Structure-Based Design and Characterization of Novel Platforms for Ricin and Shiga Toxin Inhibition

Abstract

Ribosome inhibiting proteins, RIPs, are a widespread family of toxic enzymes. Ricin is a plant toxin used as a poison and biological warfare agent; shiga toxin is a homologue expressed by pathogenic strains of E. coli. There is interest in creating effective antidote inhibitors to this class of enzymes. RIPs act by binding and hydrolyzing a specific adenine base from rRNA. Previous virtual screens revealed that pterins could bind in the specificity pocket of ricin and inhibit the enzyme. In this paper we explore a range of compounds that could serve as better platforms for inhibitor design. This establishes the importance of key hydrogen bond donors and acceptors for active-site complementarity. 8-Methyl-9-oxoguanine is a soluble compound that has the best inhibitory properties of any platform tested. The X-ray structure of this complex revealed that the inhibitor binds in an unexpected way that provides insight for future design. Several inhibitors of ricin were also shown to be inhibitors of shiga toxin, suggesting this program has the potential to develop effective antidotes to an important form of food poisoning.

Structure-Based Design of Inhibitors to the Cytotoxin Ricin

“…The IC50 is roughly 40 ^iM, about 10 times better than the 400 |iM reported earlier (Miller et al, 2002). Figure 3: Inhibition of RTA by pteroic acid in '" addition to establishing the reticuiocyte the reticuiocyte assay.…”

“…This novel compound of our design had been the most potent small molecule "platform" inhibitor previously tested (Miller et al, 2002).…”

“…This assay has been used by others for qualitative measures of Nglycosidase action (Endo et al, 1987). We observed that some potential RTA inhibitors can also inhibit ribosomes, thereby rendering the protein synthesis assay useless (Miller et al, 2002). The aniline cleavage assay only depends on the 28S rRNA being intact; depurination by ricin renders the chain susceptible to aniline cleavage which can be observed as a novel band on an acrylamide gel.…”

Development of a quantitative RT-PCR assay to examine the kinetics of ribosome depurination by ribosome inactivating proteins using Saccharomyces cerevisiae as a model

“…Many compounds, especially those modeled after adenine at the active site are significantly inhibitory by themselves in cell free translation systems presenting serious difficulties for measurement of their IC 50 values. In addition, the fairly sharp pH optimum of translation around pH 7.4 also presents problems for molecules, such as PTA, which are not water soluble (Yan et al 1997;Miller et al 2002). Furthermore, cell free translation assays cannot distinguish between compounds that affect translation by inhibiting the catalytic activity of the toxins and those that interfere with translation by toxin-independent mechanisms.…”

“…Pteroic acid (PTA) is an adenine analog which was shown by X-ray crystallography to bind to the active site of RTA and was kinetically shown to inhibit RTA with an IC 50 of 600 mM when assayed by in vitro translation using Artemia salina ribosomes (Yan et al 1997). 8-methyl-9-oxo-guanine (9OG) was previously reported as a more potent inhibitor than PTA in the Artemia translation system with an IC 50 of 400 mM (Miller et al 2002). We tested the ability of PTA and 9OG to inhibit the activity of RTA and Shiga toxin 2 (Stx2) using the qRT-PCR assay.…”