Biology subjects

Brunst, S.

Publications and source records attributed to Brunst, S..

3 recordsLinked to original sources

bioRxiv2025

Click Chemistry Enables Rapid Development of Potent sEH PROTACs Using a Direct-to-Biology Approach

The Direct-to-Biology (D2B) approach enables biological screening of crude reaction mixtures, eliminating the need for purification steps and thereby accelerating drug discovery. In this study, we developed a miniaturized D2B platform for the rapid synthesis of PROTAC degraders of soluble epoxide hydrolase (sEH). We used the copper-catalyzed azide-alkyne cycloaddition and optimized the conditions for 384-well PCR plate applications with 10 {micro}L reaction volumes on a 300 nmol scale. This approach enabled the D2B synthesis of 92 crude PROTACs from azide-functionalized CRBN-ligands and alkyne-linked sEH inhibitors. Biological screening using a HiBiT lytic degradation assay identified two hits which were resynthesized and exhibited subnanomolar DC50 values and degradation efficacy (Dmax). Thus, we established a scalable, cost-effective and time-saving D2B platform for the discovery of PROTACs in very small quantities. This methodology is particularly suitable for early-stage screening and hit validation assessing the degradability of a target.

cell biology↗

bioRxiv2024

STRUCTURE-BASED DESIGN OF PROTACS FOR THE DEGRADATION OF SOLUBLE EPOXIDE HYDROLASE

Soluble epoxide hydrolase (sEH) represents a promising target for inflammation-related diseases as it hydrolyzes highly anti-inflammatory epoxy-fatty acids (EpFAs) to the less active corresponding diols.1 sEH harbours two distinct catalytic domains, the C-terminal hydrolase domain and the N-terminal phosphatase domain which are connected by a proline-rich linker.2 Although potent inhibitors of enzymatic activity are available for both domains, sEH-PROTACs offer the unique ability to simultaneously degrade both domains, mimicking the sEH knockout phenotype associated with beneficial effects as reducing inflammation, attenuating neuroinflammation, and delaying the progression of Alzheimers disease. Herein, we report the structure-based development of a potent sEH-PROTAC as a useful tool compound for the investigation of sEH. In order to facilitate a rapid testing of the synthesized compounds a cell-based sEH degradation assay was developed based on the HiBiT-technology. A structure-activity-relationship (SAR) investigation was performed, based on the crystal structure of previously published sEH inhibitor FL217 where we identified two possible exit vectors. We designed and synthesized a set of 24 PROTACs with varying linkers in a combinatorial manner. Furthermore, co-crystallization of sEH with two selected PROTACs allowed us to explore the binding mode and rationalize the appropriate linker length. After biological and physicochemical investigation, the most suitable PROTAC 23 was identified and applied to degrade sEH in primary human macrophages, marking the successful translation and applicability to non-artificial systems.

biochemistry↗

bioRxiv2024

The transaminase-omega-amidase pathway is a redox switch in glutamine metabolism that generates alpha-ketoglutarate

Oxidative stress is caused by short-lived molecules and metabolic changes belong to the fastest cellular responses. Here we studied how the endothelial cell metabolome reacts to acute oxidative challenges (menadione or H2O2) to identify redox-sensitive metabolic enzymes. H2O2 selectively increased -ketoglutaramate (KGM), a largely uncharacterized metabolite produced by glutamine transamination and a yet unrecognized intermediate of endothelial glutamine catabolism. The enzyme nitrilase-like 2 {omega}-amidase (NIT2) converts KGM to -ketoglutarate (KG). Reversible oxidation of specific cysteine in NIT2 by H2O2 inhibited its catalytic activity. Furthermore, a variant in the NIT2 gene that decreases its expression is associated with high plasma KGM level in humans. Endothelial-specific knockout mice of NIT2 exhibited increased levels of KGM and impaired angiogenesis. Knockout of NIT2 impaired endothelial cell proliferation and sprouting and induced senescence. In conclusion, we show that the glutamine transaminase-{omega}-amidase pathway is a metabolic switch in which NIT2 is the redox-sensitive enzyme. The pathway is modulated in humans and functionally important for endothelial glutamine metabolism.

physiology↗