All small molecules to be screened were protonated or deprotonated according to physiological pH of 7.4 and their three-dimensional structures and atomic charges were obtained from AM1 semi-empirical calculations. O157:H7 is an emerging bacterial pathogen responsible for outbreaks of foodborne disease with significant morbidity and mortality in the United States [2]. O157:H7 is the most common cause of hemolytic uremic syndrome, causing more than 20,000 infections and as many as 250 deaths annually [3]. Ricin is another potent RIP isolated from the seeds of the widely available castor plant, RTA (green, 1IFT [32]) with the oligonucleotide-bound RTA at the Sigma-1 receptor antagonist 2 Michaelis-Menten state (yellow; 3HIO [13]) showing that the adenine group markedly perturbs the conformation of Tyr80; b (top right): three distinct conformations of Tyr80: conformations 1, 2, and 3 represent the conformation in green (1IFT [32]), the less populated bound conformation in magenta (1IFS [32]), and the most populated bound conformation in yellow (1FMP [31]), respectively; c (bottom left): the phenolic ring with an adenine group underneath and a docking box atop in the less populated bound conformation (1IFS [32]); d (bottom right): overlay of the oligonucleotide-bound RTA at the Michaelis-Menten state (yellow; 3HIO [13]) with Sigma-1 receptor antagonist 2 RTA in conformation 1 (green; 1IFT [32]), conformation 2 (magenta; 1IFS [32]), and conformation 3 (cyan; 1FMP [31]) showing the closeness of the Tyr80 conformations in 3HIO and Rabbit Polyclonal to HSF1 1FMP and the clash between the nucleotide and Tyr80 in 1IFS. Informed by these seminal findings and the aforementioned challenge of obtaining protein?polynucleotide-interaction inhibitors, we decided to use a doorstop approach to identify Sigma-1 receptor antagonist 2 small-molecule inhibitors of RTA and Stx2. This new approach aims to identify small molecules that work as doorstops to prevent an active-site residue of an RIP (and stereoisomers with the Z isomer being dominant, which is consistent with the stereochemistry of R22 used in our virtual screen. Open in a separate window Figure 4 Synthetic schemes for R16, R20, and R22. R20 or R20b was prepared by coupling 4-formylbenzoic acid with a substituted pyrrole in the keto form for R20 or a mixture of keto and enol forms for R20b according to a reported process [42] (Figure 4). The substituted pyrrole was obtained via cyclization of 2-amino-2-(2-chloroacetyl)butenoate [43], which Sigma-1 receptor antagonist 2 was prepared from 3-aminobutenoate using a literature procedure [44]. R20 has the stereochemistry according to the chemical structure specified by SPECS (catalog number AO-081/14455020). The proton NMR spectrum of R20 made in house matches that of R20 purchased from SPECS. Furthermore, the in vitro and ex vivo biological activities of the in-house and purchased R20 were the same. However, the NOESY spectrum shows that the in-house R20 exists in the stereochemistry because of our observed correlations of the nitrogen-attached proton with the methyl and phenyl protons in R20 (Figure 5). Consistent with the stereochemistry of R20, (stereochemistry [45]. Therefore, identification of R20 as an active RIP inhibitor resulted from sheer luck, because the stereochemistry of R20 specified by the chemical vendor was used in our virtual screen. Open in a separate window Figure 5 NOESY spectrum of R20 indicating the keto form and the stereochemistry. Evaluation of RIP Inhibitors Using in Vitro and ex Vivo Methods Firefly-luciferaseCbased cell-free translation assays with rabbit reticulocyte lysate (RRL) [46] confirmed that 22 of the 27 compounds identified in our virtual screen showed some degrees of RTA inhibition at an inhibitor concentration of 50 nM. Of the 22 active compounds, R16, R19, R20, and R22 were the most promising. Further studies of these inhibitors and their analogs (R16b, R19b, R19c, R19d, and R20b) showed a 1.1- to 1 1.7-fold increase in luciferase activity resulting from the translation in the RRL after treatment with 1 nM RTA and 1 nM inhibitor, relative to the activity after treatment with 1 nM RTA only (Table 1). R19b and R16b showed 1.7- and 1.6-fold increases in luciferase activity, respectively. Interestingly, the luciferase activity in the RRL treated with R16b alone increased as the concentration of R16b increased, whereas that of the RRL treated with RTA and R16b decreased as the R16b concentration increased (Figure 6). Other inhibitors showed similar concentration effects on luciferase activity. These concentration effects made the determination of IC50 values difficult and suggested that these inhibitors might interact with both RTA and firefly luciferase owing to the structural similarity of the inhibitors such as Sigma-1 receptor antagonist 2 R16b to D-luciferin that is the substrate of firefly luciferase [47] and to 3-(5-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzoic acid that is a known inhibitor of firefly luciferase [48] (Figure 7). Open in a separate window Figure 6 Concentration effects of R16b on the luciferase activity. Open in a separate window Figure 7 Structural similarity of R16b to D-luciferin.