Next generation risk assessment for occupational chemical safety - A real world example with sodium-2-hydroxyethane sulfonate.

Next Generation Risk Assessment (NGRA) is an exposure-led approach to safety assessment that uses New Approach Methodologies (NAMs). Application of NGRA has been largely restricted to assessments of consumer use of cosmetics and is not currently implemented in occupational safety assessments, e.g. under EU REACH. By contrast, a large proportion of regulatory worker safety assessments are underpinned by toxicological studies using experimental animals. Consequently, occupational safety assessment represents an area that would benefit from increasing application of NGRA to safety decision making. Here, a workflow for conducting NGRA under an occupational safety context was developed, which is illustrated with a case study chemical; sodium 2-hydroxyethane sulphonate (sodium isethionate or SI). Exposures were estimated using a standard occupational exposure model following a comprehensive life cycle assessment of SI and considering factory-specific data. Outputs of this model were then used to estimate internal exposures using a Physiologically Based Kinetic (PBK) model, which was constructed with SI specific Absorption, Distribution, Metabolism and Excretion (ADME) data. PBK modelling indicated a worst-case plasma maximum concentration (Cmax) of 0.8 µM across the SI life cycle. SI bioactivity was assessed in a battery of NAMs relevant to systemic, reproductive, and developmental toxicity; a cell stress panel, high throughput transcriptomics in three cell lines (HepG2, HepaRG and MCF-7 cells), pharmacological profiling and specific assays relating to developmental toxicity (Reprotracker and devTOX quickPredict). Points of Departure (PoDs) for SI ranged from 104 to 5044 µM. Cmax values obtained from PBK modelling of occupational exposures to SI were compared with PoDs from the bioactivity assays to derive Bioactivity Exposure Ratios (BERs) which demonstrated the safety for workers exposed to SI under current levels of factory specific risk management. In summary, the tiered and iterative workflow developed here represents an opportunity for integrating non animal approaches for a large subset of substances for which systemic worker safety assessment is required. Such an approach could be followed to ensure that animal testing is only conducted as a "last resort" e.g. under EU REACH.

Stable Binding of Full-Length Chemerin Is Driven by Negative Charges in the CMKLR1 N Terminus.

The adipokine chemerin is the endogenous ligand of the chemokine-like receptor 1 (CMKLR1), a member of the family of G protein-coupled receptors (GPCRs). This protein ligand plays an important role in obesity and inflammatory processes. Stable receptor-ligand interactions are highly relevant for its different physiological effects such as the migration of immune cells towards sites of inflammation. Here, we demonstrate that negative charges in the CMKLR1 N terminus are involved in the formation of strong contacts with a specific positively charged patch at the surface of full-length chemerin, which is absent in the short nonapeptide agonist chemerin-9, thus explaining its reduced affinity. Using receptor chimera of G protein-coupled receptor 1 (GPR1) and CMKLR1, we were able to identify the residues of this interaction and its relevance for stable full-length chemerin binding. This could help to develop more potent ligands for the treatment of inflammation-related diseases.

Cyclic Derivatives of the Chemerin C-Terminus as Metabolically Stable Agonists at the Chemokine-like Receptor 1 for Cancer Treatment.

Chemerin is a small chemotactic protein and a modulator of the innate immune system. Its activity is mainly mediated by the chemokine-like receptor 1 (CMKLR1), a receptor expressed by natural killer cells, dendritic cells, and macrophages. Downregulation of chemerin is part of the immune evasion strategy exploited by several cancer types, including melanoma, breast cancer, and hepatocellular carcinoma. Administration of chemerin can potentially counteract these effects, but synthetically accessible, metabolically stable analogs are required. Other tumors display overexpression of CMKLR1, offering a potential entry point for targeted delivery of chemotherapeutics. Here, we present cyclic derivatives of the chemerin C-terminus (chemerin-9), the minimal activation sequence of chemerin. Chemerin-9 derivatives that were cyclized through positions four and nine retained activity while displaying full stability in blood plasma for more than 24 h. Therefore, these peptides could be used as a drug shuttle system to target cancer cells as demonstrated here by methotrexate conjugates.

Cyclic Analogues of the Chemerin C-Terminus Mimic a Loop Conformation Essential for Activating the Chemokine-like Receptor 1.

The chemokine-like receptor 1 (CMKLR1) is a promising target for treating autoinflammatory diseases, cancer, and reproductive disorders. However, the interaction between CMKLR1 and its protein-ligand chemerin remains uncharacterized, and no drugs targeting this interaction have passed clinical trials. Here, we identify the binding mode of chemerin-9, the C-terminus of chemerin, at the receptor by combining complementary mutagenesis with structure-based modeling. Incorporating our experimental data, we present a detailed model of this binding site, including experimentally confirmed pairwise interactions for the most critical ligand residues: Chemerin-9 residue F8 binds to a hydrophobic pocket in CMKLR1 formed by the extracellular loop (ECL) 2, while F6 interacts with Y2.68, suggesting a turn-like structure. On the basis of this model, we created the first cyclic peptide with nanomolar activity, confirming the overall binding conformation. This constrained agonist mimics the loop conformation adopted by the natural ligand and can serve as a lead compound for future drug design.

A Deep Hydrophobic Binding Cavity is the Main Interaction for Different Y2 R Antagonists.

The neuropeptide Y2 receptor (Y2 R) is involved in various pathophysiological processes such as epilepsy, mood disorders, angiogenesis, and tumor growth. Therefore, the Y2 R is an interesting target for drug development. A detailed understanding of the binding pocket could facilitate the development of highly selective antagonists to study the role of Y2 R in vitro and in vivo. In this study, several residues crucial to the interaction of BIIE0246 and SF-11 derivatives with Y2 R were investigated by signal transduction assays. Using the experimental results as constraints, the antagonists were docked into a comparative structural model of the Y2 R. Despite differences in size and structure, all three antagonists display a similar binding site, including a deep hydrophobic cavity formed by transmembrane helices (TM) 4, 5, and 6, as well as a hydrophobic patch at the top of TM2 and 7. Additionally, we suggest that the antagonists block Q3.32 , a position that has been shown to be crucial for binding of the amidated C terminus of NPY and thus for receptor activation.

Unwinding of the C-Terminal Residues of Neuropeptide Y is critical for Y2 Receptor Binding and Activation.

Despite recent breakthroughs in the structural characterization of G-protein-coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double-cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T(32) to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.

Processing, signaling, and physiological function of chemerin.

Chemerin is an immunomodulating factor secreted predominantly by adipose tissue and skin. Processed by a variety of proteases linked to inflammation, it activates the G-protein coupled receptor chemokine-like receptor 1 (CMKLR1) and induces chemotaxis in natural killer cells, macrophages, and immature dendritic cells. Recent developments revealed the role of the nonsignaling chemerin receptor C-C chemokine receptor-like 2 (CCRL2) in inflammation. Besides further research establishing its link to inflammatory skin conditions such as psoriasis, functions in healthy skin have also been reported. Here, the current understanding of chemerin processing, signaling and physiological function has been summarized, focusing on the regulation of its activity, its different receptors and its controversially discussed role in diseases.

Proteolytic activation of prochemerin by kallikrein 7 breaks an ionic linkage and results in C-terminal rearrangement.

The excessive accumulation of adipose tissue in obesity is associated with multiple inflammatory dermatological diseases. Chemerin, a chemoattractant adipokine, dependent on proteolytical activation, is highly expressed in skin. Different proteases have been reported to activate prochemerin, but none is inherently expressed in human skin. In the present study, we identified a tissue-specific protease and investigated the underlying mechanism of activation at the molecular level. We characterized human KLK7 (kallikrein 7) as a prochemerin processing protease in vitro converting prochemerin into active chemerinF(156). The activating truncation by the protease might trigger a structural rearrangement leading to an increased affinity of chemerin to CMKLR1 (chemokine-like receptor 1). Molecular modelling and experimental data suggest an underlying ionic interaction in prochemerin C-terminal domains. These findings provide a general molecular basis for the necessity of C-terminal processing of prochemerin. Moreover, immunohistochemistry was used to investigate prochemerin, KLK7 and the recently identified KLK7 inhibitor vaspin expression in human skin biopsies, and distinct co-localization in psoriatic biopsies was observed. On the basis of these results, it is hypothesized that KLK7 activity may contribute to the development of psoriatic lesions as a consequence of excessive chemerin activation and impaired protease activity regulation by vaspin. Therefore this interaction represents an interesting target for psoriasis therapy and treatment of other obesity-related diseases.