New methods to modulate protein-protein interactions for drug discovery
Protein-protein interactions (PPIs) mediate all cellular processes, play a central role in disease and represent the majority of interactions in the human interactome. Thus, a major problem in life-sciences research is to understand and manipulate PPIs with molecular and temporal resolution – this would allow the identification of the transient intermediates that play key roles in the function of biomacromolecular machines, signalling, translocation and folding. In turn this will illuminate our understanding of disease development e.g. cell signalling in cancer and aggregation in amyloid disease and provide starting points for drug-discovery. Developing capability to understand and control PPIs is thus a significant challenge of immense therapeutic importance. However, methods to interrogate and manipulate PPIs are not well established. We develop and use chemical biology approaches to study and manipulate PPIs, focussing on enabling methods for drug-discovery and structural molecular biology. Prominent current targets include: p53/hDM2, BH3/BCL-2 family, HIF-1α/p300, interactions of the Aurora kinase, and interactions of 14-3-3 proteins, (all oncology), GKAP/SHANK-PDZ (synaptic function) and SIM/SUMO (SUMOylation is a regulatory post-translational modification).
Computational Tools can be enormously powerful tools to study, and inform approaches to inhibit, PPIs. Understanding the key determinants of PPI thermodynamic and kinetic stability is necessary to identify and engineer PPIs, advance understanding of biological mechanisms and mutant phenotypes, and provide a firmer foundation for inhibitor design. Moving beyond this, computational methods can be used to inform small-molecule inhibitor discovery. As part of a multi-investigator, multi-institute project – PoPPI – we are interested in in silico methods that can be used to characterize PPIs, so as to identify targets that are suitable for small-molecule inhibitor development, alongside methods that can be used to identify generic small-molecule scaffolds that can be readily functionalized to inhibit a range of PPIs. Recent studies let to development of rapid experimentally validated methods for predicting hot-residues at PPI interfaces, and an associated web-app.
Constraining peptides in a bioactive conformation can improve target affinity, stability and cell-uptake. We are interested in developing synthetic methods for constraining peptides. Recent studies have focused on the development of a new reagent (now commercialized) for constraining peptides, the introduction of reversible stapling methodology for PPI inhibition (exploiting reaction of bis-thiol containing peptides with dibromomaleimide reagents) and, through PoPPI, the use of coiled-coil assembly as a form of non-covalent stapling. In tandem, we are using physical organic chemistry to rationalise the relationships between peptide structure and protein binding that arise as a consequence of introducing the constraint. Recent studies explained unexpected enthalpy-entropy compensation for induced-fit binding of BH3-peptides to anti-apoptotic BCL-2 family proteins. Ongoing research is geared towards the application of these approaches to the discovery of cell-permeable inhibitors of therapeutically important PPIs.
Peptidomimetics exploit generic scaffolds to mimic the spatial and angular projection of "hot-spot" side chains on peptides found at PPI interfaces. These offer the potential to elaborate a rule based approach for PPI inhibition, and to access more "drug-like" small molecules. In collaboration with Prof Aitken’s group (Paris-Sud), our team employed conformationally constrained amino acids to develop α/β/γ-peptide foldamers that topologically mimic the α-helix and selectively inhibit the p53/hDM2 interaction. A particularly active area of recent research has been our introduction of topographical mimics of the α-helix; solid phase syntheses of aromatic oligoamides uniquely provided access to compounds libraries for the first time which were used as inhibitors of the p53/hDM2, NOXA-B/ MCL-1 and HIF-1a/p300 interactions, including potent cell permeable dual inhibitors of p53/hDM2 and NOXA-B/ MCL-1. Excitingly, we adapted this approach to develop a novel replacement strategy whereby a segment of protein structure (the S-peptide from RNase S) is replaced by a helix mimetic. The resultant prosthetic replacement forms a non-covalent complex with the S protein leading to restoration of catalytic function, despite the absence of a key catalytic residue. Ongoing research is geared towards the development of novel methods to mimic other secondary and tertiary structures using non-natural scaffolds.
Small Molecules remain a preferred modality in drug design and medicinal chemistry. Developing chemical probes to interrogate cell signalling pathways represents a significant challenge of immense biochemical and medical importance. However, methods to competitively inhibit PPIs using small molecules are not well established, given that they must cover 800-1100A2 of a protein surface and complement the discontinuous projection of hydrophobic and charged domains over a flat or moderately convex surface. As part of a multi-investigator, multi-institute project – PoPPI – we are interested in developing an integrated approach to the discovery of effective, low molecular weight PPI inhibitors that uses in silico design, synthesis and biophysical evaluation as its pillars. Using hot residues as templates we aim to identify small molecules and scaffolds that match the spatial orientation and composition of these residues. Ongoing research is also focussed on exploring novel fragment identification methods that can be used to target cryptic and/or dynamic binding sites and which exploit protein and/or ligand directed covalent bond formation.
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J.M. Fletcher, K.A. Horner, G.J. Bartlett, G.R. Rhys, A.J.Wilson, D.N. Woolfson: De novo coiled-coil peptides as scaffolds for disrupting protein-protein interactions, Chem. Sci., 2018, 9, 7656-7665. View Paper.
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J. A. Miles, D. J. Yeo, Philip Rowell, S. Rodriguez-Marin, C. M.Pask, S. L. Warriner, T. A. Edwards, A. J. Wilson: Hydrocarbon Constrained Peptides – Understanding Preorganization and Binding Affinity, Chem. Sci., 2016, 7, 3694-3702. View Paper.
A. Barnard, K. Long, H. L. Martin, J. A. Miles, T. A. Edwards, D. C. Tomlinson, A. Macdonald, A. J. Wilson: Selective and Potent Proteomimetic Inhibitors of Intracellular Protein-Protein Interactions, Angew. Chem. Int. Ed., 2015, 54, 2960-2965. View Paper.
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Alex Breeze (University of Leeds), Adam Nelson (university of Leeds), Thomas Edwards (University of Leeds), Christian Ottmann (TU-Eindhoven, NL), Darren Tomlinson (University of Leeds), Stuart Warriner (University of Leeds), Mike Webb (University of Leeds), Dek Woolfson (University of Bristol), Richard Sessions (University of Bristol), Xiaohui Wang (Changchun Institute of Applied Sciences, CH), Astra Zeneca, Megan Wright (University of Leeds), David Aitken (Institut de Chimie Moléculaire et des Matériaux d'Orsay, FR (ICMMO)).
Current and Recent Funding
BBSRC SLoLa Programme Grant BB/V003577/1, 'Deciphering the function of intrinsically disordered protein regions in a cellular context' (SPIDR) Project Website
EPSRC Programme Grant EP/N013573/1, ‘Integrated computational and synthetic tools to drive the discovery of orthosteric protein-protein interaction inhibitors (PoPPI)’ Project Website
EU H2020 Program Marie Curie ETN 675179, 'Targeted small-molecule Stabilization of Protein-Protein Interactions (TASSPI)' Project Website
European Research Council Starting Grant 240324: ‘Proteomimetic Foldamers: Towards Future Therapeutics and Designer Enzymes (PROTEOFOLD)’
Chemical protein labelling for structural proteomics and interactome analyses