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Orexin receptors constitute a family of class A G-protein coupled receptors. There are two subtypes of orexin receptors, namely OX1R and OX2R. OX1R and OX2R are widely distributed in the central nervous system and are the targets for the peptide neurotransmitters orexin-A and orexin-B. Orexins are involved in a plethora of key physiological functions such as regulation of the sleep/wake cycle, feeding behavior, energy homeostasis, and cognition. Dysfunction of the orexin system has been linked to various pathological conditions, such as narcolepsy, insomnia, obesity, addiction, cognitive impairment, and depression. The active state structure of OX2R has been elucidated, while the active state structure of OX1R remains unresolved. Here, we describe a method for the expression and purification of an activated OX1R bound to its native peptide ligand, orexin-A, in complex with a Dominant Negative Gsq protein and Nb35. The proteins were expressed in Hi5 insect cells and subsequently purified via two consecutive affinity chromatography steps, followed by a final polishing Size Exclusion Chromatography step. This study could stimulate further research into the activation mechanisms of OX1R and the structural determination of its active state structure.

01 Mar 2025·Biosensors and Bioelectronics

A biosensor-based phage display selection method for automated, high-throughput Nanobody discovery

Article

Author: Pardon, Els ; Kalichuk, Valentina ; De Keyser, Phebe ; Zögg, Thomas ; Steyaert, Jan ; Schenck, Stephan ; Wohlkönig, Alexandre ; Brunner, Janine

Biopanning methods to select target-specific Nanobodies® (Nbs) involve presenting the antigen, immobilized on plastic plates or magnetic beads, to Nb libraries displayed on phage. Most routines are operator-dependent, labor-intensive and often material- and time-consuming. Here we validate an improved panning strategy that uses biosensors to present the antigen to phage-displayed Nbs in a well. The use of automated Octet biolayer interferometry sensors (Sartorius) enables high throughput and precise control over each step. By playing with association and dissociation times and buffer composition, one can efficiently decrease the background of aspecific and low-affinity Nbs, reducing the rounds of panning needed for the enrichment of high-affinity binders. Octet panning also enables the use of unpurified target proteins and unpurified phage from a bacterial culture supernatant. Additionally, downscaling to a 384-well format significantly reduces the amount of protein required. Moreover, enrichment of binders can be quantified by monitoring phage binding to the target by interferometry, omitting additional phage titration steps. Routinely, up to three rounds of Octet panning can be performed in only five days to deliver target-specific binders, ready for screening and characterization using the same Octet instrument.

01 Jan 2025·Open Biology

Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide

Article

Author: Naismith, James H. ; Pardon, Els ; Le Bas, Audrey ; Steyaert, Jan ; Teelucksingh, Tanisha ; Ward, Philip N. ; El Omari, Kamel ; Lee, Micah ; Weckener, Miriam ; Liu, Huanting ; Duman, Ramona ; McMahon, Stephen A. ; Beale, John H. ; Liu, Hui ; Paterson, Neil G. ; Giltrap, Andrew M. ; Clarke, Bradley R. ; Quigley, Andrew ; Mykhaylyk, Vitaliy ; Wagner, Armin ; Harrison, Peter J. ; Davis, Benjamin G. ; Whitfield, Chris

The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.

100 Deals associated with VIB-VUB Center for Structural Biology

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Pipeline

Pipeline Snapshot as of 26 Feb 2025

The statistics for drugs in the Pipeline is the current organization and its subsidiaries are counted as organizations,Early Phase 1 is incorporated into Phase 1, Phase 1/2 is incorporated into phase 2, and phase 2/3 is incorporated into phase 3

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