0655 0.0664 0,0659 0.0007 1.0 0.1467 0.1404 0.1435 0.0045 3.1 14 0.0601 0.0645 0.0623 0.0031 5.1 0.1443 0.1445 0.1444 0.0001 0.1 15 Dec 0.0563 0.0663 0,0613 0.0071 11.5 0.1395 0.1385 0.1390 0.0007 0.5 16 0.0639 0.0590 0,0615 0.0035 5.6 0.1376 0.1295 0.1335 0,0058 4.3 17 0.0621 0.0556 0,0594 0.0039 6.5 0.1403 0.1448 0.1426 0.0032 2.2 18 0.0585 0.0606 0.0595 0.0015 2.5 0.1359 0.1302 0.1331 0,0041 3.0 19 Dec 0.0624 0.0670 0,0647 0.0032 5.0 0.1385 0.1317 0.1351 0.0048 3.6 20 May 0.0652 0.0608 0,0630 0.0031 5.0 0.1421 0.1372 0.1396 0.0035 2.5 Intra-assay (n = 20) mean CV (%) 5.1 2.5

h) AC8 Sample PI P2 RFU diameter SD CV NI N2 RFU diameter SD CV 1 0.0623 0,0731 0,0677 0.0076 11.3 0.1483 0.1508 0.1496 0.0018 1,2 2 0.0524 0,0471 0,0498 0.0038 7.6 0.1408 0.1458 0.1433 0.0035 2.4 3 0,0619 0,0557 0.0588 0.0044 7.5 0.1421 0.1453 0.1437 0,0022 1.5 4 0,0523 0,0559 0.0541 0.0025 4.7 0.1506 0.1563 0.1534 0.0040 2.6 5 0.0772 0,0634 0.0703 0,0098 13.9 0.1423 0.1473 0.1448 0.0035 2.4 6 0,0642 0.0704 0,0673 0.0044 6.5 0.1434 0.1489 0.1461 0.0039 2.7 7 0.0592 0.0512 0.0555 0.0056 10.2 0.1428 0.1466 0.1447 0,0027 1,8 8 0.0771 0,0765 0,0768 0.0005 0.6 0.1523 0.1596 0.1559 0.0052 3.3 9 0,0586 0,0568 0.0577 0,0012 2.2 0.1411 0.1573 0.1492 0.0115 7.7 10 0,0561 0,0562 0,0561 0.0001 0.1 0.1559 0.1423 0.1491 0.0096 6.5 11 0,0421 0.0404 0,0412 0,0012 2.9 0.1516 0.1486 0.1501 0,0021 1.4 12 0,0559 0,0486 0.0522 0.0052 9.9 0.1514 0.1452 0.1483 0.0044 3.0 13 0.0705 0,0661 0,0683 0.0031 4,5 0.1508 0.1531 0.1519 0.0017 1.1 14 0,0457 0,0490 0,0474 0,0023 5.0 0.1345 0.1315 0.1330 0,0021 1.6 15 Dec 0,0567 0,0479 0,0523 0,0063 12.0 0.1449 0.1440 0.1445 0.0006 0.4 16 0.0539 0.0539 0.0539 0.0000 0.0 0.1455 0.1487 0.1471 0,0022 1.5 17 0.0745 0,0612 0,0678 0.0094 13.8 0.1524 0.1472 0.1498 0.0037 2.5 18 0,0436 0.0519 0,0478 0.0059 12.3 0.1408 0.1401 0.1404 0.0005 0.3 19 Dec 0,0445 0.0541 0,0493 0.0068 13.8 0.1471 0.1489 0.1480 0,0013 0.9 20 May 0,0494 0.0531 0.0513 0,0026 5.1 0.1501 0.1467 0.1484 0,0024 1.6 Intra-assay (n = 20) mean CV (%) 7.2 2.3

(b) Inter-assay variability:

Testing was performed as ten independent analyzes. Two negative (N1 and N2 - ΟμΜ FSK) and two positive (P1 and P2 - ΙΟμΜ FSK) membrane samples were tested, each in quadruplicate according to the standard procedure above. The average fluorescence value (RFU, 665/620 nm) was calculated for each sample, the mean, the mean for the whole range, the standard deviation, the selection and the coefficient of variation for the whole measurement range were calculated (Table VII a-h). All 8 membrane-bound AC clones sensitive to forskolin stimulation (AC1-8) were included in the assay.

Table VII - Inter-assay variability measurement results, a) AC1, b) AC2, c) AC3, d) AC4, e) AC5, f) AC6, g) AC7, h) AC8

- 11 GB 33391 U1

a) AC1 Pl P2 RFU diameter NI N2 RFU diameter 1 0,0668 0,0635 0,0662 0.1496 0.1397 0.1411 0,0632 0.0711 0.1314 0.1436 2 0,0413 0,0435 0,0457 0.1446 0.1428 0.1407 0.0504 0,0478 0.1368 0.1386 3 0,0480 0,0477 0.0509 0.1408 0.1458 0.1435 0,0470 0,0611 0.1398 0.1477 4 0,0494 0.0504 0.0502 0.1446 0.1439 0.1444 0,0464 0.0546 0.1447 0.1444 5 0,0523 0.0520 0,0489 0.1475 0.1431 0.1438 0.0503 0.0408 0.1413 0.1432 6 0,0443 0.0321 0.0380 0.1461 0.1419 0.1447 0,0304 0,0453 0.1489 0.1420 7 0,0491 0,0414 0,0477 0.1368 0.1410 0.1409 0,0433 0,0569 0.1421 0.1439 8 0,0561 0.0541 0.0538 0.1414 0.1416 0.1228 0.0533 0.0518 0.1039 0.1042 9 0.0576 0,0469 0.0513 0.1512 0.1484 0.1513 0,0469 0.0539 0.1524 0.1533 10 0,0489 0,0526 0.0510 0.1462 0.1434 0.1460 0.0533 0,0492 0.1462 0.1484 Positive samples Negative samples diameter of the whole range 0.0504 diameter of the whole range 0.1419 SD for the entire range 0.0070 SD for the entire range 0.0074 CV (%) for the whole range 14.0 CV (%) for the whole range 5.2

b) AC2 PI P2 RFU diameter NI N2 RFU diameter 1 0.0579 0.0580 0,0583 0.0883 0,0947 0,0918 0.0574 0.0598 0,0949 0,0893 2 0,0553 0,0463 0.0518 0,0818 0,0861 0,0858 0,0554 0.0503 0,0872 0.0882 3 0.0757 0.0751 0.0760 0.1056 0.1110 0.1101 0,0793 0.0737 0.1122 0,1118 4 0,0683 0,0863 0,0770 0,1155 0,1123 0,1146 0,0669 0,0865 0,1175 0.1133 5 0,0742 0,0817 0.0784 0.1074 0.1016 0.1069 0,0816 0,0761 0.1061 0.1126 6 0,0831 0,0823 0,0827 0.1049 0,1134 0.1069 0,0843 0,0811 0.1047 0.1045 7 0.0710 0,0693 0.0715 0.1002 0.0992 0.0991

- 12GB 33391 U1

0.0700 0.0756 0,0984 0,0985 8 0,0769 0,0777 0.0740 0.1076 0.1067 0.1091 0,0691 0.0723 0.1081 0,1139 9 0.0706 0.0748 0,0697 0.1076 0.1074 0.1073 0,0682 0,0652 0.1071 0.1071 10 0,0682 0.0713 0.0701 0.1037 0.1027 0.1063 0.0714 0,0695 0.1095 0.1095

Positive samples Negative samples diameter of the whole range 0,0709 diameter of the whole range 0.1038 SD for the entire range 0.0094 SD for the entire range 0.0089 CV (%) for the whole range 13.2 CV (%) for the whole range 8.6

c) AC3 PI P2 RFU diameter NI N2 RFU diameter 1 0,0455 0,0423 0,0497 0.1442 0.1500 0.1477 0.0544 0,0565 0.1484 0.1482 2 0,0413 0,0448 0,0411 0.1352 0.1367 0.1358 0,0394 0,0388 0.1302 0.1411 3 0,0432 0,0435 0,0428 0.1389 0.1436 0.1419 0,0418 0,0426 0.1419 0.1433 4 0,0437 0,0439 0,0440 0.1274 0.1233 0.1288 0,0451 0,0435 0.1316 0.1327 5 0,0362 0.0350 0,0354 0.1200 0.1121 0.1217 0,0342 0,0362 0.1341 0.1207 6 0,0372 0,0376 0,0399 0.1233 0.1222 0.1234 0,0437 0,0412 0.1261 0.1221 7 0,0413 0,0430 0,0436 0.1287 0.1359 0.1338 0,0442 0,0459 0.1360 0.1347 8 0,0442 0,0432 0,0446 0.1279 0.1292 0.1328 0,0477 0,0432 0.1349 0.1393 9 0.0403 0.0402 0.0408 0.1275 0.1363 0.1334 0,0414 0,0413 0.1331 0.1367 10 0,0412 0,0438 0,0438 0.1343 0.1321 0.1328 0,0447 0,0457 0.1329 0.1320 Positive samples Negative samples diameter range all over 0,0426 diameter of the whole range 0.1332 SD for range whole 0.0037 SD for the entire range 0.0077 CV (%) for the whole range 8.7 CV (%) for the whole range 5.8

- 13GB 33391 U1

d) AC4 Pl P2 RFU diameter NI N2 RFU diameter 1 0,0628 0.0706 0,0661 0.1274 0.1211 0.1234 0,0601 0,0709 0.1215 0.1237 2 0,0690 0,0731 0,0673 0,1139 0.1249 0.1201 0.0602 0,0669 0.1211 0.1205 3 0,0643 0,0624 0,0643 0,1179 0,1193 0.1211 0,0682 0,0624 0.1217 0.1254 4 0,0791 0,0688 0,0708 0.1251 0.1351 0.1253 0,0709 0,0644 0.1297 0,1113 5 0,0699 0,0746 0,0742 0.1399 0.1486 0.1456 0,0697 0,0825 0.1494 0.1444 6 0.0734 0,0671 0,0678 0,1172 0,1170 0,1172 0,0634 0,0672 0,1155 0,1191 7 0,0647 0,0640 0,0667 0,1153 0.1132 0.1144 0.0728 0,0653 0.1144 0.1145 8 0,0635 0,0627 0,0697 0.1248 0.1102 0,1175 0.0740 0,0785 0,1198 0,1153 9 0.0788 0,0769 0.0753 0,1147 0.1124 0,1162 0.0758 0,0698 0.1208 0,1167 10 0,0643 0.0714 0,0681 0,1173 0.1180 0,1166 0,0653 0.0713 0,1123 0,1186 Positive samples Negative samples average of the whole range SD for the whole range CV (%) for the whole range 0,0690 0.0035 5.1 average of the whole range SD for the whole range CV (%) for the whole range 0.1217 0,0091 7.4

e) AC5 PI P2 RFU diameter NI N2 Diameter RFU 1 0.0505 0,0589 0,0497 0.1513 0.1547 0.1474 0,0435 0,0461 0.1384 0.1452 2 0,0482 0,0551 0,0478 0.1549 0.1489 0.1493 0,0448 0,0431 0.1464 0.1469 3 0,0489 0.0402 0,0472 0.1505 0.1513 0.1490 0,0495 0.0502 0.1476 0.1468 4 0,0429 0,0472 0,0447 0.1600 0.1549 0.1576 0,0463 0,0426 0.1568 0.1588 5 0,0469 0.0501 0,0474 0.1519 0.1499 0.1514 0,0478 0,0448 0.1538 0.1499 6 0.0576 0,0488 0.0515 0.1483 0.1465 0.1499 0,0494 0.0501 0.1524 0.1525 7 0.0515 0.0539 0.0536 0.1556 0.1404 0.1511

- 14GB 33391 U1

0.0540 0.0549 0.1566 0.1516 8 0,0459 0,0480 0,0461 0.1511 0.1413 0.1473 0,0472 0,0432 0.1469 0.1500 9 0,0492 0,0429 0,0480 0.1501 0.1404 0.1459 0,0443 0.0555 0.1420 0.1511 10 0,0462 0.0513 0,0484 0.1476 0.1434 0.1461 0,0486 0,0474 0.1438 0.1497 Positive samples Negative samples diameter of the whole range 0,0484 diameter of the whole range 0.1495 SD for the entire range 0,0026 SD for the entire range 0.0034 CV (%) for the whole range 5.3 CV (%) for the whole range 2.3

f) AC6 PI P2 RFU diameter NI N2 Diameter RFU 1 0,0684 0,0680 0,0637 0.1311 0.1319 0.1323 0,0600 0,0586 0.1314 0.1348 2 0.0535 0,0584 0,0584 0.1242 0.1207 0.1241 0.0574 0,0642 0.1243 0.1274 3 0,0679 0,0695 0.0705 0.1263 0.1284 0.1276 0,0684 0.0763 0.1287 0.1269 4 0,0662 0.0754 0,0694 0.1240 0.1276 0.1256 0,0612 0,0749 0.1239 0.1269 5 0,0639 0,0668 0,0647 0.1420 0.1400 0.1421 0,0661 0,0620 0.1419 0.1443 6 0,0693 0,0683 0,0671 0.1386 0.1349 0.1381 0,0672 0,0635 0.1372 0.1416 7 0.0511 0.0570 0,0559 0.1337 0.1308 0.1323 0.0598 0.0558 0.1303 0.1290 8 0,0479 0,0498 0,0489 0.1309 0.1206 0.1261 0,0437 0.0542 0.1290 0.1241 9 0.0724 0,0702 0.0713 0.1297 0.1381 0.1339 0,0699 0,0660 0.1374 0.1333 10 0,0742 0.0732 0.0737 0.1505 0.1529 0.1517 0,0816 0,0767 0.1482 0.1499 Positive samples Negative samples diameter of the whole range 0,0644 diameter full scale 0.1334 SD for the entire range 0.0078 SD for the entire range 0.0086 CV (%) for the whole range 12.2 CV (%) for the whole range 6.5

(g) AC7 PI P2 RFU diameter NI N2 RFU diameter 1 0,0420 0,0446 0,0420 0,1118 0.1150 0.1130 0.0404 0,0412 0.1120 0.1133 2 0.0511 0.0540 0.0530 0.1414 0.1429 0.1408

- 15GB 33391 U1

0.0507 0,0563 0.1373 0.1416 3 0,0460 0,0487 0,0473 0.1351 0.1345 0.1340 0,0443 0,0500 0.1336 0.1327 4 0,0487 0.0524 0.0505 0.1321 0.1260 0.1291 0,0431 0,0447 0.1356 0.1316 5 0,0458 0.0404 0,0439 0.1256 0.1297 0.1272 0,0461 0,0433 0.1243 0.1295 6 0,0414 0,0422 0,0437 0.1284 0.1276 0.1257 0,0478 0,0436 0.1228 0.1240 7 0,0584 0,0567 0.0560 0.1136 0.1266 0.1201 0.0520 0,0567 0.1130 0.1095 8 0,0466 0,0480 0,0473 0,1168 0,1175 0,1171 0,0465 0.0502 0,1156 0,1182 9 0,0529 0.0576 0.0555 0.1229 0.1321 0.1275 0,0458 0.0544 0.1261 0.1257 10 0,0446 0,0494 0,0470 0.1283 0,1173 0.1228 0,0447 0,0473 0.1257 0.1230 Positive samples Negative samples diameter of the whole range 0,0486 diameter of the whole range 0.1257 SD for the entire range 0,0049 SD for the entire range 0.0081 CV (%) for the whole range 10.1 CV (%) for the whole range 6.4

h) AC8 PI P2 RFU diameter NI N2 RFU diameter 1 0,0499 0,0439 0,0490 0.1460 0.1456 0.1461 0.0531 0,0490 0.1426 0.1500 2 0.0409 0,0392 0,0462 0.1203 0.1269 0.1236 0,0527 0.0519 0.1291 0,1185 3 0,0410 0,0420 0,0459 0.1319 0.1331 0.1321 0,0470 0.0536 0.1374 0.1261 4 0,0412 0,0450 0,0431 0.1260 0.1250 0.1255 0,0489 0,0460 0.1279 0.1277 5 0.0609 0.0520 0.0588 0.1432 0.1512 0.1448 0,0639 0,0583 0.1402 0.1446 6 0,0357 0,0415 0.0406 0.1342 0.1385 0.1324 0.0360 0,0494 0.1276 0.1293 7 0,0477 0.0531 0,0479 0.1363 0.1416 0.1389 0,0412 0,0496 0.1355 0.1475 8 0.0513 0.0525 0.0519 0.1254 0.1387 0.1320 0.0539 0.0608 0.1360 0.1404 9 0,0498 0,0388 0,0443 0.1234 0.1216 0.1225

- 16cz 33391 U1

10 0.0676 0.0529 0.0517 0.0470 0.0524 0.0572 0,0494 0,1174 0,1179 0.1253 0.1281 0.1298 0.1238 0.1267 Positive samples Negative samples diameter of the whole range 0,0477 diameter of the whole range 0.1325 SD for the entire range 0,0051 SD for the entire range 0,0084 CV (%) for the whole range 10.7 CV (%) for the whole range 6.4

Example 3

Examples of the composition of each kit are given below.

Kit No. 1 contains a suspension of a HEK293 cell clone having the plasmid of SEQ. Separately, it contains a lysis buffer containing 1 mmol / l hydroxyethyl piperazineethanesulfonic acid, 0.5 mmol / l EDTA, 0.5 mmol / l MgCl2 / h, 0.5 mmol / l dithiotreitol, sucrose. 100 mmol / l, (4- (2-aminoethyl) benzenesulfonyl fluoride) hydrochloride 50 mmol / l, aprotinin 40 pmol / l, N - [(2 ', 3 R) -3-amino-2-liyldoxy] - 4-phenylbutyryl] -L-lucucine 1 mmol / l, (trans-epoxysuccinyl-1-leucylamido- (4-guanidino) butane) 0.5 mmol / l, N-acetyl-L-leucyl-L-leucyl-L- argininal 0.5 mmol / l, isovaleryl-Val-Val-Sta-Ala-Sta 0.5 mmol / l. Separately, the stimulation buffer containing Hanks Balanced Salt Solution, as described above, contains 1 mmol / 1, 3-isobutyl-1-methylxanthine hydroxyethyl piperazineethanesulfonic acid, 0.1 mmol / l, bovine serum albumin at 0 0.05 mmol / l, 5 mmol / l MgCl2H2O, 0.05 mmol / l ATP. It also separately contains a detection kit that includes a europium-labeled cAMP 1 nmol / L, a cAMPspecific monoclonal antibody labeled with Seta-640-di-NHS 0.1 pg / ml acAMP calibration standard.

Kit No. 2 contains a suspension of a HEK293 cell clone having a plasmid of SEQ ID NO.4. Separately, it contains a lysis buffer containing 5 mmol / l hydroxyethyl piperazineethanesulfonic acid, 1 mmol / l EDTA, 1 mmol / l MgCl 2, 10 mmol / l dithiotreitol, 250 mmol / l sucrose, (4- (2-aminoethyl) benzenesulfonyl fluoride) 80 mmol / l, aprotinin 60 pmol / l, N - [(2 ', 3 R) -3-amino-2-liydroxy-4-phenylbutyryl] -L -lucucine 2 mmol / l, (trans-epoxysuccinyl-1-leucylamido- (4-guanidino) butane) 1 mmol / l, N-acetyl-L-leucyl-L-leucyl-L-argininal 1 mmol / l, isovaleryl- Val-Val-Sta-Ala-Sta 1 mmol / L.

Separately, the stimulation buffer containing Hanks Balanced Salt Solution, as described above, contains 5 mmol / l hydroxyethyl piperazineethanesulfonic acid and 0.5 mmol / l 3-isobutyl-1-methylxanthine, bovine serum albumin at 0 1 mmol / l, 10 mmol / l MgCl2 · H2O, 0.1 mmol / l ATP. It also separately contains a detection kit that includes a europium-labeled cAMP of 3 nmol / L, a cAMP-specific monoclonal antibody labeled with Seta-640-di-NHS 0.5 µg / ml, and a cAMP calibration standard.

Kit No. 3 contains a suspension of a HEK293 cell clone having the plasmid of SEQ. Further, separately includes a lysis buffer containing hydroxyethyl piperazineethanesulphonic acid at a concentration of 10 mmol / 1 EDTA at a concentration of 5 mM / 1 MgC12.6H ₂ O at a concentration of 5 mmol / 1 dithiothreitol at a concentration of 5 mmol / 1, sucrose at a concentration of 400 mmol / 1, (4- (2-Aminoethyl) benzenesulfonyl fluoride) hydrochloride 100 mmol / l, aprotinin 80 pmol / 1, N - [(2 ', 3 R) -3-amino-2-liidiidoxy-4-phenylbutyryl] -L-lucucine 4 mmol / l, (trans-epoxysuccinyl-1-leucylamido- (4-guanidino) butane) 1.5 mmol / l, N-acetyl-L-leucyl-L-leucyl-L-argininal in range 2 mmol / l,

- 17GB 33391 U1 isovaleryl-Val-Val-Sta-Ala-Sta 1.5 mmoFl. Separately, the stimulation buffer containing Hanks balanced salt solution of the composition as described above contains 10mmoFl hydroxyethyl piperazineethanesulfonic acid and 1 mmol / l 3-isobutyl-1-methylxanthine, 0.5 mmol bovine serum albumin. 1, 20 mmol / l MgCl2 · H2O, 0.5 mmoFl ATP. It also separately contains a detection kit that includes europium-labeled cAMP 5 nmoF1, a cAMP-specific monoclonal antibody labeled with sulfo-Cyanine7 1 µg / ml.

List of used and related literature https://www.salimetrics.com/calculating-inter-and-intra-assay-coefficients-of-variability/

Agarwal, S.R., P.-C. Yang, et al. (2014). Role of Membrane Microdomains in Compartmentation of cAMP Signaling. Pios One 9 (3).

Alasbahi, R.H. and M.F. Melzig (2012). Forskolin and derivatives as tools for studying the role of cAMP. Pharmazie 67 (1): 5-13.

Dessauer, C.W., V.J. Watts, et al. (2017). International Union of Basic and Clinical Pharmacology. WHOSE. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases. Pharmacol Rev 69 (2): 93-139.

Hylse, O., L. Maier, et al. (2017). A Concise Synthesis of Forskolin. Angew Chem Int. Ed Engl 56 (41): 12586-12589.

Pavan, B., C. Biondi, et al. (2009). Adenylyl cyclases as innovative therapeutic goals. Drug Discov Today 14 (19-20): 982–991.

Pierre, S., T. Eschenhagen, et al. (2009). Capturing adenylyl cyclases as potential drug targets. Nat Rev Drug Discov 8 (4): 321–335.

Hylse, O., L. Maier, et al. (2017). A Concise Synthesis of Forskolin. Angew Chem Int. Ed Engl 56 (41): 12586-12589.

PROTECTION REQUIREMENTS

Claims (6)

A kit for determining a specific effect on the activity of adenylate cyclase isoforms, characterized in that it comprises at least one HEK293 cell clone which selectively expresses at least one adenylate cyclase isoform and comprises a plasmid selected from the group consisting of plasmid # 1, SEQ ID NO: 1, plasmid # 1. 2 of SEQ ID No.2, plasmid No 3 of SEQ ID No.3, plasmid No 4 of SEQ ID No.4, plasmid No 5 of SEQ ID No.5, plasmid No 6 of SEQ ID NO: 2; 6, plasmid # 7 of SEQ ID NO: 7, or plasmid # 8 of SEQ ID NO: 8, further comprising separately a lysis buffer containing hydroxyethyl piperazineethanesulfonic acid, EDTA, MgCF 6 H 10, dithiotreitol, sucrose and a protease inhibitor mixture comprising ( 4- (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), aprotinin, N - [(2 S, 3 R) -3-amino-2-liyloxy-4-phenylbutyryl] -L-1-succin, (trans-epoxysuccinyl-1) -leucylamido- (4-guanidino) butane), N-acetyl-L-leucyl-L-leucyl-L-argininal, isovaleryl-Val-Val-Sta-Ala-Sta hexapeptide, further separately contains stimulation buffer containing Hanks balanced salt solution, 3-isobutyl-1-methylxanthine, bovine serum albumin, hydroxyethyl piperazineethanesulfonic acid - 18GB 33391 U1 acid, 3-isobutyl-1-methylxanthine, MgCkOTEO, ATP, and further separately contains a detection kit comprising an aqueous solution containing europium-labeled cAMP, a cAMP-specific monoclonal antibody labeled with a fluorescent dye having an emission ranging from 600 to 635 nm. Kit according to claim 1, characterized in that the HEK293 cells are in the form of a cell suspension. Kit according to claim 1 or claim 2, characterized in that in the lysis buffer the concentration of hydroxyethylpiperazine-ethanesulfonic acid is in the range of 1 to 10 mmoFl, the EDTA in the range of 0.5 to 5 mmoFl, the MgCl 3 -EO in the range of 0.5 to 5 mmol / l. , dithiotreitol in the range of 0.5 to 5 mmoFl, sucrose in the range of 100 to 400 mmol / l, hydrochloride (4- (2-aminoethyl) benzenesulfonyl fluoride) in the range of 50 to 100 mmol / l, aprotinin in the range of 40 to 80 pmol) N - [(2S, 3R) -3-amino-2-liydroxy-4-phenylbutyryl] -L-1-succin in the range of 1 to 4 mmol / l, (trans-epoxysuccinyl-1-leucylamido- ( 4-guanidino) butane) in the range of 0.5 to 1.5 mmol / l, N-acetyl-L-leucyl-L-leucyl-L-argininal in the range of 0.5 to 2 mmol / l, isovaleryl-Val-Val -Sta-Ala-Sta in the range of 0.5 to 1.5 mmol / l. Kit according to any one of claims 1 to 3, characterized in that in the stimulation buffer the concentration of hydroxyethylpiperazine-ethanesulfonic acid is in the range of 1 to 10 mmol / 1,3-isobutyl-1-methylxanthine is in the range of 0.1 to 1 mmoFl, bovine serum albumin in the range of 0.05 to 0.5 mmol / l, MgCk6EFO in the range of 5 to 20 mmoFl, ATP in the range of 0.05 to 0.5 mmol / l. Kit according to any one of the preceding claims, characterized in that the concentration of europium-labeled cAMP is in the range of 1 to 5 nmol / L and the cAMP-specific monoclonal antibody labeled with the fluorescent dye is in the range of 0.1 to 1 pg / ml . Kit according to any one of claims 1 to 5, characterized in that the detection kit comprises a cAMP calibration standard. 4 drawings