Cyclo19,31[D-Cys19]-uPA19-31 is a potent competitive antagonist of the interaction of urokinase-type plasminogen activator with its receptor (CD87)

Urokinase-type plasminogen activator (uPA) represents a central molecule in pericellular proteolysis and is implicated in a variety of physiological and pathophysiological processes such as tissue remodelling, wound healing, tumor invasion, and metastasis. uPA binds with high affinity to a specific cell surface receptor, uPAR (CD87), via a well defined sequence within the N-terminal region of uPA (uPA(19-31)). This interaction directs the proteolytic activity of uPA to the cell surface which represents an important step in tumor cell proliferation, invasion, and metastasis. Due to its fundamental role in these processes, the uPA/uPAR-system has emerged as a novel target for tumor therapy. Previously, we have identified a synthetic, cyclic, uPA-derived peptide, cyclo(19,31)uPA(19-31), as a lead structure for the development of low molecular weight uPA-analogues, capable of blocking uPA/uPAR-interaction {[}Burgle et al., Biol. Chem. 378 (1997), 231-237]. We now searched for peptide variants of cyclo(19,31)uPA(19-31) with elevated affinities for uPAR binding. Among other tasks, we performed a systematic D-amino acid scan of quPA(19-31), in which each of the 13 L-amino acids was individually substituted by the corresponding D-amino acid. This led to the identification of cyclo(19,31) {[}D-Cys(19)]-uPA(19-31) as a potent inhibitor of uPA/uPAR-interaction, displaying only a 20 to 40-fold lower binding capacity as compared to the naturally occurring uPAR-ligands uPA and its amino-terminal fragment. Cyclo(19,31)[D-Cys(19)]-uPA(19-31) not only blocks binding of uPA to uPAR but is also capable of efficiently displacing uPAR-bound uPA from the cell surface and to inhibit uPA-mediated, tumor cell-associated plasminogen activation and fibrin degradation. Thus, cyclo(19,31)[D-Cys(19)]-uPA(19-31) represents a promising therapeutic agent to significantly affect the tumor-associated uPA/uPAR-system

Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control.

Suppression of premature termination codons (PTCs) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two potent readthrough promoters-NVS1.1 and NVS2.1-that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, and activating a branch of the ribosome-associated quality control network, which involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25

The small molecule specific EphB4 kinase inhibitor NVP-BHG712 inhibits VEGF driven angiogenesis

EphB4 and its cognitive ligand ephrinB2 play an important role in embryonic vessel development and vascular remodeling. In addition, several reports suggest that this receptor ligand pair is also involved in pathologic vessel formation in adults including tumor angiogenesis. Eph/ephrin signaling is a complex phenomena characterized by receptor forward signaling through the tyrosine kinase of the receptor and ephrin reverse signaling through various protein–protein interaction domains and phosphorylation motifs of the ephrin ligands. Therefore, interfering with EphR/ephrin signaling by the means of targeted gene ablation, soluble receptors, dominant negative mutants or antisense molecules often does not allow to discriminate between inhibition of Eph/ephrin forward and reverse signaling. We developed a specific small molecular weight kinase inhibitor of the EphB4 kinase, NVP-BHG712, which inhibits EphB4 kinase activity in the low nanomolar range in cellular assays showed high selectivity for targeting the EphB4 kinase when profiled against other kinases in biochemical as well as in cell based assays. Furthermore, NVP-BHG712 shows excellent pharmacokinetic properties and potently inhibits EphB4 autophosphorylation in tissues after oral administration. In vivo, NVP-BHG712 inhibits VEGF driven vessel formation, while it has only little effects on VEGF receptor (VEGFR) activity in vitro or in cellular assays. The data shown here suggest a close cross talk between the VEGFR and EphR signaling during vessel formation. In addition to its established function in vascular remodeling and endothelial arterio-venous differentiation, EphB4 forward signaling appears to be an important mediator of VEGF induced angiogenesis since inhibition of EphB4 forward signaling is sufficient to inhibit VEGF induced angiogenesis

G-Protein-coupled Bile Acid Receptor 1 (GPBAR1, TGR5) agonists reduce the production of pro-inflammatory cytokines and stabilize the alternative macrophage phenotype

GPBAR1 (also known as TGR5) is a G protein-coupled receptor (GPCR), which triggers intracellular signals upon ligation by various bile acids. The receptor has been studied mainly for its function in energy expenditure and glucose homeostasis, and there is little information on the role of GPBAR1 in the context of inflammation. After a high-throughput screening campaign, we identified isonicotinamides exemplified by compound 3 as non-steroidal GPBAR1 agonists. We optimized this series to potent derivatives that are active on both human and murine GPBAR1. These agonists inhibited the secretion of the pro-inflammatory cytokines TNF-alpha and IL-12, but not the anti-inflammatory IL-10 in primary human monocytes. These effects translate in vivo, as compound 15 inhibits LPS induced TNF-alpha and IL-12 release in mice. The response was GPBAR1 dependent, as demonstrated using knockout mice. Furthermore, agonism of GPBAR1 stabilized the phenotype of the alternative, non-inflammatory, M2-like type cells during differentiation of monocytes into macrophages. Overall, our results illustrate an important regulatory role for GPBAR1 agonists as controllers of inflammation

The Crystal structure of the cancer osaka thyroid kinase reveals an unexpected kinase domain fold

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune disorders such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Cancer Osaka Thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a Serine-Threonine (ST) kinase and is a key regulator in the production of proinflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective and potent inhibitors for the treatment of autoimmune disorders. The production of monomeric, recombinant COT kinase has proven to be very difficult and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the X-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this publication reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The glycine-rich loop contains a 15 amino acid insert that forms a β-hairpin structure and partially covers the ATP-binding site cleft, which – together with the structurally versatile active site – significantly impacts the design of potent, low-molecular weight COT inhibitors

Discovery of new binders for DCAF1, an emerging ligase target in the Targeted Protein Degradation field

In this study, we describe the rapid identification of potent binders for the WD40 repeat domain (WDR) of DCAF1. This was achieved by two rounds of iterative focused screening of a small set of compounds selected based on internal WDR domain knowledge followed by hit expansion. Subsequent structure-based design led to nanomolar potency binders with a clear exit vector enabling DCAF1-based bifunctional degrader exploration

A novel molecule promotes readthrough at premature stop codons by inducing co-translational degradation of eRF1 involving GCN1, RNF14 and RNF25

Drugs that promote translational readthrough at premature termination codons (PTC) are a promising for treating a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two novel and potent readthrough promoters – NVS1.1 and NVS2.1 – that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect decoding of the stop codon, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Interestingly, this appears to occur by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, which activate a novel branch of the ribosome quality control (RQC) network that involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF2

G‑Protein-Coupled Bile Acid Receptor 1 (GPBAR1, TGR5) Agonists Reduce the Production of Proinflammatory Cytokines and Stabilize the Alternative Macrophage Phenotype

GPBAR1 (also known as TGR5) is a G-protein-coupled receptor (GPCR) that triggers intracellular signals upon ligation by various bile acids. The receptor has been studied mainly for its function in energy expenditure and glucose homeostasis, and there is little information on the role of GPBAR1 in the context of inflammation. After a high-throughput screening campaign, we identified isonicotinamides exemplified by compound <b>3</b> as nonsteroidal GPBAR1 agonists. We optimized this series to potent derivatives that are active on both human and murine GPBAR1. These agonists inhibited the secretion of the proinflammatory cytokines TNF-α and IL-12 but not the antiinflammatory IL-10 in primary human monocytes. These effects translate in vivo, as compound <b>15</b> inhibits LPS induced TNF-α and IL-12 release in mice. The response was GPBAR1 dependent, as demonstrated using knockout mice. Furthermore, agonism of GPBAR1 stabilized the phenotype of the alternative, noninflammatory, M2-like type cells during differentiation of monocytes into macrophages. Overall, our results illustrate an important regulatory role for GPBAR1 agonists as controllers of inflammation