Macrophage Migration Inhibitory Factor (MIF) is a multifunctional protein and a major mediator of innate immunity. The paramount importance of MIF in the pathogenesis of many inflammatory, infectious, auto-immune and oncogenic diseases led to its emergence as a viable therapeutic target. Understanding MIF' biological and structural properties is of fundamental importance to provide insights into the molecular bases underlying its mode of action, and to develop effective strategies to diagnose, prevent or treat MIF associated diseases. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo as well as the factors governing its oligomerization and stability remain poorly understood. In order to assess the structural components crucial for MIF's quaternary structure, conformational stability and function, we sought to target key interactions within MIF trimer via genetic and biochemical tools. To achieve this goal, we employed a battery of computational, biochemical, biophysical and structural biology approaches. Point mutations, insertions, and deletions MIF variants, designed in silico at the monomer-monomer interfaces of MIF's X-ray structure, were generated, expressed and characterized using a battery of biophysical methods, in vitro. In silico and molecular dynamic studies performed on MIF's X-ray structure, allowed identifying several regions crucial for MIF's structure. First, our biophysical studies demonstrate that inter-subunit interactions involving the C-terminal region 105-114 play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties (Chapter 1). Carboxy-terminus truncated mutants (ΔC5 huMIF1-109 and ΔC10 huMIF1-104), as well as the insertion-mutant P107 MIF (designed to increase the flexibility of the C-terminal region) form enzymatically inactive trimers and exhibit reduced thermodynamic stability relative to the wild Type protein. On the other hand, our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity. We also demonstrated that a stable MIF monomeric state does not populate in vitro as the monomer undergoes rapid aggregation upon dissociation from the trimer. Furthermore, we described and characterized novel intersubunit interactions, involving the packing of a Leu46 side chain from one monomer into a hydrophobic pocket from the adjacent monomer (Chapter 2). Our findings provide new insight into the role of this pocket in modulating conformational states of MIF in solution. Also, disrupting this hydrophobic interaction through point mutations (L46G, L46A and L46F), is sufficient to decrease MIF stability to modulate its catalytic activity through tuning of the enzyme's efficiency and affinity towards its substrate. In the final chapter of this theses (Chapter 3), we describe our efforts to generate an in silico protocol for virtual high throughput screening of large libraries of small molecules and the discovery of novel inhibitors of MIF targeting its tautomerase activity. Our protocol takes into account the molecular dynamic parameters of MIF and aims at combined 1Docking standalone (Surflex scoring function), 2Docking (FlexX scoring function) coupled to Molecular Dynamics Simulations (MDS), and a 3combination of Docking score from FlexX coupled to MDS and Pharmacophore Filtering. in vitro validation of our virtual protocol led to the identification of three news inhibitors of MIF, with IC50 lower than 5 µM. Together, our studies provide i) new insights into structural properties of MIF and factors governing its oligomerization; ii) novel molecular tools (MIF mutants and small molecules) that can be used to dissect the structure-function relationship of MIF in health and disease; and iii) novel technical tool to identify small molecules inhibitors of MIF. Together, these results should contribute to the development of structure-based therapeutic strategies targeting specific molecular pathway(s), or structural aspect(s) relevant for its functions and role in health and diseases.
EPFL2010
