Biology subjects

Kannt, A.

Publications and source records attributed to Kannt, A..

4 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↗

bioRxiv2025

Optimization and evaluation of complementary degrader discovery assays for application in screening

Targeted protein degradation (TPD) mediated by molecular glues is an innovative pharmaceutical paradigm. By binding to and modulating the surface of an E3-ligase component, molecular glue degraders can facilitate the recruitment of a specific target protein (or vice versa) and, ultimately, invoke target degradation. This mode of action results in specific challenges for the development of rational discovery strategies, and complex hit validation workflows may be required to reliably eliminate compounds that elicit non-specific effects. With the aim to guide screening efforts, we optimized two orthogonal cell-based, target-centric assays for degrader discovery: (1) a time-resolved FRET assay directly quantifying levels of target protein and its degradation (signal inhibition), and (2) an assay coupling TPD to cell growth (signal rescue). To enable a deeper understanding of the individual assays strengths and limitations, we compared their statistical performance as well as respective hit populations by screening a specifically designed collection of about 1000 compounds containing well annotated reference compounds and known frequent hitters. We found that the signal rescue format reliably and specifically captured active target degraders while it efficiently filtered out interfering or frequent hitter compounds. Importantly, this format achieved to retrieve lower potency hits, which might be desirable in order to confidently include as many diverse chemical starting points as possible at the start of a drug discovery project.

pharmacology and toxicology↗

bioRxiv2021

Urinary bladder enlargement across nine rodent models of diabetes: correlations with glucose and insulin levels

The urinary bladder is markedly enlarged in the type 1 diabetes mellitus model of streptozotocin (STZ)-injected rats, but much less data exist for models of type 2 diabetes (T2DM). Diabetic polyuria has been proposed to explain bladder enlargement. We have collected data on bladder weight and blood glucose from 16 studies representing 9 distinct rodent diabetes (7 T2DM) and obesity models; some included arms with diets and/or pharmacological treatments. Data were analyzed for bladder enlargement and for correlations between bladder weight on the one and glucose levels on the other hand. Our data confirm major bladder enlargements in STZ rats, minor if any enlargement in fructose-fed rats, db/db mice and mice on a high-fat diet. For the first time we report bladder weight data on 5 other models with presence and degree of bladder enlargement being heterogeneous across models. Bladder weight was correlated with plasma glucose in some but not other models, but correlations were moderate to weak except for RIP-LMCV mice. We conclude that the presence and extent of bladder enlargement varies markedly across diabetes models, particularly TD2M models; our data do not support the idea that bladder enlargement is primarily driven by glucose levels/glucosuria.

pharmacology and toxicology↗

bioRxiv2020

A 3D human liver model of nonalcoholic steatohepatitis

AO_SCPLOWBSTRACTC_SCPLOWWe have developed an in vitro preclinical 3D Non-Alcoholic SteatoHepatitis (NASH) model by co-culturing four human primary liver cell types: hepatocytes, stellate, endothelial and Kupffer cells. Cells were embedded in a hydrogel of rat collagen in 96-well plate and a NASH-like environment was induced with a medium containing free fatty acids (FFAs) and tumor necrosis factor (TNF). This model was characterized by biochemical, imaging and transcriptomics analysis. On the one hand, we succeed in defining suitable culture conditions to maintain the 3D co-culture up to 10 days in vitro with the lowest level of steatosis, and reproducible low levels of inflammation and fibrosis. On the other hand, we induced NASH disease with a custom medium mimicking NASH features (hepatocyte injury, steatosis, inflammation and fibrosis). The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets. SO_SCPLOWUMMARYC_SCPLOWWe developed a 3D human liver model which exhibits many features of non-alcoholic steatohepatitis and that could become a platform for medium throughput drug screening.

cell biology↗