
==== Front
9413298
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Bioorg Med Chem
Bioorg Med Chem
Bioorganic & medicinal chemistry
0968-0896
1464-3391

38340640
10.1016/j.bmc.2024.117614
nihpa2019052
Article
Structure-based design and optimization of a new class of small molecule inhibitors targeting the P-stalk binding pocket of ricin
Rudolph Michael J. a1
Dutta Arkajyoti b1
Tsymbal Anastasiia M. c1
McLaughlin John E. b
Chen Yang a
Davis Simon A. a
Theodorous Sophia A. a
Pierce Michael b
Algava Benjamin b
Zhang Xiaoyu b
Szekely Zoltan c
Roberge Jacques Y. c
Li Xiao-Ping b*
Tumer Nilgun E. b*
a New York Structural Biology Center, 89 Convent Ave, New York, NY 10027, United States
b Department of Plant Biology, Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ 08901, United States
c Molecular Design and Synthesis Core, Rutgers University Biomolecular Innovations Cores, Office for Research, Rutgers University, 610 Taylor Rd, Piscataway, NJ 08854, United States
1 MJR, AD, and AMT contributed equally to this work.

Authors’ contributions

NET and XPL designed the experiments. MR, YC, SAD, and SAT designed and conducted all structure determinations and characterizations. AD developed the fluorescence polarization assay and determined the affinity of the compounds. JYR designed and AMT designed, synthesized, and characterized the compounds. ZS purified the fluorescent peptide probe and characterized the peptide and the small molecules by HRMS. XPL, MP, JM, XZ, and BA conducted the depurination inhibition analysis. MR and NET wrote the paper. NET was responsible for project administration and funding acquisition.

* Corresponding authors. xpli@sebs.rutgers.edu (X.-P. Li), tumer@sebs.rutgers.edu (N.E. Tumer).
26 8 2024
15 2 2024
05 2 2024
23 9 2024
100 117614117614
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.

Ricin inhibitors
Structure-based design
Fluorescence polarization
Shiga toxin inhibitors
Ribosomal P-stalk
Ribosome inactivating protein
==== Body
pmc1. Introduction

Ricin produced by castor beans (Ricinus communis) is a worldwide problem as a biothreat agent due to its accessibility, stability, and extreme toxicity and is classified as a category B agent for bioterrorism.1 E. coli (STEC), which produce the related Shiga toxins (Stxs) are potentially fatal, foodborne pathogens responsible for the development of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS), the leading cause of kidney failure in children.2–4 Despite decades of work small molecules effective at preventing and/or treating ricin intoxication or STEC infection have not been identified and as of now only supportive care is available. Ricin and Stxs are type II ribosome inactivating proteins (RIPs), which catalyze the removal of the same adenine base from the sarcin/ricin loop (SRL) of the large rRNA, thereby inhibiting protein synthesis.5–7 Ricin contains a catalytically active A subunit (RTA) and a ricin toxin B subunit (RTB), which is a galactose/N-acetyl galactosamine-binding lectin. RTB promotes endocytosis and retrograde transport to the endoplasmic reticulum (ER). RTA is released from RTB in the ER after the reduction of the disulfide bond linking RTA and RTB, and RTA is then retrotranslocated into the cytoplasm.8–10 RTA is an N-glycosidase (EC 3.2.2.22) that hydrolyzes the N-glycosidic bond of a highly conserved adenine 4324 of the rat 28S rRNA, resulting in the inhibition of protein synthesis and cell death.11

We identified the ribosomal P-stalk as a binding site for RTA and showed that ribosome depurination occurs by binding of RTA to the ribosomal P-stalk to recruit the active site to the SRL at a remote site on the large subunit of the ribosome (Fig. 1).12,13 The P-stalk binding site of the toxin is located at the interface between RTA and RTB on the opposite face of the active site.14–16 RTA binds the P-stalk CTD through a well-defined hydrophobic pocket. This interaction anchors RTA on the ribosome and allosterically stimulates the N-glycosidase activity of RTA.14–17 The A1 subunits of Shiga toxins (Stxs)18–20 and several other RIPs also bind to the C-terminal domain (CTD) of P-stalk proteins to access the SRL for catalytic depurination of the same adenine base.21–23

The eukaryotic P-stalk is a pentameric complex composed of uL10 protein, which forms the base of the stalk, and two P1-P2 heterodimers, which are attached to uL10 (Fig. 1).24,25 Each P protein has an unstructured C-terminus with identical 11 amino acids (SDDDMGFGLFD), which are universally conserved among all eukaryotes.25 The P-stalk and the SRL are part of the GTPase center of the ribosome responsible for the recruitment of translational GTPases, such as the elongation factors and stimulation of their GTPase activity.24–27

The X-ray crystal structure analysis of RTA with a peptide corresponding to the conserved C-terminal 10 (P10) or 11 (P11) amino acids of P proteins showed that RTA binds to the last 6 amino acids (GFGLFD) at a well-defined hydrophobic pocket remote from the catalytic site (PDB ID: 5GU4).28,29 The first five residues at the N-terminus of the peptides (SDDDM) were not defined in the crystal structures.28,29 Leu9 and Phe10 of the P11 peptide are inserted into a hydrophobic pocket on RTA formed by Tyr183, Phe240, Val242, Ile247, Leu248, and Ile251 along with a positively charged segment just outside the P11-binding pocket lined by Arg234 and Arg235.28,29 Mutation analysis showed that Arg235 is the most important residue at the RTA/RTB interface for the electrostatic interactions of RTA with the P-stalk.15 When the R235A mutation was combined with mutations in the hydrophobic residues, Y183A, L232A, and F240A, the activity and cytotoxicity of RTA were eliminated in mammalian cells without altering the active site.16 Peptide mimics of the conserved CTD of P proteins, which interact with the P-stalk binding site of RTA inhibited the catalytic activity of RTA.30 These results demonstrated that the hydrophobic pocket at the P-stalk binding site was necessary for toxin activity and sufficient for the inhibition of the cytotoxicity of ricin.

As described earlier,31 our next step was to use fragment-based ligand discovery (FBLD) with surface plasmon resonance (SPR) to identify a small molecule fragment that binds at the P-stalk pocket with sufficient affinity to inhibit the catalytic activity of RTA on eukaryotic ribosomes. The crystal structure of RTA in complex with the RTA inhibitor CC10501 (PDB ID: 6URX) revealed that CC10501 binds the P-stalk pocket similar to the P11 peptide forming comparable interactions with RTA.31 Here, we developed a fluorescence polarization (FP) competition assay to evaluate a new series of compounds and characterized them by structure–activity relationships (SAR), X-ray crystallography and by in vitro and cell-based assays. We identify the first inhibitor that binds at the P-stalk binding pocket of RTA with similar affinity and potency as a five-fold larger P-stalk peptide and protects cells against ricin and Shiga toxin 2 for the first time.

2. Results

2.1. Structure-based design of RTA inhibitors

The X-ray crystal structure of the best inhibitor identified to date, RU-NT-93 in complex with RTA revealed that RU-NT-93 bound RTA in a unique manner where the thiophene ring and its carboxylate in RU-NT-93 were rotated by 77° making it perpendicular relative to the carboxylate moiety in CC10501 (Fig. S1).32 To determine if the orientation of the thiophene is important for increased affinity we wanted to analyze planar compounds where the molecule is locked in a fixed conformation (Table 1). The constrained analog of CC10501, PD00589, was commercially available. RU-NT-59 and RU-NT-61 were synthesized via 3-step synthesis (Scheme 1). Ketones 1a and 1b underwent a Vilsmeier-Haack formylation to give aldehydes 2a and 2b, which subsequently were treated with ethyl thioglycolate to produce the cyclization product 3a and 3b. Afterward, esters 3a and 3b were converted to acids RU-NT-59 and RU-NT-61 respectively using 1 M sodium hydroxide in THF and methanol. Compounds 3c, 3d, and 3e were synthesized from 3b using palladium-catalyzed cross-coupling reactions and later were converted to respective acids RU-NT-62, RU-NT-63, and RU-NT-64 using the same conditions. The difference between PD00589 and RU-NT-59, RU-NT-61, RU-NT-62, RU-NT-63, and RU-NT-64 is that they all have different groups instead of hydrogen at the C7 position (Scheme 1).

Additionally, we found the commercially available analog RU-NT-206 to help evaluate substitution on the central ring and using it as a starting material we synthesized analogs of it (Scheme 2). Amide RU-NT-253 was synthesized from acid RU-NT-206 via condensation with ammonium chloride in the presence of HATU and (i-Pr)2NEt, and treatment of this amide with Burgess’ reagent (methyl N-(triethylammoniumsulfonyl) carbamate) gave nitrile RU-NT-254.

2.2. The affinity and the potency of the inhibitors

To characterize compounds that disrupt the interaction between RTA and the P-stalk we have developed a fluorescence polarization (FP) competition assay. The FP competition assay measures the decrease in FP caused by inhibitors that displace the BODIPY TMR-X NHS labeled P11 peptide probe from RTA. First, the optimal concentration of the fluorescent P11 was determined by measuring the FP as a function of its concentration. In the second step, binding of the fluorescently labeled P11 to purified recombinant RTA was determined by dose titration of the fluorescent P11 with varying concentrations of RTA at a fixed concentration of P11 (1 μM). The FP increased with increasing concentrations of RTA, indicating the binding of BODIPY TMR-X-labeled P11 to RTA. The fluorescently labeled P11 was nearly fully bound to RTA with a KD value of 1 ± 0.2 μM (Fig. S2A). The specificity of the assay was established by competition with unlabeled P11 as a positive control and by competition with PT peptide, which binds at the active site of RTA31 and BTB13068, a small molecule, which binds RTA remote from the P-stalk site,31 as negative controls. The IC50 value determined by the FP competition assay is the concentration of the inhibitor required to replace 50 % of the fluorescent P11 probe from RTA. The IC50 values obtained from the FP competition assay were used to determine the Ki values as described in the Materials and Methods. The inhibitory constant, Ki, reflects the affinity of the inhibitor and is defined as the concentration of the inhibitor that will bind to half of the binding sites on RTA at equilibrium in the absence of labeled P11.33

As the concentration of unlabeled P11 increased, the labeled probe was displaced from RTA with an IC50 value of 4 ± 1 μM and a Ki value of 1 ± 0.3 μM, indicating that the unlabeled P11 can competitively displace the labeled probe (Fig. 2A). The PT peptide (Fig. S2B) or BTB13068 (Fig. S2C) did not show any effect, providing evidence that the binding was not due to nonspecific hydrophobic interactions with RTA. The KD value (1 μM) of the labeled P11 obtained by the direct binding assay (Fig. S2A) was the same as the Ki value of the unlabeled P11 obtained by the competition assay (1 μM) (Fig. 1A), indicating that there was no interference from the BODIPY-TMR-X dye attached to the P11 peptide and that the interaction between P11 and RTA is specific.

2.3. Structure-activity relationship of the inhibitors

Our most active compound is RU-NT-206 (entry 1, Table 1) has a Ki of 1 μM, which is 8-fold better than that observed for PD00589 that lacks the gem-dimethyl substitution. Our results indicate that the presence of carboxylic acid is essential for improved affinity. Replacement with nitrile, hydrazide, ester, or ketone completely removes activity (entries 3, 10 and 15, 12, and 16, respectively, Table 1). Substitution at the C7-position with a fluoride doubled the potency compared to no substituents (entry 4 vs 5, Table 1). Increasing the size of the substituent appears to reduce the activity (entries 5–9, Table 1). It is not clear if the basicity of RU-NT-63 or the size of the dimethylamine was responsible for the lack of activity. Based on the X-ray structures of the analogs RU-NT-59, PD00589, and RU-NT-206, the pocket where the substituents at position 7 are pointing is highly hydrophobic which may provide an alternative explanation as to why more polar substituents have lower affinity. We analyzed the commercially available compounds and found that the substitution of the 4-methylene group by an oxygen atom (RU-NT-198, R1 = H) or sulfur (RU-NT-199, R1 = 8-F) was tolerated witĥ5 to 7-fold loss in potency. The activity of these backbone-changed compounds is similar to that of the benzo[b]thieno2–51thiophene RU-NT-201 where both methylenes (position 4X and 5Y, entry 17 Table 1) are replaced by a single sulfur.

RU-NT-93, PD00589, RU-NT-59, and RU-NT-206 displaced the labeled P11 probe from RTA with 7-, 8-, 13- and 32-fold improved IC50 values relative to CC10501, respectively (Fig. 2B–F and Table 2). CC10501 had a calculated Ki value of 58 μM (Fig. 2B and Table 2). RU-NT-93, PD00589, and RU-NT-59 had 7-, 7-, and 15-fold improved Ki values relative to CC10501 respectively (Fig. 2C–F and Table 2). RU-NT-206 had the highest affinity for RTA witĥ60-fold improved Ki of 1 μM (Fig. 2F and Table 2), which was identical to the affinity of the P11 peptide for RTA (Fig. 2A and Table 2).

The IC50 values were also determined by measuring the inhibition of RTA-mediated depurination of rat liver ribosomes by RTA using the qRT-PCR assay.31 The data for the percent inhibition at different compound concentrations were fitted with Michaelis-Menten kinetics using OriginPro (Fig. 3). The IC50 value of CC10501 by the qRT-PCR assay was determined by linear regression analysis because depurination increased linearly and at 500 μM we obtained only 64 % inhibition. The IC50 values for RU-NT-93, PD00589, RU-NT-59, and RU-NT-206 by qRT-PCR improved 9-, 15-, 13-, and 18-fold relative to CC10501, respectively using rat liver ribosomes (Table 2 and Fig. 3A–D). RU-NT-206 showed the greatest potency with an IC50 value of 23 μM by the qRT-PCR assay (Fig. 3D). The IC50 values determined by the FP competition, and the qRT-PCR assay were in similar rank order and were proportional to the Ki values (Table 2), indicating that compounds optimized for binding at the P-stalk pocket more potently inhibit the activity of RTA.

The IC50 values were determined by qRT-PCR using yeast ribosomes as well.31 They were about 3-fold lower than the IC50 values obtained with rat liver ribosomes (Fig. S3) since yeast ribosomes are less sensitive to RTA than mammalian ribosomes.30,34 The previously identified inhibitor, RU-NT-93 had a 10-fold improved IC50 while PD00589 and RU-NT-59 had 15- and 16-fold improved IC50 values of 10 and 9 μM, respectively relative to CC10501 using yeast ribosomes (Fig. S3A–C). The IC50 value for RU-NT-206 showed a 17-fold improvement compared to CC10501 with yeast ribosomes (Fig. S3D). The IC50 values of RU-NT-206 measured by qRT-PCR using yeast and rat liver ribosomes (9 μM and 23 μM, respectively) were in close agreement with the IC50 values of P11 peptide using yeast or rat liver ribosomes (5 μM and 31 μM, respectively).30 The Ki and IC50 values of P11 measured by the FP competition assay were also similar to RU-NT-206 (Table 2). These results demonstrate that RU-NT-206 shows a similar affinity and inhibitory potency against RTA as the 5-fold larger P11 peptide.

2.4. Structure of RTA-inhibitor complexes

RTA was cocrystallized with RU-NT-59, PD00589, and RU-NT-206 and each RTA-inhibitor complex structure was solved by molecular replacement using PHASER (Fig. 4A–C). The RTA-RU-NT-59 complex was determined at 1.9 Å, the RTA-PD00589 structure was solved at 2.3 Å, and the RTA-RU-NT-206 complex was solved at 2.7 Å. Each structure was determined in the hexagonal P6322 space group. The electron density for each inhibitor was well defined in each structure (Fig. 4D–F). The location of RU-NT-59, PD00589, and RU-NT-206 bound within the P-stalk pocket of RTA is very similar (Fig. S4). The thiophene carboxylate in each inhibitor shared a salt-bridge with the side chain of Arg235 along with a hydrogen bond interaction with the main chain amide nitrogen of Arg235 (Fig. 5A, B, D). The cyclohexene and benzene rings in all three compounds formed similar hydrophobic interactions with Tyr183 and Phe240 in RTA. Each benzene ring in RU-NT-59 and PD00589 also hydrophobically contacted Ile247, Leu248, and Ile251 (Fig. 5A, B, D) with the fluoride atom of the fluorobenzene ring of RU-NT-59 additionally contacting Val242 in RTA while also making much closer hydrophobic contacts with Ile247 and Leu248 (Fig. 5C).

Although each inhibitor was similarly bound to the P-stalk pocket, RU-NT-206 was positioned ~1 Å closer to Arg234 and Arg235. Consequently, the thiophene carboxylate in RU-NT-206 formed an additional salt bridge with Arg234 (Fig. 5D). In addition, the dihedral angle between the carboxylate group and the thiophene ring is 43.2° in RU-NT-206. This conformational twist results in reduced conjugation and increased electron density on the carboxylate, which increases the ionic interactions with the positively charged arginines. The benzene ring in RU-NT-206 hydrophobically interacted with Ile247 and Ile251. The methyl group attached to position C5 in the cyclohexadiene ring of RU-NT-206 formed a unique hydrophobic contact with Leu207, a nonpolar residue that lined a hydrophobic cavity within the P-stalk pocket (Fig. 5D). The previously determined crystal structures of the RTA alone (PDB ID: 1RTC), RTA bound to the P11 peptide (PDB ID: 5GU4), or RTA bound to RU-NT-93 (PDB ID: 7MLP) are very similar to the RTA structures reported here. The RMSD range after the superposition of these structures ranged from 0.17 to 0.87 Å, indicating no large conformational changes in the backbone of RTA upon the binding of these inhibitors (Fig. S5).

2.5. Protection against ricin and Stx2a holotoxin in cell-based assays

Structure-based optimization led to inhibitors with submicromolar binding affinity and inhibitory potency against depurination by RTA. To evaluate their cellular protection activity, we used cultured Vero cells to determine if compounds protected cells from depurination by ricin holotoxin. In the presence of 200 pM ricin, cellular ribosome depurination increased linearly for about 4 h. We selected two hours of depurination by ricin to compare the compounds. Two hours after the simultaneous addition of the toxin and the compound, cellular RNA was isolated and used for qRT-PCR to determine the level of depurination. RNA from cells treated with the same buffer but without ricin and compound was used as a negative control to represent full protection (100 % inhibition). RNA from cells treated with ricin but no compound was used as a positive control (0 % inhibition). Cells treated with 250 μM PD00589 resulted in a 61 % inhibition of ribosome depurination and 75 % inhibition at 500 μM (Fig. 6A and Table 2). Cells treated with RU-NT-59 showed 62 % inhibition at 250 μM and 84 % inhibition at 500 μM. The greatest protection was observed with RU-NT-206, exhibiting 87 % inhibition at 250 μM and 95 % at 500 μM. We were able to determine the EC50 value of depurination inhibition for RU-NT-206 by incubation with a titration series up to 500 μM. The half-maximal effective concentration (EC50) of RU-NT-206 for inhibition of depurination by ricin holotoxin in Vero cells was 29 ± 2 μM (Fig. 6B). These results indicate that the improved affinity and in vitro potency are predictive of a protective effect in cells.

Since inhalation is the most efficient exposure route to ricin, the protective activity of the compounds against ricin was determined by challenging human lung epithelial A549 cells as well. Cells treated with 250 μM PD00589 resulted in a 53 % reduction in ribosome depurination and a 69 % reduction at 500 μM (Table 2). Cells treated with RU-NT-59 showed 51 % inhibition at 250 μM and 69 % inhibition at 500 μM. The greatest protection was observed for RU-NT-206 in A549 cells as in Vero cells, with 75 % inhibition at 250 μM and 86 % at 500 μM (Fig. S6A). RU-NT-206 protected A549 cells against ricin holotoxin with an EC50 of 92 ± 14 μM (Fig. S6B). These results demonstrated that protection from ricin holotoxin by RU-NT-206 was not restricted to a specific cell line.

The crystal structure of Stx2a with P11 (PDB ID: 6×6H) showed that as observed with RTA (PDB ID: 5GU4), only the last six residues of P11 peptide bound in a shallow pocket on Stx2a. The P11 binding site on Stx2a was differently located relative to the P11-binding site on RTA.35 Although RTA and Stx2a bind P-stalk peptides in a different manner, both pockets consist of positively charged and hydrophobic residues. Conserved hydrophobic residues (Leu9 and Phe10) and the last Asp (Asp11) of the P11 peptide play a critical role in ribosome binding to each toxin. Asp11 forms salt bridges and H-bonds with arginines at the P-stalk pocket of each toxin.35 Because of these similarities, we examined the effect of RU-NT-206 on the depurination activity of Stx2A1 using rat liver ribosomes. RU-NT-206 protected rat liver ribosomes from depurination by Stx2A1 with an IC50 value of 83 ± 30 μM (Fig. 7A). We determined if RU-NT-206 would protect against Stx2a holotoxin in Vero cells. Two hours after the simultaneous addition of Stx2a and the compound, cellular RNA was isolated and used for qRT-PCR to determine the level of depurination. A comparison of protection by RU-NT-206 against the depurination activity of ricin and Stx2a holotoxins in Vero cells is shown in Fig. 7B. RU-NT-206 showed 9 %, 30 %, and 48 % protection against Stx2a in Vero cells compared to 85 %, 95 % and 98 % protection against ricin at 125, 250 and 500 μM, respectively. Although the level of protection against Stx2a was lower than ricin, it was significantly different from the control at 250 and 500 μM. These results identified RU-NT-206 as a lead small molecule with activity against both toxins.

3. Discussion

Ricin and Stxs have been uniquely challenging drug targets. The catalytic site of ricin and Stx2a has been explored extensively as a potential target for antidotes.36,37 However, few inhibitors have been found, and none with activity in cells or in vivo.38–40 Although retrograde trafficking inhibitors have shown promising results,41 these inhibitors target the trafficking pathways, not the toxin itself, and may be detrimental to the host. Modulation of ribosome interactions by small molecules has not been fully explored as a strategy for the inhibition of ricin or Stx2a. The crystal structure of RTA in complex with the P11 peptide showed that the C-terminal Asp11 within P11 contacts the two positively charged residues Arg234 and Arg235 in RTA forming a salt bridge with Arg235 (Fig. 8A).28,29 Leu9 in P11 makes hydrophobic interactions with several residues within the nonpolar region of the P11 pocket including Phe240, Ile247, and Ile251 while the aromatic side chain of the P11 residue Phe10 forms an offsetting π-stack with RTA’s Tyr183 and Phe240 (Fig. 8A). The crystal structure of RTA in complex with the RTA inhibitor CC10501 (PDB ID: 6URX) revealed that the carboxylate moiety of CC10501 salt-bridged with Arg235 within the P-stalk binding site of RTA as Asp11 in P11 (Fig. 8B). The benzene ring in CC10501, which superpositioned near the Phe10 side chain in P11, established similar π -stacking interactions with RTA’s Tyr183 and Phe240 as Phe10 despite being offset by 56° with the side chain of Phe10 (Fig. 8C and Fig. S1). The benzene ring in CC10501 also made a comparable hydrophobic association with Ile251 as Phe10 did in P11 (Fig. 8B, C).

The best inhibitor identified in our prior study,32 RU-NT-93, is very similar chemically to CC10501 with the exception that RU-NT-93 contains an m-xylene ring instead of the benzene ring in CC10501 (Table 2) and binds with a rotated thiophene ring and carboxylate relative to CC10501 (Fig. S1). The m-Xylene ring in RU-NT-93 is positioned closer to the surface of the P stalk binding pocket relative to the benzene ring in CC10501 (Fig. 9A). Consequently, the methyl groups on the 2,6-dimethylphenyl ring of RU-NT-93 uniquely overlap with the space occupied by P11 residues Phe10 and Leu9 (Fig. 9B). The carboxylic acid of RU-NT-93 occupies the same space as the Asp11 side chain acid (Fig. 9B). CC10501 and RU-NT-93 bind the P11 pocket similarly (Fig. 9C). RU-NT-93 more completely fills the pocket compared to CC10501, indicating why RU-NT-93 is a more effective RTA inhibitor relative to CC10501 (Fig. 9C). The tighter binding of RU-NT-93 to RTA compared to CC10501 correlated well with the 7–9-fold higher RTA inhibition (Table 2) and over 10-fold greater protection of Vero cells against ricin holotoxin compared to CC10501.32

Here, we developed a new fluorescence polarization competition assay to examine the structure–activity relationships (SAR) of a new series of compounds that bind at the P-stalk pocket of RTA and demonstrated that carboxylic acid is critical for activity. To determine if the different orientation of the thiophene carboxylate in RU-NT-93 is important for inhibition, we designed planar compounds with different substituents where the molecule is locked in a fixed conformation. We identified RU-NT-59, PD00589, and RU-NT-206 with the highest affinity and potency and solved the X-ray crystal structures of each compound independently bound to RTA by co-crystallization. The structure of RTA in complex with each inhibitor revealed that all three compounds bind similarly to the P-stalk binding pocket of RTA as CC10501 but reach deeper into the pocket and establish more favorable contacts that define a new mode of interaction. RU-NT-59 and RU-NT-206 showed higher binding affinity for RTA than the leading compound, RU-NT-93, and more potent IC50 (Table 2), indicating that these inhibitors, especially RU-NT-206, bind residues in the P-stalk pocket in a way that results in more effective inhibition of RTA. The P-stalk binding pocket is quite distant from the active site of RTA at ~20 Å away (Fig. 4A–C), suggesting that the inhibitory effect of these compounds is due to the preclusion of RTA interaction with the P-stalk. The crystal structures showed that RU-NT-59 and PD00589 bind the P-stalk pocket and form most of the same contacts as RU-NT-93, including hydrophobic connections with Tyr183, Phe240, and a salt-bridge with the side chain of Arg235 (Fig. 5A, B). The cyclohexene and thiophene rings in RU-NT-59 and PD00589 occupy the same pocket space as the P11 residues Phe10 and Asp11, respectively, effectively competing with these P-stalk residues for binding to RTA as is reflected in the fluorescence polarization experiments (Fig. 2). The benzene rings from RU-NT-59 and PD00589 overlap with P11’s Leu9 upon superposition (Fig. 10A), and hydrophobically interact with RTA residues Ile247, Leu248, and Ile251 (Fig. 5 A, B). The fluoride atom within the fluorobenzene ring of RU-NT-59 contacts Val242 in RTA and forms much closer contacts with Ile247 and Leu248 (Fig. 5C) relative to PD00589, conceivably resulting in the slightly higher affinity of RU-NT-59.

RU-NT-206 binds the P-stalk pocket forming most of the same contacts as RU-NT-59 and PD00589 including hydrophobic connections with Tyr183, Phe240, Ile247, Ile251, and a stronger salt-bridge with the side chain of Arg235 (Fig. 5A, B, D). The similar pose of RU-NT-206 to RU-NT-59 and PD00589 within the P-stalk pocket equivalently placed the cyclohexene and thiophene rings in RU-NT-206 where they superposed onto P11 residues Phe10 and Asp11, respectively (Fig. 10A). The RU-NT-206 benzene ring likewise overlapped with P11’s Leu9 upon superposition (Fig. 9C and Fig. 10A). RU-NT-206 had the highest binding affinity for RTA and considerably stronger IC50 value compared to the other compounds in Table 1 either by measuring the displacement of the labeled P11 peptide from RTA by FP or by measuring the inhibition of depurination of rat liver ribosomes by RTA in vitro by qRT-PCR (Table 2). Part of the higher affinity of RU-NT-206 may be due to the near perpendicular arrangement of the carboxylate, which leads to stronger ionic interactions with arginines. Although RU-NT-206 is bound similarly to the P-stalk pocket as RU-NT-59 and PD00589, it formed an additional salt-bridge with Arg234. RU-NT-206 established nonpolar contacts with Leu207, which lines a hydrophobic cavity within the P-stalk pocket along with Leu214, Leu232, and Ile251 (Fig. 10B). Consequently, the proximity of the methyl groups on RU-NT-206 to Ser203 and Gln233 within this mostly hydrophobic cavity may be responsible for more optimal affinity and higher potency. The added contacts of RU-NT-206 within the binding site conceivably contribute to the improved binding affinity of RU-NT-206 compared to RU-NT-59 and PD00589 giving RU-NT-206 the best capacity to block P-stalk binding of RTA in vitro and in cells. The binding affinity of the three improved compounds correlated with the in vitro potency measured by two independent assays and potency in cell-based assays, indicating similar thermodynamic and biological functions. Our results demonstrate that as the affinity for RTA is improved inhibitory potency in vitro and in cells is also improved, suggesting that compounds optimized for binding to the P-stalk pocket of RTA will more potently inhibit ricin. A lead compound RU-NT-206 (MW 258 Da) showed identical affinity and similar inhibitory potency against RTA as a five-fold larger P11 peptide (MW 1218 Da) and cell protection against Stx2a holotoxin for the first time. We previously solved the X-ray crystal structure of Stx2a with P11 and showed that Stx2a binds the P-stalk CTD at a different location compared to RTA.35 Stx2a has a different binding mode and distinct requirements for the P-stalk CTD compared to RTA. The lower potency of RU-NT-206 against Stx2a compared to ricin may be due to the differences in the structure of the P-stalk binding pockets of RTA and Stx2A1. RU-NT-206 appears to be a promising candidate for further optimization. The FP competition assay will be a potent tool for further optimization of RU-NT-206 and holds great potential in serving as a rapid and robust method for efficient drug screening. Small molecule inhibitors of ricin-P-stalk interactions provide “first-in-class” compounds with potential for therapeutic intervention.

4. Conclusions

Ribosome binding sites of ricin or Shiga toxin have not been targeted by small molecules. We previously identified CC10501, which binds at the P-stalk pocket of RTA and inhibits activity. Here, we established a new fluorescence polarization assay and demonstrated that compounds, which bind at the P-stalk pocket of RTA with higher affinity cause more potent inhibition of the catalytic activity of ricin. Structure-activity relationships established that the presence of the carboxylic acid is essential for improved affinity for the P-stalk pocket of RTA. A lead compound, RU-NT-206, bound at the P-stalk pocket in a unique binding mode with similar affinity against RTA as a five-fold larger P-protein peptide and protected cells against ricin and Shiga toxin 2 for the first time. These studies validate the ribosome-binding site of ricin as a critical target for allosteric inhibition of the catalytic site.

5. Experimental section

5.1. Chemistry

The reagents were purchased and used without additional purification. LC-MS was performed on an Agilent 1100 system with a Waters Micromass ZQ spectrometer using a 5 μL injection on an XBridge C18 (3.5 μM, 4.6 × 50 mm) column at a temperature of 40 °C with a 4 min gradient from 5 % A to 95 % B (neutral method: solvent A: 10 mM ammonium formate in water, solvent B: acetonitrile; acidic method: solvent A: 0.1 % v/v formic acid in water, solvent B: acetonitrile;) at a flow rate of 2 mL/min. The detection used a diode array scanning from 190 to 600 nm or dual-wavelength detectors at 220 and 254 nm (mass detection cone voltage: 30 V). Alternatively, LC-MS and HPLC analyses were performed using the Shimadzu LCMS-2020 system with Dual Ionization Source (acidic method: solvent A: 0.1 % v/v trifluoroacetic acid in water, solvent B: acetonitrile, column at a temperature of 40 °C with a 4 min gradient from 5 % A to 95 % B).

NMR was obtained on a Varian VNMRS 300 MHz, Bruker Avance Neo 400 MHz, Varian VNMRS 500 MHz, or Bruker Avance Neo 500 MHz in DMSO-d6 (1H: δ2.50). Flash column chromatography purifications were performed using Biotage Isolera and Selekt systems. Preparative HPLC was performed on ACCQPrep HP125 system, (Waters XBridge BEH C18 column, 100 × 30 mm × 10 μm; mobile phase: A: 0.1 % v/v formic acid in water; B: acetonitrile; 10 % to 100 %, 14 min). The HRMS analyses were performed using Bruker Apex 7 T FTMS, using an ESI ion source in positive mode. Alternatively, experiments were performed using a Xevo G2-XS QTof mass spectrometer equipped with an Acquity UPLC system. The UPLC-MS system and the column were from Waters Inc. For the LC separation, the following two eluents were used: A containing 0.1 % v/v formic acid in water, and B containing neat acetonitrile. Linear gradient (5–100 % B in 3 min) was applied. The ionization method was ESI, generating [M−H]+ ions. The mass spectrometer was calibrated using a Leu-enkephalin standard. Capillary exit voltage 330 V. For further HRMS analyses, an Agilent 6546 qTOF mass spectrometer equipped with an Agilent Infinity 1290 UPLC was used. For the latter UPLC the following two eluents were used: A containing 0.1 % v/v formic acid in water, and B containing 0.1 % v/v formic acid in acetonitrile. Linear gradient (5–100 % B in 4 min) was applied. ESI ionization generated M+, [M+H]+, [M+Na]+ or [M−H]− ions as noted in HRMS tables. The mass spectrometer was calibrated using standard supplied by Agilent Inc. For the UPLC systems a 2.1 mm × 50 mm BEH C18 column (particle size 1.7 μm) was utilized. Unless otherwise stated, the purities of the final compounds were equal to or greater than 95 % by HPLC analysis. The purities were confirmed with 1H NMR to look for residual solvents or non-UV active impurities.

5.1.1. 1-Chloro-6-fluoro-3,4-dihydronaphthalene-2-carbaldehyde (2a)

DMF (0.62 mL, 8 mmol, 8 equiv.) was slowly added to a well-stirred cooled solution of POCl3 (0.66 mL, 7 mmol, 7 equiv.) at 0 °C, as soon as the reaction mixture precipitated, 6-fluoro-3,4-dihydronaphthalen-1 (2H)-one (164 mg, 1 mmol) was diluted in a small amount of DMF and was added to the reaction mixture. Afterward, the reaction mixture was warmed to room temperature and then heated to 70 °C for 2 h. After cooling to room temperature, the mixture was diluted with ethyl acetate and poured into crushed ice. The aqueous layer was extracted with ethyl acetate. The combined organic layers were successively washed with sodium bicarbonate, and brine and dried over sodium sulfate, filtered, and concentrated. The residue was used in the next step without further purification. ESI-MS: 211.0 (M+H)+.

5.1.2. 6-Bromo-1-chloro-3,4-dihydronaphthalene-2-carbaldehyde (2b)

The compound has been prepared according to the procedure for 2a. ESI-MS: 270.8 (M+H)+.

5.1.3. Methyl 7-fluoro-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate (3a)

To a stirred solution of 1-chloro-6-fluoro-3,4-dihydronaphthalene-2-carbaldehyde (210 mg, 1 mmol, 1 equiv.) in pyridine (2 mL) methyl 2-mercaptoacetate (0.103 mL, 1.15 mmol, 1.15 equiv.) and triethylamine (0.223 mL, 1.6 mmol, 1.6 equiv.) were added. The mixture was heated at 60 °C for 2 h and then left to cool down to RT. An aqueous solution of 50 % (w/w) KOH (0.165 mL) was added and the mixture was stirred for another 20 min. The medium was poured over ice and rinsed with dichloromethane before adding dropwise a solution of HCl (0.5 mL, 1 M). The organic layer was extracted and washed with 1 M HCl and water, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography to give the product (189 mg, 72 %, yellow solid). ESI-MS: 263.0 (M+H)+.

5.1.4. Methyl 7-bromo-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate (3b)

The compound has been prepared according to the procedure for 3a. ESI-MS: 322.8 (M+H)+.

5.1.5. Methyl 7-methyl-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate (3c)

A mixture of methyl 7-bromo-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate (3b, 65 mg, 0.2 mmol), methylboronic acid (36 mg, 0.6 mmol, 3 equiv.), palladium acetate (2 mg, 0.008 mmol, 0.04 equiv.), tricyclohexylphosphine (PCy3, 5 mg, 0.018 mmol, 0.09 equiv.), and K3PO4 (142 mg, 0.664 mmol, 3.3 equiv.) was dissolved in toluene (10 mL) and water (1 mL). The mixture was flushed with nitrogen for 3 min and then refluxed under nitrogen overnight. After cooling to room temperature, the reaction mixture was partitioned between water and ethyl acetate. The organic layer was washed with water and brine and dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (hexane:ethyl acetate 0 % → 15 %) to give the corresponding ester (38 mg, 75 % yield). ESI-MS: 258.9 (M+H)+.

5.1.6. Methyl 7-(dimethylamino)-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate (3d)

A mixture of methyl 7-bromo-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate (3b, 44 mg, 0.136 mmol), dimethylamine (2.0 M in THF, 0.2 mL, 0.41 mmol, 3 equiv.), palladium acetate (3 mg, 0.0136 mmol, 0.1 equiv.), BINAP (8.5 mg, 0.0136 mmol, 0.1 equiv.), and cesium carbonate (134 mg, 0.41 mmol, 3 equiv.) were dissolved in dioxane (5 mL). The mixture was flushed with nitrogen for 3 min and then refluxed under nitrogen overnight. After cooling to room temperature, the reaction mixture was partitioned between water and ethyl acetate. The organic layer was washed with water and brine and dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (hexane:ethyl acetate 0 % → 15 %) to give the corresponding ester (31 mg, 80 % yield). ESI-MS: 287.9 (M+H)+.

5.1.7. Methyl 7-methoxy-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate (3e)

A mixture of methyl 7-bromo-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylate (3b, 50 mg, 0.155 mmol), [(2-di-cyclohexylphosphino-3,6-dimethoxy-2′,4′,6′-triisopropyl-1,1′-biphenyl)-2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate (BrettPhosPdG3, 3 mg, 0.0031 mmol, 0.02 equiv.), and cesium carbonate (71 mg, 0.21 mmol, 1.4 equiv.) was dissolved in dioxane (2 mL) and methanol (2 mL). The mixture was flushed with nitrogen for 5 min and then refluxed under nitrogen for an hour. After cooling to room temperature, the reaction mixture was concentrated, and the residue was partitioned between water and ethyl acetate. The organic layer was washed with water and brine and dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography (hexane:ethyl acetate 0 % → 15 %) to give the corresponding ester (17 mg, 40 % yield). ESI-MS: 275.4 (M+H)+.

5.1.8. 5-Phenylthiophene-2-carboxylic acid (CC10501)

The compound (white solid) was purchased from Maybridge (part of Thermo Scientific since 2021) and used as is. 1H NMR: (500 MHz, DMSO-d6) δ 7.77–7.68 (m, 3H), 7.58 (d, J = 3.9 Hz, 1H), 7.50–7.43 (m, 2H), 7.43–7.38 (m, 1H). HRMS: C11H7O2S−, [M−H]−calc. 203.01722, found 203.0167, err. −2.6 ppm.

5.1.9. 4,5-Dihydronaphtho[1,2-b]thiophene-2-carboxylic acid (PD00589)

The compound (white solid) was purchased from Maybridge (part of Thermo Scientific since 2021) and used as is. 1H NMR: (300 MHz, DMSO-d6) δ 13.04 (s, 1H), 7.59 (s, 1H), 7.46–7.39 (m, 1H), 7.33–7.23 (m, 3H), 2.92 (dd, J = 9.4, 6.6 Hz, 2H), 2.84–2.75 (m, 2H). HRMS: [M+H]+ calc. 231.0480, found 231.0502.

5.1.10. 4,5-Dihydronaphtho[1,2-b]thiophene-2-carbohydrazide (PD00633)

The compound was purchased from Maybridge as a 100 mM solution in DMSO (part of Thermo Scientific since 2021) and used as is.

5.1.11. 8-Chloro-4H-thieno[3,2-c]thiochromene-2-carbohydrazide (SEW01689)

The compound was purchased from Maybridge as a 100 mM solution in DMSO (part of Thermo Scientific since 2021) and used as is.

5.1.12. Methyl 8-fluoro-4H-thieno[3,2-c]chromene-2-carboxylate (SEW01765)

The compound was purchased from Maybridge as a 100 mM solution in DMSO (part of Thermo Scientific since 2021) and used as is.

5.1.13. 1-(8-Chloro-4H-thieno[3,2-c]thiochromen-2-yl)ethan-1-one (SEW02679)

The compound was purchased from Maybridge as a 100 mM solution in DMSO (part of Thermo Scientific since 2021) and used as is.

5.1.14. 8-Chloro-4H-thieno[3,2-c]thiochromene-2-carboxylic acid (SEW01776)

The compound (bright yellow solid) was purchased from Maybridge (part of Thermo Scientific since 2021) and used as is. The purity of the sample was 85 %. 1H NMR: (500 MHz, DMSO-d6) δ 7.61–7.56 (m, 2H), 7.43 (m, 1H), 7.31 (m, 1H), 4.07 (d, J = 1.0 Hz, 2H). HRMS: C12H6ClO2S2−, [M−H]− calc. 280.95032, found 280.9513, err. 3.5 ppm.

5.1.15. 7-Fluoro-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylic acid (RU-NT-59)

To a stirred solution of methyl 7-fluoro-4,5-dihydronaphtho[1,2-b] thiophene-2-carboxylate (3a, 50 mg, 0.19 mmol, 1 equiv.) in MeOH (3 mL) and THF (3 mL) was added 1 M NaOH (4 mL). The mixture was stirred at room temperature for 2 h. After completion of the reaction by LC-MS, the reaction mixture was partitioned between 1 M HCl and ethyl acetate. The organic layer was washed with water, and brine and dried over anhydrous Na2SO4, filtered, and concentrated to afford the product as a pale-yellow solid (42 mg, 90 %). Subsequently, the product was additionally purified by prep-HPLC (ACCQPrep, C18, liquid injection, (water/formic acid 0.1 %)/CH3CN, 10 % → 100 %). Fractions, that contained the product (by LCMS), were combined to give the desired product as a white solid. 1H NMR: (500 MHz, DMSO-d6) δ 7.58 (s, 1H), 7.47 (dd, J = 8.5, 5.6 Hz, 1H), 7.20 (dd, J = 9.6, 2.7 Hz, 1H), 7.10 (td, J = 8.7, 2.8 Hz, 1H), 2.93 (dd, J = 8.8, 6.6 Hz, 2H), 2.79 (dd, J = 8.9, 6.6 Hz, 2H). 13C NMR: (126 MHz, DMSO-d6) δ 163.05, 162.76, 160.81, 140.43, 138.31, 138.25, 137.67, 133.47, 126.79, 125.25, 115.52, 113.88, 28.16, 22.75. ESI-MS: 247.2 (M–H)−. HRMS: C13H10FO2S+,[M+H]+ calc. 249.0386, found 249.0385.

5.1.16. 7-Bromo-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylic acid (RU-NT-61)

The compound (white solid) has been prepared from 3b according to the procedure for RU-NT-59. The purity of the sample was 92 %. 1H NMR: (500 MHz, DMSO-d6) δ 7.61 (s, 1H), 7.56 (dd, J = 2.1, 1.0 Hz, 1H), 7.46 (dd, J = 8.2, 2.1 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 2.93 (dd, J = 8.9, 6.5 Hz, 2H), 2.80 (dd, J = 8.9, 6.6 Hz, 2H). 13C NMR: (126 MHz, DMSO-d6) δ 162.74, 138.43, 137.68, 133.53, 130.88, 129.85, 129.11, 124.98, 27.51, 22.62. HRMS: C13H879BrO2S−[M−H]− calc. 306.94339, found 306.9436, err. 1.7 ppm.

5.1.17. 7-Methyl-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylic acid (RU-NT-62)

The compound (beige solid) has been prepared from 3c according to the procedure for RU-NT-59. 1H NMR: (500 MHz, DMSO-d6) δ 1H NMR (500 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.56 (s, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.14–7.05 (m, 2H), 2.87 (dd, J = 8.8, 6.4 Hz, 2H), 2.81–2.74 (m, 2H), 2.29 (s, 3H). 13C NMR: (101 MHz, DMSO-d6) δ 163.06, 137.84, 137.35, 135.13, 133.35, 128.99, 127.70, 127.44, 123.17, 28.09, 23.10, 20.90. HRMS: C14H11O2S− [M−H]− calc. 243.04852, found 243.0494, err. 3.6 ppm.

5.1.18. 7-(Dimethylamino)-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylic acid (RU-NT-63)

The compound (neon green solid) has been prepared from 3d according to the procedure for RU-NT-59. The purity of the sample was 92 %. 1H NMR: (500 MHz, DMSO-d6) δ 8.27 (s, 1H), 7.33 (s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 6.64 (s, 1H), 6.59 (d, J = 8.4 Hz, 1H), 2.93 (d, J = 2.3 Hz, 6H), 2.83 (d, J = 7.7 Hz, 2H), 2.71 (d, J = 7.7 Hz, 2H). HRMS: C15H15NO2S+, [M]+ calc. 273.08180, found 273.0816, err. −0.7 ppm.

5.1.19. 7-Methoxy-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylic acid (RU-NT-64)

The compound (yellow solid) has been prepared from 3e according to the procedure for RU-NT-59. The purity of the sample was 90 %. 1H NMR: (400 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.55 (s, 1H), 7.45–7.33 (m, 1H), 6.91 (d, J = 2.6 Hz, 1H), 6.83 (dd, J = 8.4, 2.6 Hz, 1H), 3.78 (s, 3H), 2.89 (dd, J = 9.0, 6.4 Hz, 3H), 2.84–2.72 (m, 2H). 13C NMR: (101 MHz, DMSO-d6) δ 163.11, 159.41, 141.83, 137.19, 136.25, 133.51, 124.64, 114.00, 112.56, 55.22, 28.47, 22.97. HRMS: C14H11O3S−, [M−H]−calc. 259.04344, found 259.0439, err. 1.8 ppm.

5.1.20. 5-(o-Tolyl)thiophene-2-carboxylic acid (RU-NT-70)

The synthesis and analysis of this compound have been previously reported.32

5.1.21. 5-Mesitylthiophene-2-carboxylic acid (RU-NT-75)

The synthesis and analysis of this compound have been previously reported.32

5.1.22. 5-(2,6-Dimethylphenyl)thiophene-2-carboxylic acid (RU-NT-93)

The synthesis and analysis of this compound have been previously reported.32

5.1.23. 4H-Thieno[3,2-c]chromene-2-carboxylic acid (RU-NT-198)

The compound (white solid) was purchased from Enamine Ltd and used as is. 1H NMR: (500 MHz, DMSO-d6) δ 7.60 (s, 1H), 7.26 (ddd, J = 8.2, 7.4, 1.6 Hz, 1H), 7.05–6.94 (m, 2H), 5.28 (s, 2H). HRMS: C12H7O3S−, [M−H] − calc. 231.01214, found 231.0110, err. −4.9 ppm.

5.1.24. 8-Fluoro-4H-thieno[3,2-c]thiochromene-2-carboxylic acid (RU-NT-199)

The compound (beige solid) was purchased from Enamine Ltd and used as is. 1H NMR: (500 MHz, DMSO-d6) δ 13.32 (br s, 1H), 7.66 (s, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.18–7.11 (m, 1H), 4.06 (s, 2H). 13C NMR: (101 MHz, DMSO-d6) δ 162.54, 161.82, 159.40, 140.53, 135.51, 133.70, 133.28, 133.10, 132.82, 130.80, 129.94, 129.42, 126.99, 124.98, 116.05, 111.82, 67.57, 24.98. HRMS:C11H5O2S2−, [M−H]− calc. 264.97987, found 264.9797, err. −0.6 ppm.

5.1.25. Benzo[b]thieno[2,3-d]thiophene-2-carboxylic acid (RU-NT-201)

The compound was purchased from Enamine Ltd. Subsequently, the compound was additionally purified by prep-HPLC to achieve the desired purity (ACCQPrep, C18, liquid injection, (water/formic acid 0.1 %)/CH3CN, 10 % → 100 %). Fractions, that contained the product (by LCMS), were combined to give the desired product as a yellow solid. 1H NMR: (500 MHz, DMSO-d6) δ 8.17 (s, 1H), 8.14–8.04 (m, 2H), 7.54–7.45 (m, 2H). HRMS:C12H6FO2S2−, [M−H]− calc. 232.97364, found 232.9732, err. −1.9 ppm.

5.1.26. 5,5-Dimethyl-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylic acid (RU-NT-206)

The compound was purchased from Enamine Ltd. Subsequently, the compound was additionally purified by prep-HPLC to achieve the desired purity (ACCQPrep, C18, liquid injection, (water/formic acid 0.1 %/CH3CN), 10 % → 100 %). Fractions, that contained the product (by LCMS), were combined to give the desired product as a white solid. 1H NMR: (500 MHz, DMSO-d6) δ 7.60 (s, 1H), 7.45 (ddd, J = 7.6, 2.8, 1.4 Hz, 2H), 7.33 (td, J = 7.5, 1.4 Hz, 1H), 7.27 (td, J = 7.5, 1.4 Hz, 1H), 2.74 (s, 2H), 1.24 (s, 6H). 13C NMR: (101 MHz, DMSO) δ 162.97, 143.53, 141.16, 136.78, 134.13, 131.37, 129.06, 128.63, 126.98, 124.82, 123.78, 38.09, 34.80, 28.09. HRMS: [M+H]+ calc. 259.0793, found 259.0817.

5.1.27. 5,5-Dimethyl-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxamide (RU-NT-253)

5,5-Dimethyl-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxylic acid (RU-NT-206, 125 mg, 0.42 mmol, 1 equiv.), ammonium chloride (23 mg, 0.42 mmol, 1 equiv.), HATU (194 mg, 0.51 mmol, 1.2 equiv.), diisopropylethylamine (0.15 mL, 0.85 mmol, 2 equiv.) were dissolved in DMF (1.6 mL) in a vial. The reaction mixture was flushed with nitrogen for 5 min. The reaction was left stirring in the heating block (50 °C) and reaction progress was monitored by LC-MS. After the completion (the next day) reaction mixture was concentrated using Biotage V-10. Subsequently, the crude mixture was purified by prep-HPLC to achieve the desired purity (ACCQPrep, C18, liquid injection, (water/formic acid 0.1 %)/CH3CN, 10 % → 100 %). Fractions, that contained the product (by LCMS), were combined to give the desired product as a white solid (71 mg, 65 %). 1H NMR: (500 MHz, DMSO-d6) δ 7.60 (s, 1H), 7.45–7.38 (m, 2H), 7.29 (td, J = 7.5, 1.5 Hz, 1H), 7.25 (td, J = 7.5, 1.5 Hz, 1H), 2.70 (s, 2H), 1.24 (s, 6H). 13C NMR: (126 MHz, DMSO-d6) δ 162.94, 143.16, 138.97, 137.49, 136.51, 129.66, 128.96, 128.58, 126.96, 124.77, 123.57, 38.35, 34.89, 28.15. HRMS: C15H16NOS+, [M+H]+ calc. 258.09471, found 258.0957, err. 3.8 ppm.

5.1.28. 5,5-Dimethyl-4,5-dihydronaphtho[1,2-b]thiophene-2-carbonitrile (RU-NT-254)

5,5-Dimethyl-4,5-dihydronaphtho[1,2-b]thiophene-2-carboxamide (RU-NT-253, 60 mg, 0.23 mmol, 1 equiv.) was dissolved in dichloromethane (1.75 mL) and methyl N-(triethylammoniumsulfonyl)carbamate (Burgess Reagent, 71 mg, 0.28 mmol, 1.2 equiv.) was added to the mixture. The reaction mixture was flushed with nitrogen for 5 min. The reaction was left stirring at room temperature and reaction progress was monitored by LC-MS. After the completion (the next day) reaction mixture was concentrated using Biotage V-10. Subsequently, the crude mixture was purified by prep-HPLC to achieve the desired purity (ACCQPrep, C18, liquid injection, (water/formic acid 0.1 %)/CH3CN, 10 % → 100 %). Fractions, that contained the product (by LCMS), were combined to give the desired product as a colourless oil (41 mg, 73 %).1H NMR: (400 MHz, DMSO-d6) δ 7.85 (s, 1H), 7.47 (dt, J = 7.6, 1.3 Hz, 2H), 7.38 (td, J = 7.5, 1.5 Hz, 1H), 7.29 (td, J = 7.4, 1.3 Hz, 1H), 2.76 (s, 2H), 1.24 (s, 6H). 13C NMR: (101 MHz, DMSO-d6) δ 144.24, 142.81, 140.02, 137.14, 130.35, 128.12, 127.63, 125.48, 124.63, 115.26, 105.19, 38.33, 35.27, 28.47. HRMS: C15H14NS+, [M+H]+ calc. 240.08415, found 240.0851, err. 4.0 ppm.

5.2. The fluorescence polarization (FP) assay

We developed a fluorescence polarization (FP) competition assay that detects competition of the fluorescently labeled P11 peptide with small molecule inhibitors to determine the binding affinity between the small molecule inhibitors and RTA.42 P11 was selected for the initial experiments based on the highest binding affinity (KD) for RTA.30 P11 was labeled with the BODIPY™ TMR-X NHS ester dye43 (ThermoFisher) at the N-terminus and separated from a free dye by semi-preparative HPLC (ACCQPrep, Gemini 30 × 150 mm C-18 column,42.5 mL/min flow rate, eluent A: 0.1 % TFA in water, eluent B 0.1 % TFA in acetonitrile; gradient: B 5 to 100 % in 40 min). The labeling of the peptide was confirmed by high resolution mass spectrometry (HRMS) analysis using an Agilent 6546 qTOF LC/MS system. The HRMS analysis of the unlabeled and BODIPY TMR-X labeled P11 are shown in Figs. S7 and S8, respectively. The HRMS value calculated for MNa+ BODIPY TMR-X labeled P11 was 1733.67927 m/z, we found 1733.6787 m/z, error: −0.33 ppm (Fig. S8). To determine the optimal concentration of the fluorescent P11 for the FP assay, we measured the fluorescence polarization of the labeled P11 as a function of its concentration. To determine the optimal concentration of RTA, binding of the fluorescently labeled P11 to purified recombinant RTA was analyzed by keeping the peptide concentration fixed at 1 μM and varying the concentration of RTA for 30 min at room temperature. The fluorescence polarization values, and the anisotropy values were calculated using the parallel and perpendicular intensities of the emitted fluorescence as shown in equations (1)–(3).

(1) FP=F∥−F⊥F∥+F⊥

(2) A=F∥−F⊥F∥+2F⊥

(3) A=2FP3−FP

The FP values obtained using equation (1) were multiplied by 1000 and expressed in millipolarization (mP) units. For quantitative analysis, we have used anisotropy values because they are additive in nature and the linear superposition principle is not valid for FP values.44,45 In a mixture of more than two interacting species, receptor protein RTA and the free and bound labeled P11 peptide, both in case of direct binding as well as competition experiments, the fraction bound (Fb) is related to the observed anisotropy values (Aobs) at a given concentration of RTA as shown in equation (4).

(4) Fb=Aobs−Afree(Abound−Aobs)Q+(Aobs−Afree)

Afree and Abound denote anisotropy values corresponding to the free and bound states of the labeled P11 peptide. The quantum yield of the fluorophore (Q) is calculated as the ratio of the total fluorescence intensity (F ‖ + 2F ⊥) of the bound and the free states of the labeled P11 peptide.46 The Fb data obtained is plotted against varying concentrations of RTA and the non-linear quadratic equation (5) is used to derive the binding constant (KD) using OriginPro (OriginLab Corporations, USA).

(5) Fb=(KD+Lt+Rt)−(KD+Lt+Rt)2−4LtRt2Lt

Lt and Rt are the total concentration of the ligand (labeled P11 peptide) and the concentration of RTA protein. In our experiments, Lt was kept constant at 1 μM and Rt varied from 0 to 40 μM. The measured KD value for binding between RTA and labeled P11 peptide is 1 ± 0.2 μM (Fig. S2).

5.2.1. Measurement of Ki values by the FP assay

To determine if small molecules can displace the fluorescently labeled P11 peptide, varying concentrations of small molecule inhibitors (5, 10, 20, 40, 80, 125, 160, 250, 500, and 1000 μM) were incubated with 3 μM of RTA and 1 μM labeled P11 in reaction buffer containing 25 mM Tris-Cl pH 8.0, 100 mM NaCl and 1 % DMSO in a black 96-well plate (Corning #3993).47 The reaction volume was made up to 40 μL along with two control sets: one with 3 μM of RTA and 1 μM of labeled P11 corresponding to the bound state dataset and another with only 1 μM of labeled P11 corresponding to the free state observables. The microplate was centrifuged at 400 × g for 3 min and was incubated in the dark for 30 min at room temperature followed by centrifugation again before scanning. After half an hour, the samples were scanned using a BioTek Synergy 4 microplate reader with an Excitation filter of 530/25 nm and an Emission filter of 590/35 nm. The experiments were repeated four times as shown in Fig. 2. The dynamic range of the assay, i.e., the difference between the Polarization values between labeled P11 peptide alone and labeled P11 peptide bound with RTA is >100 millipolarization (mP) units for all the subsequent experiments. The normalized inhibition (%) was calculated using equation (6) where Aobs are the anisotropy values obtained by varying the concentration of the competitor, Abound are the anisotropy values corresponding to the bound state dataset having RTA and labeled P11 incubated together and Afree are the anisotropy values corresponding to the free state dataset having labeled P11 only.48 (6) NormalizedBinding(%)=Abound−Aobs(Abound−Aobs)Q+(Aobs−Afree)×100

The IC50 value is the concentration of the inhibitor required to replace 50 % of the labeled P11 probe and is determined by plotting the normalized binding (%) against the concentration of the inhibitor using equation (7).49 (7) Aobs=Afree+Abound−Afree1+10(Logx-LogIC50)

(8) IC50=F0×KD(1−F0)•(2−F0)+F0×Lt2Ki(2−F0)Kd×F0+1

(9) Ki=IC50F0•KD(1−F0)(2−F0)+F0•Lt2−1KD.F02−F0

For competition-based FP assays, RTA concentration must be chosen such that the fraction of labeled P11 bound over total (F0) is between 0.5 and 0.8.50 In our experiments, RTA concentration (Rt) is kept constant at 3 μM, labeled P11 (Lt) is 1 μM and KD value is 1 μM. Substituting these values in equation (5), the F0 value is 0.7, which is in accordance with these criteria. The inhibitory constant, Ki, describes the binding affinity between the inhibitor and RTA and is defined as the concentration of the inhibitor that will bind to half of the binding sites on RTA at equilibrium in the absence of labeled P11.33 The Ki value for each compound was calculated by solving equation (8) for Ki using equation (9) where F0 is 0.7, KD is 1 μM, Lt is 1 μM and the IC50 values are obtained from equation (7).

5.3. Depurination assay in mammalian cells

Both Vero and A549 cells were used in this study. The cells were maintained in Dulbecco’s modified Eagle medium (DMEM) with penicillin, streptomycin, and 10 % fetal calf serum supplements and were incubated at 37 °C, 5 % CO2. Cells were prepared in the medium at 1.5 × 105 /mL in 24-well tissue culture plates at 500 μL per well and grown for 24 h. DMEM with penicillin and streptomycin minus serum was prepared with each toxin in volume adequate for the experiment. Ricin holotoxin was added to a final concentration of 200 pM and Stx2a was added to a final concentration of 2 nM. Aliquots of the medium containing the toxin were distributed to Eppendorf tubes and compounds in 100 % DMSO stocks were added at the designated final compound concentrations for each treatment and vortexed to dissolve well. The final concentration of DMSO in the controls and the treatments was 0.5 %. The medium containing serum was removed from each well of the 24-well culture plate and replaced with 400 μL of the compound/toxin medium lacking serum. The plate was incubated for 2 h prior to harvest. The medium was then removed, and cells were collected in 350 μL of the lysis buffer from the Qiagen RNeasy® Plus Mini kit (Qiagen). Total RNA was extracted from the cells using the Qiagen kit either immediately or after storage at −80 °C. The High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used for cDNA conversion of ~375 ng of total RNA in a 20 μL reaction.

5.4. Depurination inhibition by qRT-PCR

All qRT-PCR assays were performed with a StepOnePlus Real Time PCR System (Applied Biosystems). Optimized reactions were done in triplicate in a total volume of 20 μL using 5 μL of cDNA diluted 50-fold from the RT reaction using Power SYBR Green Master (Applied Biosystems). Forward and reverse primers used were as follows: 28S rRNA, 5′-GATGTCGGCTCTTCCTATCATTGT-3′ and 5′-CCAGCTCACGTTCCCTATTAGTC-3′; Depurinated rRNA, 5′-TGCCATGGTAATCCTGCTCAGTA-3′ and 5′-TCTGAACCTGCGGTTCCACA-3′. Primer concentrations in the final reaction volume were all at 250 nM. The comparative CT method (ΔΔCT) was used for quantification where depurinated rRNA is normalized to a total of 28S rRNA. Normalized amounts of depurinated rRNA from the experimental samples were then normalized to control samples. Data from a minimum of two biological replicates was used for analysis.

5.5. Cloning and protein production for crystallization

The PCR amplicon for RTA residues 1–267 was subcloned into the pSUMO expression vector encoding an N-terminal deca-histidine and SUMO tag. All cloning was performed using a standard ligase-independent cloning protocol. RTA was expressed in E. coli strain BL21(DE3). The transformed bacteria were grown at 37 °C in TB medium and induced at 20 °C with 0.1 mM IPTG at an OD600 of 0.6 for ~16 h. After induction, cells were harvested and resuspended in 20 mM Tris-Cl pH 7.5 and 150 mM NaCl. The cell suspension was sonicated and centrifuged at 30,000 × g for 30 min. After centrifugation, the protein-containing supernatant was purified by nickel-affinity and size-exclusion chromatography on an AKTAxpress system (GE Healthcare), which consisted of a 1 mL nickel affinity column followed by a Superdex 200 16/60 gel filtration column. The elution buffer consisted of 0.5 M imidazole in the binding buffer, and the gel filtration buffer consisted of 20 mM HEPES pH 7.6, 150 mM NaCl, and 20 mM imidazole. Fractions containing RTA were pooled and subject to TEV protease cleavage (1:10 wt ratio) for 3 h at room temperature to remove the sumo fusion tag. The cleaved RTA was passed over a 1 mL Ni-NTA agarose (Qiagen) gravity column to remove TEV protease, cleaved residues, and uncleaved fusion protein. RTA was buffer exchanged into 20 mM Hepes pH 7.5, 150 mM NaCl, and 1 mM TCEP before complexation with each fragment inhibitor.

5.5.1. Crystallization and data collection

To generate each RTA-inhibitor complex for crystallization trials, RTA was concentrated to 10 mg/ml incubated with 2 mM of each inhibitor for 30 min at room temperature, and then put into crystallization trials. All RTA-inhibitor crystals were grown by sitting drop vapor diffusion at 20 °C using a protein-to-reservoir volume ratio of 1:1 with total drop volumes of 0.2 μL. Crystallization solutions are shown in Table S2. All crystals were flash-frozen in liquid nitrogen after a short soak in the appropriate crystallization buffers supplemented with 20–25 % ethylene glycol. Data were collected at the 24-ID-E beamline at the Advanced Photon Light Source (APS), Argonne National Labs. All data was indexed, merged, and scaled using HKL2000 then converted to structure factors using CCP4 7.0.

5.5.2. Structure determination and refinement

Each RTA-inhibitor complex was solved by molecular replacement. Molecular replacement calculations were performed using the RTA coordinates (PDB ID: 1RTC) as a search model for all RTA-inhibitor complexes. The resulting phase information from molecular replacement was used to identify and place each fragment inhibitor into the resulting electron density maps using the molecular graphics program COOT 8.9.2. The electron density corresponding to the ligands bound to RTA was well defined in the original difference density maps (Fig. 4D–F). The structures of the RTA-RU-NT-59, RTA-PD00589, and RTA-RU-NT-206 complexes were solved at 1.9 Å, 2.3 Å, and 2.7 Å resolution, respectively, in the P6322 space group. Each RTA-inhibitor complex had one copy of RTA in the asymmetric unit. Structural refinement of all coordinates was performed using the PHENIX 1.20.1 package. During refinement, a cross-validation test set was created from a random 5 % of the reflections. Data collection and refinement statistics are listed in Table S1. Molecular graphics were prepared using PyMOL 4.6 (Schrodinger) (DeLano Scientific LLC, Palo Alto, CA). Each fragment inhibitor was left out of the model in the initial stages of refinement. After a few cycles of refinement, each fragment inhibitor was fitted into their respective electron densities and refined to convergence. The final refinement statistics of the structure are summarized in Table S1. B-factor analysis was done using the BAVERAGE program of the CCP4 7.0 suite. Some of the structural analysis was performed using the virtual reality software, Nanome.51

5.6. Statistical analysis

Statistical analysis (ANOVA and mean comparisons) was performed with R (version 4.3.0, R Core Team, 2023) using RStudio (version 2023.3.1.446, Posit Team, 2023). The Dunnett’s Test using the DescTools package (version 0.99.50) was used to compare treatment (ricin or Stx2 treated cells with compound) means to the control (ricin treated cells without compound). NS p > 0.05, * p < 0.05, * p < 0.01, *** p < 0.001.

Supplementary Material

Supplement

Acknowledgements

This work is based on research conducted at NE-CAT beamlines (GM124165). The Eiger 16M detector on the 24-ID-E beam line is funded by an NIH-ORIP HEI grant (S10OD021527). We thank Dr. Bin Cao and Dr. David Augeri for the synthetic route to RU-NT-59, and Dr. Melissa Egbertson for critical reading of the manuscript. The School of Environmental and Biological Sciences (SEBS) Biomolecular Interaction Analysis Core Facility is supported by an NIH Shared Instrumentation Grant, S10 OD026750, which we gratefully acknowledge. This work was supported by the National Institutes of Health grant AI072425 to NET.

Funding

This work was supported by the National Institutes of Health grant AI072425 to NET.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nilgun Tumer reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

Abbreviations:

ESI electron-spray ionization

ESI-MS electron-spray ionization-mass spectrometry

FP fluorescence polarization

FBLD fragment-based ligand discovery

HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

HRMS high resolution mass spectrometry

NTA Nitriloacetic acid

PCy3 tricyclohexylphosphine

RTA ricin toxin A subunit

RTB ricin toxin B subunit

RIP ribosome-inactivating protein

SAR structure–activity relationships

SRL sarcin/ricin loop

SPR surface plasmon resonance

STEC Shiga toxin-producing E. coli, Stx, Shiga toxin

Stx2A1 A1 subunit of Shiga toxin 2

TB Terrific Broth

TCEP tris(2-carboxyethyl)phosphine

TEV Tobacco Etch Virus

Fig. 1. Model showing the interaction of RTA with the P-stalk to access the SRL. The S. cerevisiae 60S subunit and 26S rRNA (PDB ID: 4V88) are shown in green and gray. The fitted structure shows the uL10 fragment in complex with the NMR structure of the P1-P2 heterodimers from H. sapiens (PDB ID: 4BEH) in shades of light grays and blues, interacting with RTA (PDB ID: 5GU4) in white. The hydrophobic pocket at the P-stalk binding site of RTA is colored cyan and the active site of RTA and the SRL substrate, where the adenine is removed are both colored red.

Fig. 2. Fluorescence polarization was used to measure the competitive displacement of BODIPY TMR-X-labeled P11 from RTA by the unlabeled P11 peptide (A) and small molecule inhibitors (B–F). Reactions containing labeled P11 (1 μM), and RTA (3 μM) were incubated with varying concentrations of inhibitors in 40 μL of 1X FP buffer (25 mM Tris-HCl pH 8.0 and 100 mM NaCl) for 30 min in the dark at room temperature followed by centrifugation at 400g for two minutes and scanned using BioTek Synergy 4 microplate reader. Anisotropy values were obtained using 530/25 nm excitation bandpass and 590/35 emission filters. Normalized values for the percentage of binding (%) were plotted against the inhibitor concentration and the IC50 values obtained by FP were used to calculate the Ki values as described in the Materials and Methods. The different colored data points represent 4 different measurements.

Fig. 3. In vitro depurination inhibition. The percentage of inhibition of the RTA-mediated depurination by the small molecule inhibitors was measured by qRT-PCR using rat liver ribosomes. The different colored data points represent different biological replicates (A-D). The data for the percentage of inhibition at different compound concentrations were fitted with Michaelis-Menten kinetics using OriginPro 2023 to calculate the 50 % inhibitory activity (IC50).

Fig. 4. Structures of RTA-inhibitor complexes. The structure of RTA (green) is depicted as a ribbon diagram in complex with inhibitors (A) RU-NT-59 (magenta), (B) PD00589 (cyan), and (C) RU-NT-206 (green). RTA active site residue Tyr80 is drawn as sticks and colored red. 2Fo-Fc (blue mesh) and Fo-Fc (red mesh) electron density maps of (D) RU-NT-59, (E) PD00589, and (F) RU-NT-206. The original 2Fo-Fc and Fo-Fc electron density maps were contoured at 1.0 σ and 3.0 σ levels, respectively. The maps were calculated before each inhibitor was built into the density maps. Each inhibitor is drawn as sticks with all carbon atoms in RU-NT-59 colored magenta and all carbon atoms in PD00589 colored cyan. All nitrogen oxygen atoms are colored red and sulfur atoms yellow.

Fig. 5. Key inhibitor interactions with RTA. Zoom-in of the noncovalent interactions of RTA (green) in complex with (A, C) RU-NT-59 (magenta), (B) PD00589 (cyan), and (D) RU-NT-206 (green) drawn as sticks. All nitrogen atoms are colored blue, all oxygen atoms are colored red, and all sulfur atoms are colored yellow. The salt-bridge and H-bond are represented as red dashes with the similar nonpolar contacts between RTA and each inhibitor represented as yellow dashes. The additional contacts between the fluoride atom in RU-NT-59 and RTA are colored cyan dashes in (C).

Fig. 6. Inhibition of ribosome depurination by ricin holotoxin in Vero cells. (A) Vero cells were plated at 1.5 × 105/mL and grown for 24 h. Cells were treated with each compound and 200 pM ricin as described in the Methods. The percentage of depurination was measured by qRT-PCR compared to DMSO treated cells at 2 h. Data analysis using ANOVA and Dunnett’s test was used to determine the significance of the effect of the different compounds on the percentage of inhibition of depurination of cells treated with ricin compared to the percentage of inhibition of depurination of cells treated with ricin but with no compound (control). *** p < 0.001 (B) The half maximal effective concentration (EC50) value for inhibition of depurination by ricin holotoxin in Vero cells was determined by qRT-PCR. The data for the percent inhibition at different fragment concentrations were fitted with Michaelis-Menten kinetics using OriginPro 2023. Data is from 10 different biological replicates shown in different colors.

Fig. 7. Inhibition of ribosome depurination activity of Stx2A1 and Stx2a holotoxin by RU-NT-206 in Vero cells. (A) In vitro depurination inhibition by RU-NT-206 against Stx2A1 using rat liver ribosomes. The percentage of inhibition was measured by qRT-PCR. The different colored data points represent different biological replicates. The IC50 value against Stx2A1 was determined by qRT-PCR using yeast and rat liver ribosomes. The data for the percentage of inhibition at different fragment concentrations were fitted with Michaelis-Menten kinetics using OriginPro 2023 to determine the IC50. (B) Vero cells were plated at 1.5 × 105/mL and grown for 24 h. Cells were treated with RU-NT-206 and 200 pM ricin or 2 nM Stx2a as described in the Materials and Methods. The percentage of depurination was measured by qRT-PCR compared to DMSO-treated cells at 2 h. ANOVA and Dunnett’s test were used to determine if the compound significantly reduced the percentage of inhibition of depurination in cells treated with toxin compared with cells treated with toxin and no compound (the control). NS p > 0.05, *** p < 0.001.

Fig. 8. P11 peptide contacts with RTA and similar binding mode of CC10501 and P11 with RTA. (A) Close-up of the noncovalent interactions between RTA (green) in complex with the P11 peptide (grey). RTA and P11 are all drawn as sticks. Salt-bridge is represented as red dashes with π-stacking and nonpolar contacts are represented as yellow dashes. The P11 primary sequence is depicted in bold text. The non-covalent interactions of RTA (green) in complex with (B) CC10501 (split-pea green) and (C) the RTA-CC10501 complex superposed with the P11 peptide (gray) showing the similar binding within the P stalk pocket. All molecules are drawn as sticks. All nitrogen atoms are colored blue, all oxygen atoms are colored red, and all sulfur atoms are colored yellow. The salt-bridge is represented as red dashes with the nonpolar contacts between RTA and each inhibitor represented as yellow dashes.

Fig. 9. Key interactions of RU-NT-93 with RTA in the P stalk pocket of RTA influencing inhibition. (A) Position of the 2,6-dimethylphenyl ring of RU-NT-93 (red) relative to the benzene ring in CC10501 (split pea green) within the P-stalk pocket of RTA drawn as a gray molecular surface. (B) RTA (green) bound to RU-NT-93 (red) with the superposed P11 (gray) depicting the similar space occupied by the 2,6-dimethylphenyl ring with the Phe10 and Leu9 of the P11 peptide. (C) RTA (green) bound to RU-NT-93 (red) superposed with RU-NT-206 (green) and P11 (gray) depicting the similar space occupied by the 2,6-dimethylphenyl ring in RU-NT-93 and benzene ring in RU-NT-206 and the P11 peptide Phe10 and Leu9 residues. All molecules are drawn as sticks. All nitrogen atoms are colored blue, all oxygen atoms are colored red, and all sulfur atoms are colored yellow.

Fig. 10. Interaction of RU-NT-59, PD00589, and RU-NT-206 with the P-stalk pocket of RTA. (A) The comparable binding mode of RU-NT-59 (magenta sticks), PD00589 (cyan sticks), and RU-NT-206 (green sticks) to the P-stalk pocket with the P11 peptide (gray sticks) superposed depicting each inhibitor directly blocking the interaction of Leu9, Phe10, and Asp11 with RTA. (B) The hydrophobic pocket within the P11 binding site on RTA is depicted as a gray surface. Key RTA residues lining this hydrophobic pocket were drawn as green sticks. RU-NT-206 is drawn as green sticks. The C1 and C3 methyl groups on RU-NT-206 are labeled C1 and C3, respectively. The yellow dashes depict the hydrophobic contact between the C1 and C3 methyl groups on RU-NT-206 and Leu207, Leu232, and Ile251 from RTA. All nitrogen atoms are colored blue, all oxygen atoms are colored red, and all sulfur atoms are colored yellow.

Scheme 1. Synthesis of new analogs of PD00589. i. DMF, POCl3, 70 °C; ii. Pyridine, methyl thioglycolate, Et3N, 60 °C; iii. 1 M NaOH, MeOH, THF; iv (c) MeB(OH)2, Pd(OAc)2, PCy3, K3PO4, toluene:water; iv (d) Me2NH, Pd(OAc)2, BINAP, Cs2CO3, dioxane; iv (e) BrettPhosPdG3, Cs2CO3, dioxane:MeOH.

Scheme 2. Synthesis of RU-NT-206 analogs. i. NH4Cl, HATU, (i-Pr)2NEt, DMF; ii. Burgess reagent, DCM.

Table 1 The chemical structures, affinity, and inhibitory activity of tricyclic analogs.

Tricyclic-series	R1	X	Y	R2	Ki (μiM)1	IC50(μM)2	
Entry	RU-NT	
1	206	H	CH2	CMe2	COOH	1	7	
2	253	H	CH2	CMe2	C(O)NH2	U.D.	U.D.	
3	254	H	CH2	CMe2	CN	U.D.	U.D.	
4	59	7-F	CH2	CH2	COOH	4	17	
5	PD00589	H	CH2	CH2	COOH	8	30	
6	62	7-Me	CH2	CH2	COOH	18	70	
7	61	7-Br	CH2	CH2	COOH	23	90	
8	64	7-OMe	CH2	CH2	COOH	43	166	
9	63	7-NMe2	CH2	CH2	COOH	U.D.	U.D.	
10	PD00633	H	CH2	CH2	C(O)NHNH2	U.D.	U.D.	
11	198	H	O	CH2	COOH	20	77	
12	SEW01765	8-F	CH2	O	COOMe	U.D.	U.D.	
13	199	8-F	S	CH2	COOH	28	107	
14	SEW01776	8-Cl	CH2	S	COOH	15	59	
15	SEW01689	8-Cl	CH2	S	C(O)NHNH2	U.D.	U.D.	
16	SEW02679	8-Cl	CH2	S	C(O)Me	U.D.	U.D.	
17	201	H	S	COOH	24	94		
1 The Ki values measured by fluorescence polarization (FP) were calculated as described in the Materials and Methods.

2 The IC50 values determined by FP are the concentration of the inhibitor required to replace 50 % of the fluorescent P11 probe from RTA. U.D., unable to determine.

Table 2 The Ki, in vitro IC50 measured by the FP and the qRT-PCR assay, and the EC50 for the depurination inhibition in cell-based assays by the CC10501 analogs.

Code	MW	Structure	Ki (μM)1	IC50 (μM)2	% Inh. (Vero)	% Inh. (A549)	EC50 (μM) (Vero)3	
FP	qRT-PCR	500 μM	250 μM	500 μM	250 μM	
CC10501	204		58 ± 0.3	224 ± 1	408 ± 544	0	0	U.D.	U.D.	U.D.	
RU-NT-93	232		8 ± 0.3	31 ± 1	45 ± 5	66 ± 8	52 ± 5	69 ± 7	59 ± 9	U.D.	
PD00589	230		8 ± 0.3	30 ± 1	28 ± 4	75 ± 7	61 ± 10	69 ± 15	53 ± 2	U.D.	
RU-NT-59	248		4 ± 0.3	17 ± 1	32 ± 4	84 ± 5	62 ± 5	69 ± 7	51 ± 8	U.D.	
RU-NT-206	258		1 ± 0.3	7 ± 1	23 ± 4	95 ± 3	87 ± 7	86 ± 5	75 ± 8	29 ± 2	
P11	1218	SDDDMGFGLFD	1 ± 0.3	4 ± 1	31 ± 6	U.D.	U.D.	U.D.	U.D.	U.D.	
1 The Ki values were measured by fluorescence polarization (FP) and calculated as described in the Materials and Methods.

2 The IC50 value determined by FP is the concentration of the inhibitor required to replace 50 % of the fluorescent P11 probe from RTA. The IC50 value determined by qRT-PCR is the concentration of the inhibitor required to inhibit in vitro depurination of rat liver ribosomes by RTA by 50 %.

3 The EC50 value is the half-maximal concentration required to inhibit depurination by ricin holotoxin in Vero cells as determined by qRT-PCR. U.D., unable to determine.

4 Data was from two biological replicates with R2 values of 0.97 each using linear regression analysis.

Accession codes

The structures generated in this study were deposited in the Protein Data Bank (PDB; https://www.rcsb.org/pdb/) under accession number 8T9V for the RTA-RU-NT-59 complex, 8TAB for the RTA-PD00589 complex, and 8TAD for the RTA-RU-NT-206 complex as described in Table S1. Authors will release the atomic coordinates and experimental data upon article publication.

CRediT authorship contribution statement

Michael J. Rudolph: Investigation, Formal analysis. Arkajyoti Dutta: Writing – original draft, Methodology, Formal analysis. Anastasiia M. Tsymbal: Validation, Investigation, Formal analysis. John E. McLaughlin: Methodology, Investigation, Formal analysis. Yang Chen: Investigation. Simon A. Davis: Investigation. Sophia A. Theodorous: Investigation. Michael Pierce: Investigation, Formal analysis. Benjamin Algava: Investigation, Formal analysis. Xiaoyu Zhang: Investigation. Zoltan Szekely: Investigation. Jacques Y. Roberge: Investigation, Formal analysis. Xiao-Ping Li: Writing – original draft, Formal analysis, Conceptualization. Nilgun E. Tumer: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bmc.2024.117614.
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