Tuning Mechanism‐Based Inactivators of Neuraminidases: Mechanistic and Structural Insights

Abstract

3-Fluorosialosyl fluorides are inhibitors of sialidases that function by the formation of a long-lived covalent active-site adduct and have potential as therapeutics if made specific for the pathogen sialidase. Surprisingly, human Neu2 and the Trypanosoma cruzi trans-sialidase are inactivated more rapidly by the reagent with an equatorial fluorine at C3 than by its axial epimer, with reactivation following the same pattern. To explore a possible stereoelectronic basis for this, rate constants for spontaneous hydrolysis of the full series of four 3-fluorosialosyl fluorides were measured, and ground-state energies for each computed. The alpha (equatorial) anomeric fluorides hydrolyze more rapidly than their beta anomers, consistent with their higher ground-state energies. However ground-state energies do not explain the relative spontaneous reactivities of the 3-fluoro-epimers. The three-dimensional structures of the two 3-fluoro-sialosyl enzyme intermediates of human Neu2 were solved, revealing key stabilizing interactions between Arg21 and the equatorial, but not the axial, fluorine. Because of changes in geometry these interactions will increase at the transition state, likely explaining the difference in reaction rates.

The Catalytic Mechanism of Human Parainfluenza Virus Type 3 Haemagglutinin‐Neuraminidase Revealed

“…Based on our presented data and previous reports, [19,20,25] we hypothesized that hPIV-3 HN forms a covalent intermediate with the substrate by nucleophilic attack at the anomeric carbon (C2) of the Neu5Ac glycoside. The phenolic oxygen of the key catalytic amino acid Tyr530 acts as the nucleophile.…”

“…A significant gap in potency was observed between inhibitor 5 and the other two 2,3difluoro isomers (3 and 4), where 5 was found to be 180 and 250 times more potent than 4 and 3, respectively. [20] Thus, in general, it appears that a-sialosyl fluorides (e.g. [20] Thus, in general, it appears that a-sialosyl fluorides (e.g.…”

“…Using 1 H NMR spectroscopy, the initial stereochemistry around the anomeric center (C2) of the released product, Nacetyl-d-neuraminic acid (Neu5Ac), was determined by monitoring the products from hPIV-3 HN action on the substrate MUN over 55 min. [19,20,24] Following the characterization of hPIV-3 HN as a retaining glycohydrolase, inhibitor 5 was used to further study the catalytic mechanism of hPIV-3 HN. The b-anomer, the mutarotated product characterized by H3 ax and H3 eq resonances at d = 1.7 and 2.1 ppm, respectively was initially observed after 25 min of incubation.…”

The impact of the butterfly effect on human parainfluenza virus haemagglutinin-neuraminidase inhibitor design

“…The triarginyl cluster is known to make substantial contribution, through engagement with the carboxylate of both substrates and inhibitors, in a broad range of neuraminidases (NAs) 9, 12–15 including influenza A virus (IAV) NA 16–19 . A similar contribution was observed in the hPIV-3 HN– 6 complex, in which the hPIV-3 HN triarginyl cluster (comprised of arginine residues Arg192, Arg424 and Arg502) was found to form a salt bridge and hydrogen bonds with the carboxylic acid moiety of inhibitor 6 (Fig.…”

“…The reaction mechanism of the broad NA family has been the subject of intense investigation over the last twenty years 13–15 , especially the IAV NA mechanism 16, 18, 19, 22, 23 . The triarginyl cluster–Neu5Ac carboxylate interaction is thought to have a major influence on the geometry of 1 when bound within the active site 23 .…”