19F NMR-Guided Design of Glycomimetic Langerin Ligands - ACS

Jul 26, 2016 - As mammalian carbohydrate binding sites typically display high solvent exposure and hydrophilicity, the recognition of natural monosacc...
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F NMR-Guided Design of Glycomimetic Langerin Ligands

Eike-Christian Wamhoff,†,‡ Jonas Hanske,†,‡ Lennart Schnirch,‡ Jonas Aretz,†,‡ Maurice Grube,†,‡ Daniel Varón Silva,†,‡ and Christoph Rademacher*,†,‡ †

Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, 14424 Potsdam, Germany Freie Universität Berlin, Department of Biology, Chemistry and Pharmacy, 14195 Berlin, Germany



S Supporting Information *

ABSTRACT: C-type lectin receptors (CLRs) play a pivotal role in pathogen defense and immune homeostasis. Langerin, a CLR predominantly expressed on Langerhans cells, represents a potential target receptor for the development of anti-infectives or immunomodulatory therapies. As mammalian carbohydrate binding sites typically display high solvent exposure and hydrophilicity, the recognition of natural monosaccharide ligands is characterized by low affinities. Consequently, glycomimetic ligand design poses challenges that extend to the development of suitable assays. Here, we report the first application of 19F R2-filtered NMR to address these challenges for a CLR, i.e., Langerin. The homogeneous, monovalent assay was essential to evaluating the in silico design of 2-deoxy-2-carboxamido-α-mannoside analogs and enabled the implementation of a fragment screening against the carbohydrate binding site. With the identification of both potent monosaccharide analogs and fragment hits, this study represents an important advancement toward the design of glycomimetic Langerin ligands and highlights the importance of assay development for other CLRs.

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compensated for by the multivalent presentation of either carbohydrate or CLR in heterogeneous assays.21 Yet, these methods introduce complex equilibria and surface phenomena, complicating data analysis as well as limiting the transferability of results between assay formats.22 For Langerin, heterogeneous assays have been utilized to quantify the binding specificity of Langerin for a larger set of mono- and oligosaccharides.4,5,23−25 Galactose-6-sulfate (Gal-6S, KI,app = 3.1 ± 0.4 mM) and mannose (Man, KI,app = 4.4 ± 0.1 mM) represent the most potent natural monosaccharide ligands identified. Alternatively, ITC and 1H STD NMR have served to determine the affinity for Man (KD = 6.1 ± 0.4 mM) and a heparin-derived trisaccharide (KD = 0.49 ± 0.05 mM). These biophysical methods are, however, limited with respect to throughput and material consumption. R2-filtered NMR is an additional, well-established method for the detection of ligand−receptor interactions.26 In combination with the high chemical shift anisotropy observed for 19F NMR, it provides excellent sensitivity, enabling the utilization of monovalent reporter molecules even at low affinities.27 Based on an analytical expression for the equilibrium of competitive binding experiments, the determination of KI values directly yields information on the binding site and of the stoichiometry of a glycomimetic ligand.28 The absence of background resonances from, e.g., solvent, additives, or the receptor in

-type lectin receptors (CLRs) are a family of pattern recognition receptors involved in pathogen defense and immune homeostasis.1 Langerin, a trimeric CLR, predominantly expressed on human Langerhans cells and CD103+ dermal dendritic cells (DC), has been shown to interact with self-associated as well as microbial carbohydrates.2−5 The latter comprise epitopes from, e.g., HIV, Mycobacterium leprae, and Candida albicans.6−8 Langerin is an endocytic receptor triggering antigen processing and subsequent presentation via MHC I, MHC II, and CD1a.7,9,10 These characteristics render Langerin an intriguing target receptor for the development of anti-infectives or immunomodulatory therapies.11,12 CLR binding sites are, however, typically solvent exposed and hydrophilic.13 These features contribute to the promiscuity and low affinities observed for carbohydrate recognition, generally impeding glycomimetic ligand design. Examples of high affinity ligands for CLRs are scarce with E-Selectin serving as a paradigm for the rational evolution of a carbohydrate scaffold into a drug.14 Recently, synthetic strategies to generate focused glycomimetic libraries have provided access to the three-dimensional diversity of the carbohydrate scaffold.15,16 Potent noncarbohydrate glycomimetics for DC-SIGN have been identified in a high-throughput screening.17 Alongside promising advances for additional CLRs, a systematic druggability evaluation in our laboratory justifies glycomimetic ligand design for this receptor family.18−20 However, no ligand design studies have been reported for Langerin. Likewise, assay development for CLRs remains challenging, particularly in the initial phase of the design process. Sensitivity issues and a lack of suitable reporter molecules have been © XXXX American Chemical Society

Received: June 28, 2016 Accepted: July 26, 2016

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DOI: 10.1021/acschembio.6b00561 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 1. 19F R2-filtered NMR assay for Langerin. (a) The structure of the reporter molecule served as a scaffold for the in silico design. In the presence of the Langerin ECD, the 19F NMR resonance of the trifluoroacetamido group displayed line broadening. (b) The Ca2+-dependent interaction between the reporter molecule and Langerin can be quantified via the relaxation rate R2,obs using the CPMG pulse sequence. Representative decay curves for the ECD are shown. (c) Titration experiments revealed comparable KD values of 7.9 ± 0.7 mM and 7.3 ± 1.0 mM for ECD and CRD, respectively. A significantly higher amplitude in R2,obs was observed for the ECD. R2,obs values for the ECD were determined in triplicate. (d) Competitive binding experiments yield KI values of 4.5 ± 0.5 mM and 24.9 ± 1.0 mM for Man and ManNAc, respectively. While the former were in accordance with literature data, significant deviations are observed for ManNAc. 19

While no chemical shift perturbation (CSP) was detected, the substantial line broadening observed indicates binding to Langerin. Next, the interaction between Langerin and 5.1 was quantified via the measurement of observed relaxation rates R2,obs in CPMG experiments (Figure 1b and c, eqs 1−3).30 As the validity of the analytical expressions are limited to the fast chemical exchange regime, we excluded a chemical exchange contribution R2,ex to R2,obs via relaxation dispersion experiments (Figure S2). Titration experiments served to determine the affinity of both the carbohydrate recognition domain (CRD; KD = 7.3 ± 1.0 mM) and the trimeric extracellular domain (ECD; KD = 7.9 ± 0.7 mM) for 5.1. The Ca2+ dependency of the interaction was validated by the addition of EDTA (Figure 1b and S2). Notably, an increased relaxation rate for the bound state R2,b is observed due to increased molecular weight of the native, trimeric ECD (M = 68.7 kDa) over the CRD (M = 18.0 kDa) resulting in an improved dynamic range for competitive binding experiments (Table S1). Complete competition with 5.1 binding was shown for titration with Man (KI = 4.5 ± 0.5 mM; Figure 1d). These results are in accordance with literature values, while the affinity obtained for ManNAc (KI = 24.9 ± 0.9 mM) deviates significantly from a previously published apparent KI value.24 To validate our findings, orthogonal 15N HSQC NMR titration

F NMR generally improves data quality and facilitates sample preparation. The approach can be readily applied to screen, e.g., focused libraries, providing robust ranking of the identified hits. Here, we present a monovalent 19F R2-filtered NMR assay for Langerin that meets the specific requirements of glycomimetic ligand design. The assay was utilized to determine KI values for a library of 2-deoxy-2-carboxamido-α-mannoside analogs designed in silico. The design led to the identification of an analog with a 6-fold affinity increase over natural monosaccharide ligands. Furthermore, a glycomimetic fragment was identified in an explorative fragment screening against the carbohydrate binding site of Langerin. These results represent an important advancement toward the design of glycomimetic Langerin ligands and highlight the importance of assay development for other CLRs. To implement the 19F R2-filtered NMR assay, a reporter molecule was designed based on N-acetylmannosamine (ManNAc; KI,app = 5.6 ± 0.6 mM; Figure 1a).24 The synthesis of 2-deoxy-2-trifluoroacetamido-α-mannoside analog 5.1 involved the stereospecific introduction of a propargyl group at the anomeric position (Scheme S1, Figure S1). The 19F NMR spectrum of 5.1 displays a single resonance for the trifluoroacetamido group, ensuring optimal signal-to-noise ratios (SNR). Moreover, the absence of J-coupling is a requirement for the utilization of the CPMG pulse sequence.29 B

DOI: 10.1021/acschembio.6b00561 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology Table 1. Affinities of 2-Carboxamido-2-deoxy-α-mannoside Analogs for Langerin

a

n = 3. bDetermined via 15N HSQC NMR. cDetermined via 19F R2-filtered NMR.

was conjugated to the Man scaffold in situ. The docking procedure involved a grid-based placement, as well as a force field-based pose refinement stage. A pharmacophore model served to constrain the orientation of the Man scaffold during the refinement and to filter docking poses. Retained docking poses were scored with the GBIV/WSA ΔG function and ranked according to the predicted affinity increase A over the reference molecule 5.2.32 On the basis of these simulations, 12 analogs were selected for synthesis (Scheme S1, Figure S5). The focused library was composed to explore a diverse set of structure-based binding hypotheses. Analogs 5.5 and 5.10 were selected to target the distal pocket, while substituents of low complexity were evaluated for their potential to form favorable interactions with the proximal pocket (Figure S4). The focused library was synthesized and subsequently characterized via the 19F R2-filtered NMR assay (Table 1, Figure S6). Overall, the SAR is dominated by a negative contribution of the carboxamide linker in C2. Only a minor affinity increase was observed for substituents targeting the distal pocket. By contrast, the SAR obtained for the proximal pocket revealed more potent analogs. The negatively charged substituents of 5.7 (KI = 7.1 ± 1.7 mM) and 5.11 (KI = 1.3 ±

experiments were performed, yielding comparable affinities (Table 1, Figure S3). The observed CSPs support our hypothesis of a similar binding mode for Man and 5.1 (Figure 2, Table S2). Hence, the deviations from literature may reflect the limitations of heterogeneous assays discussed above. We conclude that the 19F R2-filtered NMR assay represents a robust method to determine KI values for glycomimetic Langerin ligands. To demonstrate the applicability of the 19F R2-filtered NMR assay to different ligand design strategies, we expanded our analysis to a focused library of 2-deoxy-2-carboxamido-αmannoside analogs 5 (Figure 1a, Table 1). A structure-based in silico screening was implemented to generate this library (Figure S4).31 Man recognition by Langerin depends on the Ca2+-coordination by two equatorial hydroxyl groups and involves only few secondary interactions.3 Two pockets flanked by K299, K313, and F315 were identified in the axial direction of C2. As substituents in this direction seemed to be tolerated, we decided to target these pockets by diversification of the Man scaffold in C2. To this end, a conformation database of 24 800 commercially available carboxylic acids was generated, and each conformer C

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Figure 2. Affinity validation and binding mode analysis via 15N HSQC NMR. (a) The docking pose reveals a salt bridge formed between the sulfonate of 5.11 and K299 as well as N307. Hydrophilic regions of the receptor surface are depicted in blue while hydrophobic regions are depicted in red. (b) The 19F R2-filtered NMR assay served to determine a 6-fold affinity increase over 5.0 and a 36-fold affinity increase over 5.2 for 5.11. (c) The KI value of 1.3 ± 0.1 mM was validated via 15N HSQC NMR experiments with the CRD and a KD value of 4.3 ± 0.2 mM was obtained. Representative binding isotherms for selected resonances are shown. (d) A comparison of 15N HSQC NMR spectra in the presence or absence of different ligands reveals CSP fingerprints. The observed fingerprints suggest a similar binding mode for Man, 5.1 and 5.11.

0.1 mM) resulted in substantially improved KI values over the reference molecule 5.2 (KI = 48.0 ± 5.9 mM). With this 36-fold affinity increase, 5.11 represents the most potent member of the focused library and displays an excellent group efficiency (GE = 2.23 kJ·mol−1; Figure 2b).33 The affinity increase over Man was validated via 15N HSQC NMR (Table 1 and Figure 2c). All CSPs over 0.04 ppm observed for Man also occur upon titration with 5.11 (Table S2). The additional CSPs as well as the affinity increase may be rationalized by salt bridges between the sulfonate group and K299 as well as N307 (Figure 2a). A similar interaction has been observed for Gal6S.3 The propargyl group enables the utilization of analogs 5 as tool molecules to investigate DC immunology, e.g., via their conjugation to nanoparticles. The conjugation of mono- and oligosaccharides is typically achieved via the anomeric position. Notably, the formation of α-mannosides results in decreased affinities for Langerin as observed for the deviation between reference molecule 5.0 (KI = 7.6 ± 0.1 mM) and Man (Figure 2b). In this context, 5.11 displays an effective 6-fold affinity increase over Gal-6S and Man. The sulfonate does, furthermore, improve hydrolytic stability when compared to Gal-6S. Finally, we explored the applicability of the 19F R2-filtered NMR assay to the screening of larger libraries (Figure 3). We initially evaluated the assay performance of the original setup utilized for the characterization of the focused library of analogs 5 (setup 1). Next, the performance of an alternative setup with

improved experimental times and reduced material consumption was simulated (setup 2). The accuracy of setup 1 was assessed from KI values determined in triplicate (rel. SEM = 0.09; Table 1, Figure 3a). Additionally, an excellent Z-score was determined to quantify the quality of the assay (Z-score = 0.69).34 The simulation of binding isotherms at different KI values indicates that satisfactory sensitivity is maintained for affinities of up to 1 μM. Setup 1 involves the determination of KI values from R2,obs values at five or more ligand concentrations (Figures 1d and 2b, Figure S6). For each R2,obs value, CPMG experiments were conducted at five relaxation times (Figure 1b). To achieve a good SNR for the 19F NMR spectra, the determination of a KI value required 225 min on a regular 600 MHz spectrometer. In combination with a receptor consumption mP of approximately 1 mg per titration experiment, setup 1 is not suitable for screening larger libraries. The simulation of the alternative setup was implemented by a retrospective reduction of the analyzed data set. To this end, KI values were estimated from data at single concentrations for each ligand. Data points were selected if the concentrations fell within 1 order of magnitude of the KI value determined in setup 1 (Figure S2). The hereby-estimated KI value falls within 46% of the real KI value for 99% of the experiments (σ = 0.23; Figure 3a). The experimental time for setup 2 amounts to 45 min per ligand, and the receptor consumption is reduced to 0.2 mg. Moreover, screening a library at a concentration around the KI of the parent scaffold reduces the ligand consumption by D

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Figure 3. Assay performance in screening applications. (a) The simulated binding isotherms indicate the lower limit of accurate affinity determination Setup 1. The isotherms begin to converge at KI values below 1 μM. Setup 2 involves the estimation of KI values from single ligand concentrations and allows e.g. for the rapid elucidation of SARs. (b) Setup 2 was utilized for an explorative fragment screening. While only few hits were identified, Man was reliably detected at a concentration of 0.5 mM (n = 3 and p < 0.01, Student’s t test). (c) The fragment mixture displaying the highest competition was analyzed by 1H STD NMR. Overall, four out of six fragments were found to interact with Langerin. STD spectra are magnified 6.25-fold. (d) The deconvolution of this mixture revealed an estimated KI value of 9.7 ± 1.2 mM for fragment 8 (n = 3 and p < 0.01, Student’s t test). 15

about 1 order of magnitude compared to complete titration experiments. These features facilitate the elucidation of SARs for larger focused glycomimetic libraries. Setup 2 was then utilized for an explorative fragment screening to identify novel glycomimetic scaffolds (Figure 3b). As low to moderate hit rates are expected for carbohydrate binding sites, five to six fragments were binned in one mixture. Overall, 290 fragments were randomly selected from our inhouse library and screened at concentrations of 0.5 mM (Figure S7). While Man was reliably detected at the same concentration, only four fragment mixtures with significantly reduced R2,obs values were identified, corresponding to a low hit rate of 1.4%. Moreover, none of these mixtures displayed an apparent KI value below 5 mM. These findings highlight the challenges of glycomimetic ligand design for Langerin as well as the utility of the designed analog 5.11. The fragment mixture with the highest competition (ΔR2,obs = 0.39 Hz) was subsequently analyzed by 1H STD NMR. While no STD effect was observed in the absence of Langerin, binding was detected for four of the six fragments (Figures 3c and S7). The deconvolution of the mixture at concentrations of 1 mM revealed fragment 8 (KI,est = 9.7 ± 1.2 mM) as a potential glycomimetic, exemplifying the assay’s ability to detect low affinity fragment binding (Figure 3d and S7). In conclusion, we have demonstrated the utility of 19F R2filtered NMR for the design of glycomimetic Langerin ligands. Monovalent reporter molecule 5.1 was designed to establish a homogeneous competitive binding assay which was validated by

N HSQC NMR experiments. Next, a focused library of 2deoxy-2-carboxamido-α-mannoside analogs 5 was designed in silico and subsequently synthesized. The 19F R2-filtered NMR assay served to determine KI values for these analogs, and small, negatively charged substituents in axial direction of C2 were found to substantially increase affinities. In spite of the unfavorable ManNAc parent scaffold, the in silico design afforded a 6-fold affinity increase over natural monosaccharide ligands. Analog 5.11 (KI = 1.3 ± 0.1 mM) represents a novel tool molecule to investigate the role of Langerin in DC immunology. Furthermore, we showed that the 19F R2-filtered NMR assay is suitable for screening applications. Experiments at a single ligand concentration allow for a rapid and robust estimation of KI values at low material consumption. In an explorative fragment screening, we found a low hit rate of 1.4% and low affinities for the carbohydrate binding site of Langerin. Nevertheless, glycomimetic fragment 8 was identified (KI,est = 9.7 ± 1.2 mM), and binding was confirmed by 1H STD NMR experiments. Enabled by the assays excellent sensitivity, a comprehensive screening of our in-house fragment library will potentially yield additional glycomimetic scaffolds for a fragment-based ligand design approach and is currently ongoing in our laboratories. The presented 19F NMR-guided approach is of particular utility to glycomimetic ligand design and potentially transferrable to other CLRs. While reporter molecule 5.1 may be utilized for receptors recognizing Man or ManNAc, the E

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functionalization of other monosaccharides with, ideally, trifluoromethyl groups potentially broadens its scope.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00561. Figures S1−S7, Scheme S1, and Tables S1 and S2 as well as the Methods (Molecular Modeling, Receptor Expression and Purification, Synthetic Chemistry, 19F R2filtered NMR, 15N HSQC NMR and 1H STD NMR) (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by the German Research Foundation (RA1944/2-1), the Max Planck Society, the Beilstein Institute for the Advancement of the Chemical Sciences, the Max Planck-RIKEN Joint center for Systems Chemical Biology, and the Collaborative Research Center 765. The authors thank O. Niemeyer for technical assistance and Dr. P. H. Seeberger for support and helpful discussions.



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DOI: 10.1021/acschembio.6b00561 ACS Chem. Biol. XXXX, XXX, XXX−XXX