Small Molecule Based Approaches to Inhibit Macrophage Migration Inhibitory Factor (MIF) Activities and Elucidate its Role in Health and Disease

Macrophage migration inhibitory factor (MIF) is a major mediator in innate immunity and inflammation and a potential therapeutic target in multiple inflammatory, infectious and autoimmune diseases including cancer. Current therapeutic strategies for targeting MIF focus on modulating its biological activities using anti-MIF neutralizing antibodies or developing inhibitors of its tautomerase activity. Although the identity of its natural substrate remains unknown, several small molecule inhibitors have been reported to be effective inhibitors of MIF tautomerase activity in vitro. All these inhibitors were identified by rational design and structure-activity studies based on the structure of the catalytic site and/or non physiologic substrates of MIF. The lack of suitable high-throughput screening (HTS) assays has hindered the screening of chemical libraries and discovery of more diverse inhibitors. Motivated by the goal of discovering diverse classes of MIF inhibitors that modulate MIF activity specific targeting of its biochemical, conformation and quaternary structure properties, both activity based endpoint and kinetic HTS assays were developed, optimized and performed on diverse set of libraries. Using the endpoint assay, out of 15440 compounds screened, twelve novel inhibitors of MIF's catalytic activity (∼ 0.1% hit rate) with IC50s in the range of 1.5 to 15.5 µM were identified and validated. The interaction site and mechanism of action of all these inhibitors were defined using structure activity studies and a battery of biochemical and biophysical methods, including mass spectrometry, light scattering, and NMR. The effect on MIF biological activities was also examined on MIF-mediated glucocorticoid overriding and MIF-induced Akt phosphorylation. Using the kinetic-based activity assay, we screened 80,000 small molecules and identified and validated thirteen novel inhibitors of MIF catalytic activity with inhibition constant (Ki,app) values ranging from 0.5 to 13 µM. According to the structure and potency some of these molecules could be of great therapeutic potential. The MIF inhibitors emerging from these studies could be divided into four classes: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro1 which could be classified into 5 groups; 2) a novel class of catalytic site inhibitors, 3) a class of trimer destabilizing inhibitors. These findings demonstrate the potential of HTS assays as attractive means to identify novel classes of MIF inhibitors as lead drug candidates and/or chemical tools for elucidating the biochemical and structural bases underlying the multifunctionality of MIF's in health and disease. Having these molecules in hand, will advance our understanding of protein-protein interactions mechanism for MIF oligomerization and may help to elucidate the role of MIF enzymatic activity in health and disease.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action

Min Kyu Cho, Farah El Turk, Bruno Claude Daniel Fauvet, Hilal Lashuel, Hajer Ouertatani-Sakouhi, Magdalena Zweckstetter

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

American Society for Biochemistry and Molecular Biology2010

Elucidating the Role of Quaternary Structure in Modulating Macrophage Migration Inhibitory Factor (MIF) Functions

Farah El Turk

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

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Amer Ashrafi, Michele Cascella, Min Kyu Cho, Farah El Turk, Bruno Claude Daniel Fauvet, Hilal Lashuel, Marilisa Neri, Hajer Ouertatani-Sakouhi, Florence Pojer, Ursula Röthlisberger, Magdalena Zweckstetter

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the beta-strand beta 3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Public Library of Science2012