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 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. Low cellular expression level of human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of 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 para-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.

Chemoenzymatic synthesis of Neu5Ac9NAc-containing α2–3- and α2–6-linked sialosides and their use for sialidase substrate specificity studies

“…Arthrobacter ureafaciens sialidase, Vibrio cholerae sialidase, and Clostridium perfringens sialidase (CpNanH) were purchased from Prozyme (Hayward, CA, USA). Recombinant PmAldolase [19], NmCSS [20, 21], PmST1 M144D mutant [23], Pd2,6ST [24], Psp2,6ST A366G mutant [26], hNEU2 [28], PmST1 [22], SpNanA [30], SpNanB [30], SpNanC [32], and BiNanH2 [29], were expressed as described previously. Neu5Acα2–3Galβ p NP ( 17 ), Neu5Acα2–6Galβ p NP ( 18 ), Neu5,9Ac 2 α2–3Galβ p NP ( 19 ), Neu5,9Ac 2 α2–6Galβ p NP ( 20 ) [15], and ManNAc6NAc [11] were synthesized as reported previously.…”

“…Sialidases used include six recombinant sialidases and three commercially available sialidases. The recombinant sialidases used were human cytosolic sialidase hNEU2 [28], bacterial sialidases PmST1 (a multifunctional sialyltransferase which also has sialidase activity) [22], Bifidobacterium infantis sialidase NanH2 [29], and three Streptococcus pneumoniae sialidases SpNanA [30], SpNanB [30], and SpNanC [31, 32]. Three commercial bacterial sialidases used were those from Arthrobacter ureafaciens, Vibrio cholerae , and Clostridium perfringens .…”

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups

“…Only NEU2 has been crystallized to date (58), although the other related sialidases have been homology-modeled to this structure (59). Unlike bacterial sialidases, human NEU2 has been demonstrated to have very similar activity on Neu5Ac and Neu5Gc in ␣2-3 or ␣2-6 linkage to an underlying galactose residue (60). To our knowledge, there are no reports examining the cleavage of ␣2-8-linked Neu5Gc by vertebrate exosialidases.…”

Chemoenzymatic synthesis of sialosides containing C7-modified sialic acids and their application in sialidase substrate specificity studies

“…14 We have also shown that replacing the C9-OH group of Neu5Ac in sialosides by a fluorine atom, a hydrogen atom (deoxy-Neu5Ac), or an azido group does not significantly change the activities of all bacterial sialidases tested although human NEU2 is quite sensitive to C9-modifications on Neu5Ac 11 Here we show that both Streptococcus pneumoniae sialidase (Figure 3D) and Clostridium perfringens sialidase (Figure 3E) as well as human NEU2 (Figure 3F) are quite sensitive to Neu5Ac C7-fluorine substitution which diminishes their activities significantly. Azido-substitution at C7-OH of Neu5Ac is also not well tolerated by either human NEU2 or bacterial sialidases tested except for the α2–6-sialidase activity of Clostridium perfringens sialidase which remains a reasonable activity (50%) compared to non-modified Neu5Acα2–6Galβ p NP substrate (Figure 3E).…”

“…9–11,13 Selective sialidase inhibitors against human NEU2 or bacterial sialidases have been designed and synthesized based on the structural features of the sialic acid modification obtained from sialidase substrate specificity studies. 14,15 Nevertheless, the method has not been used for the synthesis of sialosides containing C7-modified sialic acids.…”