Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms

• Reliable SPR methods for the determination of the affinity of sulfonamide derivatives for hCAII, hCAIX and hCAXII.
• Evaluation of two immobilization approaches : amine coupling using CM chips versus biotin capture using a CAP chip.
• CM7 versus CM5 chip: a method to increase immobilization level and KD precision.
• Comparison of two approaches to validate that immobilization does not compromise the activity/functionality of proteins.

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.