Inhibition of Ricin A-Chain with Pyrrolidine Mimics of the Oxacarbenium Ion Transition State

Setu Roday, Timothy Amukele, Gary B. Evans, Peter C. Tyler, Richard H. Furneaux, Vern L. Schramm

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Ricin A-chain (RTA) catalyzes the hydrolytic depurination of a specific adenosine at position 4324 of 28S rRNA. Kinetic isotope effects on the hydrolysis of a small 10mer stem-tetraloop oligonucleotide substrate established the mechanism of the reaction as DN*AN, involving an oxacarbenium ion intermediate in a highly dissociative transition state. An inhibitor with a protonated 1,4-dideoxy-1,4-imino-D-ribitol moiety, a 4-azasugar mimic, at the depurination site in the tetraloop of a 14mer oligonucleotide with a 5 bp duplex stem structure had previously been shown to bind to RTA with a Kd of 480 nM, which improved to 12 nM upon addition of adenine. Second-generation stem-tetraloop inhibitors have been synthesized that incorporate a methylene bridge between the nitrogen of a 1-azasugar mimic, namely, (3S,4R)-3-hydroxy-4-(hydroxymethyl)pyrrolidine, and substituents, including phenyl, 8-aza-9-deazaadenyl, and 9-deazaadenyl groups, that mimic the activated leaving group at the transition state. The values for the dissociation constants (Ki) for these were 99 nM for the phenyl 10mer, 163 and 94 nM for the 8-aza-9-deazaadenyl 10- and 14mers, respectively, and 280 nM for the 9-deazaadenyl 14mer. All of these compounds are among the tightest binding molecules known for RTA. A related phenyl-substituted inhibitor with a deoxyguanosine on the 5′-side of the depurination site was also synthesized on the basis of stem-loop substrate specificity studies. This molecule binds with a Ki of 26 nM and is the tightest binding "one-piece" inhibitor. 8-Aza-9-deaza- and 9-deazaadenyl substituents provide an increased pKa at N7, a protonation site en route to the transition state. The binding of these inhibitors is not improved relative to the binding of their phenyl counterpart, however, suggesting that RTA might also employ protonation at N1 and N3 of the adenine moiety to activate the substrate during catalysis. Studies with methylated adenines support this argument. That the various stem-loop inhibitors have similar potencies suggests that an optimal one-piece inhibitor remains to be identified. The second-generation inhibitors described here incorporate ribose mimics missing the 2-hydroxy group. On the basis of inhibition data and substrate specificity studies, the 2′-hydroxyl group at the depurination site seems to be critical for recruitment as well as catalysis by RTA.

Original languageEnglish (US)
Pages (from-to)4923-4933
Number of pages11
JournalBiochemistry
Volume43
Issue number17
DOIs
StatePublished - May 4 2004

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Ricin
Rapid thermal annealing
Adenine
Ions
Substrate Specificity
Catalysis
Oligonucleotides
Ribitol
Protonation
Substrates
Ribose
Isotopes
Hydroxyl Radical
Adenosine
Hydrolysis
Nitrogen
Deoxyguanosine
Molecules
Catalyst supports
pyrrolidine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Inhibition of Ricin A-Chain with Pyrrolidine Mimics of the Oxacarbenium Ion Transition State. / Roday, Setu; Amukele, Timothy; Evans, Gary B.; Tyler, Peter C.; Furneaux, Richard H.; Schramm, Vern L.

In: Biochemistry, Vol. 43, No. 17, 04.05.2004, p. 4923-4933.

Research output: Contribution to journalArticle

Roday, S, Amukele, T, Evans, GB, Tyler, PC, Furneaux, RH & Schramm, VL 2004, 'Inhibition of Ricin A-Chain with Pyrrolidine Mimics of the Oxacarbenium Ion Transition State', Biochemistry, vol. 43, no. 17, pp. 4923-4933. https://doi.org/10.1021/bi0498499
Roday, Setu ; Amukele, Timothy ; Evans, Gary B. ; Tyler, Peter C. ; Furneaux, Richard H. ; Schramm, Vern L. / Inhibition of Ricin A-Chain with Pyrrolidine Mimics of the Oxacarbenium Ion Transition State. In: Biochemistry. 2004 ; Vol. 43, No. 17. pp. 4923-4933.
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