Development of a fluorescence screening assay for binding partners of the iron-sulfur mitochondrial protein mitoNEET.

MitoNEET belongs to the CDGSH Iron-Sulfur Domain (CISD)-gene family of proteins and is a [2Fe-2S] cluster-containing protein found on the outer membrane of mitochondria. The specific functions of mitoNEET/CISD1 remain to be fully elucidated, but the protein is involved in regulating mitochondrial bioenergetics in several metabolic diseases. Unfortunately, drug discovery efforts targeting mitoNEET to improve metabolic disorders are hampered by the lack of ligand-binding assays for this mitochondrial protein. We have developed a protocol amenable for high-throughput screening (HTS) assay, by modifying an ATP fluorescence polarization method to facilitate drug discovery targeting mitoNEET. Based on our observation that adenosine triphosphate (ATP) interacts with mitoNEET, ATP-fluorescein was used during assay development. We established a novel binding assay suitable for both 96- or 384-well plate formats with tolerance for the presence of 2% v/v dimethyl sulfoxide (DMSO). We determined the IC50-values for a set of benzesulfonamide derivatives and found the novel assay reliably ranked the binding-affinities of compounds compared to radioactive binding assay with human recombinant mitoNEET. The developed assay platform is crucial in identifying novel chemical probes for metabolic diseases. It will accelerate drug discovery targeting mitoNEET and potentially other members of the CISD gene family.

Quantification of the α2-6 Sialic Acid Linkage in Branched N-Glycan Structures with Capillary Nanogel Electrophoresis.

Sialylation and sialic acid linkage in N-glycans are markers of disease but are analytically challenging to quantify. A capillary electrophoresis method is reported that integrates a unique combination of enzymes and lectins to modify sialylated N-glycans in real time in the capillary so that N-glycan structures containing α2-6-linked sialic acid are easily separated, detected, and quantified. In this study, N-glycans were sequentially cleaved by enzymes at the head of the separation capillary so that the presence of α2-6-linked sialic acids corresponded to a shift in the analyte migration time in a manner that enabled interpretation of the N-glycan structure. Following injection, only afucosylated N-glycan structures were passed through enzyme zones that contained α2-3 sialidase, followed by ß1-3,4 galactosidase, which cleaved any terminal α2-3-linked sialic acid and underlying galactose yielding a terminal N-acetyl glucosamine. With this treatment complete, a third zone of α2-3,6,8 sialidase converted the remaining α2-6-linked sialic acid to terminal galactose. With these enzyme processing steps the α2-6-linked sialic acid residues on an N-glycan correlated directly to the number of terminal galactose residues that remained. The number of terminal galactose residues could be interpreted as a stepwise decrease in the migration time. Complex N-glycans from α-1-acid glycoprotein were analyzed using this approach, revealing that a limited number of α2-6-linked sialic acids were present with biantennary, triantennary, and tetraantennary N-glycans of α-1-acid glycoprotein generally containing 0 or 1 α2-6-linked sialic acid.