Ulrike Eggert: Big things from small molecules

Eggert uses RNAi and chemistry to develop novel tools for investigating cytokinesis

Thioglycosides Are efficient metabolic decoys of glycosylation that reduce selectin dependent leukocyte adhesion

Metabolic decoys are synthetic analogs of naturally occurring biosynthetic acceptors. These compounds divert cellular biosynthetic pathways by acting as artificial substrates that usurp the activity of natural enzymes. While O-linked glycosides are common, they are only partially effective even at millimolar concentrations. In contrast, we report that N-acetylglucosamine (GlcNAc) incorporated into various thioglycosides robustly truncate cell surface N- and O-linked glycan biosynthesis at 10-100 μM concentrations. The >10-fold greater inhibition is in part due to the resistance of thioglycosides to hydrolysis by intracellular hexosaminidases. The thioglycosides reduce β-galactose incorporation into lactosamine chains, cell surface sialyl Lewis-X expression, and leukocyte rolling on selectin substrates including inflamed endothelial cells under fluid shear. Treatment of granulocytes with thioglycosides prior to infusion into mouse inhibited neutrophil homing to sites of acute inflammation and bone marrow by ∼80%-90%. Overall, thioglycosides represent an easy to synthesize class of efficient metabolic inhibitors or decoys. They reduce N-/O-linked glycan biosynthesis and inflammatory leukocyte accumulation

Thioglycosides Are Efficient Metabolic Decoys of Glycosylation that Reduce Selectin Dependent Leukocyte Adhesion

© 2018 Elsevier Ltd Small-molecule inhibitors of glycosylation can be applied in basic science studies, and clinical investigations as anti-inflammatory, anti-metastatic, and anti-viral therapies. This article demonstrates that thioglycosides represent a class of potent metabolic decoys that resist hydrolysis, and block E-selectin-dependent leukocyte adhesion in models of inflammation

Marine Pyrrolocarbazoles and Analogues: Synthesis and Kinase Inhibition

Granulatimide and isogranulatimide are alkaloids obtained from marine sources which have been shown to inhibit cell-cycle G2-checkpoint, targeting more particularly checkpoint 1 kinase (Chk1). At a structural level, they possess a characteristic pyrrolocarbazole framework also shared by the well-known rebeccamycin and staurosporine microbial metabolites which have been described to inhibit topoisomerase I and diverse kinases, respectively. This review reports precisely on the synthesis and kinase inhibitory activities of pyrrolocarbazole-based analogues of granulatimide

HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

Cytokinesis, the final phase of cell division, is necessary to form two distinct daughter cells with correct distribution of genomic and cytoplasmic materials. Its failure provokes genetically unstable states, such as tetraploidization and polyploidization, which can contribute to tumorigenesis. Aurora-B kinase controls multiple cytokinetic events, from chromosome condensation to abscission when the midbody is severed. We have previously shown that HIPK2, a kinase involved in DNA damage response and development, localizes at the midbody and contributes to abscission by phosphorylating extrachromosomal histone H2B at Ser14. Of relevance, HIPK2-defective cells do not phosphorylate H2B and do not successfully complete cytokinesis leading to accumulation of binucleated cells, chromosomal instability, and increased tumorigenicity. However, how HIPK2 and H2B are recruited to the midbody during cytokinesis is still unknown. Here, we show that regardless of their direct (H2B) and indirect (HIPK2) binding of chromosomal DNA, both H2B and HIPK2 localize at the midbody independently of nucleic acids. Instead, by using mitotic kinase-specific inhibitors in a spatio-temporal regulated manner, we found that Aurora-B kinase activity is required to recruit both HIPK2 and H2B to the midbody. Molecular characterization showed that Aurora-B directly binds and phosphorylates H2B at Ser32 while indirectly recruits HIPK2 through the central spindle components MgcRacGAP and PRC1. Thus, among different cytokinetic functions, Aurora-B separately recruits HIPK2 and H2B to the midbody and these activities contribute to faithful cytokinesis

Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor

BACKGROUND: The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2. PRINCIPAL FINDINGS: Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases. SIGNIFICANCE: The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis

Design and investigation of bioactive ruthenium-based protein kinase inhibitors

The design and synthesis of selective small molecule inhibitors of protein kinases to modulate their activity is of great interest for chemical biologists. However, most of the small molecule inhibitors are ATP-competitive, which can limit selectivity because the ATP binding sites of kinases show significant similarity. In this thesis, I present the biological properties of a novel class of protein kinase inhibitors. Inspired from staurosporine, this approach involves introducing a metal center to an otherwise organic scaffold allowing one to explore unique chemical spaces. We envisioned that by using this approach, it should be possible to induce high selectivity and potency towards the targeted kinases. The majority of this work focuses on the studies that were conducted on GSK-3, a model kinase. Initially, structure-activity relationships were carried out on a complex with pseudotetrahedral geometry around the metal center. These studies yielded a highly selective inhibitor for GSK-3 with picomolar affinity whose remarkable activity has been confirmed in tissue culture and zebrafish embryos. In addition, this class of compounds has also shown promising anti-cancer properties. In the second half of the thesis, the binding mode of these pseudotetrahedral complexes for GSK-3 has been investigated using X-ray crystallography. The focus of these studies is to understand the nature of the potency and selectivity of this scaffold, which includes an inhibitor with a Ki ≤ 5 pM, one of the tightest binding inhibitors of protein kinases. In the last part of this thesis, two new scaffolds with octahedral geometry for GSK-3 and DAPK-1 have been investigated. These X-ray crystallographic studies have helped us to understand the interactions that promote potent binding and good selectivity and have led to new ideas for developing superior scaffolds. Overall, this thesis provides a proof of principle that ruthenium-based organometallic compounds can be used as superior scaffolds to design potent and selective protein kinase inhibitors. In addition, these complexes represent a great candidate as tools for chemical biology to unravel biological questions because they are stable in cellular environment and show cellular activities at very low concentrations

Design and investigation of bioactive ruthenium-based protein kinase inhibitors

The design and synthesis of selective small molecule inhibitors of protein kinases to modulate their activity is of great interest for chemical biologists. However, most of the small molecule inhibitors are ATP-competitive, which can limit selectivity because the ATP binding sites of kinases show significant similarity. In this thesis, I present the biological properties of a novel class of protein kinase inhibitors. Inspired from staurosporine, this approach involves introducing a metal center to an otherwise organic scaffold allowing one to explore unique chemical spaces. We envisioned that by using this approach, it should be possible to induce high selectivity and potency towards the targeted kinases. The majority of this work focuses on the studies that were conducted on GSK-3, a model kinase. Initially, structure-activity relationships were carried out on a complex with pseudotetrahedral geometry around the metal center. These studies yielded a highly selective inhibitor for GSK-3 with picomolar affinity whose remarkable activity has been confirmed in tissue culture and zebrafish embryos. In addition, this class of compounds has also shown promising anti-cancer properties. In the second half of the thesis, the binding mode of these pseudotetrahedral complexes for GSK-3 has been investigated using X-ray crystallography. The focus of these studies is to understand the nature of the potency and selectivity of this scaffold, which includes an inhibitor with a Ki ≤ 5 pM, one of the tightest binding inhibitors of protein kinases. In the last part of this thesis, two new scaffolds with octahedral geometry for GSK-3 and DAPK-1 have been investigated. These X-ray crystallographic studies have helped us to understand the interactions that promote potent binding and good selectivity and have led to new ideas for developing superior scaffolds. Overall, this thesis provides a proof of principle that ruthenium-based organometallic compounds can be used as superior scaffolds to design potent and selective protein kinase inhibitors. In addition, these complexes represent a great candidate as tools for chemical biology to unravel biological questions because they are stable in cellular environment and show cellular activities at very low concentrations

Emerging Roles of Ceramides in Breast Cancer Biology and Therapy

One of the classic hallmarks of cancer is the imbalance between elevated cell proliferation and reduced cell death. Ceramide, a bioactive sphingolipid that can regulate this balance, has long been implicated in cancer. While the effects of ceramide on cell death and therapeutic efficacy are well established, emerging evidence indicates that ceramide turnover to downstream sphingolipids, such as sphingomyelin, hexosylceramides, sphingosine-1-phosphate, and ceramide-1-phosphate, is equally important in driving pro-tumorigenic phenotypes, such as proliferation, survival, migration, stemness, and therapy resistance. The complex and dynamic sphingolipid network has been extensively studied in several cancers, including breast cancer, to find key sphingolipidomic alterations that can be exploited to develop new therapeutic strategies to improve patient outcomes. Here, we review how the current literature shapes our understanding of how ceramide synthesis and turnover are altered in breast cancer and how these changes offer potential strategies to improve breast cancer therapy