A method of indication and identification of inhibitors that inhibit the function of acetylcholinesterase and butyrylcholinesterase

Abstract

The present invention relates to a method of indication of inhibitors that inhibit the function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). It is further a method of identification of such inhibitors. The method including measuring and listing the thermal melting points of AChE or BChE and further measuring the thermal melting point of the chemical adducts formed by a reaction between said AChE or BChE and said possibly present inhibitors, whereupon the presence of an inhibitor is primarily indicated by a shift in thermal melting point for the adduct in comparison to AChE or BChE themselves.

Description

[0001]A method of indication and identification of inhibitors that inhibit the function of acetyl- cholinesterase and butyrylcholinesterase and a device for carrying out the method The present invention relates to a method of indication (i.e. proving presence of) of inhibitors that inhibit the function of acetylcholinesterase (AChE) and butyrylcholin- esterase (BChE) and a device for carrying out the method. It is further a method of identification (i.e. determining chemical identity or chemical class) of such inhibitors and a device for carrying out the method. Indication and identification of inhibitors in the form of e.g. organophosphorus nerve agents (OPNA:s) and organophosphorus pesticides (OPP:s) is a challenge, especially under situations that involves many samples, unknown OPNA:s or OPP:s and under primitive field conditions (i.e. using hand held instrumentation). The situation is complicated by the high toxicity of these substances, which in many cases means that detection levels for certain technologies are higher than the lethal dose of the OPNA:s or OPP:s. This means that a considerable effort is required to ensure that a certain item, surface or area, is safe for unprotected individuals. Despite advances in electronic instrumentation, colorimetric methods or methods using fluorescent signals are very important. Such methods are sometimes combined with a chemical reactivity that is catalysed by the enzyme acetylcholinesterase (AChE), for example in the Swedish nerve agent indicator tag 90 (NAIT90) that is used for air sampling. The armed forces also use indicator paper (called 104, 105, 16, or CALID-3) when sampling liquid. Indicator paper is based on a chemical method that does not involve the enzyme AChE. NAIT90 and similar commercially available products are based on the inhibition of AChE (or similar proteins e.g. butyrylcholinesterase, BChE) by OPNA:s or OPP:s, resulting in the AChE-catalyzed reaction not occurring (and thus no colour change happening). The interpretation of assay results is therefore somewhat illogical because a colour change indicates functional AChE and absence of OPNA:s or OPP:s, while absence of colour change indicates non-functional enzyme and presence of OPNA:s or OPP:s. NAIT90 is a very important part of the Swedish Armed Forces' ability to indicate OPNA:s and OPP:s, which is done through passive or pumped air sampling. NAIT90 has many advantages, it is inexpensive, and it is probably the most sensitive method available widely in the armed forces. It also has the ability to indicate OPNA:s and OPP:s whose chemical structure, spectroscopic signature, and physiochemical properties are unknown. One disadvantage is that it does not have the ability to identify OPNA:s or OPP:s, and thereby gain knowledge of e.g. physiochemical properties or appropriate medical treatment. NAIT90 is also sensitive to certain interferents such as solvents and other substances that affect the enzyme. It requires manual handling that involves a certain degree of skill and has limited scalability, i.e. it is time consuming to evaluate a large number of samples in parallel. Finally, the readout (presence or absence of colour change) is illogical, subjective and prone to human errors, especially under highly stressful conditions when presence of OPNA:s or OPP:s is suspected. The known solutions above have the stated disadvantages and the present invention gives a totally different solution to the problem, in that the invention has the special features that are evident from the independent claim. Other claims define suitable embodiments of the invention. The invention will be described in the following with reference to the enclosed drawing, in which Fig.1 shows a representative example of a thermal melting curve of Homo sapiens AChE (hAChE) (solid black line) that has been fitted to Boltzmann’s sigmoidal equation (dashed line). In the graph, RFU = relative fluorescence units. Fig.2a-c show a comparison of the thermal melting curves of hAChE, Fig.2a, Mus musculus AChE (mAChE), Fig.2b, and Homo sapiens BChE (hBChE), Fig.2c. Data using the GloMelt probe is shown as a solid line while data using the SYPRO®probe is shown as a dashed line. In the graph, RFU = relative fluorescence units. Fig.3a-d show the chemical structure of reversible inhibitors 1-4 used when testing an embodiment of the invention. Fig.4 shows a typical thermal melting curve of mAChE in the presence of reversible inhibitors 1-4, or with no inhibitor present, ctrl. The grey-shaded area represents the essentially linear section of the graphs where the slope was analyzed and further used to indicate / identify reversible inhibitors. This is also discussed in relation to Table 6. It has been discovered that the chemical adducts formed by a reaction between an OPNA or OPP and a protein (e.g. AChE or BChE) change the thermal melting point (Tm) of the protein. It has also been discovered that the extent of stabilization or destabiliza- tion is related to the chemical identity of the OPNA or OPP. The invention comprises a method by which OPNA:s and OPP:s can be indicated and also identified using AChE or BChE, a fluorescent probe (e.g. GloMelt, SYPRO®Orange protein gel stain or similar) and an instrument capable of monitoring a fluorescent signal for determination of the thermal melting point of proteins (e.g. instruments intended for Real Time Polymerase Chain Reaction, RT-PCR). The unknown sample containing an OPNA or OPP is mixed with the protein and the fluorescent probe, and the sample is thereafter subjected to a thermal gradient where- upon the fluorescence intensity is monitored. As the temperature increase, the protein will start to unfold (i.e. denature) and expose hydrophobic amino acids that becomes available for interactions with the probe, hence the fluorescence intensity changes. The analysis of melting curves obtained in a thermal shift assay (TSA) can be approached in multiple ways depending on the objectives. Firstly, the melting curve can be analyzed directly using untransformed raw fluorescence data. This approach provides clear visual evidence of protein unfolding through fluorescence changes as temperature increases. Alternatively, derivative methods, including first and second derivatives of the melting curve data, can be employed. The first derivative highlights the rate of change in fluorescence relative to temperature, providing enhanced precision in determining the Tmthrough clear identification of the temperature at which the maximum change occurs. The second derivative further refines this analysis by pinpointing inflection points more distinctly, which may help resolve closely spaced transitions or subtle conformational changes. Moreover, graphical approaches, such as intersection methods or tangent intersection points, can be utilized to determine the melting temperature visually. Area under curve (AUC) analysis can also be applied, evaluating overall stability changes by integrating the melting curve or defined segments. Additionally, various mathematical models beyond graphical analysis can be employed to calculate Tm, including polynomial fits, logistic regression, and sigmoidal fits such as the Boltzmann equation. The different analytical approaches are well understood by those proficient in the field. The Boltzmann equation, for example, provides a commonly adopted mathematical model that could be used to fit the sigmoidal melting curves observed in thermal shift assays. In the subsequent analyses presented herein, the Boltzmann equation has been applied in all cases. The thermal melting point of the sample (Tmsample) is thus determined by fitting the intensity of the florescence emission to the Boltzmann sigmoidal equation (Fig.1). Negative control samples (protein and probe without addition of any OPNA or OPP) are used to determine a reference value for Tm(Tmref). Each sample is compared to the negative control sample(s) and the ∆Tmvalue is calculated (∆Tm=Tmsample-Tmref). Our experiments show that OPNA:s or OPP:s are indicated by ∆Tmthat exceeds ± 0.5 °C (i.e. the Tmis increased or decreased with at least 0.5 °C) and that the chemical identity in many cases can be determined by the extent of the stabilization or destabilisation. Compared to existing methods, the invented method allows parallel processing of a large number of samples, a high sensitivity (low detection limit), requires no pre-knowledge on chemical properties of the OPNA or OPP and offers an objective readout. Thermal shift assay Enzyme based colorimetric according to the invention methods (prior art, e.g. NAIT90) Throughput 96 samples, 30 min 1 sample, 2-7 minutes analysis time Parallel processing Yes No Sensitivity High High Identification Yes, in some cases No Unknown covalent agents Yes Yes Organic solvents Tolerates, e.g.10 % (v / v) Sensitive DMSO Readout Technical, objective Visual, usually subjective Reproducibility Very high n.d. Size of technical device Pocket size 50×20×7 mm Storage stability Long (freeze dried Long (freeze dried reagents) reagents) Integration with high Easy n.d. resolution techniques (e.g. mass spectrometry) Sampling method Proven for liquid, should Designed for air, should work work with surfaces (e.g. with liquids and possibly wet swabs) and air surfaces n.d. = not determined Fluorescent probes can be used to assess the thermal stability of AChE and BChE. To investigate if commercially available fluorescent probes can be used to determine the Tmvalues of Homo sapiens AChE (hAChE), Mus musculus AChE (mAChE) and Homo sapiens BChE (hBChE), we mixed the proteins separately with either of the two probes GloMelt or SYPRO®. Please cf. information regarding the enzymes in the Materials and Methods section. Each probe was used at the concen- tration stated in Materials and Methods. The samples were transferred to a 96-well PCR plate and heated from 25 to 90 °C with a fluorescent read every 0.1 °C. The fluorescence intensity was monitored with the instrument set to the standard FAM channel excitation and emission wavelengths and when plotting the relative fluorescence units against temperature a clear sigmoidal-shaped increase in florescence intensity was observed at Tmfor all proteins, indicating that commercially available fluorescent probes can be used to assess thermal stability of both AChE and BChE (Fig.2a, 2b and 2c). To investigate fluorescent probe preferences of hAChE, mAChE and hBChE, we compared the signal (i.e. the increase of florescence intensity that occurs when the proteindenatures) at Tm. At our assay conditions, SYPRO®was the preferred probe for hBChEsince GloMelt did not even generate a fluorescent intensity peak. Both probes work on h- and mAChE, however the intensity peak was higher for GloMelt and thus we concluded that a thermal shift assay (TSA) with AChE prefer the GloMelt probe. AChE and BChE tolerate organic solvents such as DMSO and MeOH in thermal shift assays. To investigate the tolerance for solvents in the assay we included either of four commonly used organic solvents at a final concentration of 10 %: dimethyl sulfoxide (DMSO), acetonitrile (ACN), tetrahydrofuran (THF) and methanol (MeOH). The solvents affected the Tmof the samples to a varying degree compared to samples without any solvent added (Table 1). Presence of 10 % DMSO displayed the least impact on Tm, with hAChE ∆Tmat -4 °C, mAChE ∆Tmat -2.5 °C and hBChE ∆Tmat 0.6 °C. The proteins also tolerate 10 % MeOH in the thermal shift assay although it has a more destabilizing effect than DMSO, here we see ∆Tmvalues between -6 °C and -8 °C. The solvent ACN was tolerated by AChE (but not BChE) while THF was tolerated by BChE (but not AChE). Presence of 10 % ACN generated ∆Tm-values at -10 °C and -9 °C for hAChE and mAChE respectively, indicating a strong destabilization of the enzyme while 10 % THF caused a -3 °C Tm-shift for BChE. Table 1. Tmvalues for hAChE, mAChE, and hBChE in the presence of 10 % solvent. Solvent hAChE mAChE hBChE n.d. = not determined; n = number of technical replicates; SD = standard deviation AChE and BChE tolerates common buffers, salts and pH values in thermal shift assays. To find suitable assay conditions, we explored the tolerance for different pH values (Table 2), different buffers (Table 3) and different ionic strengths (Table 4) in the assay. The following properties were investigated: high and low ionic strength (varying salt concentrations from 0.05 to 1 M), acidic and basic environment (varying pH from 6.0 to 9.0), as well as the following five common buffers 4-(2-Hydroxyethyl)piperazine-1- ethanesulfonic acid (HEPES), phosphate-buffered saline (PBS), sodium phosphate buffer (SPB), Tris(hydroxymethyl)aminomethane (Tris)…, Bis-tris propane (BTP). pH was varied using a BTP buffer so that the entire pH-span could be explored using the same buffer. When evaluating how Tmfor the three enzymes was influenced by pH, we could see that both more acidic conditions, pH at 6.0, and more basic conditions, pH at 9.0, destabilized the proteins. Contrary, at pH 7.0 or 8.0, the Tmwas higher indicating an increased stability of the proteins. These results indicate that pH-values below 7 or above 8 should be avoided in the TSA. Table 2. Tmvalues for hAChE, mAChE, and hBChE when varying the pH in the assay. 6.0 52.11 0.14 4 47.06 0.02 4 41.76 0.01 4 7.0 56.96 0.05 4 54.92 0.01 4 43.61 0.04 4 8.0 56.30 0.03 4 55.15 0.03 4 43.60 0.05 4 9.0 n.d. 51.19 0.02 4 41.11 0.05 4 n.d. = not determined; n = number of technical replicates; SD = standard deviation When exploring tolerance for the different buffers BTP, HEPES, SPB, PBS, and Tris, we used a pH of 7.5, (except for PBS which has a pH of 7.4). All buffers were used at a concentration of 25 mM, except PBS that had a buffer concentration of 50 mM. The proteins tolerate all the common buffers tested in the thermal shift assay, resulting in Tmvalues that differ less than 2 °C. From these experiments we concluded that all tested buffers could be used, and for subsequent experiments we used Tris buffer at pH 7.5. Table 3. Tmvalues for hAChE, mAChE, and hBChE when varying the buffer in the assay. Buffers hAChE mAChE hBChE HEPES 56.47 0.03 4 54.30 0.03 4 41.22 0.01 4 PBS 56.29 0.06 4 54.56 0.03 4 42.15 0.02 4 SPB 55.23 0.05 4 53.52 0.03 4 41.83 0.04 4 Tris 57.48 0.02 4 55.31 0.01 4 43.08 0.50 4 n = number of technical replicates; SD = standard deviation Finally, we explored how AChE and BChE respond to variations in ionic strength in the TSA by varying the NaCl or (NH4)2SO4salt content of the buffer between 0.05 and 1 M. A variation of NaCl concentration was well tolerated by both AChE and BChE. For BChE we noticed a stabilising effect at increased salt concentrations. An increased concen- tration of (NH4)2SO4also increased the Tmfor all proteins. For the following experiments we used 100 mM NaCl. Table 4. Tmvalues for hAChE, mAChE, and hBChE when varying the salt species and salt concentration in the assay. (NH4)2SO4Tm(°C) SD n Tm(°C) SD n Tm(°C) SD n 0.05 M 57.58 0.03 4 55.37 0.01 4 43.49 0.09 4 0.1 M 57.64 0.04 4 55.71 0.01 4 44.54 0.08 4 0.5 M 58.93 0.05 4 57.14 0.01 4 48.68 0.19 4 1 M 60.49 0.02 4 58.70 0.08 4 51.84 0.11 4 n = number of technical replicates; SD = standard deviation OPNA:s and OPP:s change the Tmof AChE and BChE. To investigate whether TSA would be able to indicate the presence of OPNA or OPP in a sample, we tested 13 compounds that covalently binds to the catalytic serine of the enzymes. All compounds changed the Tmvalues of the proteins with at least 0.4 ° C (Table 5). The majority of the compounds stabilized the proteins, with a ∆Tmup to 18 ° C. We found that the assay was stable and reproducible (i.e. low standard deviation), both between technical replicates and between experiments (i.e. assays performed at different occasions). Table 5. ∆Tmvalues for OPNA- or OPP-inhibited hAChE, mAChE, and hBChE compared to uninhibited proteins. OPNA:s / OPP:s hAChE replicate 1 hAChE replicate 2 DFP (diisopropyl 1.11 0.03 4 1.16 0.08 4 phosphorofluoridate) Paraoxonethyl -0.72 0.04 4 -0.40 0.06 4 mAChE replicate 1 mAChE replicate 2 ∆Tm(°C) SD n ∆Tm(°C) SD n VX 3.88 0.01 4 3.66 0.04 4 rVX 4.68 0.01 4 4.54 0.05 4 cVX 4.82 0.04 4 4.60 0.02 4 GB, sarin 3.66 0.01 4 3.75 0.02 4 GD, soman 11.87 0.01 4 11.99 0.01 4 GF, cyclosarin 5.72 0.20 4 5.89 0.02 4 GA, tabun 0.73 0.01 4 0.61 0.01 4 A230 12.68 0.03 4 12.64 0.01 4 A242 13.38 0.01 4 13.29 0.01 4 A232 11.90 0.02 4 11.81 0.01 4 A234 10.17 0.03 4 10.05 0.01 4 DFP (diisopropyl -1.85 0.03 4 -1.36 0.02 4 phosphorofluoridate) Paraoxonethyl -1.76 0.02 4 -1.37 0.02 4 hBChE replicate 1 hBChE replicate 2 ∆Tm(°C) SD n ∆Tm(°C) SD nVX 5.52 0.06 4 5.28 0.07 4 rVX 4.16 0.05 4 3.94 0.07 4 cVX 5.99 0.12 4 6.55 0.09 4 GB, sarin 4.13 0.10 4 3.58 0.05 4 GD, soman 17.90 0.03 4 17.90 0.10 4 GF, cyclosarin 5.51 0.06 4 3.97 0.26 4 GA, tabun 2.80 0.02 4 2.90 0.14 4 A230 13.09 0.04 4 13.50 0.02 4 A242 13.04 0.05 4 13.64 0.03 4 A232 13.93 0.03 4 14.40 0.00 4 A234 15.56 0.02 4 16.19 0.03 4 DFP (diisopropyl 18.45 0.02 4 18.75 0.05 4 phosphorofluoridate) Paraoxonethyl 4.26 0.09 4 4.71 0.07 4 n = number of technical replicates; SD = standard deviation Reversible inhibitors change the slope of the Tm-curves of AChE. To investigate whether TSA would be able to indicate the presence of a reversible inhibitor in a sample, we tested four compounds 1-4 with varied affinity for the enzymes (Figure 3a-d). Compound 1 and 2 are high potency inhibitors with half maximal inhibitory concentration values (IC50) <1 µM for AChE, compound 3 and 4 are inhibitors with medium affinity and IC50-values between 10-15 µM. The inhibitors were tested at a final concentration of 100 µM. None of these compounds changed the Tmvalues of AChE more than 0.5 ° C but instead they had an obvious effect on the slope of the Tm-curve. We analysed the essentially linear section of the curve below the thermal melting point and studied the curves between degree 35 and 45, shown as a grey-shaded area in Fig. 4. We compared the slope of uninhibited protein (sloperef) to the slope of reversiblyinhibited protein (slopesample). Δslope-values are calculated as ∆slope = slopesample- sloperef. The trend is that the highly potent inhibitors get a flatter slope than the medium potency inhibitors and thus a larger Δslope (Table 6). This change in slope was only visible using the GloMelt probe and not the SYPRO®probe and the analysis was therefore not applicable on BChE. Table 6. ∆Tmand Δslope values for reversible inhibited hAChE, mAChE, and hBChE compared to uninhibited proteins. Compound hAChE replicate 1 hAChE replicate 2ΔSlope 1 0.04 0.05 4 196 -0.02 0.06 4 182 2 -0.04 0.06 4 245 0.03 0.06 4 230 3 0.09 0.05 4 85 -0.06 0.08 4 71 4 0.07 0.04 4 78 0.17 0.04 4 47 mAChE replicate 1 mAChE replicate 2∆Τm(°C) SD n ΔSlope ∆Τm(°C) SD n ΔSlope 1 0.37 0.02 0.02 4 279 2 0.35 0.02 0.02 4 195 3 0.11 0.02 0.02 4 142 4 0.07 0.02 0.01 4 114 BChE replicate replicate 2 ∆Τm(°C)SD n ΔSlope∆Τm(°C)SD n ΔSlope1 0.83 0.15 4 n. a. 1.04 0.03 4 n. a. 2 1.34 0.07 4 n. a. 1.34 0.02 4 n. a. 3 0.54 0.12 4 n. a. 0.52 0.02 4 n. a. 4 4.96 0.04 4 n. a. 5.31 0.02 4 n. a. n. a. = not applicable; n = number of technical replicates; SD = standard deviation. Materials and methods Chemicals and reagents GloMelt was obtained from Biotium (San Francisco, USA). SYPRO®Orange gel stain was supplied by Invitrogen (Waltham, USA). Nerve agents and inhibitor 1-4 were synthesised by FOI in Umeå (Sweden). All other chemicals were supplied by Sigma Aldrich (Burlington, USA). Proteins Mus musculus acetylcholinesterase (mAChE, uniprot accession number P21836) was cloned and expressed as previously described in Ekström F, Akfur C, Tunemalm AK, Lundberg S. Structural changes of phenylalanine 338 and histidine 447 revealed by the crystal structures of tabun-inhibited murine acetylcholinesterase. Biochemistry.2006 Jan 10;45(1):74-81. doi: 10.1021 / bi051286t. PMID: 16388582, which can be studied for detailed information. For the purpose of being able to carry out the present invention the following information from said document should be enough for the man skilled in the art. mAChE was amplified by PCR, and the fragment was inserted into a pcDNA3.1 expression vector (Invitrogene). The inserted open reading frame contained the native N-terminal secretion signal while the C-terminal part of the protein was truncated at position 574. HEK-293F cells (Invitrogen) were maintained in Dulbecco’s DMEM medium supplemented with 10 % fetal calf serum and appropriate antibiotics. Cells (4 × 106 / 100 mm plate) were trans- fected with 25 µg of DNA-calcium phosphate coprecipitate (Invitrogen). Transfected clones were selected by incubation with media containing geneticin (Invitrogen) at a concentration of 0.5 mg / mL. When cell death subsided, individual clones were manually transferred to 24 well plates. Clones with high expression of AChE were expanded and transferred to suspension cultures maintained in serum-free FreeStyle media (Invitrogen) for large-scale protein production. When purifying mAChE, a purification method based on affinity chromatography and size exclusion chromatography (SEC) was employed. Typically, supernatants of media containing ∼30 mg of AChE were pooled and centri- fuged at 6500g for 30 min. An appropriate amount of procainamide hydrochloride (Sigma), immobilized to epoxy-activated Sepharose 6B (Amersham Biosciences), was incubated with the cleared protein suspension for 10 min. After a brief centrifugation the procainamide Sepharose was transferred to a XK26 column (Cytiva). Following exten- sive washing with buffer A (1 mM MES, pH 6.5, 50 mM NaCl, 1 mM poly(ethylene glycol) 600) the protein was eluted in the same buffer supplemented with 50 mM procainamide. The protein was concentrated with centricons (Amicon) and further purified on a HiLoad 16 / 60 Superdex 200 column (Cytiva) equilibrated in buffer A. Fractions containing mAChE were detected using a modified Ellman assay in E-buffer composed of 100 mM sodium phosphate, pH 8.0, and 0.1% Triton X-100. Homo sapiens acetylcholinesterase (hAChE) was obtained and expressed as previously described in Allgardsson A, Berg L, Akfur C, Hörnberg A, Worek F, Linusson A, Ekström FJ. Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6. Proc Natl Acad Sci U S A.2016 May 17;113(20):5514-9. doi: 10.1073 / pnas.1523362113. Epub 2016 May 2. PMID: 27140636; PMCID: PMC4878515. For the purpose of being able to carry out the present invention the following information from said document should be enough for the man skilled in the art. The sequence MRPPQCLLHTPSLASPLLLLLLWLLGGGVGAEHHHHHHHHGSGSENLYFQGREDAELL VTVRGGRLRGIRLKTPGGPVSAFLGIPFAEPPMGPRRFLPPEPKQPWSGVVDATTFQS VCYQYVDTLYPGFEGTEMWNPNRELSEDCLYLNVWTPYPRPTSPTPVLVWIYGGGFY SGASSLDVYDGRFLVQAERTVLVSMNYRVGAFGFLALPGSREAPGNVGLLDQRLALQ WVQENVAAFGGDPTSVTLFGESAGAASVGMHLLSPPSRGLFHRAVLQSGAPNGPWA TVGMGEARRRATQLAHLVGCPPGGTGGNDTELVACLRTRPAQVLVNHEWHVLPQES VFRFSFVPVVDGDFLSDTPEALINAGDFHGLQVLVGVVKDEGSYFLVYGAPGFSKDNE SLISRAEFLAGVRVGVPQVSDLAAEAVVLHYTDWLHPEDPARLREALSDVVGDHNVVC PVAQLAGRLAAQGARVYAYVFEHRASTLSWPLWMGVPHGYEIEFIFGIPLDPSRNYTAE EKIFAQRLMRYWANFARTGDPNEPRDPKAPQWPPYTAGAQQYVSLDLRPLEVRRGLR AQACAFWNRFLPKLLSAT (SEQ ID NO 1) was synthesized into a pD603 vector (Atum, Newark, California, USA). The resulting construct was transfected into adherent HEK293F cells maintained in DMEM, Glutamax-I, 4.5 g / L D-glucose, and 25 mM Hepes- Pyruvate (Gibco) supplemented with 10% (vol / vol) FCS (Biochrome) and 50 μg / mL Gentamicin (Gibco). After 2 wk of incubation in media supplemented with 0.5 mg / mL Geneticin (Gibco), 174 individual clones were selected and expanded. The level of hAChE secretion by different clones was evaluated using the Ellman assay. The clone with the highest expression was expanded in suspension using Freestyle 293 and Glutamax (Gibco) media containing 20 μg / ml Gentamicin (Gibco). Before purification, the supernatant was centrifuged at 11,200 × g for 60 min. The cleared supernatant was sub- sequently loaded on a HisTrap HP (GE Healthcare) column and washed with 20 mM Hepes, pH 7.6 and 0.5 M NaCl, after which the purified hAChE was eluted in a buffer composed of 20 mM Hepes, pH 7.6, 0.5 M NaCl, and 500 mM imidazole. For the purpose of being able to carry out the present invention the following information based on said document should be enough for the man skilled in the art. Homo sapiens buturylcholinesterase (hBChE) was obtained via gene synthesis of the sequence MHSKVTIICIRFLFWFLLLCMLIGKSHTHHHHHHHHGSGSENLYFQSEDDIIIATKNGKVR GMQLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATKYANSCCQNIDQS FPGFHGSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQTGTSSLHVYDG KFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQWVQKNIAAFGGN PKSVTLFGESAGAASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSLYEARNRTLNLA KLTGCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLSVNFGPTVDGDFLTDMPDILLE LGQFKKTQILVGVNKDEGTAFLVYGAPGFSKDNNSIITRKEFQEGLKIFFPGVSEFGKESI LFHYTDWVDDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNNAFFYYFEHRSSK LPWPEWMGVMHGYEIEFVFGLPLERRDQYTKAEEILSRSIVKRWANFAKYGNPQETQN QSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKV (SEQ ID NO 2) into a pD603 vector (ATUM, Newark, California, USA). The sequence corresponds to the 4sugOff17 / 455 / 481 / 486 BChE∆530-construct where the C-terminal oligomerization domain has been deleted and four out of nine N- glycosylation sites has been suppressed making the recombinant hBChE more suitable for crystallization, as is described in more detail in Engineering of a monomeric and low- glycosylated form of human butyrylcholinesterase, Expression, purification, characteriza- tion and crystallization, Florian Nachon, Yvain Nicolet, Nathalie Viguie, Patrick Masson, Juan C. Fontecilla-Camps and Oksana Lockridge. The plasmid was amplified in XL10-Gold E. coli strain and subsequently transfected into adherent HEK293F cells using a Calcium Phosphate Transfection Kit (Invitrogen). HEK293F cells were maintained in Dulbecco’s DMEM medium (Gibco) supplemented with 10 % (v / v) fetal calf serum (fcs) and 50 µg / ml gentamicin (Gibco). Transfected clones were selected by incubation with media also containing 0.5 mg / ml geneticin (Gibco). Individual transfected clones were selected and allowed to multiply into small colonies, the level of hBChE secreted by different clones was evaluated by measuring the enzymatic activity using the Ellman assay as described in the paragraph Ellman assay, below. Clones with the highest hBChE expression were expanded and stepwise adapted to suspension growth in a serum-free protein expression medium (Freestyle 293 (Gibco) supplemented with 30 µg / ml gentamicin and 0.5 mg / ml geneticin) until fcs was completely out faded. hBChE protein was produced over 7 days and harvested by centrifugation of the cell culture media at 300 x g for 5 min. The supernatant was stored at -20 °C until use, and several weeks of production was pooled prior to purification. hBChE-supernatant was thawed on ice and centrifuged at 17500 x g for 30 min before loaded on a HisTrap FF Crude, 5 ml column (Cytiva) and eluted with 50 mM Tris pH 8.0, 500 mM imidazole. The high imidazole concentration was removed by a buffer exchange on a HiPrep 26 / 10 desalting column (Cytiva) and the sample were thereafter loaded on a 6 ml Resource Q anion exchange column (Cytiva) and eluted with 20 mM Tris pH 8.0, 1000 mM NaCl. Fractions containing the hBChE protein were pooled, transferred to a storage buffer (20 mM HEPES pH 7.5, 100 mM NaCl) and concentrated using an AMICON centrifugal filter unit with a molecular weight cutoff of 30 kDa. The purity of hAChE, mAChE and hBChE was analyzed by SDS-PAGE and protein concentration was determined spectrophotometrically using a nanodrop (Thermo Scientific). Aliquots of purified protein were stored at -80 °C until use. Ellman assay The colorimetric Ellman assay as previously described in ELLMAN GL, COURTNEY KD, ANDRES V Jr, FEATHER-STONE RM, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem Pharmacol.1961 Jul;7:88-95. doi: 10.1016 / 0006- 2952(61)90145-9. PMID: 13726518, was used to measure the activity of AChE and BChE of individual clones as a way to evaluate which clones had the highest protein expression. In this assay, the hydrolysis of a substrate (acetylthiocholine for AChE and butyrylthiocholine for BChE) is monitored over time in a spectrophotometer. This enzyme-catalyzed reaction produces thiocholine, which in turn reacts with the thiolcontaining reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to generate a colored product that can be quantified spectrophotometrically at 412 nm. The assay was performed in 0.1 M sodium phosphate buffer (pH 7.4) at 30°C with the final concen- tration of the substrate at 1 mM, and the reagent DTNB at 0.2 mM. Thermal Shift Assay Purified recombinant hAChE, mAChE and hBChE were thawed on ice and the samples were prepared by adding the enzyme, buffer, and probe into a master mix that were then aliquoted into 1.5 ml test tubes. The fluorescence probes GloMelt and SYPRO®Orange were kept away from light during sample preparation. Thereafter, the samples and controls were added to the tubes and finally each sample was split into four technical replicate wells in a 96 well PCR plate. Pure DMSO was used in control wells to generate a reference Tm. Final sample volume was 20 µl and final content concentration was 25 mM Tris pH 7.5, 50 mM NaCl, 0.3 mg / ml (100 pmol) AChE or 0.2 mg / ml (61 pmol) BChEenzyme, 0.5 % (GloMelt) or 1 % (SYPRO®) probe, and 10 % DMSO with or without 100µM (2 nmol) OPNA or OPP. The reversible inhibitors 1-4 was dissolved in 10 % DMSO and tested at a final concentration of 100 µM. The plate was sealed and briefly centrifuged to remove air bubbles before being placed in a CFX96 RealTime-PCR (BioRad). Samples were initially heated to 25 °C for 30 s, and the temperature was thereafter stepwise increased from 25 °C to 90 °C with a fluorescent read every 0.1 °C, total assay time was 3 h 27 min. To increase the through- put of the assay, a separate setup was explored where samples were heated from 40 °C to 80 °C with a fluorescent read every 0.5 °C, total assay time was then 27 min. To determine Tmthe data was analyzed using an open-access web server available online for rapid analysis of TSA experiments: Reys V, Kowalewski J, Gelin M, Lionne C. wTSA-CRAFT: an open-access web server for rapid analysis of thermal shift assay experiments. Bioinform Adv.2023 Sep 29;3(1):vbad136. doi: 10.1093 / bioadv / vbad136. PMID: 37822724; PMCID: PMC10562953.

Claims

Claims:

1. A method of indication of inhibitors that inhibit the function of acetylcholinesterase(AChE) or butyrylcholinesterase (BChE), c h a r a c t e r i z e d in that thermal meltingpoints of AChE or BChE are determined by a adding a fluorescent probe to a sample, the thermal melting point of which is to be determined, and using an instrument for monitoring the fluorescent signal while the sample is subjected to a thermal gradient, thereafter the corresponding measurement is carried out for possible chemical adducts formed by a reaction between said AChE or BChE and said possibly present inhibitors, whereupon the presence of an inhibitor is primarily indicated by a shift in thermal melting point for the adduct in comparison to AChE or BChE themselves.

2. A method according to claim 1, c h a r a c t e r i z e d i n that the shift in thermalmelting point is also used for identification of which inhibitor has reacted with AChE or BChE.

3. A method according to claim 1 or 2, c h a r a c t e r i z e d i n that when the shift inmelting point is too small for indication and identification, such as for some reversible inhibitors, the identification of an inhibitor is secondarily done using the change in slope of the essentially linear section of the fluorescence curve below the thermal melting point for the reversible complex in comparison to AChE or BChE themselves.

Images