Identification of Selective Nanomolar Inhibitors of the Human Neuraminidase, NEU4

The human neuraminidase enzymes (hNEU) play important roles in human physiology and pathology. The lack of potent and selective inhibitors toward these enzymes has limited our understanding of their function and the development of therapeutic applications. Here we report the evaluation of a panel of compounds against the four human neuraminidase isoenzymes. Among the compounds tested, we identified the first selective, nanomolar inhibitors of the human neuraminidase 4 enzyme (NEU4). The most potent NEU4 inhibitor (5-acetamido-9-[4-hydroxymethyl­[1,2,3]­triazol-1-yl]-2,3,5,9-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosonic acid) was found to have an inhibitory constant (<i>K</i>_<i>i</i>) of 30 ± 19 nM and was 500-fold selective for its target over the other hNEU isoenzymes tested <i>in vitro</i> (NEU1, NEU2, and NEU3). This is the first report of any inhibitor of hNEU with nanomolar potency, and this confirms that the 2,3-didehydro-2-deoxy-<i>N</i>-acetylneuraminic acid (DANA) scaffold can be exploited to develop new, potent, and selective inhibitors that target this important family of human enzymes

Identification of Selective Inhibitors for Human Neuraminidase Isoenzymes Using C4,C7-Modified 2‑Deoxy-2,3-didehydro‑<i>N</i>‑acetylneuraminic Acid (DANA) Analogues

In the past two decades, human neuraminidases (human sialidases, hNEUs) have been found to be involved in numerous pathways in biology. The development of selective and potent inhibitors of these enzymes will provide critical tools for glycobiology, help to avoid undesired side effects of antivirals, and may reveal new small-molecule therapeutic targets for human cancers. However, because of the high active site homology of the hNEU isoenzymes, little progress in the design and synthesis of selective inhibitors has been realized. Guided by our previous studies of human NEU3 inhibitors, we designed a series of C4,C7-modified analogues of 2-deoxy-2,3-didehydro-<i>N</i>-acetylneuraminic acid (DANA) and tested them against the full panel of hNEU isoenzymes (NEU1, NEU2, NEU3, NEU4). We identified inhibitors with up to 38-fold selectivity for NEU3 and 12-fold selectivity for NEU2 over all other isoenzymes. We also identified compounds that targeted NEU2 and NEU3 with similar potency

Structural Basis for Substrate Specificity of Mammalian Neuraminidases

<div><p>The removal of sialic acid (Sia) residues from glycoconjugates in vertebrates is mediated by a family of neuraminidases (sialidases) consisting of Neu1, Neu2, Neu3 and Neu4 enzymes. The enzymes play distinct physiological roles, but their ability to discriminate between the types of linkages connecting Sia and adjacent residues and between the identity and arrangement of the underlying sugars has never been systematically studied. Here we analyzed the specificity of neuraminidases by studying the kinetics of hydrolysis of BODIPY-labeled substrates containing common mammalian sialylated oligosaccharides: 3′Sia-LacNAc, 3′SiaLac, SiaLe_x, SiaLe_a, SiaLe_c, 6′SiaLac, and 6′SiaLacNAc. We found significant differences in substrate specificity of the enzymes towards the substrates containing α2,6-linked Sia, which were readily cleaved by Neu3 and Neu1 but not by Neu4 and Neu2. The presence of a branching 2-Fuc inhibited Neu2 and Neu4, but had almost no effect on Neu1 or Neu3. The nature of the sugar residue at the reducing end, either glucose (Glc) or <i>N</i>-acetyl-D-glucosamine (GlcNAc) had only a minor effect on all neuraminidases, whereas core structure (1,3 or 1,4 bond between D-galactose (Gal) and GlcNAc) was found to be important for Neu4 strongly preferring β3 (core 1) to β4 (core 2) isomer. Neu3 and Neu4 were in general more active than Neu1 and Neu2, likely due to their preference for hydrophobic substrates. Neu2 and Neu3 were examined by molecular dynamics to identify favorable substrate orientations in the binding sites and interpret the differences in their specificities. Finally, using knockout mouse models, we confirmed that the substrate specificities observed <i>in vitro</i> were recapitulated in enzymes found in mouse brain tissues. Our data for the first time provide evidence for the characteristic substrate preferences of neuraminidases and their ability to discriminate between distinct sialoside targets.</p></div

Kinetic data from substrate studies with recombinant neuraminidases.

<p>Values shown are means and standard errors of three independent experiments.</p><p>Kinetic data from substrate studies with recombinant neuraminidases.</p

Modeling of S1 and S5 binding to the Neu2 active site.

<p>(a) The substrate, S1, binds to the active site with expected contacts to the arginine triad and glycerol side chain. The GlcNAc residue B face is placed on the top of the hydrophobic side chain of Q112, forming a favorable interaction. The α2,3-glycosidic linkage between Neu5Ac and Gal allows S1 to form a relatively stable complex. (b) The same position for S1 as shown in (a), but with a surface representation of the Neu2 active site. (c) The α2,6-glycosidic linkage between the Neu5Ac and Gal residues in S5 makes it difficult for the substrate to fit into the active site without adopting an unfavorable conformation. The Neu5Ac residue adopts a twist boat conformation to preserve interactions with the Arg triad. The GlcNAc A face is interacting with a hydrophobic patch in the Neu2 active site, which is likely unfavorable. (d) The same pose for S5 as shown in (c), but with a surface representation of the Neu2 active site.</p

Chemical structures of the fluorescent substrates used in this study.

<p>Chemical structures of the fluorescent substrates used in this study.</p

Substrate specificity of mammalian neuraminidases.

<p>V_max/K_M values are plotted on a logarithmic scale. Values shown are means of three independent experiments.</p

Modeling of S1 binding to Neu3 active site.

<p>(a) Interactions of S1 with amino acid residues in the active site of the Neu3 homology model. (b) The same position of S1 shown in (a), with a surface representation of the active site of Neu3.</p

Neuraminidase activity in mouse brain tissues.

<p>The activity was measured in the homogenates of whole brain tissues of wild-type C57Bl6 mice (<i>WT</i>) and gene-targeted C57Bl6 mice deficient in Neu3 (<i>neu3</i>^−/−), Neu4 (<i>neu4</i>^−/−), Neu1 (<i>CathA^S190A-neo</i>) and double-knockout <i>neu3</i>^−/−; <i>neu4</i>^−/− mice against BODIPY-labeled sialylated oligosaccharides in 10 µM concentration. Values are shown as means (±S.E). N-value for each genotype is as follows: for WT and <i>neu3</i>^−/−; <i>neu4</i>^−/− n = 8, for <i>neu3</i>^−/− and <i>neu4</i>^−/− n = 6; for <i>CathA^S190A-neo</i> n = 4. * -significantly different from WT (P<0.05) by repeated measurements ANOVA.</p

Predicted protein-ligand contacts in Neu2 and Neu3.

<p>*Backbone interactions (carbonyl or amide).</p><p>Predicted protein-ligand contacts in Neu2 and Neu3.</p