14-3-3 Protein-Protein Interactions: From Mechanistic Understanding to Their Small-Molecule Stabilization

Abstract

Protein-protein interactions (PPIs) are of utmost importance for maintenance of cellular homeostasis. Herein, a central role can be found for 14-3-3 proteins. These hub-proteins are known to bind hundreds of interaction partners, thereby regulating their activity, localization, and/or stabilization. Due to their ability to bind a large variety of client proteins, studies of 14-3-3 protein complexes flourished over the last decades, aiming to gain greater molecular understanding of these complexes and their role in health and disease. Because of their crucial role within the cell, 14-3-3 protein complexes are recognized as highly interesting therapeutic targets, encouraging the discovery of small molecule modulators of these PPIs. We discuss various examples of 14-3-3-mediated regulation of its binding partners on a mechanistic level, highlighting the versatile and multi-functional role of 14-3-3 within the cell. Furthermore, an overview is given on the development of stabilizers of 14-3-3 protein complexes, from initially used natural products to fragment-based approaches. These studies show the potential of 14-3-3 PPI stabilizers as novel agents in drug discovery and as tool compounds to gain greater molecular understanding of the role of 14-3-3-based protein regulation.

Keywords: cooperativity; fragment-based drug discovery; molecular glues; structure-based molecule design.

Figure 1.

Proteins as drug targets : classical targets versus PPI modulators. (a) Schematic representation of proteins which are classically used as drug targets (GPRC, Nuclear Receptors, Ion Channels, Enzymes). Drug molecules (blue hexagon) bind in the well-defined pockets where normally endogenous ligands (green hexagon) would bind, thereby inhibiting these classes of proteins. (b-c) Schematic representation of PPI modulators: inhibitors and stabilizers. Crystal structures are provided for a typical PPI inhibitor (RG7112) that inhibits the p53/MDM2 interaction and a PPI stabilizer (Rapamycin) that stabilizes the interaction between FKBP12 and mTor, PDB: 1YRC, 4IPF, 1FAP

Figure 2.

14-3-3 protein/peptide complexes (a) Structure of 14-3-3ζ dimer in cartoon and surface representation. Individual helices and the binding groove are highlighted in red, PDB: 4FJ3 (b) Crystal structure of 14-3-3σ (white surface) bound to a TAZ phosphopeptide (red). Enlarged view of the peptide in the 14-3-3 binding groove with the phosphoserine in the 14-3-3 phospho-accepting pocket highlighted, PDB: 5N75. (c) Crystal structures of 14-3-3 (white surface) bound to various phosphopeptides of its binding partners, PDB: 5MHC, 4FJ3, 4JC3, 7AOG, 6QHL & 4FL5.

Figure 3.

14-3-3 protein complexes extending beyond client peptides. 14-3-3 (white/gray surface) bound to (a) yeast Nth1 pS60-pS83 (green), (b) monomeric BRAF pS365-pS729 (yellow) and MEK (cyan), (c) dimeric BRAF pS729 (yellow) and two times MEK (cyan), (d) AANAT pT31 (orange) and its substrate analogue coenzyme-A-S-acetyl tryptamine (cyan), (e) PMA2-CT52 (residues 905–956; pink) and PPI stabilizer FC-A (yellow), (f) two OsFD1 pS192 peptides (pink) and two Hd3a monomers (blue), (g) HSPB6 pS16 (red), and (h) two times Exotoxin S (green). PDB: 5N6N, 6NYB, 6Q0J, 1IB1, 2O98, 3AXY, 5LTW, 6GN0.

Figure 4.

14-3-3 modes of action. Schematic representation of four distinct mechanisms in which 14-3-3 (grey blobs) can regulate their protein partners through modulation of their (i) activity, (ii) localization, (iii) stability, or (iv) by functioning as scaffold protein.

Figure 5.

14-3-3 PPI stabilization by natural products. (a) Crystal structure of 14-3-3 (white surface) bound to PMA2 phosphopeptide (purple) and FC-A (yellow). (b) Zoom in of FC-A binding in the 14-3-3 binding groove as presented in panel a and its interactions with residues of 14-3-3 (black dashed lines), PDB: 1O9F. (c) Crystal structure of 14-3-3 dimer (white/grey surface), PMA2-CT52 (52 most C-terminal residues of PMA2, purple/pink surface) and FC-A (yellow sticks), PDB: 2O98. (d) Chemical structures of FC-A and derivatives. (e-g) Crystal structures of 14-3-3 bound to C-Raf (e, blue), NF-kB p65 subunit (f, green), and TASK3 (g, blue) which are stabilized by various FC-A variants (yellow sticks), PDB: 4IHL, 6NV2, 3SMN.

Figure 6.

Synthetic 14-3-3 PPI stabilizers. Crystal structures of 14-3-3 (white surface) in complex with five different binding partners (ERα, ChREBP, CFTR, NF-kB p65, and PIN1 in various colors) and synthetic compounds/fragments (yellow sticks) that stabilize the 14-3-3/client complex. PDB: (a) 6JTM, (b) 6YGJ, (c) 7QI1, (d) 6HMT, (e) 6YQ2, (f) 7BFW.

Figure 7.

Selective, synthetic 14-3-3 PPI stabilizers. (a) Crystal structure of 14-3-3 (white surface) in complex with a FOXO1-specific disulfide fragment (yellow sticks) and the corresponding FOXO1-derived peptide. The crystal structure of 14-3-3 in complex with an ERα-derived peptide is overlayed. An electrostatic interaction between the fragment and the +1 Trp of the FOXO1 peptide is shown as black dotted line. (b) The same structure as in (a) overlayed with the 14-3-3-complexed peptides derived from C-Raf, USP8, and SOS1 shown as semitransparent sticks. (c) Crystal structure of 14-3-3 in complex with a fragment (yellow sticks) that stabilizes both the binding of an ERα- as well as a C-Raf-derived peptide binding to 14-3-3. (d) Crystal structure of 14-3-3 in complex ERα and an ERα-selective fragment (magenta sticks). The structure of the C-Raf-derived peptide in complex with 14-3-3 is overlayed, double-sided arrows in red illustrate steric conflicts between the C-Raf peptide and the fragment.

Figure 8.

Double covalent “molecular locks”. (a) Crystal structure of 14-3-3 (white surface) in complex with a p65-derived peptide (purple sticks) and an aldehyde fragment (yellow sticks) that engages 14-3-3’s Lysine 122 via imine formation. (b) Crystal structure of 14-3-3 in complex with an ERRγ-derived peptide (green sticks) and a disulfide fragment that engages in a covalent bond with the Cysteine 180 of the ERRγ peptide. (c) Crystal structure of 14-3-3 in complex with the ERRγ-derived peptide and a double-covalent molecular lock (yellow sticks) that simultaneously engages Lysine 122 of 14-3-3 and Cysteine 180 of the ERRγ peptide.

Figure 9.

Linked fragments as starting points for molecular glue development. (a) Co-soak of a non-covalent amidine (orange sticks) and a covalent, disulfide fragment (yellow sticks), bound to the complex of 14-3-3 (white surface) and an ERα-derived peptide (green sticks). (b) and (c) two examples of non-covalent linked fragments based on the co-soaked fragments shown in (a).