Ricin A-Chain Inhibitors Resembling the Oxacarbenium Ion Transition State

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Abstract
Ricin toxin A-chain (RTA) is expressed by the castor bean plant and is among the most potent mammalian toxins. Upon activation in the cytosol, RTA depurinates a single adenine from position 4324 of rat 28S ribosomal RNA, causing inactivation of ribosomes by preventing the binding of elongation factors. Kinetic isotope effect studies have established that RTA operates via a DN*AN mechanism involving an oxacarbenium ion intermediate with bound adenine [Chen, X.-Y., Berti, P. J., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 1609−1617]. On the basis of this information, stem−loop RNA molecules were chemically synthesized, incorporating structural features of the oxacarbenium ion-like transition state. A 10-base RNA stem−loop incorporating (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-d-ribitol at the depurination site binds four times better (0.57 μM) than the 10-base RNA stem−loop with adenosine at the depurination site (2.2 μM). A 10-base RNA stem−loop with 1,2-dideoxyribitol [(2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] at the depurination site binds with a Kd of 3.2 μM and tightens to 0.75 μM in the presence of 9-deazaadenine. A similar RNA stem−loop with 1,4-dideoxy-1,4-imino-d-ribitol at the depurination site binds with a Kd of 1.3 μM and improves to 0.65 μM with 9-deazaadenine added. When (3S,4R)-4-hydroxy-3-(hydroxymethyl)pyrrolidine was incorporated at the depurination site of a 14-base RNA stem−loop, the Kd was 0.48 μM. Addition of 9-deazaadenine tightens the binding to 0.10 μM whereas added adenine increases the affinity to 12 nM. The results of this study are consistent with the unusual dissociative DN*AN mechanism determined for RTA. Knowledge of this intermediate has led to the design and synthesis of the highest affinity inhibitor reported for the catalytic site of RTA.