Inhibition of ricin by an rna stem-loop containing a ribo-oxycarbonium mimic

Ricin A-chain (RTA), the catalytic subunit of ricin, catalyzes the hydrolysis of a specific adenine from a stem-loop region of ribosomal RNA, to destroy cell protein synthesis. Conjugates of RTA are in clinical trials as toxinchemotherapy agents, but also exhibit general toxicity. The purpose of this study is to design specific inhibitors as rescue agents in toxin chemotherapy. Phenyliminoribitol (X), a transition-state analogue for N- ribohydrolases, was incorporated into a 14-base stem-loop RNA with a GXGA tetraloop. Inhibition against RTA was evaluated with a 10-base stem-loop RNA, CGC GAGA GCG (A-10) as the RTA substrate. The results demonstrated that A-10 has a A'm of 6.2 pM and a kcat of 1.7 minl under optimized reaction conditions, while the stem- loop RNA containing phenyliminoribitol at the depurination site gave an inhibition constant of 0.7 M, a 10-fold increase in binding compared to interactions with similar stem-loop substrates. The stem- loop motif is required to achieve inhibition since X alone and XG dinucleotide were not inhibitory.