ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Requirements for Specific Binding of Low Affinity Inhibitor Fragments to the SH2 Domain of pp60Src Are Identical to Those for High Affinity Binding of Full Length Inhibitors

View Author Information
Aventis Pharma, 102 route de Noisy, 93235 Romainville, France, and Aventis Pharma, Structural Biology, 65926 Frankfurt, Germany
Cite this: J. Med. Chem. 2003, 46, 24, 5184–5195
Publication Date (Web):October 28, 2003
Copyright © 2003 American Chemical Society

    Article Views





    Read OnlinePDF (821 KB)
    Supporting Info (1)»


    Abstract Image

    Results from a novel approach which uses protein crystallography for the screening of a low affinity inhibitor fragment library are analyzed by comparing the X-ray structures with bound fragments to the structures with the corresponding full length inhibitors. The screen for new phospho-tyrosine mimics binding to the SH2 domain of pp60src was initiated because of the limited cell penetration of phosphates. Fragments in our library typically had between 6 and 30 atoms and included compounds which had either millimolar activity in a Biacore assay or were suggested by the ab initio design program LUDI but had no measurable affinity. All identified fragments were located in the phospho-tyrosine pocket. The most promising fragments were successfully used to replace the phospho-tyrosine and resulted in novel nonpeptidic high affinity inhibitors. The significant diversity of successful fragments is reflected in the high flexibility of the phospho-tyrosine pocket. Comparison of the X-ray structures shows that the presence of the H-bond acceptors and not their relative position within the pharmacophore are essential for fragment binding and/or high affinity binding of full length inhibitors. The X-ray data show that the fragments are recognized by forming a complex H-bond network within the phospho-tyrosine pocket of SH2. No fragment structure was found in which this H-bond network was incomplete, and any uncompensated H-bond within the H-bond network leads to a significant decrease in the affinity of full length inhibitors. No correlation between affinity and fragment binding was found for these polar fragments and hence affinity-based screening would have overlooked some interesting starting points for inhibitor design. In contrast, we were unable to identify electron density for hydrophobic fragments, confirming that hydrophobic interactions are important for inhibitor affinity but of minor importance for ligand recognition. Our results suggest that a screening approach using protein crystallography is particularly useful to identify universal fragments for the conserved hydrophilic recognition sites found in target families such as SH2 domains, phosphatases, kinases, proteases, and esterases.


     To whom correspondence should be addressed:  Industriepark Höchst, Building G837, Tel.:  +49-69-30516207, Fax:  +49−69−305−17768, Email:  [email protected].

     Aventis Pharma, Frankfurt, Germany.


     Present address:  Bayer CropScience, Chemistry, 65926 Frankfurt, Germany.

     Present address:  Aventis Research Centre, 13 Quai Jules Guesde, 94403 Vitry, France.

     Aventis Pharma, Romainville, France.

    Supporting Information Available

    Jump To

    1H NMR spectra of compounds 4 and 9. This material is available free of charge via the Internet at

    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system:

    Cited By

    This article is cited by 40 publications.

    1. Hui Zhu, Jincai Yang, Niu Huang. Assessment of the Generalization Abilities of Machine-Learning Scoring Functions for Structure-Based Virtual Screening. Journal of Chemical Information and Modeling 2022, 62 (22) , 5485-5502.
    2. Montgomery Gray, Paige E. Bowling, John M. Herbert. Systematic Evaluation of Counterpoise Correction in Density Functional Theory. Journal of Chemical Theory and Computation 2022, 18 (11) , 6742-6756.
    3. György M. Keserű, Daniel A. Erlanson, György G. Ferenczy, Michael M. Hann, Christopher W. Murray, and Stephen D. Pickett . Design Principles for Fragment Libraries: Maximizing the Value of Learnings from Pharma Fragment-Based Drug Discovery (FBDD) Programs for Use in Academia. Journal of Medicinal Chemistry 2016, 59 (18) , 8189-8206.
    4. Ying Yang and Markus A. Lill . Dissecting the Influence of Protein Flexibility on the Location and Thermodynamic Profile of Explicit Water Molecules in Protein–Ligand Binding. Journal of Chemical Theory and Computation 2016, 12 (9) , 4578-4592.
    5. Joshua M. Ward, Nina M. Gorenstein, Jianhua Tian, Stephen F. Martin and Carol Beth Post. Constraining Binding Hot Spots: NMR and Molecular Dynamics Simulations Provide a Structural Explanation for Enthalpy−Entropy Compensation in SH2−Ligand Binding. Journal of the American Chemical Society 2010, 132 (32) , 11058-11070.
    6. Christian Steinborn, Aldo Tancredi, Christoph Habiger, Christina Diederich, Jan Kramer, Anna M. Reingruber, Bernd Laber, Jörg Freigang, Gudrun Lange, Dirk Schmutzler, Anu Machettira, Gilbert Besong, Thomas Magauer, David M. Barber. Investigations into Simplified Analogues of the Herbicidal Natural Product (+)‐Cornexistin. Chemistry – A European Journal 2023, 29 (39)
    7. Sandra Ardevines, Devan Horn, Juan V. Alegre‐Requena, Marta González‐Jiménez, M. Concepción Gimeno, Eugenia Marqués‐López, Raquel P. Herrera. Enantioselective C−P Bond Formation through C( sp 3 )−H Functionalization. Advanced Synthesis & Catalysis 2023, 365 (13) , 2152-2158.
    8. Karen Vester, Alexander Metz, Simon Huber, Bernhard Loll, Markus C. Wahl. Conformation-dependent ligand hot spots in the spliceosomal RNA helicase BRR2. Acta Crystallographica Section D Structural Biology 2023, 79 (4) , 304-317.
    9. Vibhu Jha, Marco Macchia, Tiziano Tuccinardi, Giulio Poli. Three-Dimensional Interactions Analysis of the Anticancer Target c-Src Kinase with Its Inhibitors. Cancers 2020, 12 (8) , 2327.
    10. Jonathan W. Bogart, Albert A. Bowers. Dehydroamino acids: chemical multi-tools for late-stage diversification. Organic & Biomolecular Chemistry 2019, 17 (15) , 3653-3669.
    11. David R. Hall, Dima Kozakov, Adrian Whitty, Sandor Vajda. Lessons from Hot Spot Analysis for Fragment-Based Drug Discovery. Trends in Pharmacological Sciences 2015, 36 (11) , 724-736.
    12. Dima Kozakov, David R. Hall, Stefan Jehle, Lingqi Luo, Stefan O. Ochiana, Elizabeth V. Jones, Michael Pollastri, Karen N. Allen, Adrian Whitty, Sandor Vajda. Ligand deconstruction: Why some fragment binding positions are conserved and others are not. Proceedings of the National Academy of Sciences 2015, 112 (20)
    13. Sandro Manni, Kaisa Kisko, Thomas Schleier, Jack Missimer, Kurt Ballmer‐Hofer. Functional and structural characterization of the kinase insert and the carboxy terminal domain in VEGF receptor 2 activation. The FASEB Journal 2014, 28 (11) , 4914-4923.
    14. Sambasivarao Kotha, Venkata Babu Bandarugattu, Nimita Gopal Krishna. Diversity-oriented approach to unusual amino acid derivatives and heterocycles via methyl 2-acetamidoacrylate and its congeners. Tetrahedron 2014, 70 (35) , 5361-5384.
    15. Hartmut Ahrens, Gudrun Lange, Thomas Müller, Chris Rosinger, Lothar Willms, Andreas van Almsick. 4-Hydroxyphenylpyruvatdioxygenase-Inhibitoren plus Safener: Lösungen für eine moderne und nachhaltige Landwirtschaft. Angewandte Chemie 2013, 125 (36) , 9558-9569.
    16. Hartmut Ahrens, Gudrun Lange, Thomas Müller, Chris Rosinger, Lothar Willms, Andreas van Almsick. 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors in Combination with Safeners: Solutions for Modern and Sustainable Agriculture. Angewandte Chemie International Edition 2013, 52 (36) , 9388-9398.
    17. Tomonori Kaneko, Haiming Huang, Xuan Cao, Xing Li, Chengjun Li, Courtney Voss, Sachdev S. Sidhu, Shawn S. C. Li. Superbinder SH2 Domains Act as Antagonists of Cell Signaling. Science Signaling 2012, 5 (243)
    18. Alexey A. Zeifman, Victor S. Stroylov, Fedor N. Novikov, Oleg V. Stroganov, Alexandra L. Zakharenko, Svetlana N. Khodyreva, Olga I. Lavrik, Ghermes G. Chilov. Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies. Journal of Molecular Modeling 2012, 18 (6) , 2553-2566.
    19. Michael D. Wendt. Protein-Protein Interactions as Drug Targets. 2012, 1-55.
    20. Menachem J. Gunzburg, Nigus D. Ambaye, Mark P. Del Borgo, Stephanie C. Pero, David N. Krag, Matthew C.J. Wilce, Jacqueline A. Wilce. Interaction of the non‐phosphorylated peptide G7‐18NATE with Grb7‐SH2 domain requires phosphate for enhanced affinity and specificity. Journal of Molecular Recognition 2012, 25 (1) , 57-67.
    21. Natalie J. Gunn, Michael A. Gorman, Renwick C. J. Dobson, Michael W. Parker, Terrence D. Mulhern. Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase. Acta Crystallographica Section F Structural Biology and Crystallization Communications 2011, 67 (3) , 336-339.
    22. Haitao Ji. Fragment‐Based Drug Design: Considerations for Good ADME Properties. 2011, 417-485.
    23. Daniel A. Erlanson. Fragment‐Based Drug Discovery of Kinase Inhibitors. 2009, 461-483.
    24. Sergey N. Tverdomed, Gerd-Volker Röschenthaler, Nataliya Kalinovich, Enno Lork, Alla V. Dogadina, Boris I. Ionin. New α-substituted alkylbenzene- and dialkylbenzene-1,2-diphosphonates: side-chain metalation of tetraethyl 4-methyl- and 4,5-dimethylbenzene-1,2-diphosphonates. Tetrahedron 2008, 64 (22) , 5306-5313.
    25. Freddie R. Salsbury, Stacy T. Knutson, Leslie B. Poole, Jacquelyn S. Fetrow. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid. Protein Science 2008, 17 (2) , 299-312.
    26. Jonathan D. Taylor, Philip J. Gilbert, Mark A. Williams, William R. Pitt, John E. Ladbury. Identification of novel fragment compounds targeted against the pY pocket of v-Src SH2 by computational and NMR screening and thermodynamic evaluation. Proteins: Structure, Function, and Bioinformatics 2007, 67 (4) , 981-990.
    27. Philip J. Hajduk, Jonathan Greer. A decade of fragment-based drug design: strategic advances and lessons learned. Nature Reviews Drug Discovery 2007, 6 (3) , 211-219.
    28. G. Lange. Structure-Based Drug Design – The Use of Protein Structure in Drug Discovery. 2007, 597-650.
    29. C.W. Murray, M.J. Hartshorn. New Applications for Structure-Based Drug Design. 2007, 775-806.
    30. John S. McMurray. A New Small-Molecule Stat3 Inhibitor. Chemistry & Biology 2006, 13 (11) , 1123-1124.
    31. Christian Rummey, Sonja Nordhoff, Meinolf Thiemann, Günther Metz. In silico fragment-based discovery of DPP-IV S1 pocket binders. Bioorganic & Medicinal Chemistry Letters 2006, 16 (5) , 1405-1409.
    32. Christophe Chipot, Xavier Rozanska, Surjit B. Dixit. Can free energy calculations be fast and accurate at the same time? Binding of low-affinity, non-peptide inhibitors to the SH2 domain of the src protein. Journal of Computer-Aided Molecular Design 2005, 19 (11) , 765-770.
    33. Jason Phan, Zhen-Dan Shi, Terrence R. Burke, David S. Waugh. Crystal Structures of a High-affinity Macrocyclic Peptide Mimetic in Complex with the Grb2 SH2 Domain. Journal of Molecular Biology 2005, 353 (1) , 104-115.
    34. Walter Huber. A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions. Journal of Molecular Recognition 2005, 18 (4) , 273-281.
    35. Adrian Gill, Anne Cleasby, Harren Jhoti. The Discovery of Novel Protein Kinase Inhibitors by Using Fragment‐Based High‐Throughput X‐ray Crystallography. ChemBioChem 2005, 6 (3) , 506-512.
    36. Rebecca L. Rich, David G. Myszka. Survey of the year 2003 commercial optical biosensor literature. Journal of Molecular Recognition 2005, 18 (1) , 1-39.
    37. Michael J. Hartshorn, Christopher W. Murray, Anne Cleasby, Martyn Frederickson, Ian J. Tickle, Harren Jhoti. Fragment-Based Lead Discovery Using X-ray Crystallography. Journal of Medicinal Chemistry 2005, 48 (2) , 403-413.
    38. David C. Rees, Miles Congreve,, Christopher W. Murray, Robin Carr. Fragment-based lead discovery. Nature Reviews Drug Discovery 2004, 3 (8) , 660-672.
    39. Daniel A. Erlanson, Robert S. McDowell, Tom O'Brien. Fragment-Based Drug Discovery. Journal of Medicinal Chemistry 2004, 47 (14) , 3463-3482.
    40. Christophe Chipot. Free Energy Calculations in Biological Systems. How Useful Are They in Practice?. , 185-211.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    You’ve supercharged your research process with ACS and Mendeley!

    STEP 1:
    Click to create an ACS ID

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Your Mendeley pairing has expired. Please reconnect