Paradoxically, Most Flexible Ligand Binds Most Entropy-Favored: Intriguing Impact of Ligand Flexibility and Solvation on Drug–Kinase Binding

Abstract

Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in solution prior to protein binding, and by release of these waters, the favored entropic binding is observed.

Diamondoid Amino Acid‐Based Peptide Kinase A Inhibitor Analogues

“…Herein we investigate the impact of incorporating DAAs into the native substrate-like peptidic kinase inhibitor PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] (sequencep ositions referenced as superscripts) and the interaction of these variants with their target enzyme protein kinase A (PKA). We chose this model system, as it is biochemically,c rystallographically, and NMR spectroscopically well characterized (Figure 1a nd Scheme 1).…”

“…We chose this model system, as it is biochemically,c rystallographically, and NMR spectroscopically well characterized (Figure 1a nd Scheme 1). [12] The incorporation of diamondoid aminoacids (DAAs) into peptide-like drugs is ag eneral strategyt oi mprove lipophilicity, membrane permeability,a nd metabolic stability of peptidomimetic pharmaceuticals. We designed and synthesized five novel peptidic DAA-containing kinase inhibitors of protein kinase Au sing as ophisticated molecular dynamics protocol and solid-phase peptide synthesis.…”

“…By meanso fs tructure-based design and molecular dynamics (MD) simulations, av ariety of amino acid replacements by DAAs was suggested for native PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] .T he main objective of our DAA variant design wast op rovide modified peptides that can stilla dopt ab inding mode comparable to that of native PKI 5-24 in order to maintain or even stabilizetheir complex with PKA. To suggest such variants for subsequents ynthesis and testing, we applied the following structure-based design strategy (for further details see the Experimental Section).…”

“…Startingw ith the native PKA/PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] complex structure (PDB ID:3 FJQ), [14] we used the Scaffold Replacement utility of MOE [15] to substitute six types of DAAs (Scheme 1) at appropriate sequence positions based on the PKA-bound geometry of PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] .W es ubstituted individual residues in PKI 5-24 by both enantiomeric forms of 1-3,l eadingt ov ariations in the PKI side chains. Alternatively,w es ubstituted up to six PKI residues by replacing their backbone atoms using one of the DDAs 4-6.I n total, 49 PKI variants with in each case one introduced DAA were createdl eadingt o2 3s ide chain and 26 backbonev ariations.…”

“…In addition, we selected two PKI variantsf or synthesis that did not severely destabilize the complexw ith PKA but appeared particularly interesting for the following reasons. The Thr 6 replacement by (S)-Ada Gly (1)i n PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] was assumed to be well tolerated because the un- Scheme1.Modeled and synthesized diamondoid amino acids.C ompounds 1, 2,and 4 were synthesized in the depicteds tereoisomeric form, 3, 5,and 6 were only considered as model-builtc ompounds. For computational modeling, all stereoisomerso f1-6 were considered.…”