Inhibiting protein-membrane interactions: towards new therapeutic strategies

Proteins are Nature's building blocks but not only, they are dedicated and skilled workers, each fulfilling its own specific role. They are fairly large molecules, containing thousands of atoms. When one or several key proteins are not able to fulfill their function in the human body, this might potentially result in diseases. Although proteins are large for being molecules, they are rather small in size and are difficult to observe directly. Hence it is often handy to build models, using computers, and s imulate their behavior. The models are built from physics and chemistry principles, and the simulations have proven to be useful to learn more about how, for example, proteins can bind to cell membranes or if and how drugs can bind to proteins and affect their behavior. In this project we focus on a particular type of proteins, which perform their function at the surface of the cells and have the ability to stick to the membrane of the cells for just the right amount of time to achieve their tasks there, and leave again when the job is done. Cells membranes are constituted primarily of small molecules called phospholipids, and there exist a wide range of them. Because many of these phospholipids carry a negative charge, it is believed that only proteins that are heavily positively charged on one side can interact with cell membranes. In this project we have so far studied three different proteins that perform their function at the surface of cellular membranes: two involved in the inflammation process in humans (named Proteinase 3 and HNE) and one in bacterial virulence (phosphatidylinositol-specific phospholipase C). We have described how these proteins bind to the cellular membranes and have in particular highlighted how these proteins recognize particular and different types of phospholipids, thus allowing them to be present at the cell membrane at the right place and at the right moment. We have also shown that amino acids, namely tyrosine and phenylalanine, that are often thought to behave similarly in their interactions with lipids can actually achieve totally different types of interactions and fine tune the lipid specificity of peripheral membrane binding proteins. We have also shown that proteins that do not possess many positively charged amino acids are also able to bind to cell membranes. Our results are relevant for treating disorders caused by proteins that lead to pathological disorders by sticking too much or not enough to the cells.

The goal of the present proposal is to find compounds able to hinder the binding of Proteinase 3 to the neutrophil plasma membrane in vitro. Proteinase 3 is a neutrophil enzyme, present at the surface of the neutrophil plasma membrane and which has been i dentified as a drug target for a number of inflammatory diseases and possibly leukaemia. Together with our collaborators we have studied many salient features of Proteinase 3 for many years and we recently unravelled its membrane binding mechanism using a combination of theoretical and experimental approaches. Most importantly the level of expertise we have acquired helped us build an international and multi-disciplinary, yet tight, collaborative network of excellent groups from a range of different field s using sound approaches and innovative techniques. We are thus in a world-leading position to successfully develop inhibitors of Proteinase 3 membrane binding. We will use a combination of in silico methods (high-throughput in silico screening of compou nd libraries, docking, molecular dynamics simulations) and in vitro approaches (Surface Plasmon Resonnance spectroscopy). Moreover, we will investigate a few other peripheral membrane proteins, involved in other pathologies, compare them PR3 and possibly identify general targeting strategies to modulate membrane binding. These investigations will be conducted using molecular dynamics simulations (MD) and statistical analyses on protein structure databases. Although membrane binding sites of peripheral pro teins are emerging as new promising drug targets, and are of obvious therapeutic interest, few inhibitors of such interactions have been reported yet. Our project is thus particularly timely in the international context and will contribute to the general effort in validating and establishing strategies for targeting of peripheral membrane binding in drug discovery.