参见作者原研究论文

本实验方案简略版
Jul 2018

本文章节


 

NMR waterLOGSY as An Assay in Drug Development Programmes for Detecting Protein-Ligand Interactions–NMR waterLOGSY
NMR waterLOGSY应用于药物开发项目中蛋白质-配体相互作用的检测   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.

Keywords: Drugs (药物), WaterLOGSY (WaterLOGSY), Drug discovery (药物研发), NMR (核磁共振), Ligand binding (配基结合), Protein-protein interaction (蛋白质蛋白质相互作用)

Background

As more altered protein forms are found in disease cells, there has been an increase in drug discovery programmes that rely on primary screening of target proteins with small molecule libraries followed by further medicinal chemistry campaigns to increase the potency of the chemicals emanating from the screens. After primary screens, there is a raft of orthogonal assays that contribute to verification of hit chemical matter and allow stratification of compounds for selecting the best ones to take into hit-to-lead stages and onwards to lead optimization. Among these assays is the NMR-based water Ligand Observed via Gradient SpectroscopY (waterLOGSY) method (Dalvit et al., 2000 and 2001). waterLOGSY is especially useful for detecting the binding of ligands that interact relatively weakly with the target proteins (i.e., dissociation constants in the µM to low mM range), such as would be expected to be associated with initial hits from a large chemical or fragment library screen (Lepre, 2011) prior to medicinal chemistry to improve potency and drug-like properties.

How WaterLOGSY works
The waterLOGSY method makes use of 1H NMR observation of small molecules (ligands) for the detection of ligand-macromolecule binding (Huang and Leung, 2019). It relies on the transfer of proton (1H) magnetization from excited water molecules to ligands either via 1) their direct interaction or 2) indirectly through initial transfer to protons at a protein surface and then relayed onto a protein-bound ligand. The direct interaction of the ubiquitous water molecules with free (unbound) ligands (route 1) leads to an increase in the observed ligand signal intensity due to a direct (through-space) magnetic interaction between water and ligand protons known as a positive nuclear Overhauser effect (nOe). In contrast, magnetization transfer from water to protein then relayed onto a receptor bound ligand (route 2) yields a net decrease in the ligand signal intensity (here due to a negative nOe). This difference in transfer behavior originates in the different tumbling rates of molecules in solution (i.e., their rotational correlation rates) which are “fast” for small, unbound ligands but “slow” for macromolecular receptors and for their bound ligands. Thus, a comparison of ligand signal intensities in the absence and presence of a protein receptor may indicate whether ligand binding has occurred, with a difference in signal intensity being suggestive of binding. Note that a third magnetization transfer mechanism may also occur for exchangeable (acidic) ligand protons due to their dynamic interchange with water protons, which yields the same signal response as the negative nOe regardless of the presence of protein. Thus, responses from exchangeable protons should be ignored for ligand screening purposes.

The waterLOGSY experiment detects only the signals of the ligand(s) when free in solution so relies on the dissociation of the ligand-bound complex and the release of ligand which then carries the negative nOe with it for detection. This process requires that the ligand dissociation rate is sufficiently high for transfer of the ligands into solution for nOe detection prior to it being lost through natural relaxation processes that are always operative. It similarly requires that ligand residence times on the receptor are sufficiently long for the magnetization transfer itself to take place prior to ligand release. As such, waterLOGSY is best suited to the detection of moderate to weak affinity binders, with dissociation constants in the µM to low mM range. The method has proven especially popular in the screening of libraries of small molecule fragments since these typically have weak binding affinities. Strong binding ligands (KD < μM) have residence times on the protein that are too long and their binding is less likely to be detected, leading to the possibility of false negatives in such cases.

Applications of the waterLOGSY method
In this article we present waterLOGSY protocols for evaluation of protein-ligand binding and show how protein-protein interactions can be employed to inhibit protein-ligand binding, thereby confirming the ligand location on the target protein.

WaterLOGSY is a versatile method that allows to asses qualitatively the binding of small ligands to proteins. This can be done with only one ligand present (see basic protocol) but it can also be carried out with multiple ligands present (see screening protocol), therefore allowing it to be used as a medium throughput assay. In this case, it is important to use DMSO solutions of ligand instead of the DMSO-d6 solution used in the basic protocol. Too much DMSO-d6 may prevent the NMR instrument from locking onto the D2O. DMSO could also lead to interference with the protein structure itself. Generally, a maximum of 10% v/v of DMSO-d6 or DMSO should be used.

The first step in any waterLOGSY experiment is to determine that the ligand does not aggregate (see aggregation protocol). Aggregation of small molecules causes a false positive response in the waterLOGSY experiment because the aggregate adopts the tumbling behavior of a macromolecular species and thus gives waterLOGSY responses as if the ligand were bound. This means that every ligand should be tested without protein to eliminate the presence of confounding aggregation. Conversely, this behavior leads to waterLOGSY being a very useful method to assess aggregation of small molecules.

Materials and Reagents

  1. Norell® Select SeriesTM 3 mm NMR tubes (Sigma-Aldrich, catalog number: NORS36008 )
  2. Eppendorf Tube® volume (0.5 ml), PCR clean (Sigma-Aldrich, catalog number: EP0030124537-500EA )
  3. Soda lime Pasteur pipettes unplugged, capacity 2 ml, 230 mm length (VWR, catalog number: 612-1702 )
  4. Pipette teats, MBL® natural rubber (SciLabware, catalog number: BIBBBPP004 )
  5. Pipette tips:
    10 µl graduated tips (Starlab, catalog number: S1111-3800 )
    200 µl yellow tips (Starlab, catalog number: S1111-0806)
    1,000 µl blue tips (Starlab, catalog number: S1111-6801)
  6. Phosphate buffered saline tablet (Sigma-Aldrich, catalog number: P4417 )
  7. Distilled water (in-house)
  8. D2O (deuterium oxide 99.9 atom % D) (Sigma-Aldrich, catalog number: 151882 )
  9. DMSO-d6 (dimethyl sulfoxide-d6 "100%", 99.96 atom % D) (Sigma-Aldrich, catalog number: 156914 )

Equipment

  1. Hand centrifuge without rotor 2 x 15 ml Swing Out Conical Tube Rotor 90° for Hand Centrifuge (Satorius, model: C1011 )
  2. Scientific Industries SITM Vortex-GenieTM 2 (Scientific Industries, model: SI-0266 , catalog number: 15557335 )
  3. -80 °C freezer
  4. pH meter (Jenway, catalog number: 351001 )
  5. Gilson pipettes, single channel:
    Pipetman L, 2-20 µl (Gilson, catalog number: FA10003M )
    Pipetman L, 20-200 µl (Gilson, catalog number: FA10005M )
    Pipetman L, 100-1,000 µl (Gilson, catalog number: FA10006M )

Procedure

Note: It is assumed local knowledge of NMR spectrometer operation exists for 1H NMR spectroscopy or can be sourced appropriately. Commands listed here relate to the operation of Bruker NMR spectrometers–follow equivalent protocols for other vendors.

  1. Aggregation protocol
    1. Dissolve the ligand (compound in DMSO-d6 to obtain a 10 mM stock solution).
      Weigh out the compound of interest on an accurate balance and add the DMSO-d6 using a micropipette to dissolve the compound and obtain a stock solution. If the compound was not fully soluble at first, gentle heating (around 40-50 °C) with a hair dryer can be applied for less than 1 min at a time followed by 30 s of vortexing. This can be repeated until the solid is fully dissolved. The resulting solution should be clear with no floating particles of material and not cloudy.
    2. Prepare a fresh buffer solution of 10 mM PBS buffer corrected to pH 7.4.
      Dissolve a phosphate buffered saline tablet in 100 ml of water and correct the resulting solution to pH 7.4 by mean of a pH meter.
    3. Add 10 µl of the stock 10 mM ligand solution to a 0.5 ml Eppendorf tube [the following steps (A3-A5) are intended to make a compound dilution to a final concentration of 500 µM].
      Use a micropipette and tip to add the solution of ligand to the clean new Eppendorf. Discard the tip. Put the Eppendorf on an Eppendorf stand.
    4. Add 170 µl of the buffer to the Eppendorf and vortex the solution for 30 s.
      Use a micropipette and tip to add the buffer solution to the Eppendorf containing the ligand solution. Discard the tip. Close and vortex the Eppendorf at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    5. Add 20 µl of D2O to the Eppendorf and vortex the solution for 30 s.
      Use a micropipette and tip to add D2O to the Eppendorf containing the ligand solution and buffer. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    6. Transfer the resulting solution into a 3 mm NMR tube.
      Carefully draw the solution in a long Pasteur pipette with the aid of a rubber teat. Then transfer the solution carefully to the tube, putting the pipette completely in and taking it out as the teat is pressed gently to release the solution. Flick the NMR tube a couple of time (towards the floor) to make sure that the liquid moves completely to the bottom of the tube.
    7. Cap and centrifuge the NMR tube.
      Cap the NMR tube, place it in a manual hand centrifuge and centrifuge it for a couple of minutes to ensure no bubbles at the surface (as this can cause poor field optimisation (shimming) in the NMR experiment).
    8. Place the tube in the NMR spectrometer for waterLOGSY 1-D NMR detection.
      Place the NMR tube in the 3 mm spinner (turbine) and use the appropriate NMR depth gauge to position the tube correctly in the spinner. Transfer the sample/spinner into the NMR spectrometer magnet either via manual placement or via use of a sample transfer robot attached to the magnet cryostat.

  2. waterLOGSY basic protocol
    The basic WaterLOGSY is exemplified here with using an initial stock protein solution of C = 50 µM. The concentration of the protein determines the amount of buffer that is needed in the experiment.
    Other protein concentrations can be used with buffer amount changed accordingly.
    1. Dissolve the ligand (compound in DMSO-d6 to obtain a 10 mM stock solution).
      Weigh out the compound of interest on an accurate balance and add the DMSO-d6 using a micropipette to dissolve the compound and obtain a stock solution. If the compound was not fully soluble at first, gentle heating with a hair dryer can be applied for less than 1 min at a time followed by 30 s of vortexing. This can be repeated until the solid is fully dissolved.
    2. Prepare a fresh buffer solution of 10 mM PBS, 5 mM MgCl2 buffer corrected to pH 7.4.
      This buffer is the one required for the protein used in this protocol; the appropriate buffer for the protein under study should be prepared here. Buffers with low proton content, such as PBS, or that are deuterated (e.g., Tris-d11) are preferred to minimise background signals from the buffer. Similarly, concentrations of protonated buffers should be minimised.
    3. Thaw the protein just before the preparation of the sample.
      Take the protein out of the -80 °C freezer and place it in a box of dry ice to be carried to the NMR instrument. Take the Eppendorf containing the protein out of the ice and leave it to stand at room temperature to thaw completely. Do not put the protein back in ice until the end of all experiments. In some cases the protein can be frozen back, but only if it is stable enough to do so. For this protocol, we prepared careful aliquots to prevent any protein remaining after carrying out the experiments.
    4. Add 10 µl of the ligand solution to a 0.5 ml Eppendorf tube.
      Use a micropipette and tip to add the solution of ligand to the clean new Eppendorf. Discard the tip. Put the Eppendorf on an Eppendorf stand.
    5. Add 130 µl of the buffer to the Eppendorf and vortex the solution for 30 s.
      Use a micropipette and tip to add the buffer solution to the Eppendorf containing the ligand solution. Discard the tip. Close and vortex the Eppendorf at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    6. Add 20 µl of D2O to the Eppendorf and vortex the solution for 30 s.
      Use a micropipette and tip to add D2O to the Eppendorf containing the ligand solution and buffer. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    7. Add 40 µl of the protein (concentration 50 µM) to the Eppendorf (giving an end volume of 200 µl). The final protein concentration in the assay is 10 µM, the final ligand concentration is 500 µM.
      Vortex the Eppendorf containing the protein for 30 s at setting 8. Use a micropipette and tip to add the protein to the Eppendorf containing the ligand solution, buffer and D2O. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    8. Transfer the resulting solution into a 3 mm NMR tube.
      Carefully draw the solution in a long Pasteur pipette with the aid of a rubber teat. Then transfer the solution carefully to the tube, putting the pipette completely in and taking it out as the teat is pressed gently to release the solution. Flick the NMR tube a couple of time (towards the floor) to make sure that the liquid moves completely to the bottom of the tube.
    9. Cap and centrifuge the NMR tube.
      Cap the NMR tube, place it in a manual hand centrifuge and centrifuge it for a couple of minutes to ensure no bubbles at the surface (as this can cause poor field optimisation (shimming) in the NMR experiment).
    10. Place the tube in the NMR spectrometer for waterLOGSY 1-D NMR detection.
      Place the NMR tube in the 3 mm spinner (turbine) and use the appropriate NMR depth gauge to position the tube correctly in the spinner. Transfer the sample/spinner into the NMR spectrometer magnet either via manual placement or via use of a sample transfer robot attached to the magnet cryostat.

  3. waterLOGSY protocol for library screening
    For the library screening, it is important to choose carefully the compounds that are put together in the experiment. Usually three ligands can be used at once and solutions in DMSO are used–the amount of buffer changed accordingly. The library screening protocol is exemplified here with using an initial stock protein of C = 50 µM.
    1. Dissolve the ligand (compound in DMSO-d6 to obtain a 10 mM stock solution).
      Weigh out the compound of interest on an accurate balance and add the DMSO-d6 using a micropipette to dissolve the compound and obtain a stock solution. If the compound was not fully soluble at first, gentle heating with a hair dryer can be applied for less than 1 min at a time followed by 30 s of vortexing. This can be repeated until the solid is fully dissolved.
      Repeat this step up to two more times in order to add all the ligands needed to be screened.
    2. Prepare a fresh buffer solution of 10 mM PBS, 5 mM MgCl2 buffer corrected to pH 7.4.
      This buffer is the one required for the protein used in this protocol; the appropriate buffer for the protein under study should be prepared here. Buffers with low proton content, such as PBS, or that are deuterated (e.g., Tris-d11) are preferred to minimise background signals from the buffer. Similarly, concentrations of protonated buffers should be minimised.
    3. Add 10 µl of the ligand solution to a 0.5 ml Eppendorf tube.
      Use a micropipette and tip to add the solution of ligand to the clean new Eppendorf. Discard the tip. Put the Eppendorf on an Eppendorf stand. Repeat this step for adding additional ligand solutions.
    4. Add 130 µl of the buffer to the Eppendorf and vortex the solution for 30 s (if using 3 ligands).
      Use a micropipette and tip to add the buffer solution to the Eppendorf containing the ligand solution. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    5. Add 20 µl of D2O to the Eppendorf and vortex the solution for 30 s.
      Use a micropipette and tip to add D2O to the Eppendorf containing the ligand solution and buffer. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    6. Add 40 µl of the protein (concentration 50 µM) to the Eppendorf (giving an end volume of 200 µl). The final protein concentration in the assay is 10 µM, the final ligand concentration is 500 µM (each).
      Vortex the Eppendorf containing the protein for 30 s at setting 8. Use a micropipette and tip to add the protein to the Eppendorf containing the ligand solution, buffer and D2O. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    7. Transfer the resulting solution into a 3 mm NMR tube.
      Carefully draw the solution in a long Pasteur pipette with the aid of a rubber teat. Then transfer the solution carefully to the tube, putting the pipette completely in and taking it out as the teat is pressed gently to release the solution. Flick the NMR tube a couple of time (towards the floor) to make sure that the liquid moves completely to the bottom of the tube.
    8. Cap and centrifuge the NMR tube.
      Cap the NMR tube, place it in a manual hand centrifuge and centrifuge it for a couple of minutes to ensure no bubbles at the surface (as this can cause a bad shimming in the NMR experiment).
    9. Place the tube in the NMR spectrometer for waterLOGSY 1-D NMR detection.
      Place the NMR tube in the 3 mm spinner (turbine) and use the appropriate NMR depth gauge to position the tube correctly in the spinner. It is then transferred into the NMR spectrometer magnet either via manual placement or via use of a sample transfer robot attached to the magnet cryostat.

  4. waterLOGSY protocol for competition
    For a competition experiment using Y6-ScFv VH, the preparation was carried out in a similar manner as the basic waterLOGSY protocol; it is exemplified here with using an initial stock protein of C = 312 µM and an initial stock antibody of C = 116.6 µM (protein and antibody are in a 1:1 ratio). The concentration of the proteins determines the amount of buffer that is used in the experiment. Other protein concentrations can be used with buffer amount changed accordingly.
    1. Repeat Steps B1 and B2.
    2. Thaw the protein and the antibody just before the preparation of the sample.
      Take the protein and the antibody out of the -80 °C freezer and place them in a box of dry ice to be carried to the NMR instrument. Take the Eppendorf containing the proteins out of the ice and leave it to stand at room temperature to thaw completely. Do not put the proteins back in ice until the end of all experiments. In some cases the protein can be frozen back, but only if it is stable enough to do so. For this protocol, we prepared careful aliquots to prevent any protein remaining after carrying out the experiments.
    3. Repeat Step A3.
    4. Add 146.4 µl of the buffer to the Eppendorf and vortex the solution for 30 s.
    5. Use a micropipette and tip to add the buffer solution to the Eppendorf containing the ligand solution. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    6. Repeat Step A5.
    7. Add 6.4 µl of the protein (concentration 311.8 µM) to the Eppendorf.
      Vortex the Eppendorf containing the protein for 30 s at setting 8. Use a micropipette and tip to add the protein to the Eppendorf containing the ligand solution, buffer and D2O. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    8. Add 17.2 µl of the antibody (concentration 116.6 µM) to the Eppendorf (giving an end volume of 200 µl).
      Vortex the Eppendorf containing the antibody for 30 s at setting 8. Use a micropipette and tip to add the protein to the Eppendorf containing the ligand solution, protein, buffer and D2O. Discard the tip. Close the Eppendorf and vortex it at setting 8 for 30 s. Re-open the Eppendorf and put it back on its stand.
    9. Repeat Steps A6-A8.

Data analysis

  1. Instrumentation
    Solution-phase NMR spectrometer operating at high field (400+ MHz) equipped with a probe suitable for 1H detection and with z-axis pulsed field gradient capabilities and with active sample temperature regulation (default 298 K). The probe temperature can be altered according to protein requirements and stability.
    The use of 3 mm diameter NMR tubes is optimal for 5 mm cryoprobes (or 3 mm microprobes) but can also be used with conventional 5 mm NMR probes, albeit with some sensitivity loss. If sample transport spinners (turbines) for 3 mm tubes are not available, these narrow tubes can be placed directly within conventional 5 mm tubes for transportation with 5 mm spinners. Alternatively, 5 mm tubes alone may be employed which requires all sample volumes in this protocol be scaled three-fold to yield final volumes for analysis of 600 µl.
    For library screening, a robotic sample changer is desirable to enable high sample throughput, unattended operation and overnight data collection.
    Commands listed here relate to the operation of Bruker NMR spectrometers- follow equivalent protocols for other vendors. This protocol is suitable for Bruker AVIII spectrometers running TOPSPIN 3.5, or later generations.

  2. Bruker set-up for each sample (+/- protein)
    1. Record standard 1H NMR spectrum with H2O water suppression
      1. Load parameter set WATERSUP (1D NOESY preset sequence).
      2. Perform standard instrument set-up protocol for 1H NMR detection: With sample in probe: tune 1H channel of probe (atma), lock sample (lock H2O/D2O), shim sample (gradient shim routine, e.g., topshim), calibrate 1H pulse (pulsecal).
      3. Collect 1H reference spectrum and check authenticity of ligand(s) present.
    2. Record 1H waterLOGSY NMR spectrum
      1. Load parameter set: SCREEN_WLOGSY.
      2. Load 1H pulse calibration determined from above set-up protocol.
      3. Recommended parameters for waterLOGSY screening:
        Pulse program: ephogsygpno.2 (ephogsy was an original implementation of this experiment used for protein hydration studies and known as enhanced protein hydration observed through gradient spectroscopy, the name of which is still retained by Bruker for the waterLOGSY sequence)
        Recovery delay (d1): 2 s
        Water selective 180° pulse for water excitation (p7:sp21): 7.5 ms Gaussian (Gaus1_180i.1000)
        Mixing time (d8): 1s
        Water suppression 180° selective pulse (p12:sp1): 2 ms sinc (sinc1.1000) or square profile (Squa100.1000)
        Spectral width: 16 ppm
        Acquisition time (AQ): 2 s
        Number of acquired transients (NS): ≥ 128 (dependent on instrument sensitivity)
        Do not spin samples.

  3. Data processing
    Process data with 1 Hz line broadening lb = 1 (command ef to process).
    Phase spectra such that resonances of bound small molecules are negative (inverted). Protein signals should appear with negative intensity (Figure 1).


    Figure 1. Typical result from a compound not aggregating and binding to the protein. In blue is the proton NMR of the compound. In red is the waterLOGSY of the compound alone. All peaks are still up (positive), showing no aggregation, apart from NHs and OHs (5.6 to 6.0 ppm), which exchange with the water and therefore display negative signals. In green is the waterLOGSY of the compound with protein. All the peaks from the compound are negative, indicating binding. The positive peaks seen in the spectra are the DMSO peak at 2.6 ppm and the residual water peak (still observed even after suppression at 4.7 ppm). This particular solution contained residual Tris from the protein preparation, which is observed at 3.7 ppm.

Acknowledgments

The work of CJRB and THR was supported by a grant from Bloodwise (12051) and THR also by grants from the Medical Research Council (MR/J000612/1) and the Wellcome Trust (100842/Z/12/Z).

Competing interests

The authors have no conflicts of interest to declare.

References

  1. Dalvit, C., Pevarello, P., Tato, M., Veronesi, M., Vulpetti, A. and Sundstrom, M. (2000). Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water. J Biomol NMR 18(1): 65-68.
  2. Dalvit, C., Fogliatto, G., Stewart, A., Veronesi, M. and Stockman, B. (2001). WaterLOGSY as a method for primary NMR screening: practical aspects and range of applicability. J Biomol NMR 21(4): 349-359. 
  3. Lepre, C. A. (2011). Practical aspects of NMR-based fragment screening. Methods Enzymol 493: 219-239. 
  4. Huang, R. and Leung, I. K. H. (2019). Protein-small molecule interactions by WaterLOGSY. Methods Enzymol 615: 477-500.

简介

[摘要] 在药物开发计划中,需要多种测定方法来测定蛋白质与化合物的相互作用,并评估在蛋白质与蛋白质相互作用中的潜在用途。在此协议中,我们描述了用于确认蛋白质-配体结合事件的waterLOGSY NMR方法。

[背景] 随着在疾病细胞中发现更多的蛋白质形式变化,药物发现计划不断增加,这些计划依赖于使用小分子文库对目标蛋白质进行初步筛选,然后进行进一步的药物化学运动以增加所散发的化学物质的效力。从屏幕上。经过初步筛选后,出现了一系列正交分析,这些化学分析有助于验证命中的化学物质,并可以对化合物进行分层,以选择最佳化合物以进入命中领先阶段,进而进行前导优化。在这些测定法是基于NMR的水大号igand ö 经由bserved ģ radient 小号pectroscop ý (waterLOGSY )方法(Dalvit 等人,2000和2001) 。waterLOGSY 是用于检测配体的结合是特别有用的互相作用相对弱与靶蛋白(即,在μM解离常数为低毫范围),如所预期的要与初始命中从一个大的化学或片段库相关联药物化学之前先进行筛分(Lepre,2011),以提高药效和类似药物的性能。



WaterLOGSY 如何工作

该waterLOGSY 方法利用1个小分子的1 H NMR观察(配位体),用于检测配体-高分子结合(Huang和梁,2019) 。它依赖于质子(1 H)磁化从激发的水分子到配体的转移,方法是:1)它们的直接相互作用或2)通过初始转移到蛋白质表面的质子,然后再传递到与蛋白质结合的配体上间接转移。普遍存在的水分子与游离的(未结合的)配体的直接相互作用(途径1)导致观察到的配体信号强度增加,这是由于水和配体质子之间的直接(贯穿空间)磁相互作用,称为正核Overhauser 效果(nOe )。相反,从水到蛋白质的磁化转移然后传递到受体结合的配体上(途径2),导致配体信号强度的净降低(此处归因于负nOe )。转移行为的这种差异起因于溶液中分子的不同翻转速率(即,它们的旋转相关速率),对于小的未结合配体“快速”,但对于大分子受体及其结合的配体“缓慢” 。因此,在不存在和存在蛋白质受体的情况下比较配体信号强度可以表明配体结合是否已经发生,信号强度的差异暗示了结合。注意,由于可交换的(酸性)配体质子与水质子的动态交换,第三种磁化传递机理也可能发生,无论蛋白质的存在与否,它都会产生与负nOe 相同的信号响应。因此,出于配体筛选目的,应忽略来自可交换质子的响应。

当在溶液中游离时 ,waterLOGSY 实验仅检测配体的信号,因此依赖于配体结合的复合物的解离和配体的释放,然后携带负nOe 进行检测。该过程要求配体解离速率足够高,以便在配体通过始终有效的自然弛豫过程丢失之前将其转移到溶液中进行nOe 检测。类似地,它要求配体在受体上的停留时间要足够长,以使磁化转移本身在配体释放之前发生。因此,waterLOGSY 最适合检测中度至弱亲和力的结合剂,其解离常数在µM至低mM 范围内。事实证明,该方法在筛选商城分子片段文库中特别受欢迎,因为这些分子通常具有较弱的结合亲和力。强结合配体(K d < μ 中号)有太长的蛋白上的停留时间和它们的结合是不太可能被检测到,从而导致在这样的情况下假阴性的可能性。



waterLOGSY 方法的应用

在本文中,我们介绍了用于评估蛋白质与配体结合的waterLOGSY 方案,并展示了如何利用蛋白质与蛋白质的相互作用抑制蛋白质与配体的结合,从而确定了目标蛋白质上的配体位置。

WaterLOGSY 是一种通用方法,可以定性评估小配体与蛋白质的结合。可以仅使用一种配体(请参见基本方案)进行此操作,但也可以使用多种配体(请参见筛选方案)进行此操作,因此可将其用作中等通量分析。在这种情况下,重要的是使用配体的DMSO溶液代替基本方案中使用的DMSO- d 6 溶液。太多DMSO- d 6 可以防止NMR 我nstrument从锁定在d 2 O. DMSO也可能导致与蛋白结构本身的干扰。通常,应使用最多10%v / v的DMSO- d 6 或DMSO。

任何waterLOGSY 实验的第一步都是确定配体没有聚集(请参阅聚集方案)。AGGR 小分子egation引起假阳性反应waterLOGSY 实验,因为聚集体采用了大分子种类的翻滚行为并且因此给出waterLOGSY 响应为如果配体被约束。这意味着每个配体都应在没有蛋白质的情况下进行测试,以消除混杂的聚集体的存在。相反,这种行为导致waterLOGSY 是评估小分子聚集的非常有用的方法。

关键字:药物, WaterLOGSY, 药物研发, 核磁共振, 配基结合, 蛋白质蛋白质相互作用

材料和试剂


 


诺雷尔® 选择系列TM 3毫米NMR管(西格玛- Aldrich公司,目录号:NORS36008)
Eppendorf管® 体积(0.5毫升)中,PCR清洁(Sigma-Aldrich公司,目录号:EP0030124537-500EA)
苏打石灰巴斯德移液器已拔出,容量为2 ml,长度为230 mm(VWR,目录号:612-1702)
吸管奶头,MBL ® 天然橡胶(SciLabware,目录号:BIBBBPP004)
移液器提示:
10 µl刻度吸头(Starlab ,目录号:S1111-3800)


200 µl 黄色吸头(Starlab ,目录号:S1111-0806)


1 ,000微升b 略提示(STARLAB ,目录号:S1111-6801)


磷酸盐缓冲盐水片(Sigma - Aldrich,目录号:P4417)
蒸馏水(室内)
D 2 O (氧化氘99.9原子%D )(Sigma - Aldrich,目录号:151882)
DMSO- d 6 (二甲基亚砜-d 6 “ 100%”,99.96原子%D )(Sigma - Aldrich,目录号:156914)
 


设备


 


手动离心机,不带转子2 x 15 ml,用于手动离心机的90°锥形圆锥形转子转子 (Satorius,型号:C1011)
科学工业公司SI TM Vortex- Genie TM 2(科学工业公司,型号:SI-0266,目录号:15557335 )
-80 °C 冷冻室
pH计(Jenway ,目录号:351001)
吉尔森移液器,单通道:
P ipetman L,2-20 µl (Gilson,目录号:FA10003M)


P ipetman L,20-200 µl(吉尔森,目录号:FA100 05M)


P ipetman L,100-1 ,000微升(吉尔森,目录号:FA10006M)


 


程序


 


注意:假设1 H NMR光谱学已掌握NMR光谱仪操作的本地知识,或可以适当地获取。此处列出的命令与Bruker NMR光谱仪的操作有关- 遵循其他供应商的等效协议。


 


聚合协议
溶解配体(在DMSO- d 6中的化合物以获得10 mM的储备溶液)。
在准确的天平上称出目标化合物,并使用微量移液器添加DMSO- d 6 以溶解该化合物并获得储备溶液。如果化合物最初不完全溶解,则可以用吹风机缓慢加热(约40-50°C),每次少于1分钟,然后涡旋30 s 。可以重复进行直到固体完全溶解。所得溶液应澄清,没有漂浮的物质颗粒,并且不混浊。


准备一个新的10 mM PBS缓冲液,将其pH校正为7.4 。
将磷酸磷酸盐缓冲盐水片溶于100 ml水中,并通过pH计将所得溶液的pH校正为7.4。


将10 µl的10 mM 配体储备液添加到0.5 ml的Eppendorf管中[ 以下步骤(A 3- A 5)用于将化合物稀释至最终浓度为500 µM ] 。
使用微量移液器和吸头将配体溶液添加到干净的新Eppendorf中。丢弃尖端。将Eppendorf放在Eppendorf架子上。


将170 µl缓冲液添加到Eppendorf中,并将溶液涡旋30秒钟。
使用微量移液器和吸头将缓冲液添加到含有配体溶液的Eppendorf中。丢弃尖端。在设置8处将Eppendorf关闭并涡旋30 s。重新打开Eppendorf,并将其放回原位。


向Eppendorf中加入20 µl D 2 O,并将溶液涡旋30秒钟。
使用微量移液器和吸头将D 2 O 添加到含有配体溶液和缓冲液的Eppendorf中。丢弃尖端。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf,并将其放回原位。


将所得溶液转移至3 mm NMR管中。
在橡胶奶嘴的帮助下,用长巴斯德移液器小心地吸取溶液。然后将溶液小心地转移到试管中,将移液管完全放入,然后轻轻按下奶嘴将其取出以释放溶液。轻轻摇动NMR试管几次(朝向地板),以确保液体完全移至试管底部。


盖好并离心NMR管。
盖上NMR管,将其置于手动离心机中,并离心几分钟,以确保表面没有气泡(因为这会导致NMR实验中的磁场优化不佳(匀化))。


将试管放在NMR光谱仪中进行waterLOGSY 1-D NMR检测。
将NMR管放置在3 mm旋转器(涡轮)中,并使用适当的NMR深度计将管正确放置在旋转器中。通过手动放置或通过使用与磁体低温恒温器相连的样品传输机械手,将样品/旋转体转移到NMR光谱仪磁体中。


 


waterLOGSY 基本协议
基本的WaterLOGSY 此处以C = 50 µM的初始储备蛋白溶液为例。蛋白质的浓度决定了实验中所需缓冲液的量。可以使用其他蛋白质浓度,并相应更改缓冲液量。


溶解配体(在DMSO- d 6中的化合物以获得10 mM的储备溶液)。
在准确的天平上称出目标化合物,并使用微量移液器添加DMSO- d 6 以溶解该化合物并获得储备溶液。如果化合物最初不完全溶解,则可以一次用吹风机缓慢加热少于1分钟,然后涡旋30秒。可以重复进行直到固体完全溶解。


制备10的新鲜缓冲溶液毫PBS,5 毫的MgCl 2 的buff ER校正至pH 7.4 。
该缓冲液是该规程中所用蛋白质所需的缓冲液。应该在此处准备适合所研究蛋白质的缓冲液。具有低质子含量,例如PBS,或者缓冲器氘化的(例如,三- d 11 )是优选的以最小化来自缓冲器的背景信号。同样,质子化缓冲液的浓度应降至最低。


在制备样品前解冻蛋白质。
从-80中取出蛋白质 °C冷冻机,并将其放入干冰盒中,以运送到NMR仪器。以含有蛋白质出冰的埃氏并等待其置于室温下解冻COM + pletely 。在所有实验结束之前,请勿将蛋白质放回冰中。在某些情况下,可以将蛋白质冷冻回去,但前提是必须足够稳定。对于此方案,我们准备了小心的等分试样,以防止进行实验后残留任何蛋白质。


在0.5 ml Eppendorf管中加入10 µl配体溶液。
使用微量移液器和吸头将配体溶液添加到干净的新Eppendorf中。丢弃尖端。将Eppendorf放在Eppendorf架子上。


将130 µl缓冲液添加到Eppendorf中,并将溶液涡旋30秒钟。
使用微量移液器和吸头将缓冲液添加到含有配体溶液的Eppendorf中。丢弃尖端。在设置8处将Eppendorf关闭并涡旋30 s。重新打开Eppendorf,并将其放回原位。


向Eppendorf中加入20 µl D 2 O,并将溶液涡旋30秒钟。
使用微量移液器和吸头将D 2 O 添加到含有配体溶液和缓冲液的Eppendorf中。丢弃尖端。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf,并将其放回原位。


将40 µl的蛋白质(浓度为50 µM)添加到Eppendorf(终体积为200 µl)中。测定中的最终蛋白质浓度为10 µM,最终配体浓度为500 µM。   
在设置8处使含有蛋白质的Eppendorf涡旋涡旋30 s。使用微量移液器和吸头将蛋白质添加到含有配体溶液,缓冲液和D 2 O 的Eppendorf中。丢弃吸头。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf,并将其放回原位。


将所得溶液转移到3 mm NMR管中。
在橡胶奶嘴的帮助下,用长巴斯德移液器小心地吸取溶液。然后将溶液小心地转移到试管中,将移液管完全放入,然后轻轻按下奶嘴将其取出以释放溶液。轻轻摇动NMR试管几次(朝向地板),以确保液体完全移至试管底部。


盖好并离心NMR管。
盖上NMR管,将其置于手动离心机中,并离心几分钟,以确保表面没有气泡(因为这会导致NMR实验中的磁场优化不佳(匀化))。


将试管放在NMR光谱仪中,用于waterLOGSY 1-D NMR检测。
将NMR管放置在3 mm旋转器(涡轮)中,并使用适当的NMR深度计将管正确放置在旋转器中。TRANSF ER采样/微调到NMR波谱仪磁体或者通过手动放置,或通过使用附连到磁体的低温恒温器的样品输送机器人的。


 


用于图书馆筛选的waterLOGSY 协议
对于文库筛选,重要的是仔细选择实验中放在一起的化合物。通常可以一次使用三个配体,然后使用DMSO溶液– 缓冲液的量会相应改变。文库筛选方案的示例是使用初始储备蛋白C = 50 µM。


溶解配体(在DMSO- d 6中的化合物以获得10 mM的储备溶液)。
在准确的天平上称出目标化合物,并使用微量移液器添加DMSO- d 6 以溶解该化合物并获得储备溶液。如果化合物最初不完全溶解,则可以一次用吹风机缓慢加热少于1分钟,然后涡旋30秒。可以重复进行直到固体完全溶解。


重复此步骤最多两次,以添加需要筛选的所有配体。


准备10 mM PBS,5 mM MgCl 2 缓冲液的新缓冲溶液,校正至pH 7.4 。
该缓冲液是该规程中所用蛋白质所需的缓冲液。应在此处准备适合所研究蛋白质的缓冲液。具有低质子含量,例如PBS,或者缓冲器氘化的(例如,三- d 11 )是优选的以最小化来自缓冲器的背景信号。同样,质子化缓冲液的浓度应降至最低。


将10 µl配体溶液加入0.5 ml Eppendorf管中。
使用微量移液器和吸头将配体溶液添加到干净的新Eppendorf中。丢弃尖端。将Eppendorf放在Eppendorf架子上。重复此步骤以添加其他配体溶液。


将130 µl缓冲液添加到Eppendorf中,并将溶液涡旋30秒钟(如果使用3个配体)。
使用微量移液器和吸头将缓冲液添加到含有配体溶液的Eppendorf中。丢弃尖端。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf,并将其放回原位。


向Eppendorf中加入20 µl D 2 O,并将溶液涡旋30秒钟。
使用微量移液器和吸头将D 2 O 添加到含有配体溶液和缓冲液的Eppendorf中。丢弃尖端。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf ,并将其放回原位。


将40 µl的蛋白质(浓度为50 µM)添加到Eppendorf(终体积为200 µl)中。分析中的最终蛋白质浓度为10 µM,最终配体浓度为500 µM(每种)。   
在设置8处使含有蛋白质的Eppendorf涡旋涡旋30 s。使用微量移液器和吸头将蛋白质添加到含有配体溶液,缓冲液和D 2 O 的Eppendorf中。丢弃吸头。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf,并将其放回原位。


将所得溶液转移至3 mm NMR管中。
在橡胶奶嘴的帮助下,用长巴斯德移液器小心地吸取溶液。然后将溶液小心地转移到试管中,将移液管完全放入,然后轻轻按下奶嘴将其取出以释放溶液。轻轻摇动NMR试管几次(朝向地板),以确保液体完全移至试管底部。


盖好并离心NMR管。
盖上NMR管,将其置于手动离心机中并离心几分钟,以确保表面没有气泡(因为这会在NMR实验中造成严重的匀场)。


将试管放在NMR光谱仪中,用于waterLOGSY 1-D NMR检测。
将NMR管放置在3毫米旋转器(涡轮机)中,并使用适当的NMR深度计将管正确放置在旋转器中。然后通过手动放置或通过使用与磁体低温恒温器相连的样品传输机器人将其转移到NMR光谱仪磁体中。


 


waterLOGSY 竞争协议
对于使用Y6-ScFv VH进行的竞争实验,制备方法与基本waterLOGSY 方案相似。在此通过使用C = 312 µM的初始储备蛋白和C = 116.6 µM 的初始储备抗体(蛋白质和抗体的比例为1:1)来举例说明。蛋白质的浓度决定了实验中使用的缓冲液的量。可以使用其他蛋白质浓度,并相应更改缓冲液量。


重复步骤B1和B2。
在准备样品之前解冻蛋白质和抗体。
将蛋白质和抗体从-80 °C的冰箱中取出,并将它们放入一盒干冰中,以携带到NMR仪器中。从冰中取出含有蛋白质的Eppendorf,让其在室温下静置完全融化。在所有实验结束之前,请勿将蛋白质放回冰中。在某些情况下,可以将蛋白质冷冻回去,但前提是必须足够稳定。对于此方案,我们准备了小心的等分试样,以防止进行实验后残留任何蛋白质。


重复步骤A3。
向Eppendorf中加入146.4 µl缓冲液,并将溶液涡旋30秒钟。
使用微量移液器和吸头将缓冲液添加到含有配体溶液的Eppendorf中。丢弃尖端。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf,并将其放回原位。
重复步骤A5。
将6.4 µl蛋白质(浓度311.8 µM)添加到Eppe ndorf中。
在设置8处使含有蛋白质的Eppendorf涡旋涡旋30 s。使用微量移液器和吸头将蛋白质添加到含有配体溶液,缓冲液和D 2 O 的Eppendorf中。丢弃吸头。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf,并将其放回原位。


在Eppendorf中加入17.2 µl抗体(浓度116.6 µM)(终体积为200 µl)。
在设置8处涡旋含有抗体的Eppendorf 30 s。使用微量移液器和吸头将蛋白质添加到含有配体溶液,蛋白质,缓冲液和D 2 O 的Eppendorf中。丢弃吸头。关闭Eppendorf并在设置8处涡旋30秒钟。重新打开Eppendorf,并将其放回原位。


重复步骤A6-A8 。
 


数据分析


 


仪器仪表
固相核磁共振波谱仪,在高场(400+ MHz)下运行,配备有适用于1 H检测的探针,z轴脉冲场梯度功能以及有效的样品温度调节功能(默认298 K)。探针温度可以根据蛋白质要求和稳定性来改变。


直径为3 mm的NMR管最适合5 mm的低温探针(或3 mm的微型探针),但也可以与常规的5 mm NMR探针一起使用,尽管会降低灵敏度。如果没有用于3毫米试管的样品传输微调器(涡轮),可以将这些窄管直接放置在常规5毫米试管中,以使用5毫米微调器进行运输。或者,可以单独使用5 mm的试管,这要求将本方案中的所有样品体积均按三倍比例缩放以产生用于600 µl分析的最终体积。


对于文库筛选,需要一个自动进样器以实现高样品通量,无人值守的操作和过夜数据收集。


此处列出的命令与Bruker NMR光谱仪的操作有关-遵循其他供应商的等效协议。该协议适用于运行TOPSPIN 3.5或更高版本的Bruker AVIII光谱仪。


 


每个样品的布鲁克设置(+/-蛋白)
记录具有H 2 O水抑制作用的标准1 H NMR光谱
加载参数设置WATERSUP (1D NOESY预设序列)。
执行用于1 H NMR检测的标准仪器设置方案:将样品置于探针中:调整探针的1 H通道(atma ),锁定样品(锁定H 2 O / D 2 O),匀场样品(梯度匀场例行程序,例如)。克。,吨opshim ),标定1 H脉冲(pulsecal )。
收集1 H参考光谱并检查存在的配体的真实性。
记录1 H waterLOGSY NMR光谱
罗广告参数设置:SCREEN_WLOGSY。
根据上述设置协议确定的负载1 H脉冲e标定。
waterLOGSY 筛选的推荐参数:
脉冲程序:ephogsygpno.2(ephogsy 是该实验的最初实现方式,用于蛋白质水合研究,通过梯度光谱观察到的被称为增强蛋白质水合,Bruker仍将其名称保留为waterLOGSY 序列)


恢复延迟(d1):2 s


选水180用于水激励的°脉冲(p7:sp21):7.5 ms 高斯(Gaus1_180i.1000)


混合时间(d8):1秒


水抑制180 ° 选择性脉冲(p12:sp1):2 ms sinc (sinc1.1000)或正方形轮廓(Squa100.1000)


光谱宽度:16 ppm


采集时间(AQ):2 s


获得的数量(NS):≥128(取决于仪器的灵敏度)


不要旋转样品。


 


数据处理
1 Hz线加宽lb = 1的过程数据(要执行的命令ef )。


相谱,使得结合的小分子的共振为负(反相)。蛋白质信号应以负强度出现(图1)。






D:\ Reformatting \ 2020-4-7 \ 1902587--1415 Terry Rabbitts 669671 \ Figs jpg \图1.jpg


图1 。典型的结果是化合物没有聚集并与蛋白质结合。蓝色为该化合物的质子NMR。红色为单独的化合物的waterLOGSY 。除与水交换的NHs和OHs(5.6至6.0 ppm)外,所有峰均仍向上(正),没有聚集,因此显示负信号。绿色表示化合物与蛋白质的waterLOGSY 。该化合物的所有峰均为负值,表明已结合。光谱中看到的正峰是2.6 ppm 的DMSO 峰和残留的水峰(即使在4.7 ppm抑制后仍观察到)。该特定溶液包含蛋白质制备物中的残留Tris ,在3.7 ppm处观察到。


 


ACKN owledg 发言:


 


CJRB和THR的工作得到了Bloodwise (12051)的资助,THR也得到了医学研究理事会(MR / J000612 / 1)和惠康基金会(100842 / Z / 12 / Z)的资助。


 


利益争夺


 


作者没有利益冲突要声明。


 


参考资料


 


Dalvit ,C.,Pevarello ,P.,Tato,M.,Veronesi,M.,Vulpetti ,A。和Sundstrom ,M。(2000)。通过从大量水中磁化转移来鉴定对蛋白质具有结合亲和力的化合物。Ĵ Biomol公司NMR 18(1):65-68。
Dalvit ,C.,Fogliatto ,G.,Stewart,A.,Veronesi,M。和Stockman,B。(2001)。作为基本NMR筛选方法的WaterLOGSY:实用方面和适用范围。Ĵ Biomol公司NMR 21(4):349-359。              
Lepre ,CA(2011)。基于NMR的片段筛选的实践方面。方法酶493:219-239。              
Huang,R.and Leung,IKH(2019)。通过WaterLOGSY进行的蛋白质-小分子相互作用。方法酶学方法615:477-500。  
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Bataille et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bataille, C. J. R., Rabbitts, T. H. and Claridge, T. D. W. (2020). NMR waterLOGSY as An Assay in Drug Development Programmes for Detecting Protein-Ligand Interactions–NMR waterLOGSY. Bio-protocol 10(13): e3666. DOI: 10.21769/BioProtoc.3666.
  2. Bery, N., Cruz-Migoni, A., Bataille, C. J. R., Quevedo, C. E., Tulmin, H., Miller, A., Russell, A., Phillips, S. E. V., Carr, S. B. and Rabbitts, T. H. (2018). BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions. eLife 7: e37122.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。