Method for determining the interaction between a ligand and a receptor

Abstract

The disclosure concerns a method for determining the interaction between a test compound and a receptor. The receptor may be immobilized. The disclosure also concerns a sample holder assembly including a functionalized test well wall, which may be used in combination with a Total Internal Reflection Fluorescence source.

Claims

1. A method for determining the interaction between a first ligand and a receptor, said method comprising a sequence of process steps: a) providing a first solution free from the first ligand and comprising a concentration Ci of the receptor, b) adding said first solution to a test well having a test well wall, thereby contacting said first solution with said test well wall being functionalized with a second ligand while recording the number of binding events between the receptor and the second ligand during a time interval t1, and c) adding a test solution free from said receptor and comprising a concentration Cn of the first ligand to said test well having said test well wall containing the first solution after step b) thereby providing a second solution while continuing recording the number of binding events between the second ligand and the receptor in said second solution during a time interval t2, wherein an interaction between said first ligand and said receptor alters said number of binding events between said second ligand and said receptor, wherein the interaction between the first ligand and the receptor is determined based on data from the continuous recording of the number of binding events between the second ligand and the receptor, wherein the data from the continuous recording is obtained prior to an equilibrium binding being reached between the first ligand and the receptor.

2. The method according to claim 1, wherein the time interval t.sub.2 is at least 1/k.sub.obs of the binding reaction between the first ligand and the receptor.

3. The method according to claim 1, wherein the method further comprises the step of stopping the recording of the binding events between the second ligand and the receptor takes place after the first ligand and the receptor have reached equilibrium binding.

4. The method according to claim 1, wherein the method further comprises the step of stopping the recording of the binding events between the second ligand and the receptor takes place after the first ligand and the receptor have reached equilibrium binding.

5. The method according to claim 1, further comprising a step d): d) determining the interaction between the first ligand and the receptor based on the binding events recorded in steps b) and c).

6. The method according to claim 1, wherein the sequence of process steps is performed in full in each of a plurality of test wells.

7. The method according to claim 1, wherein the sequence of process steps is performed for a number of different concentrations Cn of the first ligand in said test solution.

8. The method according to claim 1 further comprising a step e): e) repeating step c) at an increasing concentration C.sub.n of the first ligand in said test solution.

9. The method according to claim 1, wherein the receptor is combined with a vehicle, thereby providing an immobilized receptor.

10. The method according to claim 9, wherein the vehicle comprises a fluorophore.

11. The method according to claim 1, wherein the first ligand and the second ligand are identical.

12. The method according to claim 1, wherein the first ligand and the second ligand are different.

13. The method according to claim 1, wherein: the receptor is a pharmaceutical drug receptor, and/or the first ligand and/or the second ligand is/are a pharmaceutical drug.

14. The method according to claim 1, wherein step b) and/or step c) comprise(s) use of a microscope.

15. The method according to claim 1, which comprises determination for the first ligand at least one of the following: an observed rate constant k.sub.obs, an association constant k.sub.on, a dissociation constant k.sub.off, an equilibrium dissociation constant K.sub.d, a fractional occupancy.

16. The method according to claim 6, wherein the plurality of test wells form part of a sample holder assembly.

17. The method according to claim 16, wherein the sample holder assembly comprises or consists of a microtiter plate.

18. The method according to claim 16, wherein the sample holder assembly is configured to be used in combination with Total Internal Reflection Fluorescence (TIRF) microscopy, and comprises: a sample holder plate comprising a plurality of bottomless test wells, a bottom plate attached to said sample holder plate by means of a material thereby forming a well bottom wall of said plurality of test wells, said material having a refractive index (N.sub.a) that is lower than a refractive index (N.sub.g) of said bottom plate.

19. The method according to claim 18, wherein said material is UV curable and/or resistant to buffer solutions.

20. The method according to claim 18, wherein said material is an adhesive.

21. The method according to claim 16, wherein the sample holder assembly is combined with a TIRF source configured to provide a light beam into the well bottom wall such that the light beam propagates throughout the entire well bottom wall thereby creating an evanescent field in the plurality of wells.

22. The method according to claim 9, wherein the vehicle comprises a liposome, a dendrimer, a dendrone, a complexed lanthanide, a quantum dot, a nanodiamond, or a lipid disc.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing the number of binding events as a function of time for liposome immobilized thrombin before and after the addition of melagatran.

(2) FIG. 2 is a graph showing a dose response curve for melagatran added to liposome immobilized thrombin.

(3) FIG. 3 is a graph showing the observed binding rate k.sub.obs as a function of the concentration of an added melagatran solution, and linear regression providing k.sub.on between melagatran and thrombin.

(4) FIG. 4 is a graph showing the number of binding events in a single test well as a function of time for liposome immobilized thrombin before and after the addition of melagatran.

(5) FIG. 5 is a graph showing the number of binding events in a two test wells as a function of time for liposome immobilized thrombin before and after the addition of melagatran.

(6) FIG. 6 is a graph showing the number of binding events in a one hundred test wells as a function of time for liposome immobilized thrombin before and after the addition of melagatran.

(7) FIG. 7 is a cross section view of a microtiter plate comprising a plurality of test wells comprising a functionalized bottom plate attached to said microtiter plate by means of an adhesive.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(8) FIG. 7 shows a cross section of a sample holder assembly 100 comprising a sample holder plate 1 comprising a plurality of bottomless test wells 2, a bottom plate 3 attached to the sample holder plate 1 by means of an adhesive 4 thereby forming a well bottom wall 5 of said plurality of wells 2. The adhesive has a refractive index N.sub.a that is lower than the refractive index N.sub.g of said bottom plate 3.

(9) As explained herein, the adhesive 4 may be elected to allow for a TIRF light beam to propagate throughout the entire bottom plate without leaking into adjacent media such as the bottom plate. Thereby an evanescent wave is created closely to the bottom plate which may be used in the detection of binding events as described herein.

(10) The well bottom wall 5 may be functionalized with a tool compound as described herein.

(11) Further Items

(12) The present disclosure provides the following items.

(13) Item 1:

(14) A method for determining the interaction between a first ligand and a receptor, said method comprising a sequence of process steps:

(15) a) providing a first solution free from said first ligand and comprising a concentration C.sub.i of the receptor,

(16) b) contacting said first solution with a test well wall functionalized with a second ligand while recording the number of binding events between the receptor and the second ligand during a time interval t.sub.1,

(17) c) adding a test solution free from said receptor and comprising a concentration C.sub.n of the first ligand to said first solution thereby providing a second solution while recording the number of binding events between the second ligand and the receptor of said second solution during a time interval t.sub.2.
Item 2:

(18) A method according to item 1, wherein the sequence of process steps is performed in full in each of a plurality of test wells.

(19) Item 3:

(20) A method according to item 1 or 2, wherein the sequence of process steps is performed for a number of different concentrations C.sub.n of the first ligand in said test solution.

(21) Item 4:

(22) A method according to item 1 or 2 further comprising a step d):

(23) d) repeating step c) at an increasing concentration C.sub.n of the first ligand in said test solution.

(24) Item 5:

(25) A method according to any one of the preceding items, wherein

(26) the receptor is combined with a vehicle such as a liposome, a dendrimer, a dendrone, a complexed lanthanide, a quantum dot, a nanodiamond or a lipid disc,

(27) thereby providing an immobilized receptor.

(28) Item 6:

(29) A method according to any one of the preceding items, wherein the vehicle comprises a fluorophore.

(30) Item 7:

(31) A method according to any one of the preceding items, wherein the first ligand and the second ligand are the same or different.

(32) Item 8:

(33) A method according to any one of the preceding items, wherein:

(34) the receptor is a pharmaceutical drug receptor, and/or

(35) the first ligand and/or the second ligand is/are a pharmaceutical drug.

(36) Item 9:

(37) A method according to any one of the preceding items, wherein step b) and/or step c) comprise(s) use of a microscope.

(38) Item 10:

(39) A method according to any one of the preceding items, which comprises determination for the first ligand at least one of the following: an observed rate constant k.sub.obs, an association constant k.sub.on, a dissociation constant k.sub.off, an equilibrium dissociation constant K.sub.d, a fractional occupancy

(40) Item 11:

(41) A method according to any one of items 2-10, wherein the plurality of test wells form part of a sample holder assembly such as a microtiter plate.

(42) Item 12:

(43) A method according to item 11, wherein the sample holder assembly is configured to be used in combination with Total Internal Reflection Fluorescence (TIRF) microscopy, and comprises: a sample holder plate (1) comprising a plurality of bottomless test wells (2), a bottom plate (3) attached to said sample holder plate (1) by means of an adhesive (4) thereby forming a well bottom wall (5) of said plurality of wells (2),
said adhesive (4) having a refractive index (N.sub.a) that is lower than a refractive index (N.sub.g) of said bottom plate (3).
Item 13:

(44) A method according to any one of items 10-12, wherein said adhesive (4) is UV curable and/or resistant to buffer solutions.

(45) Item 14:

(46) A sample holder assembly (10) configured to be used in combination with TIRF microscopy, comprising: a sample holder plate (1) comprising a plurality of bottomless test wells (2), a bottom plate (3) attached to said sample holder plate (1) by means of an adhesive (4) thereby forming a well bottom wall (5) of said plurality of wells (2),
said adhesive (4) having a refractive index (N.sub.a) that is lower than a refractive index (N.sub.g) of said bottom glass plate (3).
Item 15:

(47) A method according to item 11 or a sample holder assembly (10) according to claim 14, wherein the sample holder assembly (10) is combined with a TIRF source configured to provide a light beam into the well bottom wall (5) such that the light beam propagates throughout the entire well bottom wall (5) thereby creating an evanescent field in the plurality of wells (2).

EXAMPLES

(48) Abbreviations

(49) CHES N-Cyclohexyl-2-aminoethanesulfonic acid

(50) HBS Hepes buffered solution

(51) HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

(52) LED Light Emitting Diode

(53) NHS N-Hydroxysuccinimid

(54) μl microliter

(55) L liter

(56) LOD lower limit of detection

(57) nm nanometer

(58) nM nanoMolar

(59) mg milligram(s)

(60) ml milliliter

(61) mM millimolar

(62) PBS Phosphate buffered solutions

(63) PEG4 Polyethylene glycol, i.e. H—(O—CH.sub.2—CH.sub.2).sub.4—OH.

(64) PC Phosphatidylcholine

(65) PEG polyethylene glycol

(66) PLL-g-PEG poly-L-lysine grafted PEG

(67) RT room temperature

(68) sec second(s)

(69) UV ultraviolet

(70) V/V volume percent

(71) Materials and Methods

(72) All lipids were bought from Avanti Polar Lipids.

(73) Pll-g-PEG (11354-X=200-2000-3.5%) and PLL-g-PEG-biotin (11835-X=200-3400-3.5%) were bought from Nanosoft Polymers.

(74) The thrombin binding peptide was synthezised upon customer specification by ThermoFisher Scientific. Melagatran was purchased from SantaCruz Biotechnology.

(75) Dymax 3025 is a product of Dymax Corporation.

(76) All other chemicals if not stated differently were bought from Sigma. All chemicals were suitable for molecular biology purposes.

(77) Preparation of Liposomes:

(78) To yield liposomes with a diameter of ˜100 nm first the required lipids were solved in chloroform and mixed. In total 5 mg 2-Oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine, 0.01 mg, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl and 0.005 mg 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) were mixed together. The lipid mixture was vacuum dried overnight. The dried lipids were hydrated in 1 ml HBS (150 mM NaCl, 20 mM/L HEPES at pH7.2) under gentle agitation. The lipid suspension was extruded through a PC-membrane with 100 nm pore size eleven times. Concentration of the liposomes was determined by light absorption at 544 nm and the concentration of the liposome solution was adjusted to 2.5 mg/ml. This equals a liposome concentration of approximately 30 nM.

(79) Preparation of Protein:

(80) To immobilize thrombin at the liposomes via click chemistry an azide-group was introduced to thrombin via NHS coupling. Therefore, 100 μL of human-thrombin at 2 mg/ml was mixed with 200 μL high salt PBS-buffer (10 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4, 400 mM NaCl+33.5% Glycerol (v/v) at pH 7.4 and 13 μL NHS-PEG4-Azide at 10 mM. The mixture was incubated for 30 minutes at RT. The reaction was stopped by the addition of 500 μL high salt PBS-buffer (10 mM Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4, 400 mM NaCl+33.5% Glycerol (v/v) at pH 6.6.

(81) Immobilization of Protein:

(82) 1.5 μL of the thrombin-azide were mixed with 48.5 μL icecold CHES-buffer (20 mM CHES, 150 mM NaCl) at pH 8.5 and subsequently 50 μL of the liposome solution is added. This mix was stored for at least 30 minutes on ice. 10 μL of the reaction mix was diluted with 990 μL icecold CHES-buffer (20 mM CHES, 150 mM NaCl) at pH 8.5.

(83) Preparation of Surfaces:

(84) A glass plate with 0.17 mm thickness was incubated in base piranha solution at 373 Kelvin for 30 minutes. The cleaned glass plate was rinsed with water and dried. An UV curable adhesive (Dymax 3025) was supplemented with ˜1% (v/v) (3-Aminopropyl)-triethoxysilane. The adhesive mixture was thinly spread at the lower side of the bottomless microplate. The glass plate was positioned on top of the bottomless microplate so it formed the bottom. After the adhesive was spread fully, it was cured according to manufacturer instructions. After curing, into each well of the microplate 10 μL of a solution containing 1 mg/ml PLL-g-PEG and 1 mg/ml PLL-g-PEG-biotin are added. The plate was incubated for at least 1 h under gentle orbital agitation. After the incubation each well was washed 10-times with HBS-buffer. The dilution ratio of every washing step was at least 1:10. After washing, into each well 10 μL of a solution containing 100 μg/mL neutravidin was added. This was incubated for at least 4 h under gentle orbital agitation. All wells were washed as previously described and 10 μL of a solution containing 10 ug/ml a thrombin binding peptide linked to biotin (GVGPRSFKLPGLA-Aib-SGFK-PEG.sub.4-biotin) was added to all wells. The microplate was incubated at least for 1 h under gentle orbital agitation. The peptide was the tool compound to bind the thrombin immobilized at the vesicles.

(85) Finally the microplate was washed as previously described and the residual buffer volume in each well is 30 μL.

(86) The Microscope Setup:

(87) The single molecule microscope was based on a Nikon Ti-E base. As light source for epifluorescence a LED-white light source is used. The Objective was a 60×APO TIRF objective. Images are recorded via a HAMAMATSU Orca-FLASH 4.0V2 sCMOS camera. The sample stage was motorized and quipped with a microwell holder.

(88) On top of the microscope liquid handling robotics were installed (Andrew, Andrew Alliance).

(89) Melagatran Dilution Series:

(90) An 8 times 1:3 dilution series of melagatran starting at 1.6 uM/L. The buffer for the dilution series was CHES-buffer (20 mM CHES, 150 mM NaCl) at pH 9.5.

Example 1

(91) The microwell plate comprising 384 wells was placed in the microwell plate holder at the microscope. Subsequently the measurement was started and conducted fully automatically.

(92) The single molecule measurement included the following steps in each well: 1. The appropriate microwell prepared as indicated above was placed over the objective and the objective was adjusted so its focal plane is placed at the inner surface of microwell. 2. The well was washed with 70 μL CHES-buffer (20 mM/L CHES, 150 mM/L NaCl) at pH 9.5. 3. 5 μL of a solution containing CHES buffer and the liposome onto which thrombin was attached was added to the well and the well content mixed. 4. The acquisition of time lapse movie with 901 images and an acquisition rate of 10 sec.sup.−1 was started 5. After 20 seconds of acquisition 5 μL solution containing the appropriate concentration of melagatran and CHES buffer was added and the well content mixed rapidly (<0.5 sec). 6. The acquisition of image data was continued till 901 frames were recorded.

(93) Steps 1-6 were repeated three times for each intended concentration of melagatran. Eight concentrations of melagatran were tested, namely 200 nM, 66.7 nM, 22.2 nM, 7.4 nM, 2.5 nM, 0.8 nM 0.3 nM and 0.1 nM. For each tested concentration of melagatran steps 1-6 above were performed in full in each well. Each method step was carried out at the same time in each well, i.e. step 1 was carried out at the same time in each well and then each consecutive step was carried out at the same time in each well.

(94) The recorded image data was analysed with the aim to determine the number of new bound liposome in each well. In a first step all objects that are similar in shape to a reference object in each well were detected.

(95) In a second step it was determined which objects were bound to the surface. As indication that an object was bound to the surface its mobility was analysed. If the mobility was below a threshold value (the object has not moved more than a pixel (here 215-304 nm between two consecutive frames) the object was considered as immobile and therefore bound to the surface. The number of bound liposomes, i.e. the number of binding events, was recorded. The number of binding events for all wells were summed up before and after the addition of melagatran, respectively, to provide a cumulative number of binding events. This was done for each concentration of melagatran.

(96) The cumulative number of bound liposomes before and after the addition of melagatran was plotted versus time as shown in FIG. 1. In Figure A the concentration of melagatran was 200 nM and the concentration of thrombin was 15 pM. In FIG. 1 t.sub.inh is the time when the solution of melagatran was added to the solution containing the liposome immobilized thrombin, and cumsum on-events [#] is the number of recorded binding events.

(97) Before the test compound melagatran was added the binding rate of liposomes to the surface was observed to be constant over time. Plotting the cumulative number of binding events versus time turned out to be a linear function where the slope equals the binding rate. During the injection and mixing of the test compound the binding rate to the surface was increased. After a short equilibration time the binding rate was normalized again. The data acquired during this mixing period was not used for analysis. The cumulative number of binding events was analysed as described herein and the observed binding rate constant k.sub.obs was extracted. Once the binding of melagatran to thrombin had reached its equilibrium the cumulative number of binding events versus time was increasing linearly again as shown in FIG. 1.

(98) The ratio of the initial slope and the final slope was calculated. This was repeated three times for each concentration of melagatran. This ratio equals the fractional occupancy of thrombin by melagatran at the respective concentration. The equilibrium dissociation constant K.sub.d was then calculated from the equation below, wherein [melagatran].sub.0 is the concentration of the added melagatran.

(99) $\mathrm{Fractional}\mathrm{occupancy}=\frac{{\left[\mathrm{melagatran}\right]}_0}{{\left[\mathrm{melagatran}\right]}_0+K_d}$

(100) The equilibrium dissociation constant K.sub.d was provided from a dose response curve wherein the fractional occupancy was plotted versus the added concentration of melagatran as shown in FIG. 2, which gave a K.sub.d value of about 3 nM.

(101) FIG. 3 shows the observed binding rate constant k.sub.obs plotted as a function of the concentration of the added test compound melagatran. Linear regression of the observed rate k.sub.obs versus the concentration of melagatran allowed for calculation of the association rate k.sub.on between melagatran and thrombin, which was found to be 21 μM.sup.−1s.sup.−1.

(102) Since the equilibrium dissociation constant K.sub.d equals the dissociation rate k.sub.off divided by the association rate k.sub.on, it was possible to calculate k.sub.off by multiplying the k.sub.on value of 21 μM.sup.−1s.sup.−1 with the K.sub.d value of 3 nM thereby providing a k.sub.off value of about 0.06 s.sup.−1.

(103) It was concluded that the method described herein allows for determining k.sub.obs, k.sub.on, k.sub.off, and K.sub.d for a test compound and also the fractional occupancy of a receptor by the test compound. Thus, the method described herein allows for determining the interaction kinetics between a test compound and a receptor.

Example 2

(104) In this example, the method steps described in Example 1 were performed in full in a single well, in each of two wells and in each of 100 wells. The thrombin concentration was 1 pM. The melagatran concentration was 7.4 nM.

(105) First, an experiment was performed in a single well. The number of binding events was plotted as a function of time as shown in FIG. 4. Due to the low number of binding events the observed rate constant k.sub.obs could not be reliably fitted. Therefore, the number of binding events were collected and summed up before and after the addition of melagatran, respectively, for two wells. The result is shown in FIG. 5, and it was found that k.sub.obs could be fitted to provide a k.sub.obs value of 0.43 sec.sup.−1. A further experiment was performed in analogy with the two well experiment but instead of two wells one hundred wells were used. The result is shown in FIG. 6, and it was found that k.sub.obs could be fitted to provide a k.sub.obs value of 0.22 sec.sup.−1. In FIGS. 4,5 and 6 t.sub.inh is the time when the solution of melagatran was added to the solution containing the liposome immobilized thrombin, and cumsum on-events [#] is the number of recorded binding events.

(106) It was concluded that the sensitivity of the method described herein is enhanced by performing the method steps in a plurality of wells and summing up the recorded number of binding events of the wells before and after the addition of the test compound.

(107) It was also concluded that the method described herein allows for reliable measurement of the observed rate constant k.sub.obs for a low receptor concentration such as a receptor concentration that is lower than the lowest concentration that corresponds to the LOD of ensemble averaging methods defining (i) how much material of the receptor that is needed to operate the assay and (ii) the tight binding regime with respect to high affinity test compounds.

REFERENCES

(108) 1. Geschwindner, S., Dekker, N., Horsefield, R., Tigerström, A., Johansson, P., Scott, C. W., and Albert, J. S. (2013). Development of a Plate-Based Optical Biosensor Fragment Screening Methodology to Identify Phosphodiesterase 10A Inhibitors. J. Med. Chem. 56, 3228-3234. 2. Motulsky, H. J., and Mahan, L. C. (1984). The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25, 1-9. 3. Schiele, F., Ayaz, P., and Fernández-Montalván, A. (2015). A universal homogeneous assay for high-throughput determination of binding kinetics. Anal. Biochem. 468, 42-49. 4. Gunnarsson, A., Snijder, A., Hicks, J., Gunnarsson, J., Höök, F., and Geschwindner, S. (2015). Drug discovery at the single molecule level: inhibition-in-solution assay of membrane-reconstituted β-secretase using single-molecule imaging. Anal. Chem. 87, 4100-4103. 5. Ola Wahlsten, Anders Gunnarsson, Lisa Simonsson Nyström, Hudson Pace, Stefan Geschwindner, and Fredrik Höök. Equilibrium-Fluctuation Analysis for Interaction Studies between Natural Ligands and Single G Protein-Coupled Receptors in Native Lipid Vesicles. Langmuir, 2015, 31 (39), pp 10774-10780. 6. Gunnarsson, A., Dexlin, L., Wallin, P.; Svedhem, S., Jönsson, P., Wingren, C. and Höök, F. (2011). J. Am. Chem. Soc. 133, 14852-14855.