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Target-specific NMR detection of protein–ligand interactions with antibody-relayed 15N-group selective STD

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Abstract

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein–ligand interactions is a key element. 1H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

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References

  • Bhunia A, Bhattacharjya S, Chatterjee S (2012) Applications of saturation transfer difference NMR in biological systems. Drug Discov Today 17:505–513. doi:10.1016/j.drudis.2011.12.016

    Article  Google Scholar 

  • Claasen B, Axmann M, Meinecke R, Meyer B (2005) Direct observation of ligand binding to membrane proteins in living cells by a saturation transfer double difference (STDD) NMR spectroscopy method shows a significantly higher affinity of integrin alpha(IIb)beta(3) in native platelets than in liposomes. J Am Chem Soc 127:916–919. doi:10.1021/ja044434w

    Article  Google Scholar 

  • Cutting B, Shelke SV, Dragic Z, Wagner B, Gathje H, Kelm S, Ernst B (2007) Sensitivity enhancement in saturation transfer difference (STD) experiments through optimized excitation schemes. Magn Reson Chem 45:720–724. doi:10.1002/mrc.2033

    Article  Google Scholar 

  • Dettmann W et al (2000) Differences in zero-force and force-driven kinetics of ligand dissociation from beta-galactoside-specific proteins (plant and animal lectins, immunoglobulin G) monitored by plasmon resonance and dynamic single molecule force microscopy. Arch Biochem Biophys 383:157–170. doi:10.1006/abbi.2000.1993

    Article  Google Scholar 

  • Di Micco S, Bassarello C, Bifulco G, Riccio R, Gomez-Paloma L (2006) Differential-frequency saturation transfer difference NMR spectroscopy allows the detection of different ligand-DNA binding modes. Angew Chem Int Edit 45:224–228. doi:10.1002/anie.200501344

    Article  Google Scholar 

  • Groves P et al (2007) Temperature dependence of ligand-protein complex formation as reflected by saturation transfer difference NMR experiments. Magn Reson Chem 45:745–748. doi:10.1002/mrc.2041

    Article  Google Scholar 

  • Harner MJ, Frank AO, Fesik SW (2013) Fragment-based drug discovery using NMR spectroscopy. J Biomol NMR 56:65–75. doi:10.1007/s10858-013-9740-z

    Article  Google Scholar 

  • Hirabayashi J, Kasai KI (1994) Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1. Glycoconj J 11:437–442. doi:10.1007/Bf00731280

    Article  Google Scholar 

  • Hoffer L, Renaud JP, Horvath D (2011) Fragment-based drug design: computational and experimental state of the art. Comb Chem High Throughput Screen 14:500–520

    Article  Google Scholar 

  • Jhoti H, Cleasby A, Verdonk M, Williams G (2007) Fragment-based screening using X-ray crystallography and NMR spectroscopy. Curr Opin Chem Biol 11:485–493. doi:10.1016/j.cbpa.2007.07.010

    Article  Google Scholar 

  • Kay LE, Torchia DA, Bax A (1989) Backbone dynamics of proteins as studied by N-15 inverse detected heteronuclear NMR-spectroscopy: application to staphylococcal nuclease. Biochemistry (Us) 28:8972–8979. doi:10.1021/Bi00449a003

    Article  Google Scholar 

  • Kobayashi M et al (2010) Target immobilization as a strategy for NMR-based fragment screening: comparison of TINS, STD, and SPR for fragment hit identification. J Biomol Screen 15:978–989. doi:10.1177/1087057110375614

    Article  Google Scholar 

  • Kovacs-Solyom F et al (2010) Mechanism of tumor cell-induced T-cell apoptosis mediated by galectin-1. Immunol Lett 127:108–118. doi:10.1016/j.imlet.2009.10.003

    Article  Google Scholar 

  • Kövér KE, Groves P, Jimenez-Barbero J, Batta G (2007) Molecular recognition and screening using a N-15 group selective STD NMR method. J Am Chem Soc 129:11579–11582. doi:10.1021/ja073291I

    Article  Google Scholar 

  • Kövér KE, Weber E, Martinek TA, Monostori E, Batta G (2010) N-15 and C-13 group-selective techniques extend the scope of STD NMR detection of weak host–guest interactions and ligand screening. ChemBioChem 11:2182–2187. doi:10.1002/cbic.201000317

    Article  Google Scholar 

  • Krishna NR, Jayalakshmi V (2006) Complete relaxation and conformational exchange matrix analysis of STD-NMR spectra of ligand–receptor complexes. Prog Nucl Magn Reson Spectrosc 49:1–25. doi:10.1016/j.pnmrs.2006.03.002

    Article  Google Scholar 

  • Krishnan VV (2005) Ligand screening by saturation-transfer difference (STD) NMR spectroscopy. Curr Anal Chem 1:307–320. doi:10.2174/157341105774573956

    Article  Google Scholar 

  • Larsson A, Jansson A, Aberg A, Nordlund P (2011) Efficiency of hit generation and structural characterization in fragment-based ligand discovery. Curr Opin Chem Biol 15:482–488. doi:10.1016/j.cbpa.2011.06.008

    Article  Google Scholar 

  • Mayer M, Meyer B (1999) Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int Edit 38:1784–1788. doi:10.1002/(Sici)1521-3773(19990614)38:12<1784:Aid-Anie1784>3.0.Co;2-Q

    Article  Google Scholar 

  • Meyer B, Peters T (2003) NMR Spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew Chem Int Edit 42:864–890. doi:10.1002/anie.200390233

    Article  Google Scholar 

  • Murray CW, Rees DC (2016) Opportunity knocks: organic chemistry for fragment-based drug discovery (FBDD). Angew Chem Int Edit 55:488–492. doi:10.1002/anie.201506783

    Article  Google Scholar 

  • Norton RS (2013) Fragment-based drug discovery. Aust J Chem 66:1463–1464. doi:10.1071/CH13492

    Article  Google Scholar 

  • Rahman M, Ismat F, McPherson MJ, Baldwin SA (2007) Topology-informed strategies for the overexpression and purification of membrane proteins. Mol Membr Biol 24:407-U416. doi:10.1080/09687860701243998

    Article  Google Scholar 

  • Silvestre HL, Blundell TL, Abell C, Ciulli A (2013) Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery. Proc Natl Acad Sci USA 110:12984–12989. doi:10.1073/pnas.1304045110

    Article  ADS  Google Scholar 

  • Viegas A, Manso J, Nobrega FL, Cabrita EJ (2011) Saturation-transfer difference (STD) NMR: a simple and fast method for ligand screening and characterization of protein binding. J Chem Educ 88:990–994. doi:10.1021/ed101169t

    Article  Google Scholar 

  • Zartler ER et al (2003) RAMPED-UP NMR: multiplexed NMR-based screening for drug discovery. J Am Chem Soc 125:10941–10946. doi:10.1021/ja0348593

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Hungarian Research Foundation OTKA projects PD83600 and K105459 and the Hungarian Academy of Sciences, Lendület program (LP-2011-009). This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/2-11/1-2012-0001 ‘National Excellence Program’. The research was supported by the EU and co-financed by the European Regional Development Fund under the projects GINOP-2.3.3-15-2016-00004 and GINOP-2.3.3-15-2016-00010.

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Authors and Affiliations

  1. Department of Medical Chemistry, University of Szeged, Dóm t. 8., Szeged, 6720, Hungary

    Anasztázia Hetényi

  2. SZTE-MTA Lendület Foldamer Research Group, Institute of Pharmaceutical Analysis Department, University of Szeged, Somogyi u. 4, Szeged, 6720, Hungary

    Zsófia Hegedűs & Tamás A. Martinek

  3. Lymphocyte Signal Transduction Laboratory, Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62, Szeged, 6726, Hungary

    Roberta Fajka-Boja & Éva Monostori

  4. Department of Inorganic and Analytical Chemistry, University of Debrecen, Egyetem tér 1., Debrecen, 4032, Hungary

    Katalin E. Kövér

Authors
  1. Anasztázia Hetényi
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  2. Zsófia Hegedűs
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  3. Roberta Fajka-Boja
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  4. Éva Monostori
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  5. Katalin E. Kövér
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  6. Tamás A. Martinek
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Corresponding author

Correspondence to Tamás A. Martinek.

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Hetényi, A., Hegedűs, Z., Fajka-Boja, R. et al. Target-specific NMR detection of protein–ligand interactions with antibody-relayed 15N-group selective STD. J Biomol NMR 66, 227–232 (2016). https://doi.org/10.1007/s10858-016-0076-3

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