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

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 D(N)*A(N), 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 K(d) 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 (K(i)) 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 K(i) of 26 nM and is the tightest binding "one-piece" inhibitor. 8-Aza-9-deaza- and 9-deazaadenyl substituents provide an increased pK(a) 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.

Transition State Structure of RNA Depurination by Saporin L3

“…VLS is caused by imperfect targeting such that excess RIP constructs, or RIP constructs lost to circulation following tumor lysis, enter the capillary bed with the loss of vascular integrity. 33 VLS is the dose-limiting toxicity of immunotherapy. As a result, there is growing interest in the design of potent inhibitors of RIP toxin activity, which can function as rescue treatments following IT therapy.…”

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

“…RTA transition state structures have guided the design and synthesis of potent RIP inhibitors (17,29). Inhibitors for RTA and SAP include 1Ј-aza-sugars with a nonhydrolyzable 9-deazaadenine to mimic the elevated pK a of the leaving group (15).…”

Vinyldeoxyadenosine in a Sarcin−Ricin RNA Loop and Its Binding to Ricin Toxin A-Chain

“…Substrate Activity of 8vdA -10 with RTA 8vdA-10 was tested as a substrate for RTA at pH 4.0 and 37 °C with assay conditions that have been reported previously for stem-loop substrates of RTA (10,11). The reaction was monitored by HPLC using dual wavelength detection at 260 nm and 305 nm.…”

“…Inhalation of aerosolized ricin causes rapid and irreversible pulmonary dysfunction. Reliable means of detection (10) and the development of antidotes (11) are important areas of research for this toxin. The activity of RTA on small 10-14mer stem-tetraloop RNA substrates at acidic pH has been demonstrated (12) and the K cat for the hydrolysis of 2'-deoxyadenosine in a hybrid GdAGA tetraloop was determined to be ~16 fold faster than that for an adenosine tetraloop (13).…”