This study focuses on design, synthesis and in vitro evaluation of inhibitory potency of two series of sialylmimetic that target an exosite ("150-cavity") adjacent to the active site of influenza neuraminidases from A/California/07/2009 (H1N1) pandemic strain and A/chicken/Nakorn-Patom/Thailand/CU-K2-2004 (H5N1). The structure-activity analysis as well as 3-D structure of the complex of parental compound with the pandemic neuraminidase p09N1 revealed high flexibility of the 150-cavity towards various modification of the neuraminidase inhibitors. Furthermore, our comparison of two methods for inhibition constant determination performed at slightly different pH values suggest that the experimental conditions of the measurement could dramatically influence the outcome of the analysis in the compound-dependent manner. Therefore, previously reported Ki values determined at non-physiological pH should be carefully scrutinized.
Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.
- MeSH
- antivirové látky chemie farmakologie MeSH
- chřipka lidská farmakoterapie enzymologie virologie MeSH
- DNA chemie MeSH
- inhibitory enzymů chemie farmakologie MeSH
- kyseliny fosforité chemie MeSH
- lidé MeSH
- neuraminidasa antagonisté a inhibitory metabolismus MeSH
- oseltamivir analogy a deriváty chemie MeSH
- preklinické hodnocení léčiv metody MeSH
- reprodukovatelnost výsledků MeSH
- virové proteiny antagonisté a inhibitory metabolismus MeSH
- virus chřipky A účinky léků enzymologie fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Neuraminidase is the main target for current influenza drugs. Reduced susceptibility to oseltamivir, the most widely prescribed neuraminidase inhibitor, has been repeatedly reported. The resistance substitutions I223V and S247N, alone or in combination with the major oseltamivir-resistance mutation H275Y, have been observed in 2009 pandemic H1N1 viruses. We overexpressed and purified the ectodomain of wild-type neuraminidase from the A/California/07/2009 (H1N1) influenza virus, as well as variants containing H275Y, I223V, and S247N single mutations and H275Y/I223V and H275Y/S247N double mutations. We performed enzymological and thermodynamic analyses and structurally examined the resistance mechanism. Our results reveal that the I223V or S247N substitution alone confers only a moderate reduction in oseltamivir affinity. In contrast, the major oseltamivir resistance mutation H275Y causes a significant decrease in the enzyme’s ability to bind this drug. Combination of H275Y with an I223V or S247N mutation results in extreme impairment of oseltamivir’s inhibition potency. Our structural analyses revealed that the H275Y substitution has a major effect on the oseltamivir binding pose within the active site while the influence of other studied mutations is much less prominent. Our crystal structures also helped explain the augmenting effect on resistance of combining H275Y with both substitutions.
- MeSH
- antivirové látky farmakologie MeSH
- chřipka lidská virologie MeSH
- inhibitory enzymů farmakologie MeSH
- kalorimetrie MeSH
- kinetika MeSH
- krystalizace MeSH
- lidé MeSH
- missense mutace MeSH
- neuraminidasa chemie genetika MeSH
- oseltamivir farmakologie MeSH
- replikace viru MeSH
- substituce aminokyselin MeSH
- termodynamika MeSH
- virová léková rezistence genetika MeSH
- virové proteiny chemie genetika MeSH
- virus chřipky A, podtyp H1N1 účinky léků enzymologie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- financování organizované MeSH
- Publikační typ
- abstrakty MeSH
