1o47 Citations

Requirements for specific binding of low affinity inhibitor fragments to the SH2 domain of (pp60)Src are identical to those for high affinity binding of full length inhibitors.

J. Med. Chem. 46 5184-95 (2003)

Cited: 20 times
EuropePMC logo PMID: 14613321


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 (pp60)src 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.

Articles - 1o47 mentioned but not cited (1)

  1. Identification of initial leads directed at the calmodulin-binding region on the Src-SH2 domain that exhibit anti-proliferation activity against pancreatic cancer. Tzou YM, Bailey SK, Yuan K, Shin R, Zhang W, Chen Y, Singh RK, Shevde LA, Krishna NR. Bioorg. Med. Chem. Lett. 26 1237-1244 (2016)

Reviews citing this publication (7)

  1. Lessons from Hot Spot Analysis for Fragment-Based Drug Discovery. Hall DR, Kozakov D, Whitty A, Vajda S. Trends Pharmacol. Sci. 36 724-736 (2015)
  2. 4-Hydroxyphenylpyruvate dioxygenase inhibitors in combination with safeners: solutions for modern and sustainable agriculture. Ahrens H, Lange G, Müller T, Rosinger C, Willms L, van Almsick A. Angew. Chem. Int. Ed. Engl. 52 9388-9398 (2013)
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  4. Survey of the year 2003 commercial optical biosensor literature. Rich RL, Myszka DG. J. Mol. Recognit. 18 1-39 (2005)
  5. A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions. Huber W. J. Mol. Recognit. 18 273-281 (2005)
  6. The discovery of novel protein kinase inhibitors by using fragment-based high-throughput x-ray crystallography. Gill A, Cleasby A, Jhoti H. Chembiochem 6 506-512 (2005)
  7. Fragment-based lead discovery. Rees DC, Congreve M, Murray CW, Carr R. Nat Rev Drug Discov 3 660-672 (2004)

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  1. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid. Salsbury FR, Knutson ST, Poole LB, Fetrow JS. Protein Sci. 17 299-312 (2008)
  2. A new small-molecule Stat3 inhibitor. McMurray JS. Chem. Biol. 13 1123-1124 (2006)
  3. 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. Chipot C, Rozanska X, Dixit SB. J. Comput. Aided Mol. Des. 19 765-770 (2005)
  4. Constraining binding hot spots: NMR and molecular dynamics simulations provide a structural explanation for enthalpy-entropy compensation in SH2-ligand binding. Ward JM, Gorenstein NM, Tian J, Martin SF, Post CB. J. Am. Chem. Soc. 132 11058-11070 (2010)
  5. Identification of novel fragment compounds targeted against the pY pocket of v-Src SH2 by computational and NMR screening and thermodynamic evaluation. Taylor JD, Gilbert PJ, Williams MA, Pitt WR, Ladbury JE. Proteins 67 981-990 (2007)
  6. In silico fragment-based discovery of DPP-IV S1 pocket binders. Rummey C, Nordhoff S, Thiemann M, Metz G. Bioorg. Med. Chem. Lett. 16 1405-1409 (2006)
  7. Superbinder SH2 domains act as antagonists of cell signaling. Kaneko T, Huang H, Cao X, Li X, Li C, Voss C, Sidhu SS, Li SS. Sci Signal 5 ra68 (2012)
  8. Ligand deconstruction: Why some fragment binding positions are conserved and others are not. Kozakov D, Hall DR, Jehle S, Luo L, Ochiana SO, Jones EV, Pollastri M, Allen KN, Whitty A, Vajda S. Proc. Natl. Acad. Sci. U.S.A. 112 E2585-94 (2015)
  9. Crystal structures of a high-affinity macrocyclic peptide mimetic in complex with the Grb2 SH2 domain. Phan J, Shi ZD, Burke TR, Waugh DS. J. Mol. Biol. 353 104-115 (2005)
  10. Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity. Gunzburg MJ, Ambaye ND, Del Borgo MP, Pero SC, Krag DN, Wilce MC, Wilce JA. J. Mol. Recognit. 25 57-67 (2012)
  11. Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase. Gunn NJ, Gorman MA, Dobson RC, Parker MW, Mulhern TD. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 67 336-339 (2011)
  12. Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies. Zeifman AA, Stroylov VS, Novikov FN, Stroganov OV, Zakharenko AL, Khodyreva SN, Lavrik OI, Chilov GG. J Mol Model 18 2553-2566 (2012)