Biology subjects

von Kries, J. P.

Publications and source records attributed to von Kries, J. P..

7 recordsLinked to original sources

bioRxiv2024

Structural basis for immune cell binding of Fusobacterium nucleatum via the trimeric autotransporter adhesin CbpF

Fusobacterium nucleatum (Fn), a commensal in the human oral cavity, is overrepresented in the colon microbiota of colorectal cancer (CRC) patients and is linked to tumor chemoresistance, metastasis, and a poor therapeutic prognosis. Fn produces numerous adhesins that mediate tumor colonization and downregulation of the hosts anti-tumor immune response. One of these, the trimeric autotransporter adhesin (TAA) CEACAM binding protein of Fusobacterium (CbpF), targets CEACAM1 on T-cells and has been associated with immune evasion of Fn-colonized tumors. Whereas the role of CEACAM1 in homophilic and heterophilic cell interactions and immune evasion is well described, the mechanistic details of its interaction with fusobacterial CbpF remain unknown due to the lack of a high-resolution structure of the adhesin-receptor complex. Here, we present two structures of CbpF alone and in complex with CEACAM1, obtained by cryogenic electron microscopy and single particle analysis. They reveal that CbpF forms a stable homotrimeric complex whose N-terminal part of the extracellular domain comprises a 64 [A] long {beta} roll domain with a unique lateral loop extension. CEACAM1 binds to this loop via its N-terminal IgV-like domain with high affinity with a nanomolar dissociation constant, as obtained by surface plasmon resonance. This study provides the first structural description of a fusobacterial TAA, illustrates a yet undescribed CEACAM1 binding mode, and paves the way for rational drug design targeting Fn in CRC.

biophysics↗

bioRxiv2024

Morphological Profiling Dataset of EU-OPENSCREEN Bioactive Compounds Over Multiple Imaging Sites and Cell Lines

Morphological profiling with the Cell Painting assay has emerged as a promising method in drug discovery research. The assay captures morphological changes across various cellular compartments enabling the rapid identification of the effect of compounds. We present a comprehensive morphological profiling dataset using the carefully curated and well-annotated EU-OPENSCREEN Bioactive Compound Set. Our profiling dataset was generated across multiple imaging sites with high-throughput confocal microscopes using the Hep G2 as well as the U2 OS cell line. We employed an extensive assay optimization process to achieve high data quality across the different imaging sites. An analysis of the four replicates validates the robustness of the generated data. We compare morphological features of the different cell lines and map the profiles to activity, toxicity, and basic compound targets to further describe the dataset as well as to demonstrate the potential of this dataset to be used for mechanism of action exploration.

cell biology↗

bioRxiv2024

Identification of drug-like molecules targeting the ATPase activity of dynamin-like EHD4

Eps15 (epidermal growth factor receptor pathway substrate 15) homology domain-containing proteins (EHDs) comprise a family of eukaryotic dynamin-related ATPases that participate in various endocytic membrane trafficking pathways. Dysregulation of EHDs function has been implicated in various diseases, including cancer. The lack of small molecule inhibitors which acutely target individual EHD members has hampered progress in dissecting their detailed cellular membrane trafficking pathways and their function during disease. Here, we established a Malachite green-based assay compatible with high throughput screening to monitor the liposome-stimulated ATPase of EHD4. In this way, we identified a drug-like molecule that inhibited EHD4s liposome-stimulated ATPase activity. Structure activity relationship (SAR) studies indicated sites of preferred substitutions for more potent inhibitor synthesis. Moreover, the assay optimization in this work can be applied to other dynamin family members showing a weak and liposome-dependent nucleotide hydrolysis activity.

biochemistry↗

bioRxiv2023

7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover, visualize and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

biochemistry↗

bioRxiv2023

An unconventional gatekeeper mutation sensitizes inositol hexakisphosphate kinases to an allosteric inhibitor

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity towards the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by HDX-MS measurements. The latter analysis suggested that displacement of the C helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the wild-type kinases.

biochemistry↗

bioRxiv2021

TSG101 Associates with PARP1 and is Essential for PARylation and DNA Damage-induced NF-κB Activation

In a genome-wide screening for components of the dsDNA-break-induced IKK-NF-{kappa}B pathway we identified scores of regulators, including tumor susceptibility protein TSG101. TSG101 is essential for DNA damage-induced formation of cellular poly(ADP-ribose) (PAR). TSG101 directly binds to PARP1 and is required for PARP1 activation. This function of TSG101 is independent of its role in the ESCRT-I endosomal sorting complex. In the absence of TSG101, the PAR-dependent formation of a nuclear PARP1-IKK{gamma} signalosome, which triggers IKK activation, is impaired. According to its requirement for PARP1 and NF-{kappa}B activation, TSG101 deficient cells are defective in DNA repair and apoptosis protection. Loss of TSG101 results in PARP1 trapping at damage sites and mimics the effect of pharmacological PARP-inhibition. We also show that loss of TSG101 in connection with inactivated tumor suppressors BRCA1/2 in breast cancer cells is lethal. Our results imply TSG101 as a therapeutic target to achieve synthetic lethality in cancer treatment.

molecular biology↗

bioRxiv2020

A semi-automated intestinal organoid screening method demonstrates epigenetic control of epithelial maturation

Intestinal organoids are an excellent model to study epithelial biology. Yet, the selection of analytical tools to accurately quantify heterogeneous organoid cultures remains limited. Here, we developed a semi-automated organoid screening method, which we applied to a library of highly specific chemical probes to identify epigenetic regulators of intestinal epithelial biology. The role of epigenetic modifiers in adult stem cell systems, such as the intestinal epithelium, is still undefined. Based on this resource data, we identified several targets that affected epithelial cell differentiation, including HDACs, EP300/CREBBP, LSD1, and type I PRMTs, which were verified by complementary methods. For example, we show that inhibiting type I PRMTs, which leads enhanced epithelial differentiation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are powerful tools to study intestinal epithelial biology and may have therapeutic potential.

cell biology↗