NMR waterLOGSY as An Assay in Drug Development Programmes for Detecting Protein-Ligand Interactions–NMR waterLOGSY

[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.

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. 3 www.bio-protocol.org/e3666  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 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.

B. 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. to make sure that the liquid moves completely to the bottom of the tube.

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.

C. 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.

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. 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. 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.

D. 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.
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.

A. Instrumentation
Solution-phase NMR spectrometer operating at high field (400+ MHz) equipped with a probe suitable for 1 H 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 threefold 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. B. Bruker set-up for each sample (+/-protein) 1. Record standard 1 H NMR spectrum with H2O water suppression a. Load parameter set WATERSUP (1D NOESY preset sequence). 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).