Fig. 1: Structural evolution and binding properties of PRS-220. | Nature Communications

Fig. 1: Structural evolution and binding properties of PRS-220.

From: Preclinical concept studies showing advantage of an inhaled anti-CTGF/CCN2 protein for pulmonary fibrosis treatment

Fig. 1

a Structural models comparing NGAL (Anticalin scaffold, PDB ID: 1DFV), an intermediate, and PRS-220 highlighting the evolution of residue variations towards high affinity binding to CCN2 and favorable biophysical properties (higher Tm). Yellow sticks: disulfide bond. Red and orange spheres: residues that were mutated in Lcn2 to generate the intermediate. Blue spheres: Cα atoms of residues that remained fixed during subsequent engineering steps towards PRS-220. Graphs were generated with Pymol. b Binding of PRS-220 to recombinant CCN2, CCN2 N-terminal fragment (IGFBP & vWC) and C-terminal fragment (TSR & CT domain) by ELISA (n = 3 experiments, mean ± SD). c Competition of PRS-220 and the Anticalin scaffold (NGAL ctrl) with pamrevlumab for binding to CCN2 (IC50 = 0.35 ( ± 0.05) nM) and competition of pamrevlumab with itself for binding to CCN2 (IC50 = 1.14 ( ± 0.03) nM) analyzed by ECLA (n = 3 experiments, mean ± SD). d Experimental setup of the competition ECLA. Created in BioRender. Pavlidou, M. (2025) https://BioRender.com/y92y274e Quantification of PRS-220 binding to CCN2 expressed by TGF-β1-activated normal human lung fibroblasts (n = 3 experiments, mean+SD). Dotted line represents background signal in presence or absence of 5 ng/mL TGF-β1 with anti-scaffold detection antibody only. f Schematic view of air-liquid interface (ALI) cell culture model for investigation of mucus interaction. Created in BioRender. Pavlidou, M. (2025) https://BioRender.com/j57y046g Confocal imaging (63x) of PRS-220 mucus penetration in ALI cultures of cystic fibrosis derived human bronchial epithelial cells (HBECs) at baseline, 0.5 min (PRS-220) or 5 min (pos. ctrl) after adding Alexa Fluor 647-labeled PRS-220 or mucus binding microspheres (positive ctrl) to the apical side. Representative images of n = 3 independent experiments with mucus = green, air surface liquid (ASL) =magenta, PRS-220/pos. ctrl  = blue. h PRS-220 mucus interaction by Quartz Crystal Microbalance with Dissipation (QCMD) monitoring of the frequency shift (=changes in mucus mass) and dissipation shift (=changes in mucus hydration). Mucus solution was perfused over a chip, followed by perfusion of 10 µM PRS-220, Poly-L-lysine (PLL) as positive control for mucus interaction and PBS. Source data are provided as a Source Data file.

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