Biology subjects

Szlachcic, A.

Publications and source records attributed to Szlachcic, A..

5 recordsLinked to original sources

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↗

bioRxiv2020

Spreading depressions and periinfarct spreading depolarizations in the context of cortical plasticity

Studies of cortical function-recovery require a comparison between normal and post-stroke conditions that lead to changes in cortical metaplasticity. Focal cortical stroke impairs experience-dependent plasticity (ExDP) in the neighboring somatosensory cortex and usually evokes periinfarct depolarizations (PiDs) - a spreading depression-like waves. Experimentally induced spreading depressions (SDs) affect gene expression and some of these changes persist for at least 30 days. However, such changes are not stroke-specific: migraine patients had prolonged protein changes after a single SD episode during migraine aura. This study investigates whether non-stroke depolarizations impair cortical ExDP similarly to the stroke. ExDP was induced in rates with SDs or PiDs by a month of contralateral partial whiskers deprivation. Cortical activity was mapped by [14C]-2-deoxy-D-glucose (2DG) incorporation during stimulation of spared and contralateral homotopic whiskers. We found that whiskers deprivation after SDs resulted in normal cortical representation enlargement suggesting that SDs and PiDs depolarization have no influence on ExDP cortical map reorganization. PiDs and the MMP-9, -3, -2 or COX-2 proteins which are assumed to influence metaplasticity in rats after stroke were compared between the SDs induced by high osmolarity KCl solution and the PiDs following cortical photothrombotic stroke (PtS). We found that none of these factors directly caused cortical post-stroke metaplasticity changes. The only significant difference between stoke and induced SD was a greater imbalance in interhemispheric activity equilibrium after stroke. The interhemispheric interactions modified by stroke may therefore be a promising target for future studies of post-stroke ExDP and for convalescence studies. HighlightsO_LIPost-stroke metaplasticity changes in an injured hemisphere are not a consequence of spreading depolarizations C_LIO_LINone of the monitored proteins (MMP-2, -3, -9; COX-2) cause modifications in poststroke cortical plasticity C_LIO_LISpreading depressions have a prolonged, global influence on the functioning of both hemispheres and of the thalamus C_LIO_LIImpaired interhemispheric interactions may underlie the post-stroke metaplasticity changes in the injured hemisphere C_LI

neuroscience↗