Biology subjects

Gajewska, D.

Publications and source records attributed to Gajewska, D..

5 recordsLinked to original sources

bioRxiv2025

A Robust Crystallographic Platform for High-Throughput β-Catenin Ligand Discovery

This study presents a robust crystallographic platform for assessing compounds binding to {beta}-catenin. We developed a standardized protein production protocol for the armadillo domain of {beta}-catenin (BC-ARM) and performed biophysical screens using Surface Plasmon Resonance (SPR) and Differential Scanning Fluorimetry (DSF). These findings led to the successful determination of the co-crystal structure of BC-ARM with compound 1 binding to previously reported site but distinct from known transcription factor binding sites. To broaden the search for novel BC binding sites, we utilized FragLites library with a cyclic peptide-stabilized BC-ARM construct. This yielded two high-resolution co-crystal structures identifying a previously unreported binding hotspot. Recognizing the limitations of the cyclic peptide-bound construct for general screening, we designed a novel, truncated BC-ARM construct. This new construct eliminates unstructured regions, reliably producing high-quality, diffracting crystals suitable for high-throughput crystallographic studies. In conclusion, the ligand-bound {beta}-catenin structures and this novel, robust BC-ARM construct establish a powerful platform for further {beta}-catenin investigation.

molecular biology↗

bioRxiv2025

Exploration of chemical probes and conformational flexibility of GID4 - the substrate receptor of human CTLH E3 ligase complex

The application of targeted protein degradation (TPD) is currently constrained by the limited availability of low-molecular-weight molecules that can recruit E3 ligases other than CRBN (Cereblon) or VHL (Von Hippel-Lindau ligase). In this study, we present the structure-based drug design (SBDD) of high-affinity ligands that engage E3 ligase GID4 (Glucose-induced degradation protein 4) in biophysical and cellular experiments. Through structural studies and molecular modeling, we identified three clusters of compounds that induce distinct conformations of GID4. We characterized potential exit vectors and used the most promising ligand as a building block to prepare bifunctional degraders in the form of proteolysis-targeting chimeras (PROTACs). Although ternary complex formation was successful in vitro, degradation of BRD4 was not observed, highlighting the need for further optimization of the degraders. Finally, we theoretically investigated the likelihood of the identified GID4 conformations participating in protein-protein interactions mediated by molecular glue mechanisms. We believe the expanded ligand diversity discovered in this study may pave the way for tuning the selectivity and efficacy of interactions involving GID4 and its neosubstrates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/662521v3_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@19187e8org.highwire.dtl.DTLVardef@171abd1org.highwire.dtl.DTLVardef@1c75a5forg.highwire.dtl.DTLVardef@fe17fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Targeted degradation of GSPT1 and NEK7 by a molecular glue prodrug for treatment of HCC

Targeted Protein Degradation (TPD) technology, in the form of CRBN-modulating molecular glues, offers numerous unprecedented therapeutic benefits as evidenced by the success of approved high-value immunomodulatory imide drugs (IMiDs) such as lenalidomide and pomalidomide. Building upon these successes, we employed a small CRBN-focused library of molecular glues in a phenotypic screen against hepatocellular carcinoma (HCC) cell lines. While the original library was primarily designed to target SALL4, we identified additional CRBN substrates, including GSPT1, NEK7, and CK1, whose degradation potently induced cell death in HCC cell lines. Subsequent lead optimization efforts yielded a compound, ABS-752, which demonstrated superior in vitro and in vivo activity through the potent degradation of GSPT1. Notably, ABS-752 does not form ternary complexes with CRBN and the neosubstrates. Further investigations revealed that ABS-752 is a prodrug activated by the monoamine oxidase, VAP-1, to an aldehyde intermediate and subsequently to the active molecule, ABT-002. VAP-1, which is overexpressed in cirrhotic liver, was identified as the primary monoamine oxidase responsible for the conversion of ABS-752. ABS-752 is currently in clinical trials for the treatment of HCC.

molecular biology↗

bioRxiv2024

Expression screen of TNFR1 R347A, MyD88, IRAK4 death domains in E. coli followed by purification and biophysical characterization of TNFR1 R347A death domain

Death domains play a crucial role in signaling pathways related to inflammation and programmed cell death, rendering them promising targets for therapeutic interventions. However, their expression as recombinant proteins often pose challenges. Here, we present expression screening of TNFR1, IRAK4, and MyD88 death domains in E. coli, followed by the biophysical characterization of TNFR1 death domain after subsequent construct optimization. The study also discusses the influence of pH and ionic strength on TNFR1R347A stability, providing statistical models to predict optimal conditions of the buffer to achieve the highest protein stability. HighlightsO_LIOptimization of expression conditions for TNFR1R347A, MyD88, IRAK4 death domains in E. coli BL21(DE3) cells. C_LIO_LIHigh-yield production of soluble monomeric TNFR1R347A death domain. C_LI

biochemistry↗

bioRxiv2023

Expression Strategies for Recombinant HECT E3 Ligases in Escherichia coli

A comparative analysis of recombinant expression in E. coli of three HECT E3 ligases reveals a consistent preference for lower expression temperatures. Lower temperatures prevent aggregation and misfolding, enhancing the efficiency and solubility of expressed HECT ligases. Isolated HECT domains generally exhibit higher expression success compared to full-length counterparts, offering improved solubility and yields. However, expression levels vary among ligases, necessitating tailored strategies. Future studies may explore full-length HECT-type E3 ligases in covalent complexes with ubiquitin as a potential, generalizable platform for biomedical research.

biochemistry↗