Biology subjects

Geschwindner, S.

Publications and source records attributed to Geschwindner, S..

5 recordsLinked to original sources

bioRxiv2026

Conformational dynamics at the pre-miR-377 Dicer site governs selective small-molecule recognition

Small molecules that target precursor microRNAs (pre-miRNAs) offer a strategy to modulate microRNA biogenesis, but rational design is limited by the intrinsic conformational dynamics of RNA. Here, we combine site-specific nuclear magnetic resonance (NMR) spectroscopy with enhanced-sampling molecular dynamics (MD) simulations to define the conformational ensemble of pre-miR-377 and its recognition by the small-molecule binder C1. Our data reveals that the Dicer cleavage site contains a dynamic adenosine bulge in which U25 shuffles between A3 and A4 with a [~]9:1 population ratio, generating two interconverting RNA triple structures. NMR-informed MD and binding simulations show that C1 engages this dynamic region through a pathway-dependent mechanism that favors minor-groove entry, stabilizes intermediate, partially reorganized Dicer-site conformations, and redistributes the conformational ensemble away from processing-competent states. A conformationally locked mutant abolishes binding, indicating that recognition depends on bulge-enabled RNA dynamics rather than a single static structure. These results define the structural and dynamic basis of small-molecule recognition at the pre-miR-377 Dicer site and highlight the importance of RNA conformational ensembles in RNA-targeted drug discovery.

biophysics↗

bioRxiv2026

Discovery of Membrane Channel Modulators via DNA-Encoded Library Screening Using Native-Like Membrane Protein Nanoparticles

Developing novel drugs against membrane proteins is a major challenge in drug discovery due to the difficulty of stabilizing these targets for high-throughput screenings. Pannexin 1 (PANX1) is a membrane channel protein involved in various physiological and pathological processes, making it a promising target for drug discovery. However, efforts to develop PANX1-targeting therapeutics have been hindered by the inherent challenges of stabilizing the protein channel and conducting effective pharmacological screening. Here, we report a proof-of-concept workflow that integrates the Salipro lipid nanoparticle platform with DNA-Encoded Library screenings in a detergent-free format. In this case study, the Salipro DirectMX method was used to generate functional PANX1 nanoparticles for drug discovery and characterisation. Using a high-stringency selection strategy and computational approaches, we identified a specific set of candidate compounds with selective PANX1 enrichment. Surface Plasmon Resonance analysis confirmed the identification of hit compounds. Cryo-Electron Microscopy of the Salipro-PANX1-Compound complex provided structural insights into a potential compound binding site. Electrophysiological recordings in PANX1-expressing Xenopus laevis oocytes demonstrated dose-dependent inhibition of PANX1-mediated ion conductance by the compounds. These findings establish a robust workflow for ligand discovery against challenging membrane protein targets and provide novel chemical starting points for the development of PANX1 modulators.

biochemistry↗

bioRxiv2024

Unveiling the Power of PROTAC Valency: Navigating Cell Type-Specific Hook Effects

Targeted protein degradation (TPD) using bivalent proteolysis-targeting chimeras (PROTAC) technology has shown potential in expanding the "druggable" proteome. In their publication in Nature Chemical Biology, Imaide et al.1 posited that augmenting PROTAC valency could potentially lead to the formation of long-lived ternary complexes between PROTAC, the protein of interest (POI), and E3 ligase, thereby constraining the formation of potent binary complexes, as evidenced by a pronounced hook effect. The authors introduced SIM1, a trivalent von Hippel-Lindau (VHL)-based PROTAC, which exhibited a superior degradation profile in comparison to its parent molecule MZ1, towards bromo and extra terminal (BET) proteins, with a predilection for BRD2. The authors attributed this heightened degradation capability of SIM1 over bivalent MZ1 as supportive evidence for their hypothesis. While we concur with the notion that increasing valency and avidity could enhance the efficacy of a PROTAC, the claim that trivalent PROTACs unequivocally eliminate the hook effect is not entirely accurate. We propose that the presence or absence of a hook effect is influenced by numerous factors beyond PROTAC valency.

cancer biology↗

bioRxiv2024

Robust prediction of relative binding energies for protein-protein complex mutations using free energy perturbation calculations

Computational free energy-based methods have the potential to significantly improve throughput and decrease costs of protein design efforts. Such methods must reach a high level of reliability, accuracy, and automation to be effectively deployed in practical industrial settings in a way that impacts protein design projects. Here, we present a benchmark study for the calculation of relative changes in protein-protein binding affinity for single point mutations across a variety of systems from the literature, using free energy perturbation (FEP+) calculations. We describe a method for robust treatment of alternate protonation states for titratable amino acids, which yields improved correlation with and reduced error compared to experimental binding free energies. Following careful analysis of the largest outlier cases in our dataset, we assess limitations of the default FEP+ protocols and introduce an automated script which identifies probable outlier cases that may require additional scrutiny and calculates an empirical correction for a subset of charge-related outliers. Through a series of three additional case study systems, we discuss how protein FEP+ can be applied to real-world protein design projects, and suggest areas of further study. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/590325v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c5e607org.highwire.dtl.DTLVardef@1810ee5org.highwire.dtl.DTLVardef@1f8f1acorg.highwire.dtl.DTLVardef@c28053_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIReliable calculation of relative binding free energy changes for most protein mutations to within [~]1 kcal/mol. C_LIO_LIAutomated Protein FEP+ Groups treatment of alternate protonation states for titratable residues. C_LIO_LIApplication of FEP+ methodology to "real-world" protein design projects. C_LI

biophysics↗

bioRxiv2022

Direct Cell Extraction of Membrane Proteins for Structure-Function Analysis

Membrane proteins are the largest group of therapeutic targets in a variety of disease areas and yet, they remain particularly difficult to investigate. We have developed a novel one-step approach for the incorporation of membrane proteins directly from cells into lipid Salipro nanoparticles. Here, with the pannexin1 channel as a case study, we demonstrate the applicability of this method for structurefunction analysis using SPR and cryo-EM.

biophysics↗