Ligand-Orientation Based Fragment Selection in STD NMR Screening

Brief Article pubs.acs.org/jmc

Ligand-Orientation Based Fragment Selection in STD NMR Screening Olivier Cala and Isabelle Krimm* §

Institut des Sciences Analytiques, UMR5280 CNRS, Université Lyon 1, Ecole Nationale Supérieure de Lyon, Université de Lyon, 5 rue de la Doua Villeurbanne 69100, France S Supporting Information *

ABSTRACT: While saturation transfer difference (STD) is a widely used NMR method for ligand screening, the selection of specific binders requires the validation of the hits through competition experiments or orthogonal biophysical techniques. We show here that the quantitative STD analysis is a reliable and robust approach to discriminate between specific and nonspecific ligands, allowing selection of fragments that bind proteins with a privileged binding mode, in the absence of any structural data for the protein.

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INTRODUCTION Rational drug design consists in using structural information to guide the development of molecules modulating the activity of therapeutic targets. One major advance in drug design in the past decade is the development of the fragment-based approaches for the generation of lead compounds.1,2 Libraries of thousands of fragment-like compounds, characterized by a molecular weight below 300 g/mol, are screened against a protein target using biophysical methods sensitive to weak interactions (μM−mM). As recently reviewed, nuclear magnetic resonance (NMR) is a powerful method for fragment screening,4−8 in particular because NMR can detect interactions at concentrations lower than the dissociation constant KD of the protein−ligand complex.3 In addition, NMR offers the possibility to check for compound aggregation and protein instability, two situations leading to false positives. Among NMR methods, the ligand-observed saturation transfer difference (STD) experiment9 has a number of advantages compared to protein-observed NMR experiments: the protein requirement is much lower, there is no need for labeling, and there is no size limitation for the protein. While the STD experiment is widely used for fragment screening, the method has one major drawback: the technique identifies both specific and nonspecific binders due its capability to accumulate binding signal from multiple binding sites. Consequently, after a STD-based screening campaign, it is necessary to discriminate between specific and nonspecific binders by performing competition STD experiments.10−12 The competitive binders must have a high affinity or a high solubility in order to efficiently displace the fragment. When such competitive reference compounds are not available, an orthogonal biophysical technique is required to validate the fragment hit.1,12,13 The STD experiment provides key information on the solvent accessibility of the ligand protons. This is the basis for © XXXX American Chemical Society

the group epitope mapping approach, where ligand protons buried into the protein structure are distinguished from ligand protons exposed toward the solvent.14 The STD-based epitope mapping is broadly applied for large and flexible ligands, such as oligosaccharides,15 but only few examples have reported STDbased epitope mapping for fragments.16−18 Here we show that STD-based screening is a robust methodology to discriminate between specific and nonspecific protein ligands without competition experiments through the identification of fragments exhibiting a privileged orientation upon interaction with the protein target. This STD-based ligand orientation represents a key criterion of fragment selection for further development.

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RESULTS Fragment screening of 500 compounds was performed against the glycogen phosphorylase GP protein, a therapeutic target in type II diabetes,19 using STD experiments. A high hit rate was obtained (26%), likely due to the identification of nonspecific binders. To discriminate between specific and nonspecific binders, the STD effects (ISTD/I0) were measured for the protons of each ligand. This allows for the distinction of fragments that exhibit similar STD effects from fragments that display STD effect variations. As an example, STD effects measured for 3-quinolinecarboxylic acid, 2-phenylphenol and 2aminopurine (fragments 1, 2 and 3, respectively) are reported in Figure 1. Only fragment 1 displayed significant variations of the STD effects (>10%). This suggests that only fragment 1 binds GP through a preferred orientation, while fragments 2 and 3 are nonspecific binders. Among the hits, 62% displayed STD effect variations. Received: July 16, 2015

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DOI: 10.1021/acs.jmedchem.5b01114 J. Med. Chem. XXXX, XXX, XXX−XXX

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ranging from 50 μM to KD > mM,17,20 indicating that STDbased epitope mapping can be observed in a large range of experimental conditions. One must nevertheless keep in mind that the average binding mode is observed, implying that fragments exhibiting two binding modes with opposite consequences for STD intensities and with similar probabilities will appear as false negative. It is well documented that the saturation time can have an influence on the epitope mapping when the STD signals of protons that display various longitudinal relaxation rates R1 are compared.15,22,23 STD signals are analyzed for aromatic protons only to minimize this issue. The influence of the saturation time was analyzed for 15 protein-fragment complexes, including both specific and nonspecific complexes, using GP, PRDX5, and the human serum albumin as protein models (SI Table S1). No significant differences larger than 10% were observed for the STD effects measured with saturation times ranging from 0.5 s to 4 s, as shown in Figure 2 for indole (fragment 4) (see also SI Figure S3).

Figure 1. STD effects measured for fragments 1 (A), 2 (B), and 3 (C) binding to GP. 1D (STD-off) spectra are displayed in blue; STD spectra are displayed in red. STD effects (ISTD/I0) are reported for each proton. Only the first ligand displayed variations of STD effects >10%, showing that only fragment 1 binds the protein target with a particular orientation.

Figure 2. STD effects measured for indole (fragment 4) upon binding to GP, with saturation times of 0.5 s (green), 2 s (blue) and 4 s (red). As shown, the saturation time does not significantly influence the STD effects measured for the aromatic protons.

Four binding pockets have been reported for GP (the active site, inhibitor site, and two allosteric sites).20 To examine the correlation between STD-based privileged orientation of the hit fragments and the displacement of the fragments by known GP ligands, competitions experiments were performed using reference molecules binding to any of the four GP binding sites.20 All the fragments exhibiting significant STD effect variations (Figure 1A) were displaced by a reference molecule (Supporting Information (SI) Figure S1). However, for fragments that did not exhibit epitope mapping (Figure 1B and 1C), two cases were observed: no displacement of the ligand by the reference molecule (29%) or displacement of the ligand by the reference compound (9%). While the displacement by a reference competitive molecule is informative about the binding site, the epitope mapping is informative about the (average) binding mode. When a ligand binds to a binding site without any preferred binding mode, this will translate into STD effects similar for all ligand protons, as observed for fragment 3. To further illustrate this issue, we report a second example with another 36 kDa protein target (PRDX5) in Supporting Information (SI Figures S2 and S3). While previous results based on 2D protein-observed NMR experiments showed that compounds bis(2-hydroxyphenyl)-methane and 4-methyl-catechol both bind to the PRDX5 active site,21 STD experiments reported here show that only the 4-methylcatechol compound binds with a preferred orientation (SI Figures S2 and S3), which is corroborated by previously published X-ray data.17 STD-based epitope mapping was observed for fragments that bound proteins with large (180 kDa, Figure 1) or moderate (36 kDa, SI Figure S2) molecular weights. In addition, protein− ligand affinities strongly varied in these examples, with KD

The influence of the protein/ligand ratio was also investigated. The STD effects observed were not altered by the ligand/protein ratio, as shown for benzoyleneurea (fragment 5) in Figure 3 (see also SI Figure S3).

Figure 3. STD effects measured for benzoyleneurea (fragment 5) upon binding to GP, with ligand/protein ratios of 66 (green), 200 (blue), and 400 (red). No significant influence of the ligand/protein ratio on the STD-based epitope mapping was observed.

In addition to the selection of fragments binding a protein target with a privileged orientation, STD screening allows for the selection of fragments that display a binding mode driven by polar interactions rather than hydrophobic interactions, as illustrated in Figure 4. The STD spectra of the 7-azaindole (fragment 6) in the presence of GP or ERK1 kinase are compared in Figure 4. The measurement of the STD effects indicated specific binding of the fragment to both proteins. Upon binding to GP, atoms H3 and H4 of ligand 6 were buried, while upon binding to the kinase, atoms H3, H4, and H5 of fragment 6 were exposed to the solvent. The STD data suggest that the recognition is driven by interactions with the nitrogens of fragment 6 in the case of ERK1 kinase. These STD B

DOI: 10.1021/acs.jmedchem.5b01114 J. Med. Chem. XXXX, XXX, XXX−XXX

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recognition toward the protein target. It is also possible to identify and select fragments that bind the protein target through polar interactions. The method can be applied for a large range of protein molecular weights, ligand/protein ratios and ligand affinities, in the absence of any structural data for the protein. Fragments exhibiting a preferred orientation upon receptor binding should be selected for follow up. Figure 4. STD spectra of fragment 6 showing binding driven by polar or hydrophobic interactions. The 1D (STD-off) spectrum of 7azaindole is shown in black. The STD spectrum in the presence of GP is shown in blue. The STD spectrum in the presence of ERK1 kinase is shown in red. STD effects for the ligand protons are displayed, showing that fragment 6 bound with a preferred orientation to both proteins, but that the binding is driven by polar interactions in case of the kinase only.

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EXPERIMENTAL SECTION

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ASSOCIATED CONTENT

Glycogen phosphorylase was purchased from Sigma. Protein peroxiredoxin 521 and kinase ERK1 were produced and purified at the platform of IBCP-Lyon “Bioengineering of proteins” using Escherichia coli. Kinase buffer was 50 mM KPO4 pH 7.1, 0,15 M NaCl, 1 mM DTT, 0.5 mM EDTA, and 2 mM MgCl2. Peroxiredoxin 5 buffer was pH 7.4, NaCl 137 mM, KCl 2.8 mM, Na2HPO4 10 mM, KH2PO4 1.8 mM, and 3 mM DTT. Fragment library was composed of commercial molecules respecting the rule of three.24,25 More details about the fragment library can be found at http://fragmentech.univlyon1.fr. Average molecular weight is 170 g/mol, average log P is 1.03, and average number of heteroatom is 3.7. NMR samples for STD experiments were prepared with 20 μM peroxiredoxin, 2 μM glycogen phosphorylase, and 5 μM kinase, respectively, with 400 μM fragment in 0.5% DMSO-d6 and 10% D2O (v/v). Standard 1D and STD NMR spectra were acquired at 20 °C with a Agilent Inova 600 MHz NMR spectrometer, equipped with a room temperature 5 mm tripleresonance inverse probe with z-axis field gradient. 1D and STD experiments were performed using identical experimental conditions (spin lock, interscan delays), and parameters for the STD experiments (saturation frequency and saturation time) were identical for all samples. Selective saturation of the protein NMR spectrum was achieved with the decoupler offset 2500 Hz upfield from the carrier frequency, and nonsaturation control was performed at 15000 Hz downfield. STD signals were measured for protons in the aromatic region only. The STD effects were measured as the ratio between the intensities of the STD signal and the 1D signal (ISTD/I1D). STD effects were then normalized by setting the largest STD effect to 100%. The error for the calculation of STD effects are estimated to be 10% (errors in NMR peak integration), and this was the cutoff used to distinguish significant STD variations.

data clearly show that the protein−ligand molecular recognition is different for the GP and ERK1 proteins.

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DISCUSSION AND CONCLUSIONS Fragment screening is the first step for the identification and development of molecules using the FBDD approach. Numerous biophysical methods exist for the identification of fragments hits.1 Among them, the NMR STD experiment is recognized as a highly robust technique for fragment screening against protein targets.3,5 Despite many advantages, the main drawback of the STD experiment is to include as fragment hits nonspecific binders and false positives due to compound aggregation. The STD factor (ISTD/I0 × ligand excess) was reported as a tool to roughly rank the ligands or discriminate between strong binders and weak binders. However, this does not prevent from the selection of nonspecific binders. Using the STD factors for ranking fragments 1, 2 and 3 would have promoted compound 2 as the best ligand (the STD factors are 2.1, 6.6, and 1.7 for fragments 1, 2, and 3, respectively). However, fragment 2 should not be selected because of the absence of observable epitope mapping indicating nonspecific binding. Rather than measuring the overall STD factor, one needs to calculate the STD effects for each ligand proton. This ensures identification of ligands with specific protein−ligand recognition. While the accurate determination of the epitope would require the measurement of the proton’s T1,22,23 we have shown here that observing significant variation of STD effects are sufficient to highlight specific binding. The method will nevertheless fail when two preferred orientations of the fragment lead to unobservable (averaged) STD effects. It was recently proposed to record the STD experiment in the presence of detergent to eliminate false positive binders due to aggregation.23 We have performed STD experiment for fragment 2 in the presence of 0.01% Tween (SI Figure S4). The binding to GP is still observed, indicating that the binding of fragment 2 to GP is not due to compound aggregation. The binding of fragment 2 is nevertheless unspecific, without any preferred orientation, and is likely due to π stacking. Another method for hit validation consists of recording competition-STD experiments.4,11,12 Yet, displacement of the fragment by a competitor does not systematically imply that the ligand binds with a privileged binding mode (Figure 1C). As shown here, analysis of STD effects for ligand protons allows the direct identification of specific binders based on the capability of the fragment to display specific molecular

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01114. Competition-STD experiments for fragments 1, 2, and 3 upon binding to the GP protein, STD effects measured for the molecule bis(2-hydroxyphenyl)-methan binding to PRDX5, STD effects measured for the molecule 4methyl-catechol binding to PRDX5, table of proteinfragment complexes.(PDF) Molecular formula strings for compounds that did not display STD-based epitope mapping but were displaced by a competitor upon binding to GP (see Figure 1c) (CSV)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +33 4 37 423 544. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. C

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(17) Aguirre, C.; ten Brink, T.; Guichou, J.-F.; Cala, O.; Krimm, I. Comparing binding modes of analogous fragments using NMR in fragment-based drug design: application to PRDX5. PLoS One 2014, 9 (7), e102300. (18) Pilger, J.; Mazur, A.; Monecke, P.; Schreuder, H.; Elshorst, B.; Bartoschek, S.; Langer, T.; Schiffer, A.; Krimm, I.; Wegstroth, M.; Lee, D.; Hessler, G.; Wendt, K.-U.; Becker, S.; Griesinger, C. A Combination of Spin Diffusion Methods for the Determination of Protein−Ligand Complex Structural Ensembles. Angew. Chem., Int. Ed. 2015, 54 (22), 6511−6515. (19) Gaboriaud-Kolar, N.; Skaltsounis, A.-L. Glycogen phosphorylase inhibitors: a patent review (2008 - 2012). Expert Opin. Ther. Pat. 2013, 23 (8), 1017−1032. (20) Krimm, I.; Lancelin, J.-M.; Praly, J.-P. Binding Evaluation of Fragment-Based Scaffolds for Probing Allosteric Enzymes. J. Med. Chem. 2012, 55 (3), 1287−1295. (21) Barelier, S.; Linard, D.; Pons, J.; Clippe, A.; Knoops, B.; Lancelin, J.-M.; Krimm, I. Discovery of fragment molecules that bind the human peroxiredoxin 5 active site. PLoS One 2010, 5 (3), e9744. (22) Yan, J.; Kline, A. D.; Mo, H.; Shapiro, M. J.; Zartler, E. R. The effect of relaxation on the epitope mapping by saturation transfer difference NMR. J. Magn. Reson. 2003, 163 (2), 270−276. (23) Kemper, S.; Patel, M. K.; Errey, J. C.; Davis, B. G.; Jones, J. A.; Claridge, T. D. Group epitope mapping considering relaxation of the ligand (GEM-CRL): including longitudinal relaxation rates in the analysis of saturation transfer difference (STD) experiments. J. Magn. Reson. 2010, 203 (1), 1−10. (24) Vom, A.; Headey, S.; Wang, G.; Capuano, B.; Yuriev, E.; Scanlon, M. J.; Simpson, J. S. Detection and Prevention of Aggregation-based False Positives in STD-NMR-based Fragment Screening. Aust. J. Chem. 2013, 66 (12), 1518−1524. (25) Congreve, M.; Carr, C.; Murray, C.; Jhoti, H. A ‘rule of three’ for fragment-based lead discovery? Drug Discovery Today 2003, 8 (19), 876−877.

ACKNOWLEDGMENTS We thank Thibaut de Smedt for reading the manuscript. We also thank Stephane Giraud (C3D, CRCL, CLB de Lyon) for discussion.

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ABBREVIATIONS USED NMR, nuclear magnetic resonance; STD, saturation transfer difference; GP, glycogen phosphorylase

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REFERENCES

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DOI: 10.1021/acs.jmedchem.5b01114 J. Med. Chem. XXXX, XXX, XXX−XXX