Rational Design, Binding Studies, and Crystal‐Structure Evaluation of the First Ligand Targeting the Dimerization Interface of the 14‐3‐3ζ Adapter Protein

Abstract

14-3-3 Proteins play a central role in signalling pathways in cells: they interact as gatekeeper proteins with a huge number of binding partners. Their function as hub for intracellular communication can explain why these adapter proteins are associated with a wide range of diseases. How they control the various cellular mechanisms is still unclear, but it is assumed that the dimeric nature of the 14-3-3 proteins plays a key role in their activity. Here, we present, to the best of our knowledge, the first example of a small molecule binding to the 14-3-3ζ dimerisation interface. This compound was designed by rational in silico optimisation of a peptidic ligand identified from biochemical screening of a peptidic library, and the binding was characterised by UV/Vis spectroscopy, microscale thermophoresis, multiscale simulations, and X-ray crystallography.

A Supramolecular Stabilizer of the 14‐3‐3ζ/ERα Protein‐Protein Interaction with a Synergistic Mode of Action

“…The recent elucidation of crystal structures of different ligands in complex with 14‐3‐3 proteins opens up opportunities to develop new supramolecular ligands to modulate 14‐3‐3 PPIs . Crystal structures of different 14‐3‐3 isoforms bound by phosphorylated peptides derived from effectors such as C‐Raf, ExoS and PKC‐ϵ improved our understanding of the structural principles that govern these PPIs.…”

“…[4,5] The recent elucidation of crystal structures of different ligands in complex with 14-3-3 proteins opens up opportunities to develop new supramolecular ligands to modulate 14-3-3 PPIs. [6][7][8][9][10][11][12][13][14] Crystal structures of different 14-3-3 isoforms bound by phosphorylated peptides derived from effectors such as C-Raf, [15,16] ExoS [19,20] and PKC-e [21] improved our understanding of the structural principles that govern these PPIs. In general, 14-3-3 binds phosphomotifs through its Arg-Arg-Tyr triad in a central binding groove, which can be broadly categorized as internal mode-I/II, RSX{pS/pT}XP, or C-terminal mode-III, {pS/pT}X-COOH motifs.…”

NMR Spectroscopy of supramolecular chemistry on protein surfaces

“…Examples for supramolecular ligands designed to recognize protein surfaces include tweezers [5][6][7][8][9][10][11][12][13][14][15][16][17][18], calixarenes , guanidinio- [75]). The guanidiniocarbonylpyrrole motif (GCP, top left) recognizes the carboxylate groups of aspartate or glutamate residues by forming an extended H-bonding network [41][42][43][44][45][46]. Supramolecular tweezers (top center) thread lysine or arginine side chains into their aromatic cavities [5][6][7][8][9][10][11][12][13][14][15][16][17][18].…”

“…Cucurbiturils (bottom right, [47][48][49][50][51]) recognize methylated lysines and arginines by binding their methylated head groups inside the macrocycle. carbonylpyrrole (GCP) ligands [41][42][43][44][45][46], cucurbiturils [47][48][49][50][51], porphyrins [52][53][54][55][56][57][58][59][60], metal complexes [61][62][63], foldamers [64][65][66][67], nanoparticles [34,68], imprinted polymers [69][70][71][72], and dendrimers [73,74].…”

“…The GCP unit provides an extended hydrogen bonding/salt bridge interface, which leads to better binding compared to its natural counterpart. The combination of two or more GCP moieties with variable linkers enables the construction of multivalent ligands geared towards a specific spot on protein surface, which by design can lead to either stabilization [42][43][44][45] or inhibition [46] of a given protein-protein interaction. The inhibition of the Survivin -Histone H3 interaction by a GCP ligand has been shown to inhibit tumor proliferation in cell culture [46].…”