The part of the influenza polymerase PA subunit featuring endonuclease activity is a target for anti-influenza therapies, including the FDA-approved drug Xofluza. A general feature of endonuclease inhibitors is their ability to chelate Mg2+ or Mn2+ ions located in the enzyme's catalytic site. Previously, we screened a panel of flavonoids for PA inhibition and found luteolin and its C-glucoside orientin to be potent inhibitors. Through structural analysis, we identified the presence of a 3',4'-dihydroxyphenyl moiety as a crucial feature for sub-micromolar inhibitory activity. Here, we report results from a subsequent investigation exploring structural changes at the C-7 and C-8 positions of luteolin. Experimental IC50 values were determined by AlphaScreen technology. The most potent inhibitors were C-8 derivatives with inhibitory potencies comparable to that of luteolin. Bio-isosteric replacement of the C-7 hydroxyl moiety of luteolin led to a series of compounds with one-order-of-magnitude-lower inhibitory potencies. Using X-ray crystallography, we solved structures of the wild-type PA-N-terminal domain and its I38T mutant in complex with orientin at 1.9 Å and 2.2 Å resolution, respectively.

• Klíčová slova
• Mannich reaction, RNA polymerase, bio-isosterism, cross-coupling, endonuclease inhibitor, flavonoids, influenza,
• MeSH
• antivirové látky chemická syntéza   farmakologie   MeSH
• endonukleasy antagonisté a inhibitory   MeSH
• katalytická doména účinky léků   MeSH
• luteolin chemická syntéza   farmakologie   MeSH
• Orthomyxoviridae účinky léků   MeSH
• virové proteiny antagonisté a inhibitory   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky MeSH
• endonukleasy MeSH
• luteolin MeSH
• virové proteiny MeSH

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.

• Klíčová slova
• DIANA, assay, crystallography, influenza neuraminidase,
• 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
• Názvy látek
• antivirové látky MeSH
• DNA MeSH
• inhibitory enzymů MeSH
• kyseliny fosforité MeSH
• neuraminidasa MeSH
• oseltamivir MeSH
• tamiphosphor MeSH Prohlížeč
• virové proteiny 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.

• Klíčová slova
• crystal structure, influenza neuraminidase, isothermal titration calorimetry, oseltamivir, resistance,
• 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
• Názvy látek
• antivirové látky MeSH
• inhibitory enzymů MeSH
• neuraminidasa MeSH
• oseltamivir MeSH
• virové proteiny MeSH