Biology subjects

Hansen, F. K.

Publications and source records attributed to Hansen, F. K..

3 recordsLinked to original sources

bioRxiv2025

Target engagement studies and kinetic live-cell degradation assays enable the systematic characterization of HDAC6 PROTACs at endogenous protein expression levels

Histone deacetylase 6 (HDAC6) is an important drug target for the treatment of cancer, inflammation, and neurodegenerative disorders. In recent years, the development of proteolysis-targeting chimeras (PROTACs) has emerged to achieve the chemical knockdown of HDAC6. Consequently, there is an urgent need to develop efficient methods for target engagement studies and to enable a thorough characterization of the degradation efficiency and kinetics of HDAC6 PROTACs. In this work, we present a simple NanoBRET assay to assess HDAC6 cellular target engagement using a HeLaHDAC6-HiBiT cell line that stably expresses the LgBiT protein. For this purpose, we successfully designed, synthesized, characterized, and utilized the cell permeable TAMRA-based fluorescent ligand 5. The key advantage of this NanoBRET assay using HeLaHDAC6-HiBiT cells is the endogenously tagged HDAC6, allowing us to study binding of inhibitors in a near-native environment. Furthermore, we succeeded in establishing a system for kinetic live cell monitoring of HDAC6 degradation. The analysis of the degradation kinetics of a set of HDAC6 PROTACs provided detailed insights into their degradation efficiency and will be helpful for the development of improved HDAC6 degraders in the future. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/646177v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@147069forg.highwire.dtl.DTLVardef@3d64eorg.highwire.dtl.DTLVardef@1eac700org.highwire.dtl.DTLVardef@aedc3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Targeting de novo lipogenesis improves gemcitabine efficacy in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few treatment options and poor survivability. In this work we sought to characterise metabolic adaptations to gemcitabine (GEMC)-based chemotherapy exposure to discover new therapeutic targets for improving treatment efficacy. We show that GEMC resistance (GEMR) upregulates de novo lipogenesis in Panc1 and MiaPaCa2 cells through increased activity and expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). We also discovered alternate fatty acid desaturase 2 (FADS2) activity in Panc1 cells, which led to the production of sapienic acid (FA 16:1n-10, cis) from palmitic acid (FA 16:0). Knockdown of key lipid synthesis enzymes sensitised cells to GEMC treatment, with FAS (both cell lines), SCD1 (MiaPaCa2 only) and SCD1+FADS2 (Panc1) knockdown showing the greatest reduction in cell growth when combined with GEMC treatment. In Panc1 cells, both desaturases upregulated their activity when the alternate was knocked down, necessitating the need for dual desaturase knockdown in this cell line. PDAC cells attenuated to grow in combination GEMC/paclitaxel (CombAT) also displayed enhanced de novo lipogenesis; however, combination chemotherapy significantly downregulated FADS2 expression and activity in Panc1 CombAT cells rendering them more sensitive to SCD1 knockdown. We conclude that co-targeting lipid synthesis in PDAC could be a viable strategy for improving the efficacy of both GEMC monotherapy and combination GEMC/PTX therapy.

cancer biology↗

bioRxiv2024

A fluorescent probe enables the discovery of improved antagonists targeting the intracellular allosteric site of the chemokine receptor CCR7

Intracellularly acting ligands of G protein-coupled receptors (GPCRs) are gaining significant interest in GPCR drug discovery. In this study, we report the development of the fluorescent ligand Mz437 (4) targeting the CC chemokine receptor CCR7 at an intracellular allosteric site. We demonstrate its experimental power by applying 4 to identify two improved intracellular CCR7 antagonists, SLW131 (10) and SLW132 (21m), developed by converting two weakly active antagonists into single- or double-digit nanomolar ligands with minimal modifications. The thiadiazoledioxide 10 was derived from the CCR7 antagonist Cmp2105 by removing a methyl group from the benzamide moiety, while the squaramide 21m was obtained from the CXCR1/CXCR2 antagonist and clinical candidate navarixin by replacing the ethyl substituent by a tert-butyl group to engage a lipophilic subpocket. We show that 10 and 21m qualify to probe CCR7 biology both in recombinant cells and in the endogenous signaling environment of immune cells. Our novel probes are expected to facilitate the design of next-generation intracellular CCR7 ligands and serve as molecular tools to interrogate CCR7 biology in human and murine endogenous settings. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/607356v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1298d55org.highwire.dtl.DTLVardef@fdebf2org.highwire.dtl.DTLVardef@8d9aecorg.highwire.dtl.DTLVardef@b6308d_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗