Small-molecule inhibitors of macrophage migration inhibitory factor (MIF) as an emerging class of therapeutics for immune disorders.

Macrophage migration inhibitory factor (MIF) is an important cytokine for which an increasing number of functions is being described in the pathogenesis of inflammation and cancer. Nevertheless, the availability of potent and druglike MIF inhibitors that are well-characterized in relevant disease models remains limited. Development of highly potent and selective small-molecule MIF inhibitors and validation of their use in relevant disease models will advance drug discovery. In this review, we provide an overview of recent advances in the identification of MIF as a pharmacological target in the pathogenesis of inflammatory diseases and cancer. We also give an overview of the current developments in the discovery and design of small-molecule MIF inhibitors and define future aims in this field.

High yield production of human invariant chain CD74 constructs fused to solubility-enhancing peptides and characterization of their MIF-binding capacities.

The HLA class II histocompatibility antigen gamma chain, also known as HLA-DR antigen-associated invariant chain or CD74, has been shown to be involved in many biological processes amongst which antigen loading and transport of MHC class II molecules from the endoplasmic reticulum to the Golgi complex. It is also part of a receptor complex for macrophage migration inhibitory factor (MIF), and participates in inflammatory signaling. The inhibition of MIF-CD74 complex formation is regarded as a potentially attractive therapeutic target in inflammation, cancer and immune diseases. In order to be able to produce large quantities of the extracellular moiety of human CD74, which has been reported to be unstable and protease-sensitive, different constructs were made as fusions with two solubility enhancers: the well-known maltose-binding domain and Fh8, a small protein secreted by the parasite Fasciola hepatica. The fusion proteins could be purified with high yields from Escherichia coli and were demonstrated to be active in binding to MIF. Moreover, our results strongly suggest that the MIF binding site is located in the sequence between the transmembrane and the membrane-distal trimerisation domain of CD74, and comprises at least amino acids 113-125 of CD74.

Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates.

A robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.

Demethionylation of Pro-1 variants of 4-oxalocrotonate tautomerase in Escherichia coli by co-expression with an engineered methionine aminopeptidase.

4-Oxalocrotonate tautomerase (4-OT) catalyzes the enol-keto tautomerization of 2-hydroxymuconate, utilizing its N-terminal proline (Pro-1) as general base catalyst. Substituting Pro-1 with bulky or charged residues will result in poor or no post-translational removal of the translation-initiating methionine by the methionine aminopeptidase (MetAP) of the Escherichia coli expression host. Here, we set out to investigate whether co-expression with previously engineered aminopeptidase MetAP-∗TG can be used to produce the P1S, P1H and P1Q variants of 4-OT in a demethionylated form. The P1S variant, which carries a small residue at the penultimate position (the first position after the initiating methionine), was found to be fully processed by wild-type MetAP. The P1S variant has low-level 2-hydroxymuconate tautomerase and promiscuous oxaloacetate decarboxylase activity. The P1Q and P1H variants of 4-OT, which carry bulky residues at the penultimate position, could only be obtained in a demethionylated form (a minor fraction of the purified protein is still composed of methionylated enzyme) by co-expression with MetAP-∗TG. Interestingly, the Gln-1 residue of the demethionylated P1Q variant undergoes intramolecular cyclization to form pyroglutamate (pE), yielding variant P1pE. Whereas the P1H/M1P2H mixture has low-level tautomerase activity, the P1pE/M1P2Q mixture has robust tautomerase activity. The substitution of Pro-1 by Gln, followed by removal of the initiating Met and cyclization of Gln-1 to form pE, is a unique way to obtain a structural analogue of proline on the N-terminus of 4-OT. This opens up new possibilities to study the importance of Pro-1 in recently discovered C-C bond-forming activities of this highly promiscuous tautomerase.

Dehalogenation of an anthropogenic compound by an engineered variant of the mouse cytokine macrophage migration inhibitory factor.

An unconventional dehalogenase: An engineered variant (I64V/V106L) of the mouse cytokine macrophage migration inhibitory factor (MIF) promiscuously catalyzes the hydrolytic dehalogenation of the xenobiotic organohalogen trans-3-chloroacrylic acid to acetaldehyde. Although the dehalogenase activity of this MIF variant is quite low, it achieves an ~10(9) -fold rate enhancement, matching those of conventional enzymes acting on their natural substrates.

Characterization of a newly identified mycobacterial tautomerase with promiscuous dehalogenase and hydratase activities reveals a functional link to a recently diverged cis-3-chloroacrylic acid dehalogenase.

The enzyme cis-3-chloroacrylic acid dehalogenase (cis-CaaD) is found in a bacterial pathway that degrades a synthetic nematocide, cis-1,3-dichloropropene, introduced in the 20th century. The previously determined crystal structure of cis-CaaD and its promiscuous phenylpyruvate tautomerase (PPT) activity link this dehalogenase to the tautomerase superfamily, a group of homologous proteins that are characterized by a catalytic amino-terminal proline and a ß-α-ß structural fold. The low-level PPT activity of cis-CaaD, which may be a vestige of the function of its progenitor, prompted us to search the databases for a homologue of cis-CaaD that was annotated as a putative tautomerase and test both its PPT and cis-CaaD activity. We identified a mycobacterial cis-CaaD homologue (designated MsCCH2) that shares key sequence and active site features with cis-CaaD. Kinetic and 1H NMR spectroscopic studies show that MsCCH2 functions as an efficient PPT and exhibits low-level promiscuous dehalogenase activity, processing both cis- and trans-3-chloroacrylic acid. To further probe the active site of MsCCH2, the enzyme was incubated with 2-oxo-3-pentynoate (2-OP). At pH 8.5, MsCCH2 is inactivated by 2-OP due to the covalent modification of Pro-1, suggesting that Pro-1 functions as a nucleophile at pH 8.5 and attacks 2-OP in a Michael-type reaction. At pH 6.5, however, MsCCH2 exhibits hydratase activity and converts 2-OP to acetopyruvate, which implies that Pro-1 is cationic at pH 6.5 and not functioning as a nucleophile. At pH 7.5, the hydratase and inactivation reactions occur simultaneously. From these results, it can be inferred that Pro-1 of MsCCH2 has a pKa value that lies in between that of a typical tautomerase (pKa of Pro-1∼6) and that of cis-CaaD (pKa of Pro-1∼9). The shared activities and structural features, coupled with the intermediate pKa of Pro-1, suggest that MsCCH2 could be characteristic of an evolutionary intermediate along the past route for the divergence of cis-CaaD from an unknown superfamily tautomerase. This makes MsCCH2 an ideal candidate for laboratory evolution of its promiscuous dehalogenase activity, which could identify additional features necessary for a fully active cis-CaaD. Such results will provide insight into pathways that could lead to the rapid divergent evolution of an efficient cis-CaaD enzyme.

Structural and functional characterization of a macrophage migration inhibitory factor homologue from the marine cyanobacterium Prochlorococcus marinus .

Macrophage migration inhibitory factor (MIF) is a multifunctional mammalian cytokine, which exhibits tautomerase and oxidoreductase activity. MIF homologues with pairwise sequence identities to human MIF ranging from 31% to 41% have been detected in various cyanobacteria. The gene encoding the MIF homologue from the marine cyanobacterium Prochlorococcus marinus strain MIT9313 has been cloned and the corresponding protein (PmMIF) overproduced, purified, and subjected to functional and structural characterization. Kinetic and (1)H NMR spectroscopic studies show that PmMIF tautomerizes phenylenolpyruvate and (p-hydroxyphenyl)enolpyruvate at low levels. The N-terminal proline of PmMIF is critical for these reactions because the P1A mutant has strongly reduced tautomerase activities. PmMIF shows high structural homology with mammalian MIFs as revealed by a crystal structure of PmMIF at 1.63 A resolution. MIF contains a Cys-X-X-Cys motif that mediates oxidoreductase activity, which is lacking from PmMIF. Engineering of the motif into PmMIF did not result in oxidoreductase activity but increased the tautomerase activity 8-fold. The shared tautomerase activities and the conservation of the beta-alpha-beta structural fold and key functional groups suggest that eukaryotic MIFs and cyanobacterial PmMIF are related by divergent evolution from a common ancestor. While several MIF homologues have been identified in eukaryotic parasites, where they are thought to play a role in modulating the host immune response, PmMIF is the first nonparasitic, bacterial MIF-like protein characterized in detail. This work sets the stage for future studies which could address the question whether a MIF-like protein from a free-living bacterium possesses immunostimulatory features similar to those of mammalian MIFs and MIF-like proteins found in parasitic nematodes and protozoa.