Structural and Functional Studies of the Human Members of the Macrophage Migration Inhibitory Factor Family

Author

Andrew Parkins, University of the PacificFollow

Date of Award

2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Georgios Pantouris

First Committee Member

Jerry Tsai

Second Committee Member

Liang Xue

Third Committee Member

Joseph Harrison

Fourth Committee Member

Geoff Lin-Cereghino

Abstract

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) are the two human members of the MIF superfamily, which are implicated in an array of autoimmune disorders, inflammatory diseases, and cancer via their pleiotropic functionality. Despite only sharing 34% sequence identity, MIF and D-DT have high structural homology and overlapping functional traits, including activation of the type II cell surface receptor CD74 and keto-enol tautomerase activity. The MIF and/or D-DT-induced activation of CD74 leads to signaling cascades pivotal for cell growth, proliferation, and inhibition of apoptosis. Such characteristics make MIF and D-DT attractive molecular targets for drug discovery.

Currently, all small molecule antagonists targeting the MIF/D-DT-CD74 axis primarily bind to the catalytic sites of these proteins. Nevertheless, the precise interplay between the catalytic residues and those crucial for CD74 activation remains enigmatic. Notably, alterations of catalytic residues, particularly the catalytic residue Pro1, have been shown to impede CD74 activation. Leveraging molecular dynamics simulations and nuclear magnetic resonance (NMR) spectroscopy, we explored the dynamic coupling between the catalytically active N-terminus of MIF and surface residues pivotal for CD74 activation. Our investigation exposed previously unseen communication between the two sites and demonstrates the important role of MIF dynamics in the modulation of CD74 activation.

The keto-enol tautomerization assay utilizing 4-hydroxyphenylpyruvate (4-HPP) as a substrate has been instrumental in screening and characterization of MIF and D-DT variants as well as small molecule inhibitors. However, discrepancies between inhibition constant (Ki) values and Michaelis-Menten parameters raised concerns about the accuracy of results from this assay and the conclusions made from them. Our rigorous analysis identified that impurities present in substrate samples impacted the kinetic parameters of wild-type (WT) MIF as well as the Ki values of ISO-1, a well-studied inhibitor. Our findings, which were validated with multiple proteins, underscore the pronounced influence of substrate impurities on enzymatic activity. Thereby emphasizing the imperative of meticulously controlled experimental conditions for robust data interpretation.

While the majority of drug discovery efforts were focused on MIF, D-DT remains relatively underexplored in this regard. The identification of 4-(3-carboxyphenyl)-2,5- pyridinedicarboxylic acid (4-CPPC) as the first reversible and selective D-DT inhibitor opened new avenues of research for the protein. Structural analysis of D-DT – 4-CPPC revealed a ligand- induced conformational change of the C-terminal region that has mechanistic value. This observation is in stark contrast to MIF, which needs a rigid C-terminal for tertiary structure stability. In order to elucidate the impact of C-terminal conformational flexibility, we employed molecular dynamics simulations and NMR experiments. We found that while the binding of 4- CPPC did not alter the folding or thermostability of the protein, it drastically altered the protein’s dynamics, allowing for the formation of new, long-range intersubunit communications.

Subsequent endeavors aimed at identifying highly selective D-DT inhibitors that did not cause a conformational change of the C-terminal region yielded 2,5-pyridinedicarboxylic acid (1). This molecule exhibits a low micromolar potency and a remarkable 79-fold specificity for D-DT over MIF. Crystallographic analysis of the D-DT-1 complex displayed that the C-terminal of D- DT was largely unperturbed by the binding of 1 and delineated structural disparities between D- DT and MIF active sites, underscoring the potential for rational drug design strategies. Further in vivo studies focusing on the cytokine activity of D-DT showed the efficacy of 1 as an inhibitor of D-DT induced activation of CD74. These findings show that 1 is a useful mechanistic tool for interrogating the pathophysiology of D-DT.

Despite these exciting discoveries, the role of the C-terminal region in the enzymatic activity and conformational flexibility of D-DT required further investigation. In-depth interrogation of seventeen protein variants and WT D-DT uncovered a previously unknown functional role of the C-terminal region. These insights deepen our comprehension of protein structure-function relationships and provides an invaluable foundation for future drug discovery studies targeting D-DT-mediated pathological conditions.

Overall, via our thorough experimental interrogations, we uncovered key structural and functional information about MIF and D-DT that will serve as the basis for future mechanistic and drug discovery projects.

Pages

242

Recommended Citation

Parkins, Andrew. (2024). Structural and Functional Studies of the Human Members of the Macrophage Migration Inhibitory Factor Family. University of the Pacific, Dissertation. https://scholarlycommons.pacific.edu/uop_etds/4267