Complementary assays helping to overcome challenges for identifying neuraminidase inhibitors

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From: Future Virology(Vol. 10, Issue 2)
Publisher: Future Medicine Ltd.
Document Type: Report
Length: 7,172 words
Lexile Measure: 1510L

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Author(s): Martina Richter aff1 , Lilia Schumann aff1 , Elisabeth Walther aff1 , Anja Hoffmann aff1 , Heike Braun aff1 , Ulrike Grienke aff2 aff3 , Judith M Rollinger aff3 , Susanne von Grafenstein aff4 , Klaus R Liedl aff4 , Johannes Kirchmair aff5 , Peter Wutzler aff1 , Andreas Sauerbrei aff1 , Michaela Schmidtke [*] aff1

KEYWORDS

assay interference; Clostridium perfringens ; influenza virus; neuraminidase inhibition assay; neuraminidase inhibitors; self-fluorescence; signal quenching; Streptococcus pneumoniae ; Vibrio cholerae

Influenza A viruses express two surface glycoproteins, hemagglutinin and neuraminidase (NA), which function in a highly balanced manner [1 ]. The binding of viral hemagglutinin to sialic acid (functioning as receptor on the cell surface) mediates the attachment of the virus to host cells, while influenza NA allows progeny virus particles to elute from infected cells by enzymatic cleavage of terminal sialic acid residues [2 ]. In this way, viral NA prevents self-aggregation of progeny virions and facilitates their spread across the respiratory tract. NA therefore represents a target of influenza therapeutics.

Because of the widespread resistance of influenza viruses to ion channel blockers, current antiviral therapy options are limited to NA inhibitors (NAIs). These include oseltamivir and zanamivir, both of which are licensed worldwide since 1999, and peramivir and laninamivir, which have been approved in Japan, China and South Korea in 2010 [3,4 ]. Resistance to NAIs is observed relatively rarely because of the essential catalytic function of these proteins. The emergence and global spread of oseltamivir-resistant H1N1 influenza viruses with H275Y substitution in the season of 2007/2008 [5,6 ] however demonstrated that resistant strains could emerge suddenly. Therefore, the search for novel NAIs is ongoing. Encouraged by the observation that oseltamivir-resistant strains maintain susceptibility to zanamivir, several research groups are investigating structural modifications of oseltamivir [7,8 ] to overcome loss of activity induced by mutation [4 ], in particular H275Y. Other groups are searching for novel scaffolds for NAI acting against oseltamivir- and zanamivir-resistant influenza viruses [ 3,9 ].

Also bacteria, such as Streptococcus pneumoniae , one of the most common causative agent of flu-associated secondary bacterial pneumonia [10 ], express three different NAs as virulence factors [11,12 ]. Pneumococcal NAs were shown to play an important role in the lethal synergism of influenza viruses and pneumococci [13 ]. According to x-ray data, the conformation of the cleavage site of pneumococcal NA resembles that of the viral enzyme [14 ]. This renders bacterial NA a pharmaceutically relevant target.

Assays most commonly used to monitor the NAI susceptibility of influenza A viruses are based on the detection of chemiluminescence (CL) or fluorescence (FL) signals [4,15-18 ]. The CL-based NA-inhibition assay (henceforth referred to as CL assay) uses NA-Star® (1,2-dioxetane derivative of sialic acid; Applied Biosystems, Darmstadt, Germany) as chemiluminescent substrate. The FL-based NA inhibition assay (henceforth referred to as FL assay) is based on 2′-(4-methylumbelliferyl)-[alpha]-d-N- acetylneuraminic acid (MUNANA) as fluorogenic substrate. Both assays are standardized and available in kit format. In addition, different protocols for in house FL assay exist. The CL assay is more sensitive than its FL counterpart [4,19 ]. Studies on the specificity...

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Gale Document Number: GALE|A407934436