Structure-Based Drug Design to Perturb Function of a tRNA-Modifying Enzyme by Active Site and Protein-Protein Interface Inhibition

Conference paper
Part of the NATO Science for Peace and Security Series A: Chemistry and Biology book series (NAPSA)

Abstract

Drug research increasingly focuses on the interference with protein-protein interface formation as attractive opportunity for therapeutic intervention. The tRNA-modifying enzyme Tgt, a putative drug target to fight Shigellosis, is only functionally active as a homodimer. To better understand the driving forces responsible for assembly and stability of the formed homodimer interface we embarked onto a computational and mutational analysis of the interface-forming residues. We also launched spiking ligands into the interface region to perturb contact formation. We controlled by non-degrading mass spectrometry the actual ratio of monomer-dimer equilibrium in solution and used crystal structure analysis to elucidate the geometrical changes resulting from the induced perturbance. A patch of four aromatic amino acids, embedded into a ring of hydrophobic residues and further stabilized by a network of H-bonds is essential for the dimer contact. Apart from the aromatic hot spot, the interface shows an extended loop-helix motif, which exhibits remarkable flexibility. In the destabilized mutant variants and the complexes with the spiking ligands, the loop-helix motif adopts deviating conformations in the interface region. This motivated us to follow a strategy to raise small molecule binders against this motif to mould the loop geometry in a conformation incompatible with the interface formation.

References

  1. 1.
    Klebe G (2013) Drug design, Chapter 21, Springer Reference, Heidelberg, New York, Dordrecht, London. doi:10.1007/978-3-642-17907-5
  2. 2.
    Romier C, Ficner R, Reuter K, Suck D (1996) Purification, crystallization, and preliminary x-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis. Proteins Struct Funct Genet 24:516–519CrossRefPubMedGoogle Scholar
  3. 3.
    Grädler U, Gerber HD, Goodenough-Lashua DM, Garcia GA, Ficner R, Reuter K, Stubbs MT, Klebe G (2001) A new target for shigellosis: rational design and crystallographic studies of inhibitors of tRNA-guanine transglycosylase. J Mol Biol 306:455–467CrossRefPubMedGoogle Scholar
  4. 4.
    Stengl B, Reuter K, Klebe G (2005) Mechanism and substrate specificity of tRNA – guanine transglycosylases (TGTs): tRNA modifying enzymes from thee three different kingdoms of life seem to share a common mechanism. ChemBioChem 6:1–15CrossRefGoogle Scholar
  5. 5.
    Brenk R, Naerum L, Grädler U, Gerber HD, Garcia GA, Reuter K, Stubbs MT, Klebe G (2003) Virtual screening for submicromolar leads of TGT based on a new unexpected binding mode detected by crystal structure analysis. J Med Chem 46:1133–1143CrossRefPubMedGoogle Scholar
  6. 6.
    Meyer EA, Donati N, Guillot M, Schweizer B, Diederich F, Stengl B, Brenk R, Reuter K, Klebe G (2006) Synthesis, biological evaluation, and crystallographic studies of extended guanine-based (lin-benzoguanine) inhibitors for tRNA-guanine transglycosylase (TGT). Helv Chim Acta 89:573–597CrossRefGoogle Scholar
  7. 7.
    Hörtner S, Ritschel T, Stengl B, Kramer C, Klebe G, Diederich F (2007) Potent inhibitors of tRNA-guanine transglycosylase, an enzyme linked to the pathogenicity of the Shigella bacterium: charge-assisted hydrogen bonding. Angew Chem Int Ed 46:8266–8269CrossRefGoogle Scholar
  8. 8.
    Ritschel T, Kohler PC, Neudert G, Heine A, Diederich F, Klebe G (2009) How to replace the residual solvation shell of polar active site residues to achieve nanomolar inhibition of tRNA-guanine transglycosylase. ChemMedChem 4:2012–2023CrossRefPubMedGoogle Scholar
  9. 9.
    Kohler PC, Ritschel T, Schweizer WB, Klebe G, Diederich F (2009) High-affinity inhibitors of tRNA-guanine transglycosylase replacing the function of a structural water cluster. Chem Eur J 15:10809–10817CrossRefPubMedGoogle Scholar
  10. 10.
    BarandunL IF, Kohler PC, Ritschel T, Heine A, Orlando P, Klebe G, Diederich F (2013) High-affinity inhibitors of Zymomonas mobilis tRNA–guanine transglycosylase through convergent optimization. Acta Crystallogr D69:1798–1807Google Scholar
  11. 11.
    Stengl B, Meyer EA, Heine A, Brenk R, Diederich F, Klebe G (2007) Crystal structures of tRNA-guanine transglycosylase (TGT) in complex with novel and potent inhibitors unravel pronounced induced-fit adaptations and suggest dimer formation upon substrate binding. J Mol Biol 370:492–511CrossRefPubMedGoogle Scholar
  12. 12.
    Xie W, Liu X, Huang RH (2003) Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate. Nat Struct Biol 10:781–788CrossRefPubMedGoogle Scholar
  13. 13.
    Ritschel T, Atmanene C, Reuter K, Van Dorsselaer A, Sanglier-Cianferani S, Klebe G (2009) An integrative approach combining noncovalent mass spectrometry, enzyme kinetics and X-ray crystallography to decipher Tgt protein-protein and protein-RNA interaction. J Mol Biol 393:833–847CrossRefPubMedGoogle Scholar
  14. 14.
    Gohlke H, Kiel C, Case DA (2003) Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes. J Mol Biol 330:891–913CrossRefPubMedGoogle Scholar
  15. 15.
    Jakobi S, Nguyen TXP, Debaene F, Metz A, Sanglier-Cianferani S, Reuter K, Klebe G (2014) Hot-spot analysis to dissect the functional protein–protein interface of a tRNA-modifying enzyme. Proteins Struct Funct Bioinform 82:2713–32PubMedGoogle Scholar
  16. 16.
    Bogan AA, Thorn KS (1998) Anatomy of hot spots in protein interfaces. J Mol Biol 280:1–9CrossRefPubMedGoogle Scholar
  17. 17.
    Immekus F, Barandun LJ, Betz M, Debaene F, Petiot S, Sanglier-Cianferani S, Reuter K, Diederich F, Klebe G (2013) Launching spiking ligands into a protein–protein interface: a promising strategy to destabilize and break interface formation in a tRNA modifying enzyme. ACS Chem Biol 8:1163–1178CrossRefPubMedGoogle Scholar
  18. 18.
    Arkin MR, Wells JA (2004) Small-molecule inhibitors of protein–protein interactions: progressing towards the dream. Nat Rev Drug Discov 3:301–317CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Department of Pharmaceutical ChemistryUniversity of MarburgMarburgGermany

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