Functional mapping of the 14-3-3 hub protein as a guide to design 14-3-3 molecular glues

Abstract

Molecular glues represent an evolution in drug discovery, however, targeted stabilization of protein complexes remains challenging, owing to a paucity of drug design rules. The functional mapping of hotspots has been critical to protein-protein interaction (PPI) inhibitor research, however, the orthogonal approach to stabilize PPIs has not exploited this information. Utilizing the hub protein 14-3-3 as a case study we demonstrate that functional mapping of hotspots provides a triage map for 14-3-3 molecular glue development. Truncation and mutation studies allowed deconvoluting the energetic contributions of sidechain and backbone interactions of a 14-3-3-binding non-natural peptide. Three central 14-3-3 hotspots were identified and their thermodynamic characteristics profiled. In addition to the phospho-binding pocket; (i) Asn226, (ii) Lys122 and (iii) the hydrophobic patch formed by Leu218, Ile219 and Leu222 were critical for protein complex formation. Exploiting this hotspot information allowed a peptide-based molecular glue that elicits high cooperativity (α = 36) and selectively stabilizes the 14-3-3/ChREBP PPI to be uniquely developed.

Fig. 1. (a) Conceptual representation of PPI inhibitor design based on hotspot residue analysis, using the MDM2/p53 PPI as an example. Hotspot residues (red region) for MDM2 (white surface) binding were determined for the p53 protein (orange). Based on these hotspots small molecule inhibitors such as RG7112 (blue) were designed to inhibit the MDM2/p53 interaction. (b) Enlarged and detailed view on MDM2 (white surface)/p53 (orange cartoon) interaction with hotspot residues Phe19, Trp23 and Leu26 and the MDM2/RG7112 (blue sticks) interaction is depicted (PDB: 1YCR & 4IPF). (c and d) Conceptual representation of this work where a PPI stabilizer is designed based on hotspot residue analysis. Hotspot residues for 14-3-3 (white surface) binding were determined for peptide 1 (orange). Based on these hotspots a peptide-based stabilizer (2d, blue) was designed for the 14-3-3/ChREBP PPI (PDB: 6TCH, 7ZMU & 5F74). (e) Schematic representation of the functional mapping of hotspots, based on truncation studies and point mutations, to determine hotspot residues in 14-3-3 binding peptide, for the design of a 14-3-3 PPI stabilizer.

Fig. 2. (a) Schematic representation of peptide 1 with the naming of individual amino acids and the according numbering system. Lys = lysine; Nva = norvaline; Nph = nitrophenylalanine; Thz = thiazolylalanine; pS = phosphoserine; βSer = beta-serine; βAla = beta-alanine. (b) Chemical structure of peptide 1. (c) Crystal structure of 14-3-3σ (white surface) bound to peptide 1 (orange sticks) (PDB: 6TCH). (d) Isothermal titration calorimetry (ITC) raw thermogram of 100 μM 14-3-3γ binding to 20 μM 1. (e) Fluorescence anisotropy (FA) binding curve of 14-3-3γ titration to fluorescein-labelled 1 (10 nM), including a schematic representation of FA concept. (f) Bar plot representation of binding affinity (K_D) and thermodynamic parameters (ΔG, −TΔS, ΔH) parameters as obtained from ITC and FA experiments.

Fig. 3. Truncation studies. (a) Fluorescence anisotropy assay of peptide 1 (orange) and N-terminally truncated peptides 2a–d (blue) in which 14-3-3γ is titrated to fixed concentrations fluorescein-labelled peptide (10 nM). (b) Fluorescence anisotropy assay of peptide 1 (orange) and C-terminally truncated peptides 2e–i (green) in which 14-3-3γ is titrated to fixed concentrations of fluorescein-labelled peptide (10 nM). (c and d) Bar plot representation from obtained binding affinities (K_D) of truncated peptides and the fold change in affinity between different constructs. Data shows K_D values obtained from three independent experiments (n = 3). (e and f) Energy contribution analysis of individual amino acids of peptide 1 (blue/green) and the overall N- and C-terminal (red), based on the binding affinities observed in (a–d) and represented as ΔΔG. A positive value indicates enhanced 14-3-3 binding whereas a negative value represents a reduction in 14-3-3 binding affinity. (g) Crystal structure analysis of amino acid Thz(−1) of 1 (orange sticks) and Thz(−1) of truncated peptide 2c (blue sticks) bound to 14-3-3σ (white cartoons and sticks). Favorable hydrogen bond and electrostatic interactions are shown with black or blue dashed lines (PDB: 6THC & 7ZMW). (h and i) Crystal structure analysis of amino acids Nph(+1) and Nph(+4) of the 1 (orange sticks) bound to 14-3-3 (white cartoon, surface and sticks). Favorable hydrogen bond and electrostatic interactions are shown with black dashed lines (PDB: 6TCH).

Fig. 4. Mutational studies around Thz(−1), Nph(+1) and Nph(+4) residues. (a) Energy contribution analysis of point mutated Thz(−1) (3a, blue), Nph+1 (4a–c, dark green) and Nph+4 (5a–c, light green) residues in 1 to tyrosine, phenylalanine and alanine residues, represented as ΔΔG. Data represents both results from fluorescence anisotropy (FA) and isothermal titration calorimetry (ITC). (b) Thermodynamic parameters (ΔH, −TΔS and ΔG in kcal mol⁻¹) determined by ITC assays for peptide 1 and Nph+1 mutants 4b,c. Thermodynamic parameters are reported from two independent experiments (n = 2). (c) Thermodynamic parameters (ΔH, −TΔS and ΔG in kcal mol⁻¹) determined by ITC assays for peptide 1 and Nph+1 mutants 5b-c. Thermodynamic parameters are reported from two independent experiments (n = 2). (d) Hotspot analysis map of 14-3-3σ binding groove showing most relevant residues for the interaction with 14-3-3 binding partners. Red amino acids form hydrogen bonds with a binding partner, blue residues facilitate electrostatic interactions and yellow amino acids represent residues that form hydrophobic contacts.

Fig. 5. 14-3-3γ/ChREBP PPI stabilization by non-natural truncated peptide 2d. (a) Crystal structure of 14-3-3 (white surface) bound to ChREBP peptide (salmon sticks and cartoon), (PDB: 5F74). (b) Crystal structure of 14-3-3 (white surface) bound to 2d (blue sticks). The final 2Fo-Fc electron density map is represented as a blue mesh contoured at 1σ (PDB: 7ZMU). (c) Structural overlay of 14-3-3 bound to 2d and ChREBP showing a strong binding complementary in the 14-3-3 binding groove. An enlarged view is given of the phosphate group of peptide 2d interacting with the phospho-accepting pocket of 14-3-3 (black dashes) and potential interactions between 2d and ChREBP (salmon dashes) (PDB 7ZMU & 5F74). (d) Fluorescence anisotropy data from ChREBP titration to a preformed complex of 14-3-3γ and FITC-labelled 2d and 2c. (e) Fluorescence anisotropy data from ChREBP titration to FITC-labelled 2d and 2c (f) 2D fluorescence anisotropy data of 14-3-3γ titration to FITC-labelled 2d (20 nM) with varying ChREBP peptide concentrations (n = 2). (g) Bar plot representation of obtained apparent K_D values in the 2D fluorescence anisotropy assay. The calculated K^II_D and cooperativity factor α from a thermodynamic model are given. (h) Fluorescence anisotropy based selectivity study in which eight partner peptides of 14-3-3 are titrated to a preformed complex of 14-3-3γ (2 μM) and FITC-labelled 2d (20 nM) showing strong selectivity for 14-3-3/ChREBP stabilization (n = 2).