Developing Novel Tools for Elucidating the Structure-Function Relationship of Macrophage Migration Inhibitory Factor

The macrophage migration inhibitory factor (MIF) is an important cytokine of the innate immune system. It has a broad range of pro-inflammatory activities and possesses a keto/enol tautomerase and thiol-protein oxidoreductase activities that might be linked to its cytokine function. MIF was first crystallized in 1996 as a homotrimer; nevertheless, there is still a controversy concerning its native oligomeric state and the role of oligomerization in modulating its function(s) in health and disease. Therefore, elucidating its structure-function relationship is an important step towards understanding the biology of MIF and its role in disease. This work attempts to address this question through the use of two different though complementary approaches to generate novel molecular tools. First, we used protein engineering to design and express mutant MIF (N110C) that exists only as a homotrimer stabilized by disulphide bonds. Using biophysical and biochemical techniques, we optimized covalent trimer formation, characterized the mutant, and used it to prove the mechanism of action of a recently discovered MIF inhibitor. The second approach consists of finding new MIF enzymatic activity inhibitors. The implications of such a discovery go beyond fundamental research about MIF, given its role in many inflammatory diseases. For this part of the project, a virtual high-throughput screening was performed on a diverse library of compounds, which are currently being tested experimentally by other members of the laboratory. The purpose of this exercise is to optimize the in silico screening approaches and facilitate the in silico screening of large libraries and restrict the costly in vitro screening to small numbers of potentially interesting molecules. The protocol was optimized to take into account protein and ligand flexibility to model ligand ibnding as faithfully as possible. Based on this analysis, we proposed a list of potential MIF inhibitors to be tested experimentally