Amplex red assay, a standardized in vitro protocol to quantify the efficacy of autotaxin inhibitors

Summary

Autotaxin (ATX), a secreted lysophospholipase D responsible for the extracellular production of the bioactive phospholipid lysophosphatidic acid (LPA), is a therapeutic target in idiopathic pulmonary fibrosis and pancreatic cancer, among other disorders, promoting the synthesis of novel ATX inhibitors. Here, we present a protocol for detecting and characterizing ATX inhibitors using a fluorometry-based microplate assay. We describe steps for a first screening of compounds, half-maximal inhibitory concentration (IC₅₀) quantification of initial hits, screening for false positives, and identification of the hits’ mode of inhibition.

For complete details on the use and execution of this protocol, please refer to Stylianaki et al.¹

Graphical abstract

Highlights

• •In vitro fluorescence-based assay for screening of potential autotaxin inhibitors
• •Complete analysis of enzyme kinetics and inhibitor parameters (IC₅₀, K_m, V_max, K_i, and k_cat)
• •Exclusion of false positives, highly reproducible, low cost/sample, and easy to scale up

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Autotaxin (ATX), a secreted lysophospholipase D responsible for the extracellular production of the bioactive phospholipid lysophosphatidic acid (LPA), is a therapeutic target in idiopathic pulmonary fibrosis and pancreatic cancer, among other disorders, promoting the synthesis of novel ATX inhibitors. Here, we present a protocol for detecting and characterizing ATX inhibitors using a fluorometry-based microplate assay. We describe steps for a first screening of compounds, half-maximal inhibitory concentration (IC₅₀) quantification of initial hits, screening for false positives, and identification of the hits’ mode of inhibition.

Before you begin

Autotaxin (ATX) is a protein that was first discovered by Stracke et al. in 1992 when it was identified as a motility-stimulating factor isolated from the conditioned medium of human melanoma cells.² The main function of ATX is the catalysis of the hydrolysis of Lysophosphatidylcholine (LPC) to the bioactive lipid Lysophosphatidic acid (LPA). This was discovered in 2002, when an enzyme with plasma Lysophospholipase D (lysoPLD) activity was found to be identical with ΑTΧ.³ Since then, a large number of studies have showcased ATX’s role in the initiation of various pathologies when upregulated, e.g., idiopathic pulmonary fibrosis (IPF), hepatic fibrosis, rheumatoid arthritis and lung cancer.^4,5,6,7,8 These findings were appreciated both by the academic sector and the pharmaceutical industry and prompted the design of several classes of small-molecule ATX inhibitors which were extensively utilized in preclinical research.^9,10 ATX activity abrogation by such inhibitors turned out to be successful in alleviating the aforementioned diseases’ manifestations,^6,11,12 among many others, and additionally, it was also demonstrated that ATX abrogation is well tolerated in healthy adult mice.¹³ ATX was, thus, established as a safe and efficacious pharmacological target, and provided the proof-of-concept of a novel, promising strategy for combating such pathologies that could lead to a new investigational drug. This evidence was translated to the development of five different types of ATX inhibitors^14,15 by the pharmaceutical industry and the academic sector, such as PF8380¹⁶ and GLPG1690¹⁷ among others, that served as a stepping stone for developing new structures endowed with improved pharmacological properties. This accumulation of knowledge has now culminated in two major clinical trials, among others, assessing the profoundly effective inhibitor BBT-877 in preclinical studies against IPF, and IOA-289 which has already received orphan drug designation for pancreatic ductal adenocarcinoma.^{18,19,20,21,22}

The early success of these two investigational drugs has reignited the interest in ATX as a promising drug target and, concomitantly, has given rise to a need to develop cheap and robust enzymatic assays that assess ATX lysoPLD activity against experimental modulators, so as to discover further ATX inhibitors. Such assays can be divided in two categories; those that utilize ATX’s natural substrate, LPC, and those that employ artificial substrates. The current assay belongs to the first category and utilizes the Amplex red reagent (IUPAC nomenclature: 10-acetyl-3,7-dihydroxyphenoxazine) as a surrogate probe to estimate ATX activity. This reagent has been widely employed combined with horseradish peroxidase (HRP) to detect hydrogen peroxide in biological samples or in mixes of known composition at which this oxygen species is generated.²³ In the presented protocol, ATX first catabolizes LPC to yield LPA and choline and, next, choline becomes the substrate of choline oxidase to produce betaine and hydrogen peroxide. The latter reacts with Amplex red in the presence of HRP, to produce the fluorescence-emitting probe resorufin, allowing indirect quantification of ATX activity (Figure 1). This assay poses an advantage compared to assays that employ artificial substrates of ATX such as FS-3, a synthetic fluorescent analog of LPC. As FS-3 bears both a fluorophore and a quencher group, the relevant assay may not fully recapitulate the interaction of ATX with its native substrate. Indeed, discrepancies regarding ATX inhibitor potencies have been reported between assays that measure the produced LPA in the presence of the inhibitor and the FS-3 assay.²⁴ Two other assays which utilize ATX’s natural substrate are the Homovanillic acid (HVA) assay and the TOOS ((N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine) assay.^1,25 Despite the fact that both of them are reliable, the HVA assay is more expensive than the current assay and the TOOS assay is less sensitive than the Amplex Red assay since it is a colorimetric and not a fluorescence-based assay. Moreover, recently, a cell-based assay for the detection of ATX activity and the discovery of ATX inhibitors was established.²⁶ Compared to simple biochemical assays, this assay requires equal cell seeding between experiments in order to have reliable inhibitor characterization and, additionally, it is expected to be more expensive. Therefore, the Amplex Red assay represents an efficient, cheap and physiologically relevant method for identifying potential inhibitors (or activators) against ATX within a set of investigatory compounds. Specifically, it is suitable for quantifying their in vitro potential (IC₅₀ or AC₅₀), as well as distinguishing their mode of action (K_i, V_max). ATX can be also assessed against various substrates (LPx species), even without the presence of a putative modulator, so as to quantify the enzyme’s chemical affinity (K_m) and turnover rate (k_cat) of that specific substrate. Since the discovery of inhibitors/activators against an enzyme is a heuristic process, several sets of candidate structures may have to be screened until a hit arises. Herein, the detailed characterization procedure of such hit modulators is described, segmented as individual steps in the protocol below, which is suitable for further scaling up with straightforward modifications.

Figure 1.

Chemical principle of the protocol

First, Autotaxin (ATX) catalyzes the transformation of Lysophosphatidylcholine (LPC) to Lysophosphatidic acid (LPA) and Choline. Choline is, then, oxidized by Choline oxidase to Betaine and Hydrogen peroxide. In the presence of Hydrogen peroxide and Horseradish peroxidase (HRP) the probe Amplex Red transforms to fluorescent Resorufin. Compounds that are used as reactants are in bold letters.

Stock reagent solutions preparation

• 1.Amplex Red Assay buffer

Reagents	Final concentration	Amount
Tris-base	50 mM	1211 mg
CaCl2	5 mM	111 mg
HCl 37.2%	0.087%	470 μL
ddH2O	N/A	Up to 200 mL
Total	N/A	200 mL

HCl is added so as to set pH at 8. Store at 4°C, protected from light.

Note: Another option is to make stock solutions of, e.g., 1 M Tris-base pH 8 and 2 M CaCl₂, and utilize the respective volume of these to prepare the Amplex Red Assay buffer.

Note: This buffer is prone to gradual desalting and, therefore, it will be unsuitable for use after two weeks. Covering the bottle with aluminum foil protects the assay buffer by eliminating hydrogen peroxide generation produced by exposure of water to light.

• 2.
  10 mM solution of Amplex Red reagent.

  • a.
    Dilute 5 mg in 1943 μL of DMSO.
  • b.
    Prepare 100 μL aliquots. Store at −20°C, protected from light.

CRITICAL: Aliquots should be covered individually with aluminum foil to avoid photooxidation²⁷ and should not be thawed more than twice. If the reagent turns pink, the background of the assay is increased, therefore it is recommended that pinkish aliquots are avoided.

• 3.
  530 U/mL HRP solution.

  • a.
    Prepare 100 mM phosphate buffer pH 6.

    Reagents	Amount
    NaH2PO4·H2O	119.1 mg
    Na2HPO4·7H2O	36.7 mg
    ddH2O	Up to 10 mL
    Total	10 mL

  • b.
    Dilute 2650 U HRP in 5 mL of 100 mM phosphate buffer pH 6.

    Note: The exact U/mg ratio depends on the batch of the enzyme. It is recommended to make 38 μL aliquots and store them at −20°C.

    Note: The residual quantity of phosphate buffer can be stored at 4°C for up to 2 weeks.

• 4.
  200 U/mL of Choline Oxidase.

  • a.
    Dilute 500 U in 2.5 mL of 50 mM Tris-base/5 mM CaCl₂ pH 8.
  • b.
    Make aliquots of 10 μL and store them at −20°C.

Note: The exact U/mg ratio depends on the batch of the enzyme.

CRITICAL: Choline oxidase solution is very sensitive to temperature fluctuations, such as repetitive freeze-thaw cycles. If exposed to such fluctuations, choline oxidase will precipitate to form a yellowish aggregate separated from the liquid phase. Do not thaw or re-use an aliquot more than once.

• 5.
  800 nM of human recombinant ATX.

  • a.
    Dilute 50 μg ATX in 500 μL of 50 mM Tris-base/5 mM CaCl₂ solution pH 8.
  • b.
    Prepare aliquots of 40 μL and store them at −20°C.

Note: As stated by Sino Biological, the calculated Mr of this recombinant human ATX is ∼96 kDa, but it becomes ∼120–130 kDa due to glycosylation. The calculations are made with a Mr of 125 kDa for ATX.

CRITICAL: ATX batches may differ in terms of resulting activity in the assay. It is strongly recommended to assess its activity by comparing your old batch with the new at the pre-selected flashes/gain value pair (see section below) to ensure matching activities.

• 6.200 mM of 16:0 LPC in 2:1 chloroform:methanol solution.

Reagent	Final concentration	Amount
LPC	200 mM	25 mg
chloroform	66.6% v/v	168 μL
methanol	33.3% v/v	84 μL
Total	N/A	252 μL

Note: Aliquot the solution in glass vials with PTFE septa on the lid to prevent sticking of the lipid to the plastic and corrosion of the lid by chloroform and store at −20°C.

CRITICAL: First add chloroform to the vial containing the LPC powder, mix thoroughly and then add methanol and mix thoroughly again for optimal dilution. Dilution of LPC should be done in a hood to account for carcinogenic properties of both methanol and chloroform. Screw cap should be further sealed with Parafilm to prevent evaporation of the volatile solvents.

• 7.All compounds to be tested should be diluted at a desired concentration (e.g., 50 mM), preferably in DMSO.

CRITICAL:In vitro drug-screening assays require properly diluted compounds in their stock solution to ensure precise assessment. Make sure that the concentration and the selected solvent suit the chemical properties of the compounds.

• 8.
  2 mM choline chloride.

  • a.
    Dilute 2.5 mg in 8.95 mL of distilled water.
  • b.
    Make aliquots of desired volume, e.g., 1 mL, and store them at −20°C.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Dimethyl sulfoxide	Sigma-Aldrich	Cat#589569
Calcium dichloride	Sigma-Aldrich	Cat#C5670
Hydrochloric acid 37.2%	Sigma-Aldrich	Cat#258148
Choline chloride	Sigma-Aldrich	Cat#C1879
Sodium phosphate dibasic heptahydrate	Sigma-Aldrich	Cat#S9390
Sodium phosphate monobasic monohydrate	Sigma-Aldrich	Cat#S9638
Chloroform	Merck Millipore	Cat#102447
Methanol	Merck Millipore	Cat#1026002
Amplex red reagent	Thermo Fisher Scientific	Cat#A12222
16:0 lysophosphatidylcholine	Sigma-Aldrich	Cat#855675C
Tris-base	Sigma-Aldrich	Cat#10708976001
GLPG1690	MedChemExpress	Cat#HY-101772
Human autotaxin	Sino Biological	Cat#11308-H07H
Horseradish peroxidase	Sigma-Aldrich	Cat#P8125
Choline oxidase from Alcaligenes sp.	Sigma-Aldrich	Cat#C5896
Software and algorithms
GraphPad Prism	GraphPad	https://www.graphpad.com
Other
Black 96-well plate, flat bottom with lid	Corning	Cat#3916
Glass screw cap vials (N8)	Isolab	Cat#095.00.001
Caps & septa suitable for N8 vials without slit, PTFE	Isolab	Cat#096.00.001
Parafilm	Sigma-Aldrich	Cat#P7543
pH meter	Adwa	AD1020
Monochromator-based microplate fluorometer with heating platform	Tecan	Infinite M200
Analytical balance	Radwag	AS 62.R2
Non-CO2 incubator	HYBAID	Shake ‘n’ Stack

Materials and equipment

• •Prepare the reading protocol at the microplate reader with at least six 5-minute-step reading cycles with the excitation/emission pair set at 530 nm/590 nm at a standard pre-selected excitation intensity (number of flashes/flashes’ intensity) and gain (manual gain value/integration time).

Note: A standard pre-selected gain is useful to track and evaluate overall assay performance past time, thus, enzymatic activity levels and reagents’ stability. Similar to microscopy, there is not one specific pair of values of flashes and gain that would be suitable for signal detection. One way to select these values for the first time that this protocol is being employed, is to utilize fresh reagents, prepare a plate including only positive and negative controls (see section below), keep the pre-selected flashes value of the instrument, and select the “optimal gain” option (or anything similar). Keep this automatically selected value and assign it manually in the next measurements. In the Tecan Infinite M200 instrument a good starting point is to set 25 as a number of flashes and 60 as a manual gain value.

• •Right before performing the assay, pre-heat a non-CO₂ incubator at 37°C and ensure stabilized temperature.
• •Cover all falcon/eppendorf tubes to be filled with assay buffer with aluminum foil to protect from generation of H₂O₂ produced by exposure of H₂O to light.
• •Keep enzymes on ice during the preparation procedure. Do not keep compounds on ice to allow better dissolution in their solvent.
• •Pre-heat the microplate reader at 37°C and ensure stabilized temperature prior to reading.

Step-by-step method details

Discovery of a potential candidate inhibitor

Timing: ∼3 h

This step refers to the initial screening of multiple compounds for their potential inhibitory (or activation) properties. ATX is being assessed against a high concentration of these compounds. In case of existing remaining ATX activity, there are few chances of this compound being an actual hit without further structural modification.

The actual number of pre-selected compounds to be tested may vary. Here, a procedure examining 10 potential inhibitors will be described. A maximum of 42 investigational compounds can be simultaneously assessed in a 96-well plate. The well-known ATX inhibitor GLPG1690 is also added as a positive control. The proposed layout of the plate and the final concentrations of the compounds are the following.

	1–2	3–4
A	no ATX - no Compounds (DMSO only)	100 μM Compound 7
B	no ATX - no Compounds (DMSO only)	100 μM Compound 8
C	100 μM Compound 1	100 μM Compound 9
D	100 μM Compound 2	100 μM Compound 10
E	100 μM Compound 3	10 μM GLPG1690
F	100 μM Compound 4	10 μM GLPG1690
G	100 μM Compound 5	no Compound (DMSO only)
H	100 μM Compound 6	no Compound (DMSO only)

• 1.
  Prepare the substrate-containing assay buffer.

  • a.
    Add 2.5 μL of stock 16:0 LPC solution in 2.5 mL of assay buffer to make a 200 μM solution.

    Note: Due to increased cohesive forces applied between this solution and its container, the volume of the mix to be prepared should exceed by at least 10% the minimum volume needed to account for increased spreading of the liquid across the container surface.

    CRITICAL: Upon LPC addition, the solution should be immediately mixed. If not, the bottom of the polystyrene falcon or eppendorf tube will be corroded by LPC and its solvent and result in suboptimal preparation of the mix. Immediate mixing by inverting rapidly the tube is sufficient to prevent potential corrosion.

  • b.
    Place the tube with the substrate-containing assay buffer in a non-CO₂ incubator at 37°C until its usage.

    CRITICAL: Substrate-containing assay buffer should remain at 37°C for at least 15 min in order to aid the dilution of LPC.

• 2.Prepare 10 mM working stocks of the compounds to be tested and a 1 mM working stock of GLPG1690 in DMSO.
• 3.
  Prepare the conditions to be tested and add them to the wells of the plate.

  • a.
    Make 120 μL of 400 μM of each of the compounds diluted in assay buffer.
  • b.
    Make 220 μL of 40 μM of GLPG1690 in assay buffer.
  • c.
    Make 420 μL of DMSO-containing-only assay buffer by adding 16.8 μL DMSO in 403.2 μL assay buffer.

    Alternatives: Any working stock concentration of the compounds will suit, given that the working stock of the positive control, GLPG1690, is at a 10-fold lower concentration. This will ensure the presence of an equal concentration of DMSO in all conditions to account for the effect of DMSO on fluorescence intensity.

    Alternatives: Any other known ATX inhibitor, such as PF8380, could serve as a positive control at a desired concentration.

    CRITICAL: Mix thoroughly by pipetting up and down the dilutions of the compounds in the assay buffer and examine carefully whether they have diluted completely. Solubility issues will be revealed during the analysis procedure. Troubleshooting 1.

  • d.
    Add 50 μL of each of the mixes in the respective wells.
• 4.
  Prepare the ATX mix and add it to the plate wells.

  • a.
    Add 12 μL of the 800 nM ATX stock solution to 1.5 mL of assay buffer to make the working solution of 6.4 nM of ATX.
  • b.
    Add 50 μL of ATX working solution to the wells by utilizing a multichannel pipette, or plain assay buffer to the “no ATX” wells.
  • c.
    Cover the plate with aluminum foil and place it for 15 min in a non-CO₂ incubator set at 37°C to allow the putative inhibitors to interact and attach to the enzyme.
• 5.
  Add the substrate-containing assay buffer to the plate wells.

  • a.
    Add 50 μL of the substrate-containing assay buffer to all the wells.
  • b.
    Cover the plate with aluminum foil and place it for 30 min in a non-CO₂ incubator set at 37°C, to allow the enzyme to initially interact with its substrate.

    Note: ATX exhibits a∼10 minute-long lag phase period²⁸ where LPC is bound to the active site but it is not yet catabolized. The 30-minute period allows the adequate production of LPA and choline, which will be used as a substrate in the next reaction.

• 6.
  Prepare and add the detection mix to the plate wells.

  • a.
    Add 40 μL of the Amplex Red, 4 μL of Choline Oxidase and 15 μL of HRP stock solutions to 2 mL of plain assay buffer and mix well by pipetting up and down until complete dilution in the assay buffer.
  • b.
    Add 50 μL of the detection mix to all the plate wells.
  • c.
    Cover the plate with aluminum foil, transfer it to the fluorometer, pre-warmed at 37°C, and initiate measurement protocol.

Note: Final concentrations in the detection mix are 200 μM Amplex Red, 0.4 U/ml Choline Oxidase and 3.975 U/ml HRP.

CRITICAL: The detection mix must be prepared just before its use.

• 7.Calculate the percentage of the remaining ATX activity for each of the compounds assessed (refer to the “quantification and statistical analysis” section).

Pause point: In case no compound leads to an abolished enzyme activity when incubated with ATX, the procedure ends here. In case of the detection of a primary hit, the procedure could be paused at this point for an indefinite amount of time.

IC₅₀ quantification of the candidate inhibitor

Timing: ∼3 h

In this step the IC₅₀ value of a potential primary hit is quantified. Since the actual number of hits may vary, here, the scenario of one hit is exemplified. The plate layout showing the final concentrations of the compounds assessed is the following.

	1–2	3–4	5–6
A	10 μM GLPG1690	100 μM Compound X	no ATX - no Compound (DMSO only)
B	1 μM GLPG1690	50 μM Compound X	no ATX - no Compound (DMSO only)
C	0.1 μM GLPG1690	25 μM Compound X
D	0.01 μM GLPG1690	10 μM Compound X
E	0.001 μM GLPG1690	1 μM Compound X
F	0.0001 μM GLPG1690	0.1 μM Compound X
G	0.00001 μM GLPG1690	0.01 μM Compound X
H	no Compound (DMSO only)	no Compound (DMSO only)

• 8.
  Prepare the substrate-containing assay buffer.

  • a.
    Add 2.5 μL of stock 16:0 LPC solution in 2.5 mL of assay buffer to make a 200 μM solution.
  • b.
    Place the tube in a non-CO₂ incubator at 37°C until subsequent addition to the plate wells.

Note: Same notes apply as in step 1.

• 9.
  Prepare the conditions to be tested and add them to the plate wells.

  Note: There are multiple strategies to prepare the serial dilutions described above. The approach adopted in this protocol, to directly dilute the compounds in the assay buffer and then add the respective volume to the wells, minimizes deviation among duplicates as it allows working with larger volumes, which is less error-prone, and allows the researcher to easier notice any occasional poor solubility of the compounds in the assay buffer while preparing the dilutions.

  • a.
    Add 80 μL of DMSO in 1920 μL of assay buffer and mix well. This will be the DMSO-containing assay buffer.

    Note: This will serve both as a condition to be tested and as a solution containing the compensatory volume of DMSO required while performing the serial dilutions of the compounds.

  • b.
    Make a 1 mM working dilution of GLPG1690 in DMSO and follow the serial dilution strategy described below. In the end of this preparation, a sufficient volume of each of the dilutions will be available to add to the wells.

    Intermediate GLPG1690 dilutions
    40 μM	10 μL of the 1 mM working solution	240 μL of plain assay buffer
    4 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer
    0.4 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer
    0.04 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer
    0.004 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer
    0.0004 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer
    0.00004 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer

  • c.
    Make a 10 mM working dilution of Compound X in DMSO and follow the serial dilution strategy described below. In the end of this preparation, a sufficient volume of each of the dilutions will be available to add to the wells.

    Intermediate compound X dilutions
    400 μM	10 μL of the 10 mM stock solution	240 μL of plain assay buffer
    200 μM	125 μL of the previous dilution	125 μL of DMSO-containing assay buffer
    100 μM	100 μL of the previous dilution	100 μL of DMSO-containing assay buffer
    40 μM	60 μL of the previous dilution	90 μL of DMSO-containing assay buffer
    4 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer
    0.4 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer
    0.04 μM	15 μL of the previous dilution	135 μL of DMSO-containing assay buffer

  • d.
    Add 50 μL of each of the mixes to the respective wells.

    Note: Same notes apply as in step 3.

• 10.
  Prepare the ATX mix and add it to the plate wells.

  • a.
    Add 16 μL of the ATX stock solution to 2 mL of assay buffer to make the working solution of 6.4 nM of ATX.
  • b.
    Add 50 μL of ATX working solution to the wells by utilizing a multichannel pipette. Instead, add plain assay buffer to the “no ATX” wells.
  • c.
    Cover the plate with aluminum foil and place it for 15 min in a non-CO₂ incubator set at 37°C to allow the putative inhibitors to interact and attach to the enzyme.
• 11.
  Add the substrate-containing assay buffer to the plate wells.

  • a.
    Add 50 μL of the substrate-containing assay buffer to all the wells with a multichannel pipette.
  • b.
    Cover the plate with aluminum foil and place it for 30 min in a non-CO₂ incubator set at 37°C to allow the enzyme to initially interact with its substrate.

Note: Same note applies as in step 5.

• 12.
  Prepare and add the detection mix to the plate wells.

  • a.
    Add 40 μL of the Amplex Red, 4 μL of Choline Oxidase and 15 μL of HRP stock solutions in 2 mL of assay buffer and mix well.
  • b.
    Add 50 μL of the detection buffer to all the plate wells with a multichannel pipette.
  • c.
    Cover the plate with aluminum foil and transfer it to the fluorometer, pre-warmed at 37°C, and initiate measurement protocol.

Note: Same note applies as in step 6.

• 13.Calculate the IC₅₀ for each of the compounds assessed (refer to the “quantification and statistical analysis” section). Troubleshooting 2.

Pause point: In case of not identifying an inhibitor with IC₅₀ in the nanomolar or in the lowest micromolar range, the procedure ends here; otherwise, in case of discovering a lead compound, it may go on or pause for indefinite time.

Screening for potential off-target inhibition of choline oxidase or HRP

Timing: ∼2 h

Provided that the present assay consists of three individual enzymic reactions, where the product of the first enzyme becomes the substrate of the second enzyme to yield a second product that is in turn used by the third enzyme until eventual fluorescence emission; the decreased fluorescence intensity observed upon completion of the previous steps might not be due to inhibition of the first enzyme, ATX, but a result of inhibition of either the second, Choline oxidase, or the third, HRP, enzyme. To validate the discovery of a lead compound that indeed inhibits ATX and to minimize the possibility of a false-positive, another major step is added to the protocol. In this step, fluorescence emission is measured in the absence of ATX’s substrate, LPC, but in the presence of its product, choline, and the subsequent enzymes, Choline oxidase and HRP. In this case, the first reaction (ATX) does not take place, whereas the second (choline oxidase) and third reaction (HRP) take place. Therefore, any detected decrease in fluorescence intensity will be the result of an off-target inhibition event. Additionally, fluorescence intensity decrease might be also due to potential structural properties of the primary hit that will result in an inner filter effect, where the emitted fluorescence is being quenched by the hit compound. Both of these conditions are assessed in this step of eliminating false positives making it, thus, essential in the process of identifying real ATX inhibitors.

The concentrations of the Compound X to be assessed include the initial high concentration tested (100 μΜ), and the concentration equal to its IC₅₀. The plate layout showing the final concentrations of the compound will be the following.

	1 - 2
A	No Compound X (choline chloride and DMSO)
B	No Compound X (choline chloride and DMSO)
C	100 μM Compound X
D	IC50 Compound X
E	No Compound X - no choline chloride (only DMSO)
F	No Compound X - no choline chloride (only DMSO)
G
H

• 14.
  Prepare the required Compound X dilutions and DMSO dilution by following a similar strategy to the one described in step 9 and add 50 μL of each condition to the respective wells.

  • a.
    Use the 10 mM working dilution of Compound X in DMSO to prepare the 400 μM intermediate dilution with plain assay buffer (which corresponds to the 100 μM final concentration),
  • b.
    Prepare DMSO-containing assay buffer by adding 40 μL of DMSO to 960 μL plain assay buffer and
  • c.
    Prepare the intermediate dilution that corresponds to the IC₅₀ final concentration using the DMSO-containing assay buffer.
  • d.
    Add 50 μL of the dilutions to the respective wells. Add 50 μL of DMSO-containing assay buffer to the remaining wells.

Optional: Other concentrations between the maximum and the IC₅₀ concentration could be also utilized.

• 15.
  Prepare the ATX mix and add it to the plate wells.

  • a.
    Add 6 μL of the ATX stock solution to 750 μL of assay buffer to make the working solution of 6.4 nM of ATX.
  • b.
    Add 50 μL of ATX working solution to all the wells by utilizing a multichannel pipette.
  • c.
    Cover the plate with aluminum foil and place it for 15 min in a non-CO₂ incubator set at 37°C to allow the interaction of the putative inhibitors with the enzyme.

Note: ATX is being included in this step in order to simulate the full reaction and to quantitatively measure the percentage of fluorescent emission decrease which is due to inhibition of either HRP or Choline oxidase as a result of the relative binding of the compound to all of the enzymes.

• 16.
  Prepare the choline chloride-containing assay buffer and add it to the plate wells.

  • a.
    Add 2 μL of stock choline chloride solution to 500 μL of assay buffer to make an 8 μM working solution.
  • b.
    Add 50 μL of the choline chloride-containing assay buffer or plain assay buffer to the respective wells and incubate for 30 min at 37°C.

Note: The final concentration of 2 μM of choline chloride is close to the choline produced normally in the assay during the 30-minute incubation period with LPC when ATX is not inhibited or activated.

• 17.
  Prepare and add the detection mix to the plate wells.

  • a.
    Add 20 μL of Amplex Red, 2 μL of Choline Oxidase and 7.5 μL of HRP stock solutions to 1 mL of assay buffer and mix well.
  • b.
    Add 50 μL of the detection mix to all the plate wells.
  • c.
    Transfer the plate covered with aluminum foil to the fluorometer pre-warmed at 37°C and initiate measurement protocol.
• 18.Calculate the percentage of off-target inhibition (refer to the “quantification and statistical analysis” section).

Note: It is of great importance to examine whether the dilution equal to the compound’s IC₅₀ results in off-target inhibition as this would mean that the discovered “hit” is not an actual ATX inhibitor.

Pause point: In case of significant off-target inhibition, the initially considered lead compound is validated as being a false-positive hit and the procedure stops here. In the absence of off-target inhibition, the compound could be further characterized and this point could serve as a pause point for an indefinite period.

Identification of the mode of action of the inhibitor

Timing: ∼3 h

Here, the lead inhibitor, Compound X, is assessed against various concentrations of the substrate to identify its mode of binding to ATX. The exact concentrations of the inhibitor depend on the properties of the hit, but it is highly recommended to test 5 concentrations, with the middle one being equal to the IC₅₀, plus the positive control not containing inhibitor, assessed against a wide range of substrate concentrations. The overall plate layout below shows the final concentrations of the compound being assessed against a range of concentrations of the substrate, LPC.

Compound X concentrations	0 nM	c1 (<c2)	c2 (<IC50)	c3 (=IC50)	c4 (>IC50)	c5 (>c4)
	1–2	3–4	5–6	7–8	9–10	11–12
A	no LPC
B	3.125 μM LPC
C	6.25 μM LPC
D	12.5 μM LPC
E	25 μM LPC
F	50 μM LPC
G	100 μM LPC
H	150 μM LPC

• 19.
  Prepare the substrate-containing assay buffers.

  • a.
    Add 12 μL of stock 16:0 LPC solution in 4 mL of assay buffer to make the starting working solution of 600 μM. Next, follow the serial dilution strategy shown below.

    Intermediate concentrations of LPC
    600 μM
    400 μM	2 mL of the previous dilution	1 mL of plain assay buffer
    200 μM	1.5 mL of the previous dilution	1.5 mL of plain assay buffer
    100 μM	1.5 mL of the previous dilution	1.5 mL of plain assay buffer
    50 μM	1.5 mL of the previous dilution	1.5 mL of plain assay buffer
    25 μM	1.5 mL of the previous dilution	1.5 mL of plain assay buffer
    12.5 μM	1.5 mL of the previous dilution	1.5 mL of plain assay buffer

    Note: Adding a compensatory volume of 2:1 chloroform:methanol will not be well diluted and will interfere with the assay.

  • b.
    Place the tubes with the substrate-containing assay buffers in a non-CO₂ incubator at 37°C until subsequent addition to the plate wells.

    Note: Same notes apply as in step 1.

• 20.Prepare the described Compound X dilutions and DMSO dilution by following a similar strategy to the one described in step 9 and add 50 μL of each condition to the respective wells.
• 21.
  Prepare the ATX mix and add it to the plate wells.

  • a.
    Add 40 μL of the ATX stock solution to 5 mL of assay buffer to make the working solution of 6.4 nM of ATX.
  • b.
    Add 50 μL of ATX working solution to the wells by utilizing a multichannel pipette.
  • c.
    Cover the plate with aluminum foil and place it for 15 min in a non-CO₂ incubator set at 37°C to allow the interaction between the inhibitor and the enzyme.
• 22.
  Add the substrate-containing assay buffers to the plate wells.

  • a.
    Add 50 μL of the substrate-containing assay buffers to the respective wells. Add plain buffer to the wells not containing LPC.
  • b.
    Cover the plate with aluminum foil and place it for 30 min in a non-CO₂ incubator set at 37°C, to allow the initial interaction of the enzyme with its substrate.

Note: Same note applies as in step 5.

• 23.
  Prepare and add the detection mix to the plate wells.

  • a.
    Add 110 μL of the Amplex Red, 11 μL of Choline Oxidase and 41 μL of HRP stock solutions in 5.5 mL of assay buffer and mix well.
  • b.
    Add 50 μL of the detection buffer to all the plate wells.
  • c.
    Transfer the plate covered with aluminum foil to the fluorometer pre-warmed at 37°C and initiate measurement protocol.

Note: Same note applies as in step 6.

• 24.Create a Lineweaver-Burk plot to identify the mode of action of the inhibitor and the parameters related to enzyme kinetics (refer to the “quantification and statistical analysis” section).

Expected outcomes

Enzyme modulators identification is a repetitive trial and error process that, by definition, produces many negative results along the way of discovery until a hit arises. So, in this section, results that exemplify an actual inhibitor discovery process that led to the identification of a potent hit will be presented. The fluorescence data acquired from the current protocol can be plotted against time to create a reaction kinetics graph for each compound and condition tested (Figure 2A). In the step “discovery of a potential candidate inhibitor”, the value of the remaining activity of ATX in each condition is measured, which can be plotted against the compounds (Figure 2B).From this plot, one can decipher the presence or absence of a hit, either inhibitor or activator. A potent inhibitor worth of further characterization would completely nullify ATX activity, whereas a potential activator would appreciably enhance it. The discovered inhibitor is further assessed to estimate its in vitro potency, as described in the step “IC₅₀ quantification of the candidate inhibitor”. Plotting the remaining activities of ATX against the logarithm of the concentrations of the compound assessed, results in a sigmoidal curve by which IC₅₀ can be inferred using a curve-fitting model (Figure 2C). Chances of a false-positive are eliminated by following the “screening for potential off-target inhibition” step. In this step one can plot the fluorescence over time and extrapolate the percentage of inhibition of the reaction due to compound off-target binding, which, for a true hit, must be equal to zero (Figure 2D). Lastly, in the “characterization of the mode of action” step, one plots 1/[V] values with respect to 1/[S] for each of the inhibitor concentrations in the same graph to create a Lineweaver-Burk plot (Figure 2E). This plot reveals the mode of inhibition of the compound and, by utilizing the enzyme kinetics equations, the V_max of the reaction, the K_m of the substrate in the presence of the inhibitor, the k_cat of the enzyme and the K_i of the inhibitor.

Figure 2.

Representative figures of the screening procedure

(A) Kinetics of the reactions in the “Discovery of a potential candidate inhibitor” step. The diagram shows fluorescence (in relative fluorescence units, RFU) in the presence, or absence, of each compound versus time.

(B) Diagram showing the remaining activity of ATX in the presence of each compound at a final concentration of 100 μM.

(C) Non-linear regression diagram with inhibition curves for the two inhibitors (GLPG1690 and Compound 4).

(D) Kinetics of the reactions in the step “Screening for potential off-target inhibition of Choline Oxidase or HRP”. The kinetics of the reactions in the presence of DMSO or Compound 4 (at a concentration equal to its IC₅₀) are shown.

(E) Lineweaver-Burk diagram for Compound 4.

Given that the assay is performed correctly, the inter-plate relative standard deviation (RSD) is about 3.1%. Due to its high signal to background ratio and the respective assessment of off-target inhibition, this assay exhibits low false-negative and false-positive outcomes. Its long-lasting linear phase, along with the increased precision that fluorescence-based assays show, allow the accurate quantification of ATX remaining activity. Additionally, since the substrate to Km ratio (S/Km) is equal to 1, there is no bias towards detecting more frequently competitive or uncompetitive inhibitors.²⁹ As this protocol is fast, scalable (thus, easy to miniaturize), robust, with a low cost per compound and with limited hazardous material, it can be also performed in a high-throughput format in laboratories with liquid handling devices and automated lab procedures.

Quantification and statistical analysis

The emission data should be initially plotted in GraphPad Prism or any other similar software. Plot emission values (in relative fluorescent units, RFUs) with respect to time to create a graph showing the kinetics of the reaction for each well. The “no Compound” wells should have the maximum fluorescence. Troubleshooting 2. The linear phase of the reaction (with stable reaction velocity -de/dt) should be analyzed as below.

For the first part, of discovering a potential candidate inhibitor, follow the procedure below.

• 1.Calculate the de/dt ratio (where e is the fluorescence emission) of the linear phase of the kinetic reaction for each of the wells. The de/dt ratio is the reaction velocity, V.
• 2.Calculate the mean of the de/dt ratio of the “no ATX” wells.
• 3.Subtract the calculated mean value of the “no ATX” wells from each of the rest well values. Exclude “no ATX” wells from this background subtraction step and the following steps.
• 4.Calculate the mean of the subtracted de/dt ratio of the “no Compound” wells.
• 5.Divide all well values by the calculated mean value of the “no Compound” wells. Exclude the individual “no Compound” wells.
• 6.Multiply the calculated values of each well with 100 to reveal the percentage of remaining ATX activity for each of the wells containing potential inhibitors.
• 7.Calculate the mean value of each of the duplicates. Putative inhibitors showing ATX remaining activity close to 0% will be the ones selected to proceed to the next step of characterization.
• 8.Plot the percentage of ATX remaining activity for each of the putative inhibitors in a standard dot plot in GraphPad Prism (Figure 2B).

For the second part, of quantifying IC₅₀ of the potential candidate inhibitor, follow the procedure below.

• 9.Calculate the de/dt ratio of the linear phase of the kinetic reaction for each of the wells.
• 10.Calculate the mean of the de/dt ratio for the “no ATX” wells.
• 11.Subtract the calculated mean value of the “no ATX” wells from each of the rest well values. Exclude “no ATX” wells from this background subtraction step and the following steps.
• 12.Calculate the mean of the subtracted de/dt ratio of the “no Compound” wells.
• 13.Divide all well values by the calculated mean value of “no Compound” wells. Exclude the individual “no Compound” wells.
• 14.Multiply the calculated values of each well with 100 to reveal the percentage of remaining ATX activity for each of the wells containing potential inhibitors.
• 15.Calculate the mean value of each of the duplicates.
• 16.Transfer to an “XY” datasheet in GraphPad Prism the mean percentage of the ATX remaining activity for each of the putative inhibitors with respect to their final concentrations in the wells.
• 17.Transform the inhibitor concentration values to their log₁₀ value. As there is no log value for zero concentration (corresponding to the “no Compound” wells), substitute “0” with “1∗10^-10“ prior to the transformation.
• 18.Create a non-linear regression diagram (variable slope logistic curve), with a top to bottom curve (Figure 2C). Troubleshooting 3.
• 19.Calculate IC₅₀ values for each of the compounds.

For the third part, of screening for potential off-target inhibition, follow the procedure below.

• 20.Calculate the de/dt ratio of the linear phase of the kinetic reaction for each of the wells.
• 21.Calculate the mean of the de/dt ratio of the “no choline chloride” wells.
• 22.Subtract the calculated mean value of “no choline chloride” wells from each of the rest well values. Exclude “no choline chloride” wells from this background subtraction step and the following steps.
• 23.Calculate the mean of the subtracted de/dt ratio of the “no Compound” wells.
• 24.Divide all well values by the calculated mean value of “no Compound” wells. Exclude the individual “no Compound” wells.
• 25.Multiply the calculated values of each well with 100 to transform the above value to a percentage value. This percentage is the remaining activity of the complete reaction.
• 26.Subtract the remaining activity percentage from 100 to reveal the percentage of inhibition due to compound off-target binding.
• 27.Calculate the mean value for each of the duplicates. If the percentage of inhibition due to compound off-target binding is equal to zero, then the discovered inhibitor is a real ATX inhibitor.

For the fourth part, of identifying the mode of action of the inhibitor, follow the procedure below.

• 28.Calculate the de/dt ratio of the linear phase of the kinetic reaction for each of the wells.
• 29.Calculate the mean of the de/dt ratio of the “no LPC” wells.
• 30.Subtract the calculated mean value of “no LPC” wells from each of the rest well values. This is the reaction velocity. Exclude “no LPC” wells from this background subtraction step and the following steps.
• 31.Calculate the 1/[LPC concentration] (1/[S] or 1/μM), utilized in the experiment, and 1[/reaction velocity] (1/[V] or min/RFU) values for each well.
• 32.Plot 1/[V] values with respect to 1/[S] for each of the inhibitor concentrations in the same graph to create a Lineweaver-Burk plot in GraphPad Prism (Figure 2E).
• 33.Examine the curves to distinguish the inhibitor type based on Grant et al.³⁰
• 34.Use the following enzyme kinetics equations to calculate values of the enzyme kinetics parameters regarding V_max, K_m, K_i and k_cat.

Note: IC₅₀ of the inhibitor could be recalculated so as to verify its pre-quantified potential.

Enzyme kinetics equations

$$\mathrm{V}={\mathrm{V}}_{\max }\left[\mathrm{S}\right]/({\mathrm{K}}_{\mathrm{m}}+\left[\mathrm{S}\right])$$

$${\mathrm{V}}_{\max }={\mathrm{k}}_{\text{cat}}\left[\mathrm{E}\right]$$

Where:

[S] is the concentration of the substrate.

[E] is the concentration of the enzyme.

V is the velocity of a reaction under given conditions.

V_max is the maximum velocity of a reaction under given conditions.

K_m is the substrate concentration (in the absence of inhibitor) at which the velocity of the reaction is half-maximal; also called Michaelis-Menten constant.

k_cat is the turnover number or catalytic constant (the number of substrate molecules that can be converted to product in a given period of time under conditions where the enzyme is completely saturated with substrate).

K_i is the inhibitor dissociation constant (both IC₅₀ and K_i have to do with the concentration needed to reduce the activity of the enzyme by half).

In the presence of an inhibitor the value of K_m appears to change, thus it is called K_m apparent (K_m,app). The same applies to Vmax, which is then called V_max apparent (V_max,app). K_i can be inferred from the following equations depending on the type of inhibition.

Inhibition type	Km,app	Vmax,app	IC50
None	Km	Vmax	N/A
Competitive	Km(1+[I]/Ki)	Vmax	(1+[S]/Km)Ki
Noncompetitive	Km	Vmax/(1+[I]/Ki)	Ki
Uncompetitive	Km/(1+[I]/Kiu)	Vmax/(1+[I]/Kiu)	(1+Km/[S])Ki
Mixed	Different from Km	Different from Vmax	([S]+Km)/(Km/Ki1+[S]/Ki2)

Where:

[I] is the concentration of the inhibitor.

IC₅₀ is the concentration of the inhibitor required to produce 50% inhibition.

Limitations

The current assay is only suitable for measuring ATX activity in vitro, in a sample of known ingredients. ATX activity cannot be assessed in biological samples through this method, due to the presence of endogenous choline and albumin that interferes with fluorescence assays (among other components). Furthermore, different batches of the recombinant ATX show different activity levels and must be examined each time a new batch is being utilized. These differential activity levels do not affect final IC₅₀ quantification or mode of binding.

Troubleshooting

Problem 1

Poor solubility of the compounds assessed (related to Steps 2 and 3) that results in negative de/dt values.

Potential solution

Use a heating sonicator for a couple of minutes or as long as needed until complete dilution of the compound.

Problem 2

Low RFUs or small difference in the slopes between the kinetic of “no compound” wells and the kinetic of the “no ATX” wells (related to the “quantification and statistical analysis”).

Potential solution

Change batch of ATX or prepare fresh assay buffer or prepare fresh enzyme stocks for the detection mix.

Problem 3

No fit of the remaining ATX activity, in the presence of a compound, to a logistic curve (related to the “quantification and statistical analysis”; IC₅₀ quantification; Step 18).

Potential solution

A narrower range of concentrations could be tested in order to minimize the error among dilutions.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Christiana Magkrioti (magkrioti@fleming.gr).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Christiana Magkrioti (magkrioti@fleming.gr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate or analyze datasets or code.

Acknowledgments

This protocol paper was supported by the Hellenic Foundation for Research and Innovation (HFRI) under the “2nd Call for HFRI Research Projects to support Faculty Members & Researchers” (project number 3565 to V.A.) and under the “2nd Call for HFRI Research Projects to support Post-Doctoral Researchers” (project number 01144 to C.M.).

Author contributions

Conceptualization, V.A. and E.K.; investigation, E.-A.S., E.K., A.N.M., and C.M.; writing – original draft, E.-A.S.; writing – review and editing, E.-A.S., C.M., A.N.M., and V.A.; funding acquisition, C.M. and V.A.; supervision, C.M., A.N.M., and V.A.

Declaration of interests

The authors declare no competing interests.