Structural Basis for Streptococcus pneumoniae NanA Inhibition by Influenza Antivirals Zanamivir and Oseltamivir Carboxylate

Abstract

The human pathogen Streptococcus pneumoniae is the major cause of bacterial meningitis, respiratory tract infection, septicemia, and otitis media. The bacterium expresses neuraminidase (NA) proteins that contribute to pathogenesis by cleaving sialic acids from host glycoconjugates, thereby enhancing biofilm formation and colonization. Recent in vivo experiments have shown that antiviral compounds, widely used in clinics and designed to inhibit influenza NA, significantly reduce biofilm formation and nasopharyngeal colonization of S. pneumoniae in mice. Here, we present the structural basis for the beneficial effect of these compounds against pneumococcal infection. Crystal structures of pneumococcal NanA in complex with zanamivir and oseltamivir carboxylate are discussed, correlated with measured inhibitory constants K_i, and compared with the binding modes of the inhibitors in the viral enzyme. Inhibitor structures show for the first time how clinically approved anti-influenza compounds interact with an NA of the human pathogen S. pneumoniae and give a rational explanation for their antibacterial effects.

Introduction

Neuraminidases (NAs), or sialidases, catalyze the cleavage of terminal sialic acids from a variety of glycoconjugates and have been shown to play a role in many pathogenic processes in infectious diseases.¹ NAs can differ in substrate specificity of the glycosidic linkage and are also known to release chemically different products. Most NAs simply hydrolyze sialoglycosides and release N-acetylneuraminic acid (Neu5Ac), while the trypanosomal enzymes can also transfer Neu5Ac to a sugar acceptor.² An exception is the leech intramolecular trans-sialidase that exhibits strict specificity for α2,3-linked sialic acids and releases 2,7-anhydro-Neu5Ac.³

The human pathogen Streptococcus pneumoniae is a major health concern worldwide, being responsible for respiratory tract infections, meningitis, and septicemia. The S. pneumoniae genome encodes three different NAs (NanA, NanB, and NanC) that are expressed in a strain-dependent manner and that are established virulence factors contributing to infection and virulence.4, 5, 6 NanA has a wide substrate specificity and cleaves α2,3-, α2,6-, and α2,8-linked sialic acids, while NanB and NanC show only considerable activity toward α2,3-linked substrates.7, 8, 9 The three NAs also differ in their catalytic reaction and produce different products; NanA releases Neu5Ac, while NanB produces 2,7-anhydro-Neu5Ac similar to the leech enzyme, and NanC initially produces 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA, Neu5Ac2en).7, 8, 9 Crystal structures of both NanA and NanB, without ligand or in complexed form (Neu5Ac, DANA for NanA; 2,7-anhydro-Neu5Ac, DANA for NanB) are available, while the NanC structure has not yet been determined.7, 8, 10, 11 NanA was shown to play a critical role in pneumococcal endothelial brain invasion via its lectin domain,12, 13 but less is known about the role of NanA, NanB, and NanC enzymatic activity in pneumococcal infection. Neu5Ac, the product of the NanA reaction, was shown to enhance streptococcal biofilm formation in vitro, and inoculation of Neu5Ac intranasally in mice increased pneumococcal colonization in the nasopharynx and enhanced translocation to the lungs. Both the in vitro and the in vivo effects were successfully suppressed using the broadband NA inhibitor DANA and the influenza NA-specific inhibitors zanamivir (Relenza; GlaxoSmithKline) and oseltamivir carboxylate (OC) (Tamiflu; Roche).5, 14, 15, 16 These influenza inhibitors are prescribed worldwide to treat influenza infections and play an important role in the treatment and control of potential influenza pandemics. A wealth of information is available about how the inhibitors zanamivir and OC act on the influenza NA, but there are no data that describe how they inhibit bacterial NA that can provide an explanation for the beneficial effects of treatment with these drugs in mouse models of pneumococcal infection. Here, we describe the crystal structures of NanA in complex with the influenza NA inhibitors zanamivir and OC at 1.9 Å and 1.75 Å resolutions and correlate structural findings with experimentally determined inhibitory constants K_i.