Inhibition and detection of ribosome inactivating proteins

Abstract

Ricin is a highly toxic ribosome inactivating protein (RIP) and is abundant in castor beans. Ricin A-chain (RTA) catalyzes the hydrolytic depurination of A4324, the first adenosine of the GAGA loop portion of 28S eukaryotic ribosomal RNA resulting in the loss of elongation factor binding, inhibition of protein synthesis, and cell death. Inhibitors to RTA could serve as a ricin antidote or rescue agent in immunotoxin cancer therapeutics. Small RNA hairpin versions of the 28S ribosomal RNA are RTA substrates and have provided insight in understanding RTAs binding specificity and enzymatic mechanism for transition state (TS) inhibitor studies. The RNA hairpin substrate A-10 (5'-CGCGAGAGCG-3') has been used as a scaffold for inhibitor design to yield several nanomolar inhibitors of RTA. TS mimic sugars replace the first adenine in the GAGA A-10 tetraloop motif. We investigated small circular DNA/RNA molecules as promising new compounds for RTA inhibitor design. A 70 nM competitive inhibition constant ( Ki) on RTA was measured for cyclic G(N-Bn)GA, a circular tetraloop TS mimic. We also developed a sensitive luminescent assay for the initial rate measurement of RTA reactions with sub-picomole sensitivity. Kinetics on eukaryotic ribosomes are followed in continuous, high-throughput assays. TS inhibitor efficacy at physiologic pH was investigated in competition assays with ribosomes and RTA. We also describe the potent inhibition of saporin-L1 ribosome inactivating protein (RIP) with RTA TS mimics. Saporin-L1 inhibition into the low nanomolar range has been achieved by substituting the first adenosine of the GAGA tetraloop with TS mimic 9-deazaadenine-9-methylene- N-hydroxypyrrolidine (DADMeA). Inhibitors bound saporin-L1 up to 40,000-fold tighter than substrate A-10 RNA. TS inhibitors to saporin-L1 directly competed with ribosome depurination in physiologic conditions and prevented RIP toxicity to in vitro protein translation mixtures. Inhibitors of RIPs may have use in advancing cancer therapy based on RIPs conjugated to ligands specific for tumor cells. Crystal structures of saporin-L1 and RTA with bound cyclic tetraloop TS inhibitors revealed novel interactions for future inhibitor design.