DISSECTING AND EXPLOITING MOLECULAR RECOGNITION AT PROTEIN-PROTEIN INTERFACES

Proteins regulate many of the processes that are crucial for the activity of a living cell. In order to successfully carry out their biological function, proteins often interact in complex with other proteins. The principal subject of the proposed research concerns the development of new approaches to advance our understanding of protein-protein interactions and of how we could disrupt these interactions using the binding of a small molecule. In this project, I focus on studying the binding of small molecules to protein interfaces. I pose the following questions: what features of protein interfaces determine binding and activity (or lack thereof) of a small molecule? Can we modify protein interfaces so that a small molecule can 'stick' to the surface better and better? What can we learn from these new interfaces? How can we use such information to discover new compounds that could function in the cell by binding tightly to these sites? To interrogate protein interfaces, I will first use protein engineering, a technique to generate mutations on a protein by changing amino acids, the building blocks of proteins, from one type to another. I will make mutations in a defined manner, by replacing large amino acids at the protein interface with smaller and smaller ones, hence creating larger and larger cavities. The location and strength of small molecules bound to these engineered pockets will be determined. This will provide useful information to find other protein interfaces that may be functional in living organisms, and that may have the potential to be disrupted using small molecules. Secondly, I will exploit this knowledge further to facilitate identification of small molecules that function by disrupting a protein interface. I will develop new methods to detect small molecules that bind together to adjacent sites of the interface. For this purpose, I will use nuclear magnetic resonance (NMR) spectroscopy, a technique that allows monitoring the hydrogen atoms of small molecules and that can report if these are bound to a protein close to one another. I will also make crystals of the protein in which the interface is accessible to small molecules, and let these molecules react together as they are bound close to each other at the protein surface. Since the structure of a small molecule bound to the protein can be determined directly by shooting X-ray radiations at the protein crystal, a technique called X-ray crystallography, this is a rapid way of identifying any compound that has successfully assembled at the protein interface. The research is important and exciting for the following reasons: 1. Protein interfaces tend to be relatively flat and featureless, as they were not 'evolved' by nature to bind small molecules. The modulation of protein-protein interactions using small molecules is therefore a challenging task, and is at the forefront of molecular recognition. 2. A new scientific horizon is to advance our understanding of biological systems by disrupting pathways and networks in a selective fashion inside the cell. As protein-protein interactions occur widely within the cell, their modulation using small molecules offers an opportunity to interrogate and discover new biology. 3. The disruption of protein-protein complexes offers a novel and general mechanism to develop new medicines. In conclusion, this research has the potential to significantly impact on the way new biology and new drugs will be discovered in the future, with wider benefits to society and exciting opportunities in the fight against disease.

The overall objective of this fellowship application is to advance our fundamental understanding of molecular recognition at protein interfaces by small organic compounds. The project will employ a unique combination of molecular biology and biophysical studies of protein-ligand interactions to dissect the nature of non-covalent interactions at protein interfaces, and to exploit such knowledge with the goal of aiding small molecule design. I will study the interaction of small peptides and fragment-like compounds (MW < 300 Da) to the tumour suppressor Von Hippel-Lindau (pVHL), a protein which is part of a complex responsible for the degradation of the Hypoxia-Inducible Factor (HIF-1) inside the cell. I will use protein engineering to make larger and larger cavities at the pVHL:HIF-1 interface with the aim to bind small molecule with increasingly higher affinities. I will study the fundamental changes to the recognition of the small molecules in terms of structure (using X-ray crystallography) and thermodynamics (using low c value isothermal titration calorimetry). This information will provide a platform for future studies in the following two directions: 1) to find structurally similar protein interfaces that may be druggable, using computational tools and structural databases; 2) to facilitate discovery of novel small molecules that bind with high affinity and specificity to a protein interface, using intermolecular Nuclear Overhauser Effect to identify fragments binding at adjacent sites by NMR spectroscopy, and Dynamic Combinatorial X-ray Crystallography to assemble fragments together in the presence of a protein crystal, with the aim of selecting the highest affinity compound. Together these studies will provide significant new insights into the principles that govern the modulation of protein-protein interactions using small molecules, and new tools for studying systems of biological importance and therapeutic potential.