Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus

Tiralongo, Joe; Pegg, Michael S.; Itzstein, Mark von

doi:10.1016/0014-5793(95)00967-e

Cited by 20 publications

(7 citation statements)

References 13 publications

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“…The inverse kinetic isotope effect at pH 6.0 was not seen with a different substrate and the mechanistic aspects of influenza NA hydrolysis were proposed to involve the formation of an A-lactone intermediate, instead of a sialosyl cation [31]. The difference in results obtained by the two groups [11,31], leading to different transition states, was later shown to be due to the different substrates used [32].…”

Section: Discussionmentioning

confidence: 97%

Site‐directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design

Ghate

Air

1998

European Journal of Biochemistry

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The neuraminidase of influenza virus is a surface glycoprotein that catalyzes the hydrolysis of glycosidic linkages between terminal sialic acids and adjacent sugar moieties. Neuraminidase function is critical for the spread of virus to new cells, and if the enzyme activity is inhibited, then virus infection is abrogated. The neuraminidase active site is conserved in all influenza type-A and type-B isolates, which makes it an excellent target for drug design. To determine the potential for resistance to develop against neuraminidase inhibitors, we have constructed mutations in seven of the conserved active-site residues of a type B (B/Lee/40) neuraminidase and analyzed the effect of the altered side chains on enzyme activity. There is a reduction in k cat in all our mutants. A transition-state analogue inhibitor shows variation in K i with the mutant neuraminidases, allowing interpretation of the effects of mutation in terms of transition-state binding and product release. The results show that Tyr409 is the most critical residue for enzyme activity, but that Asp149, Arg223, Glu275 and Arg374 also play important roles in enzyme catalysis. Based on the pH profile of neuraminidase activity of the D149E mutant protein, we conclude that Asp149 is not a proton donor, but is involved in stabilizing the transition state. If designed inhibitors are targeted to these residues where mutations are highly deleterious, particularly Tyr409, Glu275 and Asp149, the virus is unlikely to generate resistance to the drug.

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Section: Discussionmentioning

confidence: 97%

Site‐directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design

Ghate

Air

1998

European Journal of Biochemistry

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“…(1C)). Instead, the extent of bond-breaking and cationic character of the reaction intermediate may depend on the nature of the leaving group, as discussed by Tiralongo et al [18].…”

Section: Na Active Site Structure and Catalytic Mechanismmentioning

confidence: 97%

“…Schematic representation of steps in the influenza neuraminidase mechanism of hydrolysis of oligosaccharides with terminal sialic acid residues[8,18]. NA active site interaction pulls the carboxylic acid of sialic acid into an equatorial position and the glycosidic bond into an axial position.…”

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confidence: 99%

Optimization of Small Molecule Drugs Binding to Highly Polar Target Sites: Lessons from the Discovery and Development of Neuraminidase Inhibitors

Klumpp

Graves²

2006

CTMC

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Binding affinity optimization of small molecules interacting with polar binding sites on target proteins is a formidable, but not uncommon challenge in drug discovery. The challenge relates to the difficulty of integrating favourable and unfavourable polar, non-polar and conformation contributions into overall favourable binding energies. This review describes the surprising breakthrough findings leading to the development of Tamiflu, a clinically efficacious orally bioavailable drug targeting the active site of influenza neuraminidase (NA). The NA active site is highly polar and formed mostly by arginine, aspartate and glutamate residues. This active site structure evolved for efficient interaction with charged sialic acid moieties on glycoproteins and stabilization of an oxocarbonium ion in the transition state of the neuraminidase reaction. The initial strategy of optimizing polar interactions in transition state analogs led to NA inhibitors (NAIs) with sub-nanomolar binding affinities, but such compounds were highly polar and lacked oral bioavailability. The realization of the possibility to achieve high affinity binding in a highly polar active site through optimization of non-polar and van-der-Waals interactions initially appeared counterintuitive and required a few serendipitous findings, but was key to reduce the polarity of drug candidates, avoid large desolvation penalties and achieve oral bioavailability.

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“…Based on kinetic isotope effect measurements [70,71] and molecular modeling studies [72], the mechanism is then envisaged to proceed through a semiplanar sialosyl cation (oxocarbenium ion) intermediate (2, Scheme 22.1) that could be stabilized by a negatively charged environment in the surrounding area of the active site [63]. A strictly conserved tyrosine residue then acts as a nucleophile to attack the anomeric cen ter, forming a covalent enzyme intermediate (3, Scheme 22.1) [73].…”

Section: Influenza Virus Sialidase: Structure and Mechanismmentioning

confidence: 99%

Development of Influenza Virus Sialidase Inhibitors

Pascolutti

Thomson

Itzstein

2016

Methods and Principles in Medicinal Chemistry

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Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus

Abstract: The effect of substrate aglycon on enzyme mechanism of sialidase from influenza virus was investigated by kinetic isotope effects using the substrates 4-methylumbelliferyi-N-acetyla-l)-neuraminic acid (

Cited by 20 publications

References 13 publications

Site‐directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design

Site‐directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design

Optimization of Small Molecule Drugs Binding to Highly Polar Target Sites: Lessons from the Discovery and Development of Neuraminidase Inhibitors

Development of Influenza Virus Sialidase Inhibitors

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