Peptide-derived Inhibitors of Protein-Protein Interactions

Transcription

1 Peptide-derived Inhibitors of Protein-Protein Interactions Sven Hennig Department of Chemistry and Pharmaceutical Sciences Vrije Universiteit Amsterdam 1

2 Biomolecular recognitions Classification via interaction areas small molecule protein interaction protein protein interaction PDB 3up3 PDB 1lfd interaction area Å 2 deep cavities interaction area Å 2 flat surface 2

3 Total human proteins 19,613 Intrinsically 16 % dissordered proteins undruggable Intracellular proteins without hydrophobic pockest 3 % approved drugs 7 % potential targets 74 % not or not yet disease-relevant Human Protein Atlas, DrugBank, KS analysis 3

4 Ligands based on protein binding epitopes PDB 1ldf 4

5 Peptidomimetics, Loop α-helix β-sheet 5

6 Peptidomimetics, Loop α-helix β-sheet Review: M Pelay-Gimeno et al. Angew. Chem. Int. Ed. 2015, 54,

7 Peptidomimetics Review: M Pelay-Gimeno et al. Angew. Chem. Int. Ed. 2015, 54,

8 Stabilization of secondary and tertiary structures Monocycles Bicycles crystal structure PDB ID: 4n7y crystal structure PDB ID: 4djs model based on PDB ID: 3cwz 10 aa 15 aa 20 aa 25 aa 30 aa 35 aa 150 aa model based on PDB ID: 1t2w crystal structure unpublished 8

9 Stabilization of secondary and tertiary structures Monocycles Bicycles crystal structure PDB ID: 4n7y crystal structure PDB ID: 4djs model based on PDB ID: 3cwz 10 aa 15 aa 20 aa 25 aa 30 aa 35 aa K d : 1 µm 40 nm b-catenin K d : 5 µm 8 nm Small GTPase (Rab) K d : >100 µm 8 µm Angew. Chem. Int. Ed Cell Chem. Biol ChemBioChem 2016 Chem. Eur. J J. Med. Chem Angew. Chem. Int. Ed Nat. Commun ACS Chem. Biol

10 Hydrocarbon stapled peptides CE Schafmeister, J Po, GL Verdine JACS 2000, 122, , PM Cromm, J Spiegel, TN Grossmann, ACS Chem. Biol. 2015, 10,

11 Fmoc based SPPS: Solid Phase Peptide Synthesis Hydrocarbon stapled peptides Grubbs Catalyst 1 st Generation YW Kim, TN Grossmann, GL Verdine, Nature Prot. 2011, 6,

12 Wnt signaling pathway 12

13 Inhibition of protein-protein interaction (PPI) Inhibition of PPI TCF4 β-catenin PDB 2gl7 J. Sampietro et al. Mol. Cell 2006, 24,

14 β-catenin in protein-protein interactions (PPI) TCF4 β-catenin PDB 2gl7 Axin BCL9 APC PDB 1qz7 PDB 2gl7 PDB 1th1 ICAT LHR1 E-cadherin PDB 1m1e PDB 3tx7 PDB 3ifq 14

15 Scaffolds for peptide stapling destruction complex transcrip. activator complex J. Sampietro et al. Mol. Cell 2006, 24, Y. Xing et al. Genes Dev. 2003, 17,

16 Rational design of stapled inhibitors FP: Fluorescent Polarization T.N. Grossmann et al. PNAS 2012, 109,

17 Rational design of stapled inhibitors CD: Circular Dichrosim PDB 1QZ7 FP: Fluorescent Polarization T.N. Grossmann et al. PNAS 2012, 109,

18 Phage Display Affinity maturation Computational, here: Docking T.N. Grossmann et al. PNAS 2012, 109, D. Krüger et al., J. Med. Chem. 2017, 60, Sequence maturation eg. phage display, in silico design 18

19 Affinity maturation and sequence optimization Pull-Down Variants: here: Competition Pull-Down Variants: here: IP from Lysates - phage display results suggest variants - enrich Arg to trying to increase cellular uptake (in analogy to cell penetrating peptides - CPPs) T.N. Grossmann et al. PNAS 2012, 109,

20 Cellular Uptake in cellulo, here: confocal microscopy, fixed - phage display results suggest variants - enrich Arg to trying to increase cellular uptake (in analogy to cell penetrating peptides - CPPs) - Careful: microscopy is prone to false positives (peptides are still mobile) - Better: live cell and/or flow cytometry in cellulo: see better T.N. Grossmann et al. PNAS 2012, 109,

21 Cellular Activity in cellulo, here: LUC-Reporter gene assay - phage display results suggest variants - enrich Arg to trying to increase cellular uptake (in analogy to cell penetrating peptides - CPPs) - Careful: microscopy is prone to false positives (peptides are still mobile) - Better: live cell and/or flow cytometry - Cellular activity correlates with uptake rather than pure binding affinity T.N. Grossmann et al. PNAS 2012, 109,

22 Crystal structure at 3.0 Å resolution RRWPQS 5 ILDS 5 HVRRVWR Atomic structure, here: X-ray crystallography T.N. Grossmann et al. PNAS 2012, 109,

23 Crystal structure at 3.0 Å resolution RRWPQS 5 ILDS 5 HVRRVWR Atomic structure, here: X-ray crystallography T.N. Grossmann et al. PNAS 2012, 109,

24 Lessons learned from CPPs? stapled peptide StAx L Dietrich et al., Cell Chem. Biol. 2017, 24,

25 Ability of penetrate cells Peptidomimetics Review: M Pelay-Gimeno et al. Angew. Chem. Int. Ed. 2015, 54,

26 Summary Sven Hennig Department of Chemistry and Pharmaceutical Sciences Vrije Universiteit Amsterdam General peptidomimetics: M Pelay-Gimeno et al. Angew. Chem. Int. Ed. 2015, 54, (review) YH Lau et al., Chem. Soc. Rev., 2015, 44, Hydrocarbon stapled peptide: PM Cromm et. al., ACS Chem. Biol. 2015, 10, (review) YW Kim et al., Nature Prot. 2011, 6, (method) Affinity maturation: TN Grossmann et al. PNAS 2012, 109, D Krüger et al., J. Med. Chem. 2017, 60, P Diderich et al., ACS Chem. Biol. 2016, 11, Cellular uptake: L Dietrich et al., Cell Chem. Biol. 2017, 24, Q Chu et al., Med. Chem. Commun. 2015, 6, R Brock, Bioconjugate Chem. 2014, 25,