Occupying a flat subpocket in a tRNA-modifying enzyme with ordered or disordered side chains: Favorable or unfavorable for binding?
Graphical abstract
Introduction
Concepts in rational drug design try to optimize a given ligand scaffold to accommodate and interact optimally with a certain target protein. Our hypotheses and thus the followed design strategies are usually strongly biased by the idea to achieve perfect complementarity between protein-binding pocket and ligand in terms of geometry and experienced interaction patterns. We assume that this strategy leads to energetically favored interactions and a strong binding affinity. By contrast, any residual mobility or intrinsic flexibility of the bound ligand are intuitively assumed as unfavorable for binding. However, binding affinity is a Gibbs energy value and thus composed by an enthalpic and entropic component. With respect to a favorable entropic component to binding, residual mobility or pronounced ligand disorder can be beneficial as a smaller number of degrees of freedom are lost by the ligand upon complex formation.
Residual mobility of ligand side chains or attached substituents is usually observed in flat, solvent-exposed binding pockets where significant contacts to the neighboring solvent environment are given. In a congeneric series of thermolysin inhibitors with varying P2′ substituents of increasing size and hydrophobicity, we observed on the one hand a growing influence imposed by the structural arrangement of the surrounding first and second solvent layer but on the other hand by an enhanced residual flexibility particularly given for larger substituents.1, 2
As a second example, we studied the binding of potent inhibitors blocking the function of the tRNA-modifying enzyme tRNA-guanine transglycosylase (TGT) by attaching hydrophobic substituents of growing size at the parent lin-benzoguanine scaffold.3, 4, 5, 6, 7 The target protein performs a complete nucleobase exchange at the wobble position of specific tRNAs.8 Inhibition of bacterial TGT may serve as a putative therapeutic concept for drug development as its function has been linked to the pathogenicity of Shigella, the causative agent of bacterial dysentery.9 In developing countries, Shigellosis is a major threat as infections with these bacteria are responsible for more than 100,000 lethal cases every year.10, 11, 12 In previous studies, we attached substituents at the 2- and 4-position of the parent scaffold and in combination of both.7 The 2-substituents orient into an open, bowl-shaped pocket that recognizes uracil 33 with its adjacent ribose ring of the natural substrate tRNA.13 In addition, we have developed derivatives, exhibiting substituents at the 4-position, designed to occupy the ribose-34 pocket. In the subsequently determined crystal structures, the position of the 4-substituent could be assigned unequivocally to the difference electron density, while the substituents, anchored at C2, indicated in most of the determined crystal structures enhanced mobility or at least a scatter across multiple conformational states to varying extend.4, 7, 13, 14 This observation suggests that the 2-substituent, depending on its chemical composition, experiences pronounced flexibility in the bound state.
Closer inspection of the architecture of the uracil-33 pocket indicates the exposure of polar and non-polar residues in this binding cavity. In consequence, lin-benzoguanines with alternative 2-substituents were synthesized following the design hypothesis to fix the regarded ligand portion in the uracil-33 pocket by addressing the available polar contacts of the protein. In the present study, we investigated ligands exhibiting an extended phenethyl substituent in 2-position by molecular dynamics (MD) simulations, crystal structure analysis, isothermal titration calorimetry (ITC), and by a radioactive washout-binding assay. At their terminal end, the phenethyl substituents were decorated with different polar functional groups, principally capable to establish the desired contacts with the protein. The consequences of these modifications are described with respect to the obtained binding poses, dynamics, and energetic properties.
Section snippets
Ligand design
We recently reported on the crystal structures of the parent 2-methylamino-lin-benzoguanine (1), the 2-thienylmethyleneamino (2), 2-piperidylethylenamino- (3), and 2-morpholinoethylenamino (4) derivatives (Scheme 1).13, 14
All crystal structures showed the tricyclic ring system in ordered state with full occupancy. In the complex crystal structure with 1, a network of five water molecules is found wrapping around the terminal methyl group. The two derivatives 3 and 4 with an ethylene linker show
Discussion
In the present study, we investigated the conformational properties and the residual mobility of extended substituents oriented into a flat bowl-shaped binding pocket that recognizes the nucleobase uracil-33 with its attached ribose sugar ring of the natural tRNA substrate. Crystal structure analysis suggests residual mobility of the placed substituents in the protein bound state. This residual dynamic property is indicated by enhanced B-factors attributed to the atoms of the substituents. Not
Conclusions
Protein-ligand binding occurs overwhelmingly in surface-exposed depressions and cavities on a protein. The available pockets are of different shape, burial, and physicochemical composition. The affinity of ligand binding to such pockets depends on the strength of the interactions established in these pockets, the change in the dynamic properties and thus in the degrees of freedom of the system, and the modulation of the local solvent structure.
Usually in deep and highly buried binding pockets,
Enzyme assay
The enzyme kinetic characterization of TGT is based on a method described by Grädler et al.28 and Stengl et al.29 Due to low solubility, the ligands were dissolved in 100% DMSO and subsequently diluted to the desired concentration with 5% DMSO with the assay buffer. The protein dissolved in the same buffer was added to the various ligand samples with a final concentration of 9 nm and incubated for 10 min at 37 °C. Additionally, a reference without the ligand only containing DMSO and buffer was
Acknowledgements
We are grateful to the beamline staff at BESSY II (Helmholtz-Zentrum Berlin) and Petra in Hamburg (Desy, EMBL) for providing outstanding support during data collection. We also thank the Helmholtz-Zentrum Berlin for the travel support. We are grateful to the Chemical Computing Group (Montreal, Canada) for making the MOE program available to us. The usage of the AMBER program suite is kindly acknowledged. The present study was supported by the ERC Advanced Grant no. 268145-DrugProfilBind awarded
References and notes (53)
- et al.
J. Mol. Biol.
(2007) - et al.
Int. J. Antimicrob. Agents
(2012) - et al.
Synlett
(2008) - et al.
J. Mol. Biol.
(2006) Acta Crystalogr., Sect. D
(2010)- et al.
ChemBioChem
(2005) - et al.
Proteins: Struct., Funct., Bioinf.
(1996) Acta Crystallogr., Sect. D
(2010)- et al.
J. Comput.-Aided Mol. Des.
(1997) - et al.
Proteins: Struct., Funct., Bioinform.
(2006)
ChemMedChem
Angew. Chem., Int. Ed.
Chem. Eur. J.
ChemMedChem
ChemBioChem
Acta Crystallogr., Sect. D
Nat. Struct. Biol.
WHO Bull.
J. Bacteriol.
Nat. Rev. Microbiol.
J. Med. Chem.
J. Med. Chem.
Chem. Eur. J.
Angew. Chem.
Angew. Chem., Int. Ed.
Cited by (8)
Profiling Drug Binding by Thermodynamics: Key to Understanding
2017, Multi-Scale Approaches in Drug Discovery: From Empirical Knowledge to In silico Experiments and BackStrategies for Late-Stage Optimization: Profiling Thermodynamics by Preorganization and Salt Bridge Shielding
2019, Journal of Medicinal ChemistryApplication of ITC-based characterization of thermodynamic and kinetic association of ligands with proteins in drug design
2018, Frontiers in PharmacologyParadoxically, Most Flexible Ligand Binds Most Entropy-Favored: Intriguing Impact of Ligand Flexibility and Solvation on Drug-Kinase Binding
2018, Journal of Medicinal Chemistry
- †
Equally contributing authors.