Effects of the Surface Densities of Glycoclusters on the Determination of Their IC50 and Kd Value Determination by Using a Microarray

• Published in: Chembiochem : a European journal of chemical biology Vol. 16; no. 16; pp. 2329 - 2336
• Main Authors: Dupin, Lucie, Zuttion, Francesca, Géhin, Thomas, Meyer, Albert, Phaner-Goutorbe, Magali, Vasseur, Jean-Jacques, Souteyrand, Eliane, Morvan, François, Chevolot, Yann
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 02.11.2015; Wiley Subscription Services, Inc; Wiley-VCH Verlag
• Subjects: Adhesins, Bacterial - chemistry; Adhesins, Bacterial - metabolism; Bacterial Adhesion; Biofilms; Cystic fibrosis; dissociation constants; Engineering Sciences; Fluorescence Resonance Energy Transfer; glycoconjugates; Glycoproteins - chemistry; Glycoproteins - metabolism; Inhibitory Concentration 50; Kinetics; Lectins; Materials; Micro and nanotechnologies; Microarray Analysis; microarrays; Microelectronics; Microscopy, Atomic Force; Optics; Photonic; Protein Binding; Pseudomonas aeruginosa; Pseudomonas aeruginosa - genetics; Pseudomonas aeruginosa - metabolism; surface density; Virulence Factors; Pseudomonas aeruginosa; surface density; glycoconjugates; lectins; dissociation constants; microarrays
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201500371

Pseudomonas aeruginosa (PA) is an opportunistic bacterium involved in 10–30 % of nosocomial diseases. It causes severe lung injury to cystic fibrosis patients, often leading to patient death. PA strains are multidrug resistant, thus making the design of new therapeutics a challenge for public health. One promising therapeutic option is to design glycoclusters that target the virulence factor of PA. LecA is a galactose‐specific lectin that might be involved in adhesion and biofilm formation by PA. The DNA‐directed immobilization (DDI) microarray is a powerful tool for screening and understanding of structure–activity relationships between glycoclusters and lectins. High‐throughput and multiplexed analysis of lectin–glycocluster interactions on a DDI microarray allows measurement of IC50 and dissociation constant (Kd) values with minute amounts of material. In order to study the robustness of the DDI microarray in determination of IC50 and Kd values, the impact of glycocluster surface density was investigated. The data obtained show that measured IC50 values were influenced by glycocluster surface density: as the density of glycoclusters increases, the measured IC50 values increase too. In contrast, the measured Kd values were not affected by glycocluster surface density, provided that the experimental conditions allow interaction between glycocluster and lectin at single‐molecule level (no surface cluster effect). Making sense of dense: The glycocluster surface densities on microarrays were modulated to investigate the impact on measuring IC50 and Kd values. The results show that measured IC50 values increase proportionally with glycocluster surface density. In contrast, measured Kd values are not influenced by glycocluster surface density.