Filling Blank Spots on the Map: Identification of Ligand Binding Modes

Oct 30, 2017 - Filling Blank Spots on the Map: Identification of Ligand Binding Modes and Interacting Water Molecules for Brd4-BD1 by WaterLOGSY Titra...
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Filling Blank Spots on the Map: Identification of Ligand Binding Modes and Interacting Water Molecules for Brd4-BD1 by WaterLOGSY Titrations Ann-Christin Pöppler* Institute of Organic Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany ABSTRACT: Fragment-based drug discovery and continuous improvement of existing protein inhibitors rely on the knowledge of exactly how and how strongly a range of small molecules bind to their respective protein targets. By increasing the (perdeuterated) protein concentration, WaterLOGSY titration experiments give access to ligand binding modes even in the case of weak binders as well as to the location of protein-bound water in the surroundings of the ligand. On the basis of these findings, specific chemical modifications of the ligand could be shown to yield significantly enhanced binding affinities.

n order to fight diseases and discover more potent and active molecules, fragment-based drug design has been a successful pathway for the past 20 years. By screening of small molecules for their bioactivity targeting specific proteins, more than 30 drug candidates have been developed, two of which are approved and several others are currently undergoing advanced trials.1 As the investigated fragments or molecules contain only few sites for possible interactions, the observed binding affinities are usually lower than for larger drug molecules so that the detection of this weak binding event can be a significant hurdle in the screening process. Subsequently, the goal is to modify the respective compounds such that the specific binding affinity is increased by several orders of magnitude and thus develop suitable drug candidates.2 As could be shown, for example, by Grzesiek et al. for HIV protease, water molecules in the binding pocket often also play crucial roles in protein−ligand interactions.3 Therefore, one way to systematically improve existing weakly binding molecules could focus on identifying and later chemically modifying those moieties with a particular orientation or proximity to water molecules in the protein−ligand complex. As water molecules can be difficult to locate by XRD methods, especially when they are not tightly bound, NMR spectroscopy can be a valuable toolkit for the assessment of those water molecules bound to large biological molecules.4 In this context, the authors present a ligand-based NMR spectroscopic approach using the slope of WaterLOGSY titration experiments with increasing protein concentration to determine (i) the ligand binding modes and (ii) protein-bound water in the proximity of the ligand for two different ligands interacting with the bromodomain of the protein Brd4-BD1 (Figure 1).5 By doing so, they could not only characterize the hydration shell of the ligands but also distinguish between differently tightly bound water molecules,

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Figure 1. Information obtained from WaterLOGSY titrations. The titration experiments give access to the binding modes with high LOGSY factors (red) representing deeply buried and lower LOGSY factors (blue) more solvent accessible sites of the ligand. Titration with perdeuterated proteins indicates interactions with differently ordered water molecules, a potential starting point for chemical modifications to increase the binding affinity to the target protein.

identify “disordered” water molecules, and use this information to improve the binding affinity of three different ligands by a factor of up to 100. Particularly at early stages of the drug discovery process, this might be of great help to medicinal chemists and to adjacent areas of research. Received: October 10, 2017 Published: October 30, 2017 8706

DOI: 10.1021/acs.jmedchem.7b01497 J. Med. Chem. 2017, 60, 8706−8707

Journal of Medicinal Chemistry

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significantly increased affinities. So overall, WaterLOGSY titration experiments represent a valuable addition to the toolbox of medicinal chemists in their search for new drugs that target and thus block specific interaction sites.

From the various tools NMR spectroscopy offers, the concepts developed by Geist et al. are based on WaterLOGSY experiments,6,7 where bulk water is selectively excited in the course of the experiment and the resulting magnetization then transferred to the ligand of the protein−ligand complex. This process is nuclear Overhauser effect (NOE) mediated, and it is possible to discriminate between binders or nonbinders for a specific protein target based on the sign of the observed NMR signals (binders, positive signal; nonbinders, negative signal). In addition, WaterLOGSY experiments also allow studying of the orientation of a ligand bound to a target (SALMON).8 However, if the binding is weak and the general protein size small, it may be difficult to use the sign of the signal as the sole source of information, as positive WaterLOGSY cross peaks are rarely observed in those cases (as can be seen for the LOGSY titration in Figure 1b of the work by Geist et al.).5 To circumvent these drawbacks and use the WaterLOGSY approach for a broader range of protein−ligand combinations, this paper presents WaterLOGSY titration experiments as an improved procedure. In such a LOGSY titration, the protein concentration is gradually increased and the normalized intensity for each of the ligand’s proton signals is measured. As the ligand shows a specific binding to the protein, each ligand proton will experience a different surrounding and thus will show a different effect in the LOGSY titration. On the basis of this titration experiment, a set of curves can be extracted and the resulting slope is analyzed and fitted linearly. So even if no zero crossing occurs, the steepness of the slope gives straightforward and very useful information on the binding situation: ligand protons in close proximity to the protein will have stronger LOGSY titration slopes compared to more solvent-accessible ligand protons in less contact with the protein. In this way, the authors could differentiate between different binding modes of two ligands embedded in the protein Brd4-BD1. This was known from XRD data but could not be explained based on saturation transfer difference (STD) experiments as these gave very similar transfer difference values for both ligands. In fully hydrated proteins, the NOE transfer consists of direct cross-relaxation between the ligand and protein-bound water as well as indirect contributions due to spin diffusion (water protons and protons from the protein inside the binding pocket). Therefore, an additional important insight can be gained by performing the above-described experiments with perdeuterated proteins discriminating between the two contributions. Under these experimental conditions, the LOGSY slopes show again very distinct trends and it is possible to distinguish solvent-accessible ligand protons with smaller values from protons in proximity to very ordered water molecules, e.g., in the binding pocket of the protein. With the help of these LOGSY factors, the authors could not only identify proximities of the respective ligands to tightly incorporated water molecules but also extend the characterization of the hydration environment to more loosely bound and thus positionally less well-defined water molecules. So even without previous knowledge of the complete structure of the protein−ligand complex, WaterLOGSY titration experiments enable mapping of protein-bound water in the proximity of the ligand, which is a very valuable starting point for systematic chemical modifications to increase the binding affinity of a specific ligand. This was successfully used in this work by introducing amino and methylamino groups to a set of three different ligands with all postmodification structures showing



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ann-Christin Pöppler: 0000-0002-0624-1708 Notes

The author declares no competing financial interest.

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ACKNOWLEDGMENTS I kindly thank Dr. Simone Kosol and Marvin Grüne for their helpful discussions. ABBREVIATIONS USED Brd4-BD1, bromodomain 1 of bromodomain containing protein 4; NOE, nuclear Overhauser effect; SALMON, solvent accessibility, ligand binding, and mapping of ligand orientation by NMR; STD, saturation transfer difference; WaterLOGSY, water ligand observed via gradient spectroscopy



REFERENCES

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DOI: 10.1021/acs.jmedchem.7b01497 J. Med. Chem. 2017, 60, 8706−8707