Toward target 2035: EUbOPEN - a public-private partnership to enable & unlock biology in the open.

Target 2035 is a global initiative that seeks to identify a pharmacological modulator of most human proteins by the year 2035. As part of an ongoing series of annual updates of this initiative, we summarise here the efforts of the EUbOPEN project whose objectives and results are making a strong contribution to the goals of Target 2035. EUbOPEN is a public-private partnership with four pillars of activity: (1) chemogenomic library collections, (2) chemical probe discovery and technology development for hit-to-lead chemistry, (3) profiling of bioactive compounds in patient-derived disease assays, and (4) collection, storage and dissemination of project-wide data and reagents.

Wnt/β-catenin and NFκB signaling synergize to trigger growth factor-free regeneration of adult primary human hepatocytes.

Background And Aims: The liver has a remarkable capacity to regenerate, which is sustained by the ability of hepatocytes to act as facultative stem cells that, while normally quiescent, re-enter the cell cycle after injury. Growth factor signaling is indispensable in rodents, whereas Wnt/β-catenin is not required for effective tissue repair. However, the molecular networks that control human liver regeneration remain unclear.

Target 2035 - an update on private sector contributions.

Target 2035, an international federation of biomedical scientists from the public and private sectors, is leveraging 'open' principles to develop a pharmacological tool for every human protein. These tools are important reagents for scientists studying human health and disease and will facilitate the development of new medicines. It is therefore not surprising that pharmaceutical companies are joining Target 2035, contributing both knowledge and reagents to study novel proteins.

Deep Annotation of Donated Chemical Probes (DCP) in Organotypic Human Liver Cultures and Patient-Derived Organoids from Tumor and Normal Colorectum.

Well-characterized small molecules are essential tools for studying the biology and therapeutic relevance of a target protein. However, many compounds reported in the literature and routinely studied in biomedical research lack the potency and selectivity required for mechanistic cellular studies on the function of a given protein. Furthermore, commercially available compounds often do not include useful tools developed by industry as part of their research and development efforts, as they frequently remain proprietary.

Comparative structural analyses of the NHL domains from the human E3 ligase TRIM-NHL family.

Tripartite motif (TRIM) proteins constitute one of the largest subfamilies of the RING-type E3 ubiquitin ligases that play a role in diverse processes from homeostasis and immune response to viral restriction. While TRIM proteins typically harbor an N-terminal RING finger, a B-box and a coiled-coil domain, a high degree of diversity lies in their C termini that contain diverse protein interaction modules, most of which, both structures and their roles in intermolecular interactions, remain unknown. Here, high-resolution crystal structures of the NHL domains of three of the four human TRIM-NHL proteins, namely TRIM2, TRIM3 and TRIM71, are presented.

Target 2035 - update on the quest for a probe for every protein.

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function.

A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5.

The transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases, and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases, and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems.

Biglycan, a novel trigger of Th1 and Th17 cell recruitment into the kidney.

Th1 and Th17 cells, T helper (Th) subtypes, are key inducers of renal fibrosis. The molecular mechanisms of their recruitment into the kidney, however, are not well understood. Here, we show that biglycan, a proteoglycan of the extracellular matrix, acting in its soluble form as a danger signal, stimulates autonomously the production of Th1 and Th17 chemoattractants CXCL10 and CCL20 in macrophages.

Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants.

In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-β (TRIF)-dependent manner in murine primary macrophages.

Nitric oxide mediates prolyl hydroxylase 3 expression in mesangial cells and in glomerulonephritis.

Unlabelled: Renal mesangial cells are regarded as main players in glomerular inflammatory diseases. To investigate a possible crosstalk between inflammatory and hypoxia-driven signaling processes, we stimulated cultured mouse mesangial cells with different inflammatory agents and analyzed the expression of prolyl hydroxylase domain containing proteins (PHDs), the main regulators of hypoxia-inducible factor (HIF) stability. Administration of IL-1β (1 nM) and TNF-α (1 nM), a combination further referred to as cytokine mix (CM), resulted in a fivefold increase in PHD3 but not PHD1 and PHD2 mRNA expression compared to untreated controls.

Proteoglycan neofunctions: regulation of inflammation and autophagy in cancer biology.

Inflammation and autophagy have emerged as prominent issues in the context of proteoglycan signaling. In particular, two small, leucine-rich proteoglycans, biglycan and decorin, play pivotal roles in the regulation of these vital cellular pathways and, as such, are intrinsically involved in cancer initiation and progression. In this minireview, we will address novel functions of biglycan and decorin in inflammation and autophagy, and analyze new emerging signaling events triggered by these proteoglycans, which directly or indirectly modulate these processes.

Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains.

Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library.

Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains.

Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library.

Bimodal role of NADPH oxidases in the regulation of biglycan-triggered IL-1β synthesis.

Biglycan, a ubiquitous proteoglycan, acts as a danger signal when released from the extracellular matrix. As such, biglycan triggers the synthesis and maturation of interleukin-1β (IL-1β) in a Toll-like receptor (TLR) 2-, TLR4-, and reactive oxygen species (ROS)-dependent manner. Here, we discovered that biglycan autonomously regulates the balance in IL-1β production in vitro and in vivo by modulating expression, activity and stability of NADPH oxidase (NOX) 1, 2 and 4 enzymes via different TLR pathways.

Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and β.

Zinc metalloproteinases meprin α and meprin β are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools, we have developed high throughput screening assays to enable discovery of inhibitors of both meprin α and meprin β and screened a collection of well characterized pharmaceutical agents (library of pharmaceutically active compounds, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin β.

LC-MS based cleavage site profiling of the proteases ADAM10 and ADAM17 using proteome-derived peptide libraries.

A Disintegrin and Metalloproteinase 10 (ADAM10) and ADAM17 catalyze ectodomain shedding of a number of cell surface proteins important for embryonic development and tissue homeostasis. Changes in the expression levels or dysregulated proteolytic activity of ADAM10 and ADAM17 have been shown to play important roles in multiple diseases such as inflammation, cancer, and neurodegenerative disorders. Despite the well documented substrate repertoire of ADAM10 and ADAM17, little is known about their cleavage site specificity.