title: Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies.
abstract: Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA. chen@chem.ucdavis.edu.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Li
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Cao
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.forename: Kam
authors.initials: K
authors.lastname: Lau
authors.forename: Jingyao
authors.initials: J
authors.lastname: Qu
authors.forename: Vireak
authors.initials: V
authors.lastname: Thon
authors.forename: Go
authors.initials: G
authors.lastname: Sugiarto
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology; D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D015195:Drug Design; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D002412:Cations; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; C403285:NEU2 protein, human; D009439:Neuraminidase