The N-terminus of MIF Controls the Flexibility of Specific β-sheet Residues Resulting in Dynamic Regulation of CD74 Activation

Macrophage migration inhibitory factor (MIF) is a pleiotropic protein with catalytic, CD74, CXCR2, CXCR4, and nuclease activities that contribute to the pathology of various inflammatory disorders, cardiovascular diseases, and cancer. The majority of MIF-triggered pathological conditions are associated with the activation of CD74, MIF’s cell surface receptor. The mechanistic details of MIF-induced activation of CD74 were mostly unknown until recently where it was shown that intramolecular dynamic signals transmitted from an allosteric center regulate the CD74activation site on MIF’s surface. Via backbone dynamic signals, the same center also controls the enzymatic pocket of MIF and more specifically the catalytically active residue Pro1, which serves as the sole target for the discovery and development of CD74 antagonists. Through these findings, the need to explore the dynamic communication between the enzymatic and CD74 activation sites became apparent. By utilizing molecular dynamics(MD) simulations, nuclear magnetic resonance (NMR) and circular dichroism (CD) experiments, we investigated how changes of the N-terminal flexibility influence the surface of MIF. Our findings support that dynamic signals transmitted from the enzymatic site reach the surface of MIF, affecting CD74 activation. For the first time, such data were able to provide dynamic profiles that explain the differences between MIF variants serving as CD74 agonists or antagonists

Insights into inhibition of heme-dependent dioxygenases

Tryptophan 2,3-dioxygenase (TDO), along with indoleamine 2,3-dioxygenase (IDO) and indoleamine 2,3-dioxygenase-2 (IDO2) are the three enzymes that catalyse oxidation of L-tryptophan (L-Trp) in the first step of the kynurenine pathway. Despite the fact that all three catalyse the same reaction, they were detected and characterized in different chronological periods; TDO, IDO and IDO2 were discovered in 1936, 1967 and 2007 respectively. Years of studies showed that abnormal regulation of L-Trp, in the first step of kynurenine pathway, is related with several disorders, including cancer. Regardless of their distinct dissimilarities, TDO, IDO and IDO2 were all detected in various cancers, supporting tumour escape and survival. The early identification of IDO immunomodulatory action (1990s) led to intense research for the development of IDO inhibitors, but not TDO. Despite this effort, the most pharmacologically suitable IDO inhibitor, 1-methyltryptophan (1- MT), appears to be ineffective as monotherapeutic drug. Discovery of IDO2 showed that 1-MT action is not fully understood, raising questions about the biological significance of IDO2. The ultimate goal of the current study was to address the problems outlined above. Because TDO and IDO are two druggable molecular targets, the discovery of a new class of effective inhibitors was pursued. Plate screening of ~2800 potential inhibitor compounds obtained from National Cancer Institute (NCI), USA, indicated seven promising compounds that inhibit both TDO and IDO in either nanomolar or low micromolar range. Interestingly, of these seven inhibitors, six have been identified to have cytotoxic action against several types of tumour cell lines (NCI data). NSC 26326, known as b-lapachone, is a natural occurring quinone and the strongest inhibitor of all seven. This NCI compound inhibits both TDO and IDO with inhibition constants of ~30-70 nM and 97 ± 14 nM respectively. Like NSC 26326, NSC 36398 is another natural occurring product and the only compound that showed selectivity against TDO with inhibition constant of 16.3 ± 3.8 μM. Among the seven compounds that displayed promise as inhibitors of TDO and IDO was mitomycin C. Mitomycin C, which is an approved oncology drug and a known inhibitor of IDO (Ki = 24.2 ± 1.2 μM), is also inhibitor of TDO with inhibition constant of 2.86 ± 0.03 μM. Another major goal of the current work was the discovery of isatin derivatives as inhibitors of TDO and IDO. Using the tryptophan-like structure of isatin as starting point, a number of structural modifications were carried out (structureactivity relationship (SAR)) succeeding the optimization of their inhibition activity. This new family of TDO and IDO inhibitors demonstrated inhibition potencies in the low micromolar range with 5,7-dicholoisatin to reach the nanomolar range (in the case of TDO). Halogenation of isatin and its derivatives was found to increase noticeably the inhibition potencies of these molecules by 12fold and 6fold for TDO and IDO respectively while breakdown of isatin’s pyrrolidine ring had a disastrous result on the inhibition of both enzymes. Combinations of 1-MT with either the newly-identified NCI inhibitors or the isatin derivatives were also examined. The in vitro combinations of 1-MT with either the NCI inhibitors or the isatin derivatives revealed an additive effect without though excluding the possibility of synergistic effect in vivo. The specificity of TDO, IDO and IDO2 against the two stereoisomers of 1- MT was also investigated, with interesting results. While IDO is inhibited only by the L-isoform of 1-MT (Ki = 18.0 ± 3.4 μM), IDO2 is inhibited by both 1-Me-L-Trp and 1-Me-D-Trp with inhibition constants of 306 ± 17 μM and 3419 ± 259 μM respectively. Biochemical characterization of human IDO2 was another goal of the current thesis, which completed successfully. Kinetic, redox and inhibition study of human IDO2 indicated significant differences in comparison with human IDO something which suggests the potential implication of IDO2 in an identified biological pathway (other than tryptophan catabolism function).The findings presented herein help to solve the mystery of 1-MT action, at least in vitro, give answers in regards to IDO2 function, and provide a number of new, promising inhibitors for TDO and IDO

Protocol for purification and enzymatic characterization of members of the human macrophage migration inhibitory factor superfamily

Summary: Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT or MIF-2) are two proteins serving a key role in the pathogenesis of multiple disorders, including cancer.1 Here, we present a protocol for the purification and enzymatic characterization of MIF and D-DT using keto-enol tautomerase activity. This approach measures enzymatic activity through the formation of an enol-borate complex. We describe steps for expressing and purifying proteins, preparing the 96-well microplate, and assay implementation including monitoring of keto-enol tautomerase activity.For complete details on the use and execution of this protocol, please refer to Parkins et al.2,3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

A novel enzymatic assay for the identification of 4-hydroxyphenylpyruvate dioxygenase modulators

Summary: 4-hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme involved in the pathogenesis of tyrosinemia III and cancer. Herein, we describe a spectroscopy-based assay to detect HPPD dioxygenase activity in the presence or absence of small-molecule modulators. We describe steps for transformation, expression, and purification of HPPD and preparation of the assay plate. We detail initiation and completion of the enzymatic reaction followed by detection of remaining substrate in the form of enol-HPP/borate complex. This assay is applicable for high-throughput screening.For complete details on the use and execution of this protocol, please refer to Parkins et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Insights into the mechanism of inhibition of tryptophan 2,3-dioxygenase by isatin derivatives

<p>Tryptophan 2,3-dioxygenase (TDO) is a cytosolic protein with a proven immunomodulatory function that promotes tumoral immune resistance and proliferation. Despite the interest in TDO as a therapeutic target in cancer treatment, the number of biologically useful inhibitors is limited. Herein, we report isatin derivatives as a new class of TDO inhibitors. Through structure–activity relationships and molecular docking studies, we optimized the inhibition potency of isatin derivatives by >130-fold and elucidated the mechanistic details that control their mode of action. Hydrogen bond interactions between the compound and key active site residues of TDO, freedom upon rotation of the C3 chemical moiety and the presence of chlorines in the benzene ring of the compound comprise the properties that an isatin-based inhibitor requires to effectively inhibit the enzymatic activity of TDO.</p

2,5-Pyridinedicarboxylic acid is a bioactive and highly selective inhibitor of D-dopachrome tautomerase

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT) are two pleotropic cytokines, which are coexpressed in various cell types to activate the cell surface receptor CD74. Via the MIF/CD74 and D-DT/CD74 axes, the two proteins exhibit either beneficial or deleterious effect on human diseases. In this study, we report the identification of 2,5-pyridinedicarboxylic acid (a.k.a. 1) that effectively blocks the D-DT-induced activation of CD74 and demonstrates an impressive 79-fold selectivity for D-DT over MIF. Crystallographic characterization of D-DT-1 elucidates the binding features of 1 and reveals previously unrecognized differences between the MIF and D-DT active sites that explain the ligand's functional selectivity. The commercial availability, low cost, and high selectivity make 1 the ideal tool for studying the pathophysiological functionality of D-DT in disease models. At the same time, our comprehensive biochemical, computational, and crystallographic analyses serve as a guide for generating highly potent and selective D-DT inhibitors

Nanosecond Dynamics Regulate the MIF‐Induced Activity of CD74

Macrophage migration inhibitory factor (MIF) activates CD74, which leads to severe disorders including inflammation, autoimmune diseases and cancer under pathological conditions. Molecular dynamics (MD) simulations up to one microsecond revealed dynamical correlation between a residue located at the opening of one end of the MIF solvent channel, previously thought to be a consequence of homotrimerization, and residues in a distal region responsible for CD74 activation. Experiments verified the allosteric regulatory site and identified a pathway to this site via the MIF β-strands. The reported findings provide fundamental insights on a dynamic mechanism that controls the MIF-induced activation of CD74