A selective small-molecule inhibitor of macrophage migration inhibitory factor-2 (MIF-2), a MIF cytokine superfamily member, inhibits MIF-2 biological activity

Abstract

Edited by Dennis R. Voelker Cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a recently characterized second member of the MIF cytokine superfamily in mammalian genomes. MIF-2 shares pro-inflammatory and tumorigenic properties with the clinical target MIF (MIF-1), but the precise contribution of MIF-2 to immune physiology or pathology is unclear. Like MIF-1, MIF-2 has intrinsic keto-enol tautomerase activity and mediates biological functions by engaging the cognate, common MIF family receptor CD74. Evidence that the catalytic site of MIF family cytokines has a structural role in receptor binding has prompted exploration of tautomerase inhibitors as potential biological antagonists and therapeutic agents, although few catalytic inhibitors inhibit receptor activation. Here we describe the discovery and biochemical characterization of a selective small-molecule inhibitor of MIF-2. An in silico screen of 1.6 million compounds targeting the MIF-2 tautomerase site yielded several hits for potential catalytic inhibitors of MIF-2 and identified 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) as the most functionally potent compound. We found that 4-CPPC has an enzymatic IC 50 of 27 M and 17-fold selectivity for MIF-2 versus MIF-1. An in vitro binding assay for MIF-1/MIF-2 to the CD74 ectodomain (sCD74) indicated that 4-CPPC inhibits MIF-2-CD74 binding in a dosedependent manner (0.01-10 M) without influencing MIF-1-CD74 binding. Notably, 4-CPPC inhibited MIF-2-mediated activation of CD74 and reduced CD74-dependent signal transduction. These results open opportunities for development of more potent and pharmacologically auspicious MIF-2 inhibitors to investigate the distinct functions of this MIF family member in vivo. Macrophage migration inhibitory factor (MIF or MIF-1) 2 is a widely expressed multifunctional cytokine that mediates the immune response to infection, contributes to the development of autoimmune disorders, and has role in the inflammatory pathogenesis of cancer. MIF-1's pathogenic role is supported by both experimental and clinical studies, including human genetic findings that link a commonly occurring, functional promoter polymorphism with different autoimmune disorders (1-3), infectious conditions (4-6) and tumors (7). The threedimensional X-ray crystal structure of MIF-1 was elucidated in 1996 and led to the description of a new structural superfamily with MIF-1 as its defining member (8, 9). A second family member, D-dopachrome tautomerase (D-DT or MIF-2), with 34% sequence identity and a three-dimensional structure nearly identical to MIF-1, was defined structurally by Sugimoto et al. (10) and recently characterized biologically (11, 12). Both MIF-1 and MIF-2 are released from activated monocytes/ macrophages and signal through the surface receptor CD74, leading to recruitment of CD44 into a signaling complex and subsequently initiating the ERK1/2 mitogen-activated protein kinase pathway (13, 14). In addition, MIF-1 exerts chemokinelike functions thro...

Separating cytokine twins with a small molecule

“…E-mail: juergen.bernhagen@ med.uni-muenchen.de. This is where the current study by Tilstam et al (9) comes in. The authors begin their search for MIF-2 inhibitors with an in silico screen of 1.6 million compounds docked into the MIF-2 catalytic pocket (9), leading to 1821 hits that bound to two conformational states.…”

“…In summary, whereas 4-CPPC is the first identified MIF-2specific small molecule, it certainly will require extensive optimization by medicinal chemistry approaches to improve selectivity, affinity, and potency. The study by Tilstam et al (9) lays out a novel translational avenue for MIF protein familydirected therapies in particular and cytokine homolog-specific strategies in general and provides the scientific community with a long-awaited research tool to elucidate the functions of the MIF ligand/receptor network in physiology and pathophysiology.…”

A structurally preserved allosteric site in the MIF superfamily affects enzymatic activity and CD74 activation in D-dopachrome tautomerase

“…18 This active site has been the target of many MIF inhibitors, yet it has also been demonstrated that certain small molecules can selectively inhibit MIF or MIF-2. 19 Importantly, there is a connection between the enzymatic site and CD74 binding and activation, shown by MIF mutants 20 and molecular dynamics (MD) simulations. 21 Whether a similar connection between the enzymatic and CD74 sites exists in MIF-2 is unknown, but it is important to highlight that predicted models of MIF-CD74 and MIF-2-CD74 have similarities (and differences) in regions that interact with CD74.…”

MIF-2/D-DT is an atypical atherogenic chemokine that promotes advanced atherosclerosis and hepatic lipogenesis

“…To further confirm the role of MIF-2 in early atherogenesis and address the potential role of compensatory effects caused by global gene deficiency, we employed a pharmacological blockade approach, using 4-CPPC, a specific small molecule inhibitor of MIF-2 that exhibits a 13-fold selectivity against MIF-2 over MIF (32,51). Apoe -/mice were placed on a 4.5-week HFD and 4-CPPC versus vehicle control was administered in parallel to the HFD (intraperitoneal (i.p.)…”