Functionalized pyrrolidine inhibitors of human alpha- mannosidases II in cancer: optimizing the fit to the active site

Refining the chemical structure of functionalized pyrrolidine-based inhibitors of Golgi α- mannosidase II (GMII) to optimize binding affinity provided a lead molecule that demonstrated nanomolar competitive inhibition of α-mannosidases II and an optimal fit in the active site of Drosophila GMII by X-ray crystallography. Esters of this lead compound also inhibited the growth of human glioblastoma and brain-derived endothelial cells more than the growth of non-tumoral human fibroblasts, suggesting their potential for anti-cancer therapy.

Development of new anticancer agents based on [alpha]-mannosidase inhibition

Hélène Fiaux

Enzymes that are involved in the synthesis and processing of oligosaccharides, such as glycosidases, are important catalysts for the specific assembly of oligosaccharide structures on proteins. Design and preparation of selective inhibitors of these enzymes are of high interest as these molecules can be used to modulate cellular functions. Moreover, they may provide potential drugs in new therapeutic strategies. In particular, swainsonine (Swa), a natural inhibitor of Golgi α-mannosidase II, reduces certain tumors and hematological dysfunctions. Nevertheless, some side effects resulted in limitations for the development of this compound in medicinal treatments. Recently, our laboratory has developed a new combinatorial methodology for the disclosure of glycosidase inhibitors based on the dihydroxypyrrolidine scaffold found in Swa. This method allowed the preparation of a new family of selective and competitive a-mannosidase inhibitors with inhibitory activities (Ki values) ranging from 3 µM to 135 nM for the best congeners toward α-mannosidase from jack bean, a reliable model enzyme for mammalian Golgi α-mannosidase II. The aim of the present work was to design and synthesise optimized inhibitors of α-mannosidases II based on a 2-aminomethyl-3,4-dihydroxypyrrolidine scaffold and to assess their potential as new anti cancer leads. In a first time, a structural study was carried out through the analysis of x-ray structures of the best inhibitors previously developed in our laboratory complexed to Golgi α-mannosidase II from Drosophila melanogaster (dGMII), which presents a high sequence identity with the human enzyme. The binding mode of these inhibitors could be thus established. This study combined with docking experiments allowed us to design inhibitors of α-mannosidase II, with improved efficacy. The best congener (3R,4R,5R)-3,4-dihydroxy-5-({[(1R)-2-hydroxy-1-phenylethyl]amino}methyl)-1-methylpyrrolidin-2-one displayed an enhanced inhibitory potency against dGMII (IC50 = 0.5 µM vs 80 µM for the first generation inhibitor). Introduction of a polar substituent on the C(5) position of the pyrrolidine ring accounted for this improved activity as revealed by the x-ray structure of this new derivative complexed to dGMII which displayed additional interactions with active site residues. In a second time, these new α-mannosidase inhibitors have been evaluated as anti cancer agents and their effects were determined on human cancer cells from glioblastoma and melanoma, two tumors associated with a high proliferative and invasive potential, multiple resistance toward conventional chemotherapeutic agents, and poor prognosis. As these compounds were not able to inhibit cellular growth, probably due to a poor cellular uptake, derivatization of our inhibitors into more lipophilic esters, ethers and amides derivatives was envisaged. In particular, introduction of a 4-bromobenzoate moiety resulted in an improvement of cellular uptake, leading to a complete inhibition of cellular growth at 300 µM with inhibitors (2R)-2-({[(2R,3R,4S)-3,4-dihydroxypyrrolidin-2-yl]methyl}amino)-2-phenylethyl 4-bromobenzoate and (2R)-2-({[(2R,3R,4R)-3,4-dihydroxy-1-methyl-5-oxopyrrolidin-2-yl]methyl}amino)-2-phenylethyl 4-bromobenzoate. Under the same conditions, Swa didn't provide any significant growth inhibition. Moreover, exposure of human fibroblasts, as model of healthy cells, to our lead derivatives revealed some selectivity toward tumoral cells. It was hypothesized that cellular esterases were able to hydrolyse the ester moiety and thus release the more hydrophilic parent inhibitors within the tumoral cells. Finally, in order to get more insights into the mode of action of our derivatives, their inhibitory activity on α-mannosidases from human glioblastoma extracts was determined and compared to the activity of Swa. The first generation inhibitor presented a moderate inhibitory potency on α-mannosidases whereas the optimized derivative displayed potent inhibitory activity on human α-mannosidases (IC50 = 50 µM decreased to 0.5 µM). Nevertheless, Swa presented higher potency than our new lead (IC50 = 10-50 nM). It was also determined by gel filtration that the cellular α-mannosidases targeted by our pyrrolidine based inhibitors displayed apparent molecular weight of about 120 kD.

EPFL2007

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

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