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, WaterLOGSY, Drug discovery, NMR, Ligand binding, Protein-protein interaction
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 worksThe 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 methodIn 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
Equipment
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.
Data analysis
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
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