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Jun 2019

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A Radioactive-free Kinase Inhibitor Discovery Assay Against the Trypanosoma brucei Glycogen Synthase Kinase-3 short (TbGSK-3s)
一种无放射性布氏锥虫糖原合成酶激酶-3抑制剂探究实验   

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Abstract

The identification of small molecules possessing inhibitory activity in vitro, against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay for the identification of inhibitors for the short isoform of the Trypanosoma brucei’s Glycogen Synthase Kinase-3 (TbGSK-3s). TbGSK-3s represents a potential drug target as it is essential for parasite survival. Small molecules used in our study are indirubin analogues possessing substitutions in different positions in the bis-indole backbone. Presently, the standard laboratory practice for the kinase assays is the incorporation of radiolabeled phosphate from [gamma-32P]ATP as the efforts for developing non-radioactive assays (ELISA-based assays, fluorescence quenching assays, etc.) exhibit limitations such as lack in sensitivity or limitations for broad applications. This protocol can be a useful starting point for lead discovery, as it surpasses the drawbacks of radioactive kinase assays and it allows for relatively sensitive measurements of kinase inhibition for TbGSK-3s.

Keywords: Kinase assay (激酶分析), Non-radioactive assay (非放射性分析), Trypanosoma brucei (布氏锥虫), TbGSK-3s (布氏锥虫糖原合成酶激酶-3), Lead discovery (先导物), Inhibitors (抑制剂), Indirubins (靛玉红类)

Background

Kinases are enzymes that play a crucial role in biological processes including differentiation, cell proliferation and apoptosis, by putting in motion signaling pathways upon catalyzing the transfer of the γ-phosphate from ATP to substrate (Jia et al., 2008; Efstathiou et al., 2019). Deregulation of kinases can frequently lead to a variety of diseases (Ways and Sheetz, 2000; Cohen and Goedert, 2004; Mazitschek and Giannis, 2004; Resnick and Fennell, 2004) and therefore, they are considered one of the largest classes of drug targets (Cohen 1999; Manning et al., 2002). The first step of the lead kinase inhibitor discovery is the establishment of an in vitro kinase assay. The radioactive assays are the standard laboratory practice due to their high sensitivity (Jia et al., 2008; Lilienthal et al., 2010). However, drawbacks of kinase-based radioactivity assays include the need of special handling and the restriction in flexibility because of the short half-life of 32P. To resolve these limitations, new non-radioactive technologies have been created that are based on fluorescence or luminescence (Jia et al., 2008). Herein, we describe a non radioactive protocol using luciferase-based ATP assay, for the identification of inhibitors for the short isoform of the Trypanosoma brucei’s Glycogen Synthase Kinase-3 (TbGSK-3s). In the bloodstream form of T. brucei, TbGSK-3s is essential for survival (Ojo et al., 2008) and therefore it is a molecular target for the discovery of new anti-trypanosomal agents (Ojo et al., 2008; Oduor et al., 2011; Woodland et al., 2013; Urich et al., 2014; Swinney et al., 2016). Mammalian GSK-3 has been related to a wide range of diseases and thus small molecular weight GSK-3 inhibitors has been developed (Woodland et al., 2013; Gaboriaud-Kola et al., 2015; Masch and Kunick, 2015). Amongst GSK-3 inhibitors, there are the indirubins, a family of natural bis-indole derivatives (Hoessel et al., 1999, Polychronopoulos et al., 2004, Vougogiannopoulou et al., 2008, Myrianthopoulos et al., 2013). In this protocol, a luminescent kinase assay based on the Kinase-Glo® reagent of Promega, is described. This method is straightforward, radioactive-free, fast and it doesn’t lack sensitivity. While the protocol described below is specific for the recombinant TbGSK-3s expressed in baculovirus system as described before (Efstathiou et al., 2019), it can be applicable to any kinase with the appropriate alterations for the specific kinase (substrate, ATP concentration, buffer, etc.).


Figure 1. Scheme for the luminescent kinase assay based on the Kinase-Glo® reagent. The kinase reaction is conducted under the appropriate conditions with or without inhibitors. The remaining ATP at the time that the reagent is added, is used as a substrate by the Kinase-Glo® Luciferase to catalyze the mono-oxygenation of luciferin. The luciferase reaction produces one photon of light per turnover. Luminescence is inversely related to kinase activity (Promega).

Materials and Reagents

Materials

  1. Pipettes tips: 0.5-10 μl, 10-200 μl, 200-1,000 μl (Greiner Bio-One, catalog numbers: 771291, 739290, 740290)
  2. Eppendorf tubes (Greiner Bio-One, catalog number: 616201)
  3. Amicon® Ultra-4 centrifugal filters

Reagents

  1. GSK-3 peptide substrate YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE (HQ), 1 mg (Biaffin GmbH & Co KG, proteinkinase.de, catalog number: PEP-GSK-001, storage temperature: -20 °C)
  2. Kinase-Glo® Luminescent Kinase Assay, 10 ml (Promega, catalog number: V6711, storage temperature: -20 °C)
  3. Kinase Glo® Substrate, 1 vial (lyophilized) (Promega, catalog number: V378A)
  4. Kinase Glo® Buffer, 10 ml (Promega, catalog number: V379A)
  5. Adenosine 5′-triphosphate (ATP) disodium salt hydrate, 1 mg (Sigma-Aldrich, catalog number: FLAAS, storage temperature: -20 °C)
  6. TbGSK-3s expressed in a baculovirus expression system as previously described (Efstathiou et al., 2019) (storage temperature: immediate usage after expression or glycerol stock at -80 °C)
    Notes:
    1. TbGSK-3s is not available commercially. In order to use it, it must be expressed in the laboratory following the protocols described in bibliography (Efstathiou et al., 2019). Briefly, as mentioned in Efstathiou et al., 2019, the pTriEx-1.1-TbGSK3s plasmid was cotranfected with the BaculoGold DNA into Spodoptera frugiperda (Sf9) insect cells and upon production of the TbGSK3s, the kinase was purified on Ni2+-nitrilotriacetate (Ni-NTA) resin according to the manufacturer’s instructions (Qiagen).
    2. Kinase fractions should be used immediately to ensure maximum activity. If they are stored as glycerol stocks at -80 °C, they should be used up to 3-5 days upon isolation to avoid complete loss of the kinase activity.
  7. 32 indirubin analogs that were synthesized as previously described (Meijer et al., 2003; Polychronopoulos et al., 2004; Ferandin et al., 2006; Vougogiannopoulou et al., 2008), (storage temperature: 4 °C, away from sunlight)
  8. MOPS (3-(N-morpholino)propanesulfonic acid) (Applichem, catalog number: A2947, storage temperature: RT)
  9. MgCl2 (Magnesium chloride hexahydrate) (Applichem, catalog number: A1036, storage temperature: RT)
  10. EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid) (Sigma-Aldrich, catalog number: E4378, storage temperature: RT)
  11. PierceTM DTT (Dithiothreitol) (ThermoFisher Scientific, catalog number: 20290, storage temperature: 4 °C)
  12. Ni-NTA Agarose (25 ml) (QIAGEN, catalog number: 30210, storage temperature: 4 °C)
  13. Amicon® Ultra-4 Centrifugal Filter Unit (Merck-Millipore, catalog number: UFC801008, storage temperature: RT)
  14. Imidazole for molecular biology (Applichem, catalog number: A1378,0050, storage temperature: RT)
  15. Kinase assay solution (10x) (see Recipes)

Equipment

  1. Water bath (Julabo, ED-13 Open Heating Bath Circulator/discontinued product)
  2. GloMax® 20/20 Luminometer (Promega, model/catalog number: 2030-100/E5311)
  3. Eppendorf 5417R Refrigerated Centrifuge (Marshall Scientific, Product code: EP-5417R)

Software

  1. Microsoft® Office Excel 2013
  2. GraphPad Prism v6 software

Procedure

  1. Preparation of Kinase-Glo® reagent
    1. Mix the 10 ml Kinase Glo® Buffer with the lyophilized Kinase Glo® Substrate.
    2. Make sure that the dilution of the substrate powder is complete and the solution is clear.
    3. Aliquot the Kinase-Glo® reagent and store it at -20 °C until usage.

  2. Preparation of kinase assay solution 10x
    Prepare the kinase assay solution 10x as described in the Recipes, aliquot and store it at -20 °C until usage.

  3. Determination of the TbGSK-3s fraction with the highest enzymatic activity
    1. Perform the kinase assay for the different isolated fractions of the kinase (TbGSK-3s). The isolated fractions have been purified on Ni2+-nitrilotriacetate (Ni-NTA) agarose according to the manufacturer’s instructions (Qiagen) with elutions of different imidazole concentrations (Fractions A, B, C and D using 50, 150, 250 and 400 nM imidazole respectively) as previously described (Efstathiou et al., 2019).
      Note: Isolated fractions of the TbGSK-3s were subsequently concentrated using Amicon® Ultra-4 Centrifugal Filters and rediluted in kinase assay solution 1x in order to remove excess imidazole from the samples. Imidazole in high concentrations could interfere during the kinase assay providing false results.
    2. Adjust the temperature of the water bath to 30 °C.
    3. Defreeze the Kinase-Glo® reagent from -20 °C to 4 °C.
    4. Defreeze the kinase assay solution 10x from -20 °C to 4 °C.
    5. Prepare master mix solution (containing a final concentration of 1x kinase assay solution, 10 μΜ ATP, 10 μΜ GSK-3 peptide substrate) as described below (final volume 40 μl per sample):
      4 μl x [number of samples] of 10x kinase assay solution
      1 μl x [number of samples] of 400 μΜ ATP
      1.2 μl x [number of samples] of 333 μΜ GSK-3 peptide substrate
      23.8 μl x [number of samples] of ddH2O
    6. Mix well the master mix by pipetting up and down.
    7. Add 30 μl of the master mix in each Eppendorf.
    8. Add 10 μl of each TbGSK-3s isolated fraction (A, B, C and D fractions) in each tube.
    9. Make duplicates or triplicates for each fraction.
    10. Mix well the samples by pipetting up and down.
    11. Spin down the samples.
    12. Incubate the samples for 30 min in the water bath at 30 °C.
    13. Stop the reaction by adding 40 μl of cold Kinase-Glo® reagent (4 °C).
    14. Spin down the samples.
    15. Measure the luminescence in the GloMax® 20/20 Luminometer for 1 s.
      Note: Read the luminescence with the ‘ready to use’ Promega protocol named ‘Kinase-Glo’.
    16. Determine the elution fraction of TbGSK-3s with the optimal enzymatic activity (see Data analysis A).
    17. Use the kinase immediately for the kinases assays or store it in a 50% (v/v) glycerol stock at -80 °C.
    Notes:
    1. Purified elution fraction C of TbGSK-3s (initially eluted with 250 mM imidazole) was determined in our analysis as the one with higher enzymatic activity (see Data analysis A).
    2. It is recommended to use the kinase TbGSK-3s fractions immediately after the elution to ensure maximum activity. The 50% (v/v) glycerol stock of the kinase has diminished activity depending on the time period that it is stored at -80 °C and they should be used up to 3-5 days upon isolation to avoid complete loss of the kinase activity.

  4. Determination of the optimal protein kinase (TbGSK-3s) concentration
    1. Perform the kinase assay with the appropriate elution fraction (elution fraction C) of TbGSK-3s which was identified above as the one with higher enzymatic activity.
    2. Follow Steps C2-C4.
    3. Prepare master mix solution (containing a final concentration of 1x kinase assay solution, 10 μΜ ATP, 10 μΜ GSK-3 peptide substrate) as described below (final volume 40 μl per sample):
      4 μl x [number of samples] of 10x kinase assay solution
      1 μl x [number of samples] of 400 μΜ ATP
      1.2 μl x [number of samples] of 333 μΜ GSK-3 peptide substrate
      23.8 μl x [number of samples] of ddH2O
    4. Follow Steps C6-C7.
    5. Add 10 μl of TbGSK-3s isolated fraction C in different concentrations (containing 0, 2, 5, 10, 15, 20, 40, 100 and 200 ng TbGSK-3s, diluted with ddH2O).
      Note: Different concentrations of kinase can be used if needed, depending on the parameters of the experiment.
    6. Follow Steps C9-C15.
    7. Determine the optimal concentration of TbGSK-3s for the kinase assays (see Data analysis B).
    Note: 40 ng of TbGSK-3s (Fraction C) was determined in our analysis as the optimal concentration of TbGSK-3s for the kinase assays (see Data analysis B). Fraction C contains 20 ng/μl of TbGSK-3s (see Data analysis B).

  5. Determination of the ATP concentration
    1. Perform the kinase assay with the TbGSK-3s isolated fraction C.
    2. Follow Steps C2-C4.
    3. Prepare master mix solution (containing a final concentration of 1x kinase assay solution, 40 ng of TbGSK-3s isolated fraction C, 10 μΜ GSK-3 peptide substrate) as described below (final volume 40 μl per sample):
      4 μl x [number of samples] of 10x kinase assay solution
      2 μl x [number of samples] of TbGSK-3s isolated fraction C
      1.2 μl x [number of samples] of 333 μΜ GSK-3 peptide substrate
      23.8 μl x [number of samples] of ddH2O
    4. Follow Steps C6-C7.
    5. Add 10 μl of ATP in different concentrations of (0, 0.5, 1, 2, 5, 7.5, 10 and 50 μΜ).
      Note: Different concentrations of ATP can be used if needed, depending on the parameters of the experiment. However, for concentrations of ATP up to 10 μΜ, you can use the Kinase-Glo® Assay (Promega), for 0-100 µM of ATP, you can use the Kinase-Glo® Plus Assay (Promega) and for 0-500 µM, you can use the Kinase-Glo® Max Assay (Promega).
    6. Follow Steps C9-C15.
    7. Plot the diagram of the rate of reaction DY/DX versus the ATP concentration and determine the Vmax and Km of ATP (see Data analysis C).
    Notes:
    1. DY = [(RLU x 106 upon 30 min of reaction) – (RLU x 106 upon 0 min of reaction)]
    2. DX = time of reaction = 30 min
    3. Vmax is the maximum rate of reaction and represents the rate of reaction when the enzyme is saturated with substrate.
    4. The value of the Michaelis-Menten constant (Km) is numerically equal to the substrate concentration at which the reaction rate is half of Vmax.
    5. Km of ATP was determined equal to 6.3 μΜ using 40 ng of TbGSK-3s fraction C (see Data analysis C).
    6. A standard ATP concentration, identical in all kinase assays performed with TbGSK-3s, was used and it was equal to the Km for the ATP (6.3 μΜ).

  6. Determination of the GSK-3 peptide substrate concentration
    1. Perform the kinase assay with the TbGSK-3s isolated fraction C.
    2. Follow Steps C2-C4.
    3. Prepare master mix solution (containing a final concentration of 1x kinase assay solution, 40 ng of TbGSK-3s isolated fraction C, 10 μΜ ATP) as described below (final volume 40 μl per sample):
      4 μl x [number of samples] of 10x kinase assay
      2 μl x [number of samples] of TbGSK-3s isolated fraction C
      1.2 μl x [number of samples] of 400 μΜ ATP
      23.8 μl x [number of samples] of ddH2O
    4. Follow Steps C6-C7.
    5. Add 10 μl of GSK-3 substrate in different concentrations of (0, 1, 2, 5, 10 and 50 μΜ).
      Note: Different concentrations of substrate can be used if needed, depending on the parameters of the experiment.
    6. Follow Steps C9-C15.
    7. Plot the diagram of the rate of reaction DY/DX versus the GSK-3 concentration and determine the Vmax and Km of GSK-3 substrate (see Data analysis C).
    Notes:
    1. Km of GSK-3 substrate was determined equal to 5.8 μΜ using 40 ng of TbGSK-3s (Fraction C) (see Data analysis C).
    2. A standard GSK-3 substrate concentration, identical in all kinase assays performed with TbGSK-3s, was used and it was equal to the Km for the GSK-3 substrate (5.8 μΜ).

  7. Identification of TbGSK-3s inhibitors
    1. Perform the kinase assay with the TbGSK-3s isolated fraction C.
    2. Follow Steps C2-C4.
    3. Prepare master mix solution (containing a final concentration of 1x kinase assay solution, 40 ng of TbGSK-3s isolated fraction C) as described below (final volume 40 μl per sample):
      4 μl x [number of samples] of 10x kinase assay solution
      2 μl x [number of samples] of TbGSK-3s isolated fraction C
      22.7 μl x [number of samples] of ddH2O
    4. Follow Steps C6-C7.
    5. Add 10 μl of inhibitor in different concentrations (0, 0.01, 0.03, 0.1, 0.33, 1 and 3.33 μΜ).
      Note: Different concentrations of inhibitors can be used if needed, depending on the parameters of the experiment.
    6. Add 5.8 μΜ GSK-3 peptide substrate in each sample (0.7 μl of 333 μΜ GSK-3 peptide substrate).
    7. Add 6.3 μΜ ATP in each sample (0.6 μl of 400 μΜ ATP).
    8. Follow Steps C9-C15.
    9. Plot the diagram of the % inhibition of TbGSK-3s activity versus the inhibitor’s concentration and determine the IC50 of the inhibitor (see Data analysis D).
    Note : The sample with no inhibitor serves as a positive control. Moreover, use a sample that contains no ATP as a negative control (4 μl kinase assay solution 10x, 2 μl TbGSK-3s isolated fraction C containing 40 ng of kinase, 0.7 μl GSK-3 peptide substrate 333 μΜ in order to obtain 5.8 μΜ final concentration, and 33.3 μl ddH2O).

Data analysis

  1. Determination of the TbGSK-3s fraction with enzymatic activity:
    1. The fraction that contains the active kinase will utilize the ATP to phosphorylate the GSK-3 substrate and therefore the concentration of the ATP in the sample will reduce. As a result, the luciferin will no longer have the same amount of ATP to interact with and the reaction will produce less light. Concequently, the luminescence (RLU) measurement of the sample will be lower than the one of the negative control (sample containing no kinase).
    2. Collect the luminescence readings for the elution fractions of TbGSK-3s.
    3. Calculate the average value of luminescence and the standard deviation for each fraction.
    4. Plot the diagram of the luminescence values (Figure 2).
    5. The fraction with the lowest value of luminescence is the one with the optimal enzymatic activity and can be used for further experiments for kinase assays.


      Figure 2. Luminescence values for isolated TbGSK-3s fractions A, B, C and D. The TbGSK-3s fractions A, B, C and D were purified on Ni2+-nitrilotriacetate (Ni-NTA) resin according to the manufacturer’s instructions (Macherey-Nagel) with elutions of different imidazole concentrations (Fractions A, B, C and D using 50, 150, 250 and 400 nM imidazole respectively) as previously described (Efstathiou et al., 2019) and subsequently they were concentrated using Amicon® Ultra 0.5 ml Centrifugal Filters and re-diluted in kinase assay solution 1x in order to remove excess imidazole from the samples. Kinase assay solution 1x (no kinase) was used as a negative control.

  2. Determination of the optimal protein kinase (TbGSK-3s) concentration
    1. Determine the amount of kinase present in each fraction using Bradford protein assay (He, 2011) and by a semi-quantification method based on the intensity of the signal in the Western blot upon comparison with already known amount of protein in the marker bands and given that the volume of the kinase fraction put in the Western blot is constant and known.
      Notes:
      1. The protocol and analysis procedure for the Western blot, including the reagents and the materials used, can be found in bibliography (Mahmood and Yang, 2012; Heiber et al., 2014).
      2. The Bradford protein assay is used for the quantification of the total protein amount in each eluted fraction, while the Western plot was used in order to illustrate the existence of TbGSK-3s in each fraction.
    2. Isolated fraction C (Lane 5 in Figure 3–TbGSK-3s fraction which was eluted from the Ni2+-nitrilotriacetate (Ni-NTA) resin Ni2+ beads using 250 mM imidazole) was estimated to contain 20 ng/μl of TbGSK-3s (Figure 3).
    3. Collect the luminescence readings for the samples containing different concentrations of TbGSK-3s.


      Figure 3. Western blot of isolated TbGSK-3s fractions A, B, C and D. 30 μl of TbGSK-3s fractions A. (lane 3), B. (lane 4), C. (lane 5) and D. (lane 6), which were purified on Ni2+-nitrilotriacetate (Ni-NTA) resin with elutions of different imidazole concentrations (50, 150, 250 and 400 nM imidazole respectively), and the remaining Ni2+-nitrilotriacetate (Ni-NTA) resin upon the elutions (lane 2), were identified in a Western blot using an affinity purified polyclonal antibody raised against LGSK-3s. The Western blot showed a unique band at the theoretical molecular mass of TbGSK-3s (40 kDa). In lane 1, 30 μl of kinase assay solution 1x were also used as a negative control.

      Table 1. TbGSK-3s used in kinase assay, expressed in ng, when using different volumes of isolated fraction C. In the column entitled ‘Fraction C’, the volume of the eluted kinase TbGSK-3s (expressed in μl) which was added in each sample, is given. In the column entitled ‘TbGSK-3s’, the corresponding concentration of the kinase that corresponds to the volume of Fraction C added in each sample, is calculated. The calculation of the corresponding kinase concentration was based on the protein concentration provided by Bradford protein assay as mentioned in paragraph B1 of Data Analysis Section.


    4. Calculate the average value of luminescence for each TbGSK-3s concentration (the concentration of TbGSK-3s in each sample depending on the added volume of fraction C (μl) is shown in Table 1).
    5. Plot the diagram of the luminescence values versus the amount of TbGSK-3s containing in the sample (Figure 4).
    6. The sample which diminishes the luminescence value at the highest portion while it has not yet been saturated is circled in red in the plot and it is the one containing 2 μl of fraction C (40 ng TbGSK-3s) (Figure 4).


      Figure 4. Plot of luminescence values versus the amount of TbGSK-3s. Luminescence (RLU) in the Y axis represents the value of each sample when measured in the GloMax® 20/20 Luminometer and it is expressed in RLU x 106. The X axis represents the volume of the kinase (TbGSK-3s) in μl which is added in each sample.

  3. Determination of the Km for ATP and GSK-3 substrate
    1. Collect the luminescence readings for the samples containing different concentrations of GSK-3 substrate or ATP.
    2. Calculate the average value of luminescence (RLU) and the standard deviation for each sample.
    3. Calculate the rate of reaction DY/DX for each sample (as shown in Table 2 for the ATP and in Table 3 for the GSK-3 substrate),
      where DY = [(RLU x 106 upon 30min of reaction) – (RLU x 106 upon 0 min of reaction)]
      DX = time of reaction = 30 min

      Table 2. Calculations for the rate of reaction DY/DX for different concentrations of ATP

      RLU stands for luminescence. a is for the RLU (luminescence) measured in the beginning of the reaction (t0 = 0 min). b is for the RLU (luminescence) measured in the end of the reaction (t1 = 30 min). DY is the difference between the luminescence measurements in the beginning and at the end of the reaction. DX represents the time of reaction between the two luminescence measurements (total time of reaction, 30 min).

      Table 3. Calculations for the rate of reaction DY/DX for different concentrations of GSK-3 substrate

      RLU stands for luminescence. a is for the RLU (luminescence) measured in the beginning of the reaction (t0 = 0 min). b is for the RLU (luminescence) measured in the end of the reaction (t1 = 30 min). DY is the difference between the luminescence measurements in the beginning and at the end of the reaction. DX represents the time of reaction between the two luminescence measurements (total time of reaction, 30 min).

    4. Plot the diagram of the rate of reaction DY/DX versus the ATP concentration (Figure 5) and the GSK-3 substrate (Figure 6) concentration.
    5. Determine the Vmax in the plot.
    6. Find the Vmax/2 in the plot and determine the Km of ATP and of GSK-3 substrate.


      Figure 5. Plot of the reaction rate DY/DX versus the ATP concentration. In X axis, the ATP concentration (μΜ) for each sample is presented. In Y axis, the reaction rate DY/DX that corresponds to each sample with different ATP concentration is presented, as calculated in Table 2. The Vmax for the reaction is calculated in the plot and it equals to 0.51 (Vmax/2 = 0.255). The Km for ATP after plotting the corresponding ATP concentration of the Vmax/2 value, was determined to be equal to 6.3 μΜ.


      Figure 6. Plot of the reaction rate DY/DX versus the GSK-3 substrate concentration. In X axis, the GSK-3 substrate concentration (μΜ) for each sample is presented. In Y axis, the reaction rate DY/DX that corresponds to each sample with different GSK-3 substrate concentration is presented, as calculated in Table 3. The Vmax for the reaction is calculated in the plot and it equals to 0.48 (Vmax/2 = 0.238). The Km for GSK-3 substrate after plotting the corresponding GSK-3 substrate concentration of the Vmax/2 value, was determined to be equal to 5.8 μΜ.

  4. Identification of TbGSK-3s inhibitors and calculation of their IC50 values
    1. Collect the luminescence readings for the samples containing different concentations of each possible TbGSK-3s inhibitors.
    2. Collect the luminescence readings for negative and positive control.
    3. Calculate the average value of luminescence (RLU) and the standard deviation for each sample.
    4. Substract the average RLU value of the negative control from the average RLU value of all the samples and from the positive control.
    5. The positive control’s RLU value (after the substraction of the negative control RLU value) corresponds to 100% TbGSK-3s activity.
    6. Calculate the % inhibition of TbGSK-3s activity for each concentration of the inhibitor using the formula below:
      % inhibition = (RLU value of inhibitor/RLU value of positive control) x 100
      Note: The RLU values used in the formula are the ones after the substraction of the negative control RLU value.
    7. Plot the diagram of the % inhibition of TbGSK-3s activity versus the inhibitor’s concentration. Indicative plots for an analog with no inhibition and for analogs presenting weak, moderate and strong activity against the TbGSK-3s are shown in Figures 7, 8, 9 and 10 respectively.
    8. Determine the IC50 value of the inhibitor. The IC50 value represents the concentration of the inhibitor that inhibits the kinase activity at a 50% rate, therefore the concentration of the inhibitor that increases 50% of the sample’s luminescence compared to the positive sample (positive control is the sample that contains no inhibitor, but contains the kinase, the GSK-3 substrate and the ATP).


      Figure 7. Indicative plot of an analog with no inhibitory activity against TbGSK-3s. In X axis, the concentration of the inhibitor (μΜ) in each sample, is displayed. In Y axis, the % inhibition of TbGSK-3s activity is expressed. The IC50 (in μΜ) is calculated in the plot by corresponding the concentration of the inhibitor that causes 50% inhibition of the TbGSK-3s activity. The IC50 of the compound used for the current indicative plot is calculated > 3.5 μΜ.


      Figure 8. Indicative plot of an analogue with weak inhibitory activity against TbGSK-3s. In X axis, the concentration of the inhibitor (μΜ) in each sample, is displayed. In Y axis, the % inhibition of TbGSK-3s activity is expressed. The IC50 (in μΜ) is calculated in the plot by corresponding the concentration of the inhibitor that causes 50% inhibition of the TbGSK-3s activity. The IC50 of the compound used for the current indicative plot is calculated 0.73 μΜ.


      Figure 9. Indicative plot of an analogue with modearte inhibitory activity against TbGSK-3s. In X axis, the concentration of the inhibitor (μΜ) in each sample, is displayed. In Y axis, the % inhibition of TbGSK-3s activity is expressed. The IC50 (in μΜ) is calculated in the plot by corresponding the concentration of the inhibitor that causes 50% inhibition of the TbGSK-3s activity. The IC50 of the compound used for the current indicative plot is calculated 0.59 μΜ.


      Figure 10. Indicative plot of an analog with strong inhibitory activity against TbGSK-3s. In X axis, the concentration of the inhibitor (μΜ) in each sample, is displayed. In Y axis, the % inhibition of TbGSK-3s activity is expressed. The IC50 (in μΜ) is calculated in the plot by corresponding the concentration of the inhibitor that causes 50% inhibition of the TbGSK-3s activity. The IC50 of the compound used for the current indicative plot is calculated 0.06 μΜ.

Recipes

  1. Kinase assay solution (10x)
    MOPS (pH = 7.5) 500 mM
    MgCl2 200 mM
    EGTA 100 mM
    DTT 20 mM
    Note: Aliquot in Eppendorfs and store at -20 °C.

Acknowledgments

The authors would like to thank Prof. George A.M. Cross (Rockefeller University, New York) for donating the T. brucei BSF 90-13 strain, Prof. Pevelope Mavromara for her advice and support on the baculovirus expression system. We would also like to thank for financial support IKY-SIEMENS Postdoctoral scholarship of excellence 2016-2017 ‘contract number 2016-017-0173-10398' and the COST Action BM0801. This protocol is based on the TbGSK-3s kinase assay presented in Efstathiou et al. (2019) and is a modified version of the kinase assay protocol performed against leismanial GSK-3s in Xingi et al. (2009).

Competing interests

Both authors declare that they have no conflicts of interest.

References

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简介

[摘要 ] 在体外,针对给定的靶激酶,具有抑制活性的小分子的鉴定是药物开发过程的第一步。本文中,我们描述了一种非放射性方案,该方案使用基于萤光素酶的ATP分析来鉴定布鲁氏锥虫糖原合酶激酶3(Tb GSK-3s)短异构体的抑制剂。Tb GSK-3s代表了潜在的药物靶标,因为它对于寄生虫生存至关重要。在我们的研究中使用的小分子是靛玉红类似物,在双吲哚骨架中的不同位置具有取代基。目前,激酶测定法的标准实验室操作是将[γ- 32 P] ATP 放射性标记的磷酸盐掺入,这是开发非放射性测定法(基于ELISA的测定法,荧光猝灭测定法等)的努力,例如对广泛应用缺乏敏感性或局限性。该协议可以克服铅放射性测定的不足,并且可以相对敏感地测量Tb GSK-3 对激酶的抑制作用,因此是发现铅的有用起点。
[背景 ] 激酶是一种酶发挥一个在生物学过程,包括分化,细胞增殖和细胞凋亡的关键作用,通过将在运动信号传导途径在催化的转移γ 磷酸从ATP到基板(佳等人,2008 ; Efstathiou 等等,2019)。激酶失调通常会导致多种疾病(Ways和Sheetz ,2000;Cohen和Goedert ,2004;Mazitschek和Giannis ,2004;Resnick和Fennell ,2004),因此,它们被认为是最大的药物靶标之一(Cohen 1999;Manning 等人,2002)。发现先导激酶抑制剂的第一步是建立体外激酶测定法。放射性测定具有很高的灵敏度,因此是实验室的标准操作(Jia 等,2008;Lilienthal 等,2010)。然而,基于激酶放射性检测的缺点包括特殊处理的需要,因为缺乏灵活性的限制半- 生活的32 ,新的非放射性技术已创建P.要解决这些限制是基于荧光或发光(Jia 等,2008)。在此,我们描述了一种基于荧光素酶ATP分析的非放射性方案,用于鉴定布鲁氏锥虫糖原合酶激酶3(Tb GSK-3s)短异构体的抑制剂。在T. brucei 的血流形式中,Tb GSK-3s 是生存所必需的(Ojo 等,2008),因此它是发现新的抗锥虫病药物的分子靶标(Ojo 等,2008;Oduor 等人,2011;Woodland 等人,2013;Urich 等人,2014;Swinney 等人,2016)。哺乳动物GSK-3与 广泛的疾病,因此开发了小分子量的GSK-3抑制剂(Woodland 等,2013;Gaboriaud-Kola 等,2015;Masch和Kunick ,2015)。当中GSK-3抑制剂,也有炮弹树碱,一个家庭天然双吲哚衍生物的(Hoessel 等人,1999年,Polychronopoulos 等人,2004年,Vougogiannopoulou 等人,2008年,Myrianthopoulos 等人,2013)。在这个协议中,发光激酶测定基于所述激酶-Glo ® 试剂Promega公司的,进行说明。该方法简单,无放射性,快速且不缺乏敏感性。尽管下文所述的方案是针对杆状病毒系统中表达的重组Tb GSK-3的特异性(如前所述(Efstathiou 等,2019),但它可以适用于对特定激酶有适当改变的任何激酶(底物,ATP浓度) ,缓冲区等)。





图1方案基础上的激酶-Glo发光激酶测定® 试剂。激酶反应在适当的条件下,在有或没有抑制剂的条件下进行。在该试剂的添加了时剩余的ATP,用作由激酶-Glo衬底® 萤光素酶催化荧光素的单氧合。荧光素酶反应每转换产生一个光子光。发光与激酶活性(Promega)成反比。

关键字:激酶分析, 非放射性分析, 布氏锥虫, 布氏锥虫糖原合成酶激酶-3, 先导物, 抑制剂, 靛玉红类

材料和试剂


 


用料


1. 移液器提示:0.5-10 微升,10-200 微升,200-1,000 微升(格雷纳生物一,产品目录号:771291,739290,740290)      


2. Eppendorf管(Greiner Bio-One,目录号:616201)      


3. 的Amicon ® 超4离心过滤器      


试剂种类


GSK-3肽底物YRRAAVPPSPSLSRHSSPHQ(pS )EDEEE(HQ),1 mg( Biaffin GmbH&Co KG,proteinkinase.de,目录号:PEP-GSK-001,储存温度:-20 °C)
激酶-Glo ® 发光激酶测定法,10个ml的(Promega公司,目录号:V6711,存储温度:-20 ℃下)
激酶格洛® 底物,1小瓶(冻干)(Promega公司,目录号:V378A)
激酶格洛® 缓冲液,10 ml的(Promega公司,目录号:V379A)
5'-三磷酸腺苷(ATP)水合二钠盐,1 mg(Sigma-Aldrich,目录号:FLAAS,储存温度:-20 °C )
Tb GSK-3在杆状病毒表达系统中表达,如先前所述(Efstathiou 等,2019)(储存温度:表达后立即使用或在-80°C 甘油储备)。
笔记:


TbGSK-3s不能从市场上买到。为了使用它,必须按照参考书目(Efstathiou et al。,2019)中描述的协议在实验室中表达它。简而言之,如Efstathiou等人所述。,2019中,pTriEx-1.1-TbGSK3s质粒cotranfected 与的BaculoGold DNA导入草地贪夜蛾(的Sf9)昆虫细胞中和制造时的TbGSK3s,激酶是在Ni纯化2+ -nitrilotriacetate(的Ni-NTA)树脂根据制造商的说明(Qiagen)。
激酶级分应立即使用以确保最大活性。如果它们在-80°C下作为甘油原液存储,则应在分离后最多3-5天使用,以避免完全丧失激酶活性。
32靛玉红模拟小号THA 吨合成如先前所描述(梅杰等人,2003 ; Polychronopoulos 等人,2004 ; Ferandin 等人,2006 ; Vougogiannopoulou 等人,2008年),(储存温度:4 ℃,远来自阳光)
MOPS(3-(N-吗啉代)丙烷磺酸)(Applichem ,目录号:A2947,储存温度:RT)
MgCl 2 (六水合氯化镁)(Applichem ,目录号:A1036,储存温度:RT)
EGTA(乙二醇-双(2-氨基乙基醚)-N,N,N 'N ' - 四乙酸)(Sigma-Aldrich公司,目录号:E4378,贮存温度:RT)
Pierce TM DTT(二硫苏糖醇)(ThermoFisher Scientific,目录号:20290,储存温度:4 °C )
Ni-NTA琼脂糖(25毫升)(QIAGEN,目录号:30210,储存温度:4 °C )
的Amicon ® 超4离心过滤单元(默克-Millipore公司,目录号:UFC801008,贮存温度:RT)
咪唑用于分子生物学(Applichem ,目录号:A1378,0050,储存温度:RT)
激酶测定溶液(10x)(请参阅食谱)
 


设备


 


水浴(Julabo ,ED-13开放式加热浴循环器/停产产品)
GloMax ® 20/20光度计(Promega公司,型号/目录号:2030-100 / E5311)
Eppendorf 5417R冷冻离心机(Marshall Scientific,产品代码:EP-5417R)
 


软件




微软® Office Excel中2013
GraphPad Prism v6软件
 


程序


 


的制备激酶-Glo ® 试剂
混合至10毫升激酶格洛® 与冻干激酶格洛缓冲® 底物。
确保底物粉末稀释完全,溶液澄清。
等分试样中的激酶-Glo ® 试剂,并将其存储在-20 ℃下直到使用。
 


P 的赔偿激酶测定溶液10倍
按照食谱中的说明准备10倍激酶测定溶液,等分并保存在-20 °C 直至使用。


 


测定具有最高酶促活性的Tb GSK-3s馏分
对激酶的不同分离部分(Tb GSK-3s)进行激酶测定。将分离的级分已对Ni纯化2 -nitrilotriacetate(镍- NTA)根据制造商的说明书(Qiagen)用琼脂糖洗脱不同的咪唑浓度(的馏分小号A,B,C和d使用50,150,250和400 纳米分别咪唑)如先前所述(Efstathiou 等人,2019)。
注:隔离的TbGSK-3的级分使用随后浓缩的Amicon ® 超4离心过滤器和再稀释在激酶测定溶液1 X ,以从样品中除去过量的咪唑。高浓度的咪唑可能会干扰激酶测定,从而提供错误的结果。


将水浴温度调节至30 °C。
解冻激酶-Glo ® -20试剂℃下于4 ℃。
从-20 °C 到4 °C 解冻激酶测定溶液10倍。
制备主混合物溶液(含有1x终浓度激酶测定溶液,10 μΜ ATP,10 μΜ GSK-3的肽底物),如下(最终体积40描述μ 每个样品1):
4 微升* [样本数]的10×激酶测定溶液


1 微升* [样本数]的400 μΜ ATP


1.2 微升* [样本数]的333 μΜ GSK-3的肽底物


23.8 微升* [样本数]的的DDH 2 ö


上下移液均匀混合主混合物。
在每个Eppendorf中添加30μl 的预混液。
在每个试管中添加10μl 每个Tb GSK-3s分离的级分(A,B,C和D 级分s )。
为每个分数重复或三份。
通过上下移液将样品充分混合。
旋转样品。
将样品在30 °C的水浴中孵育30分钟。
通过添加40停止反应微升冷的激酶-Glo ® 试剂(4 ℃下)。
旋转样品。
测量在发光GloMax ® 20/20光度计1秒。
注意:使用名为“ Kinase-Glo”的“即用型” Promega协议阅读发光。


确定具有最佳酶促活性的Tb GSK-3 的洗脱比例(请参阅数据分析A)。
立即将激酶用于激酶测定,或将其储存在-80 °C 的50%(v / v)甘油中。
笔记:


在我们的分析中,TbGSK-3s的纯化洗脱级分C(最初用250 mM咪唑洗脱)确定为具有较高酶促活性的级分(请参阅数据分析A)。
建议洗脱后立即使用激酶TbGSK-3s馏分,以确保最大活性。激酶的50%(v / v)甘油储备根据其在-80°C下储存的时间段而降低了活性,分离后应使用长达3-5天,以避免激酶完全损失活动。
 


确定最佳蛋白激酶(Tb GSK-3s)浓度
用Tb GSK-3s 的适当洗脱级分(洗脱级分C)进行激酶测定,以上确定为具有较高酶促活性的Tb GSK-3s。
遵循步骤C2-C4。
制备主混合物溶液(含有1x终浓度激酶测定溶液,10 μΜ ATP,10 μΜ GSK-3的肽底物),如下(最终体积40描述μ 每个样品1):
4 微升* [样本数]的10×激酶测定溶液


1 微升* [样本数]的400 μΜ ATP


1.2 微升* [样本数]的333 μΜ GSK-3的肽底物


23.8 微升* [样本数]的的DDH 2 ö


遵循步骤C6-C7。
加入10 微升的铽在不同浓度(含0,2,5,10,15,20,40,100和200纳克分离馏分C GSK-3 Tb的GSK-3,用双蒸水稀释2 O)。
注意:可以根据需要,可以使用不同浓度的激酶依赖于参数的实验。


遵循步骤C9-C15。
确定用于激酶测定的Tb GSK-3s 的最佳浓度(请参阅数据分析B)
注意:在我们的分析中,确定40 ng TbGSK-3s(组分C)为激酶测定的最佳TbGSK-3s浓度(请参阅数据分析B)。馏分C含有20纳克/ μ 升TbGSK-3脂肪酸(参见数据分析B)的。


 


ATP浓度的测定
用Tb GSK-3s分离的级分C 进行激酶测定。
遵循步骤C2-C4。
制备主混合物溶胶ution(含有1X激酶测定溶液的最终浓度,40纳克的Tb的GSK-3分离馏分C,10 μΜ GSK-3的肽底物),如下(最终体积40描述μ 每个样品1):
4 微升* [样本数]的10×激酶测定溶液


2 微升* [样本数]的Tb的GSK-3中分离馏分C


1.2 微升* [样本数]的333 μΜ GSK-3的肽底物


23.8 微升* [样本数]的的DDH 2 ö


遵循步骤C6-C7。
加入10 微升的ATP在不同浓度的(0,0.5%,1,2,5,7.5,第10和50 μΜ )。
注意:如果需要,可以根据实验参数使用不同浓度的ATP。然而,对于ATP浓度高达10 μΜ ,您可以用激酶-Glo ® 试验(Promega),0-100为μM的ATP,您可以用激酶-Glo ® 加试验(Promega)和0-500 μM,您可以用激酶-Glo ® 最大试验(Promega)。


遵循步骤C9-C15。
                                                                                    绘制反应速率DY / DX与ATP浓度的关系图,并确定ATP的Vmax和Km(请参见数据分析C)
注意小号:


DY = [ (RLU×10 ^ 6 在30 反应的分钟)- (RLU×10 ^ 6 时REAC的0分钟和灰)]
DX =反应时间= 30分钟
Vmax是最大反应速率,代表当酶被底物饱和时的反应速率。
Michaelis-Menten常数(Km)的值在数值上等于反应速度为Vmax一半时的底物浓度。
使用40ng TbGSK-3s级分C 确定ATP的Km等于6.3μM (参见数据分析C)。
使用在用TbGSK-3s进行的所有激酶测定中相同的标准ATP浓度,其等于ATP的Km(6.3μM )。
 


 


GSK-3肽底物浓度的测定
用Tb GSK-3s分离的级分C 进行激酶测定。
遵循步骤C2-C4。
制备主混合物溶液(含有1x终浓度激酶测定溶液,40 纳克的Tb的分离馏分C,10 GSK-3 μΜ ATP)如下(最终体积40描述μ 每个样品1):
4 微升* [数量的样本] 10X激酶测定的


2 微升* [样本数]的Tb的GSK-3中分离馏分C


1.2 微升* [样本数]的400 μΜ ATP


23.8 微升* [样本数]的的DDH 2 ö


遵循步骤C6-C7。
添加10 微升GSK-3底物的不同浓度的(0,1,2,5,10和50 μΜ )。
注意:根据实验参数,可以根据需要使用不同浓度的底物。


遵循步骤C9-C15。
绘制反应速率DY / DX与GSK-3 c 浓度的关系图,并确定GSK-3底物的Vmax和Km(请参见数据分析C)
注意小号:


GSK-3底物的公里被确定等于5.8 μΜ 使用40纳克TbGSK-3的(级分C)(见数据分析C) 。
使用在用TbGSK-3s进行的所有激酶测定中相同的标准GSK-3底物浓度,其等于GSK-3底物的Km(5.8μM )。
 


Tb GSK-3s抑制剂的鉴定
用Tb GSK-3s分离的级分C 进行激酶测定。
遵循步骤C2-C4。
制备主混合物溶液(含有1x终浓度激酶测定溶液,40的纳克Tb的GSK-3分离馏分C)以下(终体积40如所描述的μ 每个样品1):
4 微升* [样本数]的10×激酶测定溶液


2 微升* [样本数]的Tb的GSK-3中分离馏分C


22.7 微升* [样本数]的ddH 2 O


遵循步骤C6-C7。
加入10μl 不同浓度的抑制剂(0、0.01、0.03、0.1、0.33、1 和3.33μM )。
注意:如果需要,可以根据实验参数使用不同浓度的抑制剂。


添加5.8 μΜ 各样品(0.7中GSK-3的肽底物微升的333 μΜ GSK-3的肽底物)。
添加6.3 μΜ ATP每个样品(0.6 微升的400 μΜ ATP) 。
遵循步骤C9-C15。
绘制Tb GSK-3s活性抑制百分率与抑制剂浓度的关系图,并确定抑制剂的IC 50 (数据分析D)
注意:没有抑制剂的样品用作阳性对照。此外,使用包含没有ATP,作为阴性对照(4样品微升激酶测定溶液10倍,2 微升含TbGSK-3分离馏分C 40 纳克Ò ˚F激酶,0.7 微升GSK-3肽底333 μΜ 为了获得5.8 μΜ 终浓度,33.3 μ 升的DDH 2 O)。             


 


数据分析


 


具有酶活性的Tb GSK-3s馏分的测定:
包含活性激酶的部分将利用ATP磷酸化GSK-3底物,因此样品中ATP的浓度将降低。结果,荧光素将不再具有与之相互作用的相同量的ATP,并且该反应将产生较少的光。因此,样品的发光(RLU)测量值将低于阴性对照(不含激酶的样品)之一。
收集Tb GSK-3s 洗脱级分的发光读数。
计算每个部分的发光平均值和标准偏差。
绘制发光值图(图2)。
发光值最低的部分是具有最佳酶活性的部分,可用于激酶测定的进一步实验。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-1-20 \ 3493--1902945--1257 Antonia Efstathiou 810565 \图JPG \图2-updated.jpg


图2.分离的发光值Tb的GSK-3级分A,B,C和D 的Tb的GSK-3级分A,B,C和d在Ni纯化2 -nitrilotriacetate(镍- NTA)树脂根据制造商的说明(马歇雷-Nagel)与洗脱不同的咪唑浓度(FR 动作小号A,B,C和d使用50,150,250和400 nM的分别咪唑)如先前所述(Efstathiou 等人,2019),并且随后它们使用浓缩的Amicon ® 超0.5 ml的离心过滤器并重新在激酶测定溶液1X为了从样品除去过量的咪唑稀释。激酶测定溶液1x(无激酶)用作阴性对照。


 


 


确定最佳蛋白激酶(Tb GSK-3s)浓度
使用Bradford蛋白测定法(He ,2011年),并基于W 酯印迹法中信号的强度,并与标记条带中已知蛋白的量进行比较,通过半定量方法确定每个馏分中存在的激酶量。鉴于激酶级分放入的体积W¯¯ 西部时代印迹是恒定的并且是已知的。
笔记:


W 酯印迹的方案和分析程序,包括所用的试剂和材料,可在参考书目中找到(Mahmood 和Yang,2012 ;Heiber 等人,2014 )。
Bradford蛋白测定用于定量每个洗脱级分中的总蛋白量,而W 酯图用于说明每个级分中TbGSK-3的存在。
(泳道5分离的馏分C 在图3 - 铽ģ 其从洗脱的SK-3级分的Ni 2 -nitrilotriacetate(镍- NTA)树脂的Ni 2+ 使用珠250 mM咪唑)估计为含有20 纳克/ μ 升的铽GSK-3(图3)。
收集包含不同浓度的Tb GSK-3 的样品的发光读数。
 






图3.分离的蛋白质印迹Tb的GSK-3级分A,B,C和D 30 μ 的升Tb的GSK-3级分A 。(第3道),B 。(第4泳道),C 。(泳道5)和d 。(泳道6),将其在Ni纯化2 -nitrilotriacetate(镍- NTA)树脂与洗脱不同咪唑浓度(50,150,250和400的纳米分别咪唑),而其余的Ni 2 -nitrilotriacetate(镍- 使用针对L GSK-3 的亲和纯化多克隆抗体,在W 酯印迹中鉴定洗脱时的NTA)树脂(泳道2)。该w ^ 西部时代印迹在的理论分子量表现出独特的带Tb的GSK-3(40 kDa的)。在泳道1中,30 μ 的激酶测定溶液升1 X 也被用作阴性对照。






表1. 当使用不同体积的分离级分C 时,激酶测定中使用的Tb GSK-3s以ng表示。在列标题为“组分C”,将洗脱的激酶的量Tb的GSK-3(在表示μ 升),将其在每个样品中加入,给出。在标题为“ Tb GSK-3s” 的栏中,计算了与每个样品中添加的馏分C的体积相对应的激酶的相应浓度。相应激酶浓度的计算基于数据分析部分第B1段所述的Bradford蛋白质测定法提供的蛋白质浓度。


分数C (μl)


TB GSK-3s(ng)


0


0


0.1


2


0.25


5


0.5


10


0.75


15


1个


20


2


40


5


100


10


200


 


计算发光的每个平均值Tb的GSK-3脂肪酸浓度(浓度Tb的GSK-3在每个样品中根据馏分C(的加入体积上μ 升)示于钽BLE 1)。
绘制发光值与样品中Tb GSK-3含量的关系图(图4)。
在尚未饱和的最高位置减小发光值的样品在图中用红色圈出,它是包含2μl 馏分C(40 ng Tb GSK-3s)的样品(图4)。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ 2020-1-20 \ 3493--1902945--1257 Antonia Efstathiou 810565 \图JPG \图4-updated.jpg


图4.发光值与Tb GSK-3s 数量的关系图。在测量时,发光(RLU)在Y轴表示每个样品的值GloMax ® 20/20光度计,它是在RLU * 10表示6 。X轴代表所述激酶的体积(铽在GSK-3)μ ,其在每个样品中加入升。


ATP和GSK-3底物Km的测定
收集包含不同浓度的GSK-3底物或ATP的样品的发光读数。
计算每个样品的发光平均值(RLU)和标准偏差。
计算每个样品的DY / DX反应速率(如表2所示为ATP,表3所示为GSK-3底物),
其中DY = [((反应30分钟时RLU x 10 6 )– (反应0分钟时RLU x 10 6 )]]


DX = 反应时间= 30 分钟


 


表2. 不同ATP浓度下DY / DX反应速率的计算


ATP(μΜ)


RLU a
[RLU x 10 6


t0 = 0 分钟]


RLU b
[RLU x 10 6


t1 = 30 分钟]


DY
(RLU a -RLU b )


DX
(反应分钟)


反应速率(DY / DX)


0


0


0


0


30


0


0.5


2.52


1.21


1.31


30


0.04 4


1个


6.31


0.86


5.45


30


0.18 2


2


9.38


3.38


6 .00


30


0.2 00


5


18.81


11.61


7.2 0


30


0.24 0


7.5


23.66


15.26


8.4 0


30


0.28 0


10


28.84


19.84


9 .00


30


0.3 00


20


68.37


54.33


14.04


30


0.468


50


121.65


106.3 0


15.35 0


30


0.51 2


RLU代表发光。a 是在反应开始时(t0 = 0分钟)测得的RLU(发光)。b 是在反应结束时(t1 = 30分钟)测得的RLU(发光)。DY是反应开始和结束时发光测量之间的差。DX表示两次发光测量之间的反应时间(反应总时间,30分钟)。






表3.不同浓度的GSK-3底物的DY / DX反应速率的计算


GSK-3底物(μΜ )


RLU a
[RLU x 10 4


t0 = 0 分钟]


RLU b
[RLU x 10 4


t1 = 30 分钟]


DY
(RLU a -RLU b )


DX
(反应分钟)


反应速率(DY / DX)


0


0


0


0


30


0


1个


29.36


24.86


4.50


30


0.150


2


28.81


23.35


5.46


30


0.182


5


26.44


19.84


6.60


30


0.220


10


27.67


18.07


9.60


30


0.320


20


28.69


14.59


14.10


30


0.470


50


28.99


14.29


14.70


30


0.490


RLU代表发光。a 是在反应开始时(t0 = 0分钟)测得的RLU(发光)。b 是在反应结束时(t1 = 30分钟)测得的RLU(发光)。DY是反应开始和结束时发光测量之间的差。DX表示两次发光测量之间的反应时间(反应总时间,30分钟)。


 


绘制反应DY / DX速率与ATP浓度(图5)和GSK-3底物(图6)浓度的关系图。
确定图中的Vmax 。
在图中找到Vmax / 2,并确定ATP和GSK-3底物的Km 。
 






图5.反应速率DY / DX与ATP浓度的关系图。在X轴上,给出了每个样品的ATP浓度(μM )。在Y轴上,显示了对应于具有不同ATP浓度的每个样品的反应速率DY / DX ,如表2所示。在图中计算反应的Vmax,它等于0.51(Vmax / 2 = 0.255)。 。在绘制对应的ATP浓度的Vmax / 2值之后,ATP的Km被确定为等于6.3μM 。


 






图6.反应速率DY / DX与GSK-3底物浓度的关系图。在X轴上,给出了每个样品的GSK-3底物浓度(μM )。在Y轴上,显示了对应于具有不同GSK-3底物浓度的每个样品的反应速率DY / DX,如表3所示。在图中计算反应的Vmax,它等于0.48(Vmax / 2 = 0.238)。在绘制相应的Gmax-3值的GSK-3底物浓度后,确定GSK-3底物的Km等于5.8μM 。


 


              Tb GSK-3s抑制剂的鉴定及其IC 50 值的计算
收集包含每种可能的Tb GSK-3s抑制剂浓度不同的样品的发光读数。
收集阴性和阳性对照的发光读数。
计算每个样品的发光平均值(RLU)和标准偏差。
。减去来自所有样品的平均RLU值和从阳性对照阴性对照的平均RLU值。
阳性对照的RLU值(减去阴性对照RLU值之后)对应于100%Tb GSK-3s活性。
使用以下公式计算每种浓度的抑制剂对Tb GSK-3s活性的抑制百分比:
抑制百分比=(抑制剂的RLU值/阳性对照的RLU值)x 100


注意:Ť 他RLU公式中使用的值是后的那些减法阴性对照RLU值的。


绘制Tb GSK-3s活性抑制百分率与抑制剂浓度的关系图。图7、8、9和10分别显示了没有抑制作用的类似物以及对Tb GSK-3具有弱,中和强活性的类似物的指示性批次。
确定抑制剂的IC 50 值。IC 50 值代表以50%的速率抑制激酶活性的抑制剂的浓度,因此与阳性样品相比,该抑制剂的浓度增加了样品发光50%(阳性对照是不含抑制剂的样品,但包含激酶,GSK-3底物和ATP)。
 






图7.对Tb GSK-3s 无抑制活性的类似物的指示图。在X轴上,显示了每个样品中抑制剂的浓度(μM )。在Y轴上,表示了Tb GSK-3s活性的%抑制。通过对应于引起Tb GSK-3s活性50%抑制的抑制剂的浓度,在曲线图中计算IC 50 (以μM计)。计算用于当前指示图的化合物的IC 50 > 3.5μM 。


 






图8.对Tb GSK-3s 具有弱抑制活性的类似物的指示图。在X轴上,显示了每个样品中抑制剂的浓度(μM )。在Y轴上,表示了Tb GSK-3s活性的%抑制。通过对应于引起Tb GSK-3s活性50%抑制的抑制剂的浓度,在曲线图中计算IC 50 (以μM计)。该IC 50 用于当前的化合物的指示情节被计算0.7 3 μΜ 。


 






图9.具有对Tb GSK-3s的modearte 抑制活性的类似物的指示图。在X轴上,显示了每个样品中抑制剂的浓度(μM )。在Y轴上,表示了Tb GSK-3s活性的%抑制。通过对应于引起Tb GSK-3s活性50%抑制的抑制剂的浓度,在曲线图中计算IC 50 (以μM计)。计算用于当前指示图的化合物的IC 50 为0.59μM 。


 






图10.对Tb GSK-3s 具有强抑制活性的类似物的指示图。在X轴上,显示了每个样品中抑制剂的浓度(μM )。在Y轴上,表示了Tb GSK-3s活性的%抑制。通过对应于引起Tb GSK-3s活性50%抑制的抑制剂的浓度,在曲线图中计算IC 50 (以μM计)。计算用于当前指示图的化合物的IC 50 为0.06μM 。






菜谱


 


激酶测定液(10x )
MOPS(p ħ = 7.5)的500mM


的MgCl 2 的200mM


EGTA 100毫米


DTT 20毫米


注:分装于eppendorf管,并储存在- 20 ℃的。


 


致谢


 


作者在此感谢纽约洛克菲勒大学的George AM Cross 教授捐赠了布鲁氏T. brucei BSF 90-13株,Pevelope Mavromara 教授对杆状病毒表达系统的建议和支持。我们还要感谢IKY-SIEMENS博士后卓越奖学金2016-2017'合同号2016-017-0173-10398'和COST行动BM0801的财政支持。该协议是基于Tb的GSK-3激酶测定中提出(Efstathiou 等人。,2019 ),反对进行激酶测定协议的修改版本leismania 升在GSK-3 Xingi 等。,2009年。


 


利益争夺


 


两位作者都宣称他们没有利益冲突。


 


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引用:Efstathiou, A. and Smirlis, D. (2020). A Radioactive-free Kinase Inhibitor Discovery Assay Against the Trypanosoma brucei Glycogen Synthase Kinase-3 short (TbGSK-3s). Bio-protocol 10(2): e3493. DOI: 10.21769/BioProtoc.3493.
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