Healthy working in inclusive companies - a study protocol of the GAIN project.

BACKGROUND: The research project GAIN (working healthy in inclusion companies) deals with the topics of health and work in inclusive companies. Due to a great need for research on (occupational) health (e.g. physical and mental health status) and workplace design in companies employing people with disabilities, this project pursues the primary goal of generating information for the development and implementation of health-preserving measures within the framework of occupational health and safety, and risk assessment, for employees with and without impairments in inclusive companies. METHODS: Within the framework of the project, the employees of three inclusive companies will be examined with the help of an interdisciplinary and triangulative approach. Using quantitative and qualitative methods, specific physical workloads and hazards will be investigated by means of baseline screening methods and measurement techniques, specifically among employees with physical disabilities and impairments. In the statistical analysis, descriptive methods will be used to record the current state, while inferential statistical methods will be used to evaluate health maintenance measures. Inferential statistics for continuous data with confidence intervals based on the statistical parametric mapping (SPM) method will also be performed. The significance level will be set at 5%. Qualitative methods will be used to analyse structures and working conditions within the companies, with particular attention to the specific construction of the relationship between work, health and disability. CONCLUSIONS: The structures in inclusion companies must be specifically designed to support and promote the understanding of work and health in relation to the idea of one's own body, its positioning in space and its performance. These characteristics are to be identified in the course of the project and bundled into best-practice recommendations. Furthermore, it is the aim of the research project to derive recommendations for action at its conclusion and to present further advice for the promotion of health in inclusive companies.

Leveraging Ligand Affinity and Properties: Discovery of Novel Benzamide-Type Cereblon Binders for the Design of PROTACs.

Immunomodulatory imide drugs (IMiDs) such as thalidomide, pomalidomide, and lenalidomide are the most common cereblon (CRBN) recruiters in proteolysis-targeting chimera (PROTAC) design. However, these CRBN ligands induce the degradation of IMiD neosubstrates and are inherently unstable, degrading hydrolytically under moderate conditions. In this work, we simultaneously optimized physiochemical properties, stability, on-target affinity, and off-target neosubstrate modulation features to develop novel nonphthalimide CRBN binders. These efforts led to the discovery of conformationally locked benzamide-type derivatives that replicate the interactions of the natural CRBN degron, exhibit enhanced chemical stability, and display a favorable selectivity profile in terms of neosubstrate recruitment. The utility of the most potent ligands was demonstrated by their transformation into potent degraders of BRD4 and HDAC6 that outperform previously described reference PROTACs. Together with their significantly decreased neomorphic ligase activity on IKZF1/3 and SALL4, these ligands provide opportunities for the design of highly selective and potent chemically inert proximity-inducing compounds.

Accessing three-branched high-affinity cereblon ligands for molecular glue and protein degrader design.

The Petasis borono-Mannich reaction was employed for an alternative entry towards three-branched cereblon ligands. Such compounds are capabable of making multiple interactions with the protein surface and possess a suitable linker exit vector. The high-affinity ligands were used to assemble prototypic new molecular glues and proteolysis targeting chimeras (PROTACs) targeting BRD4 for degradation. Our results highlight the importance of multicomponent reactions (MCRs) in drug discovery and add new insights into the rapidly growing field of protein degraders.

Cereblon neo-substrate binding mimics the recognition of the cyclic imide degron.

In targeted protein degradation, immunomodulatory drugs (IMiDs) or cereblon (CRBN) E3 ligase modulatory drugs (CELMoDs) recruit neo-substrate proteins to the E3 ubiquitin ligase receptor CRBN for ubiquitination and subsequent proteasomal degradation. While the structural basis of this mechanism is generally understood, we have only recently described the recognition mode of the natural CRBN degron. In this communication, we reveal that the IMiD- or CELMoD-mediated binding of neo-substrates closely mimics the recognition of natural degrons. In crystal structures, we identify a conserved binding mode for natural degron peptides with an elaborate hydrogen bonding network involving the backbone of each of the six C-terminal degron residues, without the involvement of side chains. In a structural comparison, we show that neo-substrates recruited by IMiDs or CELMoDs emulate every single hydrogen bond of this network and thereby explain the origins of the largely sequence-independent recognition of neo-substrates. Our results imply that the V388I substitution in CRBN does not impair natural degron recognition and complete the structural basis for the rational design of CRBN effectors.

Synthesis of novel glutarimide ligands for the E3 ligase substrate receptor Cereblon (CRBN): Investigation of their binding mode and antiproliferative effects against myeloma cell lines.

To expand the chemical toolkit for targeted protein degradation, we report the generation of a new series of non-thalidomide Cereblon (CRBN) ligands. Readily available 2-methylidene glutarimide was converted to a series of 2-((hetero)aryl(methyl))thio glutarimides via the thio-Michael addition reaction. The compounds thus synthesized were evaluated for their affinity to the thalidomide-binding domain of human CRBN and their binding modes studied via X-ray crystallography. This helped identify several promising glutarimide derivatives which bind stronger to CRBN compared to thalidomide and contain a functional group which permits further chemical conjugation. Oxidation of the sulfur atom in a select group of 2-((hetero)aryl(methyl))thio glutarimides produced the respective sulfones which were found to possess a markedly stronger antiproliferative profile against multiple myeloma cell lines and a sophisticated structural binding mode with additional hydrogen bonding interactions. The newly identified Cereblon ligands form the basis for the synthesis of novel PROTAC protein degraders.

Identification and structural basis of C-terminal cyclic imides as natural degrons for cereblon.

Cereblon (CRBN) is a ubiquitously expressed E3 ligase substrate receptor and a key player in pharmaceutical targeted protein degradation. Despite substantial insight gained into its chemical ligand space that is exploited in small-molecule protein degraders, its cellular role and native mechanism of substrate recognition remained elusive so far. In this communication, we report the discovery of C-terminal aspartimide and aminoglutarimide residues as natural degron motifs that are recognized by CRBN with high specificity. These C-terminal cyclic imides are known to form in ageing proteins as a result of spontaneous chain breaks after an attack of an asparagine or glutamine side chain amide on the adjacent peptide bond, and thereby mark potentially malfunctional protein fragments. In crystal structures, we uncover that these C-terminal cyclic imides are bound in the same fashion as small-molecule CRBN modulators, and that the residues preceding the cyclic terminus contribute to the interaction with a sequence-unspecific backbone hydrogen bonding pattern with strictly conserved residues in CRBN. We postulate that C-terminal aspartimide and aminoglutarimide residues resulting from chain breaks are largely underappreciated protein damages and represent the native degrons of CRBN.

Basic Motor Competencies of 6- to 8-Year-Old Primary School Children in 10 European Countries: A Cross-Sectional Study on Associations With Age, Sex, Body Mass Index, and Physical Activity.

Basic motor competencies (BMC) are a prerequisite for children to be physically active, participate in sports and thus develop a healthy, active lifestyle. The present study provides a broad screening of BMC and associations with age, sex, body mass index (BMI) and extracurricular physical activity (PA) in 10 different European countries. The different country and regional contexts within Europe will offer a novel view on already established BMC associations. The cross-sectional study was conducted in 11 regions in 10 European countries in 2018. The motor competence areas, object movement (OM) and self-movement (SM), were assessed using the MOBAK-1-2 test instrument in 3758 first and second graders (age: M = 6.86 ± 0.60 years; 50% girls) during Physical Education classes. Children were questioned about their extracurricular PA and age. Their body weight and height were measured in order to calculate BMI. Statistical analyses included variances and correlations. The results showed significant differences in BMC levels between countries (OM: F = 18.74, p < 0.001, η2 = 0.048; SM: F = 73.10, p < 0.001, η2 = 0.163) whereas associations between BMC and correlates were similar. Boys performed significantly better in OM while girls performed better in SM. Age was consistently positively related to OM and SM with older children reaching higher levels of BMC than younger ones. While participation rates for extracurricular PA differed widely, participation in ball sports was correlated with OM and SM. Participation in individual sports showed a significant association with SM. In summary, BMC levels of children seem to depend on where they live and are strongly related to their participation in extracurricular PA. Therefore, education and health policies, in order to enhance motor competence development and PA participation, are recommended. Further research on country-specific Physical Education frameworks and their influence on BMC will provide more insights into structural factors and cultural characteristics of BMC development. On a school level, support tools and educational materials for teachers about BMC may enable children to achieve a basic level of motor competencies through Physical Education, contributing to lifelong participation in PA.

High-resolution structures of the bound effectors avadomide (CC-122) and iberdomide (CC-220) highlight advantages and limitations of the MsCI4 soaking system.

Cereblon (CRBN) is the substrate receptor of the CRL4CRBN E3 ubiquitin ligase and is a central player in targeted protein degradation. It is the target of the thalidomide-derived immunomodulatory drugs (IMiDs) and is one of the most widely employed receptors for proteolysis-targeting chimeras (PROTACs), both of which induce the ubiquitination and subsequent proteasomal degradation of target proteins. Structural studies of ligand binding to CRBN are crucial to elucidate the mechanisms of action and for mediation of side effects, ultimately aiding the development of next-generation IMiDs and PROTACs. With this aim, a crystal-soaking system based on the single-domain bacterial homologue MsCI4 has previously been established and used to delineate the binding modes of several classes of small molecules, including FDA-approved drugs, at the molecular level. Here, this system was used to characterize the binding of the next-generation IMiDs avadomide (CC-122) and iberdomide (CC-220) at high resolution, highlighting the advantages and limitations of the MsCI4 system and its implications for the development of future cereblon effectors.

PotN represents a novel energy-state sensing PII subfamily, occurring in firmicutes.

PII proteins are signal processor proteins that regulate the cellular metabolism of Bacteria, Archea and plant chloroplasts typically in response to the cellular nitrogen status. Here, we report the first biochemical characterization of a novel PII-like protein PotN from Lentilactobacillus hilgardii. PotN is encoded in an operon together with the potABCD genes, encoding the ABC transporter for spermidine/putrescine. Like canonical PII proteins, the native PotN has a trimeric structure and competitively binds ATP and ADP, but it does not bind 2-oxoglutarate. Immunoprecipitation and pull-down experiments revealed that PotN is associated in vivo with the transcriptional regulator GlnR and the beta-subunit of pyruvate/2-oxoglutarate/acetoin dehydrogenase AcoB. Moreover, in vitro assays revealed that the ATPase domain of PotA also is able to interact with PotN. Interaction analyses demonstrated that PotN preferentially associates with PotA in the ADP state, whereas it binds to GlnR at elevated ATP levels. This suggests that PotN regulates the transport of polyamines and GlnR-dependent gene expression in response to the energy availability for the cell.

Replacing the phthalimide core in thalidomide with benzotriazole.

The advent of proteolysis-targeting chimaeras (PROTACs) mandates that new ligands for the recruitment of E3 ligases are discovered. The traditional immunomodulatory drugs (IMiDs) such as thalidomide and its analogues (all based on the phthalimide glutarimide core) bind to Cereblon, the substrate receptor of the CRL4ACRBN E3 ligase. We designed a thalidomide analogue in which the phthalimide moiety was replaced with benzotriazole, using an innovative synthesis strategy. Compared to thalidomide, the resulting "benzotriazolo thalidomide" has a similar binding mode, but improved properties, as revealed in crystallographic analyses, affinity assays and cell culture.

Correction to "Sweet and Blind Spots in E3 Ligase Ligand Space Revealed by a Thermophoresis-Based Assay".

[This corrects the article DOI: 10.1021/acsmedchemlett.0c00440.].

Sweet and Blind Spots in E3 Ligase Ligand Space Revealed by a Thermophoresis-Based Assay.

Repurposing E3 ubiquitin ligases for targeted protein degradation via customized molecular glues or proteolysis-targeting chimeras (PROTACs) is an increasingly important therapeutic modality. Currently, a major limitation in the design of suitable molecular glues and PROTACs is our fragmentary understanding of E3 ligases and their ligand space. We here describe a quantitative assay for the discovery and characterization of E3 ligase ligands that is based on the thermophoretic behavior of a custom reporter ligand. Thereby, it is orthogonal to commonly employed fluorescence-based assays and less affected by the optical properties of test compounds. It can be employed for the high-throughput screening of compound libraries for a given ligase but also for hit validation, which we demonstrate with the identification of unexpected well-binders and non-binders, yielding new insights into the ligand space of cereblon (CRBN).

On the correlation of cereblon binding, fluorination and antiangiogenic properties of immunomodulatory drugs.

Cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, is a target of thalidomide and thalidomide-derived immunomodulatory drugs (IMiDs). The binding of these IMiDs to CRBN alters the substrate specificity of the ligase, thereby mediating multiple effects that are exploited in cancer therapy. However, to date, it is not clear which other possible targets might be involved in the efficacy of IMiDs. One especially prominent effect of a number of thalidomide analogs is their ability to inhibit angiogenesis, which is typically enhanced in fluorinated analogs. So far, the involvement of CRBN in antiangiogenic effects is under debate. Here, starting from a systematic set of thalidomide analogs and employing a quantitative in vitro CRBN-binding assay, we study the correlation of fluorination, CRBN binding and antiangiogenic effects. We clearly identify fluorination to correlate both with CRBN binding affinity and with antiangiogenic effects, but do not find a correlation between the latter two phenomena, indicating that the main target for the antiangiogenic effects of thalidomide analogs still remains to be identified.

De-Novo Design of Cereblon (CRBN) Effectors Guided by Natural Hydrolysis Products of Thalidomide Derivatives.

Targeted protein degradation via cereblon (CRBN), a substrate receptor of an E3 ubiquitin ligase complex, is an increasingly important strategy in various clinical settings, in which the substrate specificity of CRBN is altered via the binding of small-molecule effectors. To date, such effectors are derived from thalidomide and confer a broad substrate spectrum that is far from being fully characterized. Here, we employed a rational and modular approach to design novel and minimalistic CRBN effectors. In this approach, we took advantage of the binding modes of hydrolyzed metabolites of several thalidomide-derived effectors, which we elucidated via crystallography. These yielded key insights for the optimization of the minimal core binding moiety and its linkage to a chemical moiety that imparts substrate specificity. Based on this scaffold, we present a first active de-novo CRBN effector that is able to degrade the neo-substrate IKZF3 in the cell culture.

Construct and correlates of basic motor competencies in primary school-aged children.

BACKGROUND: A central aim of physical education is the promotion of basic motor competencies (in German: Motorische Basiskompetenzen; MOBAK), which are prerequisites for children's active participation in sports culture. This article introduces the MOBAK-1 test instrument for 6- to 8-year-old children and determines the construct validity of this test instrument. In addition, the relationship between MOBAK and motor ability (i.e., strength) as well as body mass index (BMI), sex, and age is investigated. METHODS: We analyzed data of 923 first and second graders (422 girls, 501 boys, age = 6.80 ± 0.44 years). The children's basic motor competencies were assessed by the MOBAK-1 test instrument. Besides analyses of frequency, correlation, and variance, 3 confirmatory factor analyses with covariates were performed. RESULTS: We found 2 MOBAK factors consisting of 4 items each. The first factor, locomotion, included the items balancing, rolling, jumping, and side stepping; the second factor, object control, included the items throwing, catching, bouncing, and dribbling. The motor ability strength had a significant influence on the factors locomotion (ß = 0.60) and object control (ß = 0.50). Older pupils achieved better results than younger pupils on object control (ß = 0.29). Boys performed better on object control (ß = -0.44), whereas girls achieved better results in locomotion (ß = 0.07). Pupils with a high BMI achieved lower performance only on the factor locomotion (ß = -0.28). CONCLUSION: The MOBAK-1 test instrument developed for this study meets psychometric validity demands and is suitable to evaluate effects of sports and physical education.

Design-Considerations regarding Silicon/Graphite and Tin/Graphite Composite Electrodes for Lithium-Ion Batteries.

An analytical model is proposed to investigate properties of composite electrodes that utilize more than one active material. We demonstrate how the equations can be applied to aid in the design of electrodes by comparing silicon-graphite and tin-graphite composite negative electrodes as examples with practical relevance. Based on simple assumptions, the results show how volume expansion tolerance and initial porosity are important factors for the achievable gravimetric and volumetric capacities as well as volumetric energy density. A Si-alloy/graphite composite electrode is used as an experimental system to corroborate the formulated analysis. Kinetic limitations are also addressed based on a novel heuristic approach.

Chemical Ligand Space of Cereblon.

The protein cereblon serves as a substrate receptor of a ubiquitin ligase complex that can be tuned toward different target proteins by cereblon-binding agents. This approach to targeted protein degradation is exploited in different clinical settings and has sparked the development of a growing number of thalidomide derivatives. Here, we probe the chemical space of cereblon binding beyond such derivatives and work out a simple set of chemical requirements, delineating the metaclass of cereblon effectors. We report co-crystal structures for a diverse set of compounds, including commonly used pharmaceuticals, but also find that already minimalistic cereblon-binding moieties might exert teratogenic effects in zebrafish. Our results may guide the design of a post-thalidomide generation of therapeutic cereblon effectors and provide a framework for the circumvention of unintended cereblon binding by negative design for future pharmaceuticals.

Lactoferrin Is an Allosteric Enhancer of the Proteolytic Activity of Cathepsin G.

Protease-mediated degradation of proteins is critical in a plethora of physiological processes. Neutrophils secrete serine proteases including cathepsin G (CatG), neutrophile elastase (NE), and proteinase 3 (PR3) together with lactoferrin (LF) as a first cellular immune response against pathogens. Here, we demonstrate that LF increases the catalytic activity of CatG at physiological concentration, with its highest enhancing capacity under acidic (pH 5.0) conditions, and broadens the substrate selectivity of CatG. On a functional level, the enzymatic activity of CatG was increased in the presence of LF in granulocyte-derived supernatant. Furthermore, LF enhanced CatG-induced activation of platelets as determined by cell surface expression of CD62P. Consequently, LF-mediated enhancement of CatG activity might promote innate immunity during acute inflammation.

The risk of involuntary firearms discharge.

OBJECTIVES: To investigate whether unintended muscle activity induced by motor overflow can result in the unintentional discharge of a firearm. BACKGROUND: In the past few years an increasing number of people have been injured, sometimes fatally, as a result of police weapons being discharged involuntarily. METHODS: Two experiments were performed using a pistol fitted with sensors to register the pressure exerted on the hand stock and the trigger. In Study 1, 34 police officers reacted to a set case using the prepared pistol. In Study 2, 25 participants performed 13 tasks requiring the use of different limbs. RESULTS: In Study 1, about 20% of participating officers made contact with the trigger, thus violating police regulations to keep the trigger finger outside the trigger guard at all times until a conscious decision to fire has been made. Study 2 showed that motor activity in different limbs leads to a significant increase in grip force exerted on a firearm (significant results for 12 of 13 trials, Bonferroni-corrected p < .004). Furthermore, pressure on the trigger resulting from involuntary muscle contractions was, in certain cases, sufficient to unintentionally discharge a weapon. CONCLUSION: Unintentional discharges attributable to involuntary muscle activity can be a real danger for police officers when carrying out their duties. APPLICATION: Results may help improve police training with the aim of reducing the danger of involuntary discharges in real-life situations.