(NAS Colloquium) Proteolytic Processing and Physiological Regulation (1999) / Chapter Skim
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Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes
Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes
Pages 11000-11007
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From page 11000... ...
Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has y~elded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity.
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From page 11001... ...
The side chain of Asn-165 is positioned directly above the benzene of CBZ, with its carboxamide NH pointing into the face of the aromatic ring, suggesting that some additional binding energy probably derives from this favorable amino-aromatic interaction (17~. The affinity of peptide aldehyde inhibitors for trypsin-like serine proteases has been attributed to their ability to form, with the active-site serine, hemiacetals that resemble the transition state in amide hydrolysis, with the oxyanion stabilized in a structurally conserved oxyanion hole.
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From page 11002... ...
that hydrogen bonds to ordered water molecules at the back of the S2 pocket. In summary, the available crystallographic and amino acid sequence data suggest that inhibitors of rhinovirus 3C protease could be expected to show efficacy against the enzyme from multiple viral serotypes provided they do not depend on binding determinants at the back of the S2 specificity pocket where structural variability between serotypes may be most pronounced.
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From page 11003... ...
Irreversible Michael Acceptors as Inhibitors of 3C Protease Peptidic substrates in which the scissile amide carbonyl is replaced by a Michael acceptor were first introduced as specific irreversible inhibitors of the cysteine protease papain by Hanzlik and coworkers (20, 21~. We reasoned that, although this reaction is probably facilitated by the especially nucleophilic thiolateimidazolium ion pair in papain-like cysteine proteases, suitably activated Michael acceptors might also undergo addition by the presumably less nucleophilic catalytic cysteine of 3C.
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From page 11004... ...
The rate of chemical inactivation presumably depends on not only the intrinsic electrophilic character of the inhibitor, but on how the reactive vinyl group is oriented in the active site relative to Cys-147 before nucleophilic attack and on the extent to which the transition state for the reaction can be stabilized by the enzyme. Mechanism-based activation of an inherently weak Michael acceptor as a means of increasing the rate of the chemical step, and thus kobs/I' is conceptually more attractive than attempting to achieve a similar effect by simply increasing intrinsic electrophilic reactivity, which would likely impart undesirable properties to such compounds.
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From page 11005... ...
compared with that of compound III. AG7088, a 3C Protease Inhibitor with Potent Antiviral Activity Against Multiple Human Rhinovirus Serotypes For each position in the N-terminal protected tripeptide portion of compound III, modifications were identified that imparted increased activity against 3C protease and better antiviral properties compared with those of the parent molecule.
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From page 11006... ...
groups also create additional van der Waals contacts with backbone atoms of residues 143 and 144, which, compared with a P1 Gin, may further reduce the flexibility and conformational heterogeneity that is observed for this region in the absence of bound inhibitors. Particularly noteworthy is that, for AG7088 bound to 3C protease, the peptide bond 144-145 has its NH pointing in toward the oxyanion hole where it may play a role in hydrogen bonding to the carbonyl oxygen of the Michael acceptor in the transition state for Michael addition.
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From page 11007... ...
= l.9~o. Protein atomic coordinates from the cocrystal structure of type 2 3C protease with compound I (15)
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