Biomedical subjects
M S Pegg
Publications and source records attributed to M S Pegg.
A study of the active site of influenza virus sialidase: an approach to the rational design of novel anti-influenza drugs.
The development of sialidase inhibitor-based potential anti-influenza drugs using rational drug design techniques has been of recent interest. The present study details as investigation of the active site of influenza virus sialidase by using the program GRID in an attempt to design more potent inhibitors in the hope they will eventually lead to anti-influenza drugs. A number of different probes (amino, carboxy, hydroxy, methyl, etc) have been used in an effort to determine the functional groups most likely to improve the binding of the starting template 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en). The data have correctly predicted the binding regions for the carboxylate, acetamido (NH and methyl), and glycerol (OH) groups of N-acetylneuraminic acid. Moreover, the data suggest that the addition of certain functionalities (amino group) at the C-4 position should enhance the overall binding.
Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus.
The effect of substrate aglycon on enzyme mechanism of sialidase from influenza virus was investigated by kinetic isotope effects using the substrates 4-methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid (Neu5Ac alpha 2MU) and p-nitrophenyl-N-acetyl-alpha-D-neuraminic acid (Neu5Ac alpha 2PNP). The kinetic isotope effect on Vmax (beta DV), at pH 6.0, as revealed by direct comparison of rates obtained with Neu5Ac alpha 2MU and the [3,3-2H]-substituted substrate analogue, was shown to be inverse. This indicates that sialidase-catalysed hydrolysis of Neu5Ac alpha 2MU proceeds with substantial positive charge development at the reaction centre in the transition state for the formation of the glycosyl cation-enzyme intermediate. However, no such inverse effect on Vmax at pH 6.0 was observed when using Neu5Ac alpha 2PNP and the [3,3-2H]-substituted substrate. A mechanism by which hydrolysis proceeds through an alpha-lactone intermediate has been proposed by Guo et al. [8]. We propose that the differences in beta DV for the substrates investigated are due primarily to the differing properties of the aglycon leaving groups, which may result in influenza virus sialidase catalysing substrate hydrolysis by a similar mechanism with alternative stabilisation of transition state.
Slow-binding inhibition of sialidase from influenza virus.
Sialidase from influenza virus A (Tokyo/3/67, N2) is inhibited in slow-binding fashion by 2,3-didehydro-2,4-dideoxy-4-guanidino-N-acetyl-D-neuraminic acid. The Ki observed for the tightly-bound form at steady-state is 3 x 10(-11) M. Slow-binding, which is a consequence of the guanidinyl moiety of the inhibitor, is observed only for influenza virus A sialidase and not for influenza virus B or any other viral, bacterial, or mammalian sialidase investigated. The different results obtained for sialidases from influenza virus A and B, whose active sites are conserved, point to the involvement of the expulsion of a structural water molecule in the slow-binding mechanism.
Rational design of potent sialidase-based inhibitors of influenza virus replication.
Two potent inhibitors based on the crystal structure of influenza virus sialidase have been designed. These compounds are effective inhibitors not only of the enzyme, but also of the virus in cell culture and in animal models. The results provide an example of the power of rational, computer-assisted drug design, as well as indicating significant progress in the development of a new therapeutic or prophylactic treatment for influenza infection.
Inhibition of sialidases from viral, bacterial and mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid modified at the C-4 position.
The inhibition of sialidase activity from influenza viruses A and B, parainfluenza 2 virus, Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, and sheep liver by a range of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified at the C-4 position has been studied. All substitutions tested resulted in a decrease in the degree of inhibition of the bacterial and mammalian sialidases. For sialidases from influenza viruses A and B, on the other hand, most of the substitutions tested either had no significant effect on binding or, in the case of the basic amino and guanidino substituents, resulted in significantly stronger inhibition. The results for parainfluenza 2 virus sialidase were mostly intermediate, in that inhibition was neither significantly increased nor decreased by most of the modifications. We conclude that only the influenza A and B sialidase active sites possess acid groups correctly positioned to participate in charge-charge interactions in the region of C-4 of bound substrate, and that the C-4 binding pockets of the bacterial and mammalian sialidases examined are considerably smaller than is observed for either the influenza virus or parainfluenza virus sialidases.
Evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza virus.
The enzyme mechanism of sialidase from influenza virus has been investigated by kinetic isotope methods, NMR, and a molecular dynamics simulation of the enzyme-substrate complex. Comparison of the reaction rates obtained with the synthetic substrate 4-methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid and the [3,3-2H]-substituted substrate revealed beta-deuterium isotope effects for V/Km ranging over 1.09-1.15 in the pH range 6.0-9.5, whereas the effects observed for V in this pH range increased from 0.979 to 1.07. In D2O, beta DV/Km was slightly increased by 2% and 5% at pD 6.0 and 9.5 respectively, while beta DV was unchanged. Solvent isotope effects of 1.74 were obtained for both beta DV/Km and beta DV at pD 9.5, with beta DV/Km decreasing and beta DV remaining constant at acidic pD. 1H-NMR experiments confirmed that the initial product of the reaction is the alpha-anomer of N-acetyl-D-neuraminic acid. Molecular dynamics studies identified a water molecule in the crystal structure of the sialidase-N-acetyl-D-neuraminic acid complex which is hydrogen-bonded to Asp151 and is available to act as a proton donor source in the enzyme reaction. The results of this study lead us to propose a mechanism for the solvent-mediated hydrolysis of substrate by sialidase that requires the formation of an endocyclic sialosyl cation transition-state intermediate.
Influenza virus sialidase: effect of calcium on steady-state kinetic parameters.
Ca2+ increases the initial rate of activity of sialidase from influenza virus (A/Tokyo/3/67). Increasing ionic strength also activates influenza virus sialidase. When ionic strength is controlled, smaller but still significant Ca2+ effects are observed, with Vmax/Km increased from 0.8.10(5) to 1.4.10(5) M-1 s-1 and Vmax increased from 6.3 to 9.5 s-1 by saturating Ca2+. The Ki of the competitive inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was decreased from 2.7.10(-6) to 1.15.10(-6) M after the addition of saturating Ca2+. The data show that Ca2+ exerts a specific effect on Vmax/Km, leading to an increased rate of interaction of substrate with the enzyme. The Kd-app for the Ca2(+)-sialidase complex is 2 mM. Except for Mg2+ which behaves similarly to Ca2+, other mono- and divalent cations have little specific effect on sialidase kinetics. Sequence analysis of a range of subtypes of sialidases from influenza virus supports the proposal that Ca2+ binds at the subunit interface transmitting a conformational change to the enzyme active site. Ca2+ activation may have a physiological role in switching on sialidase activity during the release of newly synthesised virions from the host cell surface.
Characterisation of an ionisable group involved in binding and catalysis by sialidase from influenza virus.
The effect of pH on the kinetics of sialidase purified from influenza virus (A/Tokyo/3/67, H2N2) was investigated. A pK of 9.0 for inhibition of the enzyme by three competitive inhibitors, due to an ionisable group in the active site, was observed. A similar pK was observed for V/Km for the fluorogenic substrate 2-(4-methylumbelliferyl)-N-acetyl-alpha-D-neuraminic acid. However, the shape of the V/Km profile indicates that this substrate is sticky. Solvent perturbation experiments indicated that the observed ionisable active site group is likely to be a cationic amino acid. The results provide evidence against the hypothesis that Glu 276 acts as a proton donor in the enzyme reaction and supports the proposal of a role for one of the active site cationic amino acids in binding and catalysis.
Potency changes of intravenous induction agents in the first ten weeks of life: an experiment using beagle dogs.
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Hypotension following metoclopramide injection.
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