The Search for Antidotes Against Ricin.

The castor plant (Ricinus communis) is primarily known for its seeds, which contain a unique fatty acid called ricinoleic acid with several industrial and commercial applications. Castor seeds also contain ricin, a toxin considered a chemical and biological warfare agent. Despite years of investigation, there is still no effective antidote or vaccine available. However, some progress has been made, and the development of an effective treatment may be on the horizon. To provide an updated overview of this issue, we have conducted a comprehensive review of the literature on the current state of research in the fight against ricin. This review is based on the reported research and aims to address the challenges faced by researchers, as well as highlight the most successful cases achieved thus far. Our goal is to encourage the scientific community to continue their efforts in this critical search.

Identification of novel potential ricin inhibitors by virtual screening, molecular docking, molecular dynamics and MM-PBSA calculations: a drug repurposing approach.

Ricin is a potent cytotoxin with no available antidote. Its catalytic subunit, RTA, damages the ribosomal RNA (rRNA) of eukaryotic cells, preventing protein synthesis and eventually leading to cell death. The combination between easiness of obtention and high toxicity turns ricin into a potential weapon for terrorist attacks, urging the need of discovering effective antidotes. On this context, we used computational techniques, in order to identify potential ricin inhibitors among approved drugs. Two libraries were screened by two different docking algorithms, followed by molecular dynamics simulations and MM-PBSA calculations in order to corroborate the docking results. Three drugs were identified as potential ricin inhibitors: deferoxamine, leucovorin and plazomicin. Our calculations showed that these compounds were able to, simultaneously, form hydrogen bonds with residues of the catalytic site and the secondary binding site of RTA, qualifying as potential antidotes against intoxication by ricin.Communicated by Ramaswamy H. Sarma.

Discovery of RTA ricin subunit inhibitors: a computational study using PM7 quantum chemical method and steered molecular dynamics.

Ricin is a potent toxin derived from the castor bean plant and comprises two subunits, RTA and RTB. Because of its cytotoxicity, ricin has alarmed world authorities for its potential use as a chemical weapon. Ricin also affects castor bean agribusiness, given the risk of animal and human poisoning. Over the years, many groups attempted to propose small-molecules that bind to the RTA active site, the catalytic chain. Despite such efforts, there is still no effective countermeasure against ricin poisoning. The computational study carried out in the present work renews the discussion about small-molecules that may inhibit this toxin. Here, a structure-based virtual screening protocol capable of discerning active RTA inhibitors from inactive ones was performed to screen over 2 million compounds from the ZINC database to find novel scaffolds that strongly bind into the active site of the RTA. Besides, a novel score method based on ligand undocking force profiles and semi-empirical quantum chemical calculations provided insights into the rescore of docking poses. Summing up, the filtering steps pointed out seven main compounds, with the SCF00-451 as a promising candidate to inhibit the killing activity of such potent phytotoxin.

Ligand-Based Virtual Screening, Molecular Docking, Molecular Dynamics, and MM-PBSA Calculations towards the Identification of Potential Novel Ricin Inhibitors.

Ricin is a toxin found in the castor seeds and listed as a chemical weapon by the Chemical Weapons Convention (CWC) due to its high toxicity combined with the easiness of obtention and lack of available antidotes. The relatively frequent episodes of usage or attempting to use ricin in terrorist attacks reinforce the urge to develop an antidote for this toxin. In this sense, we selected in this work the current RTA (ricin catalytic subunit) inhibitor with the best experimental performance, as a reference molecule for virtual screening in the PubChem database. The selected molecules were then evaluated through docking studies, followed by drug-likeness investigation, molecular dynamics simulations and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) calculations. In every step, the selection of molecules was mainly based on their ability to occupy both the active and secondary sites of RTA, which are located right next to each other, but are not simultaneously occupied by the current RTA inhibitors. Results show that the three PubChem compounds 18309602, 18498053, and 136023163 presented better overall results than the reference molecule itself, showing up as new hits for the RTA inhibition, and encouraging further experimental evaluation.

A New Method for Extraction and Analysis of Ricin Samples through MALDI-TOF-MS/MS.

We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.

Determining the Relative Binding Affinity of Ricin Toxin A Inhibitors by Using Molecular Docking and Nonequilibrium Work.

Ricin is a ribosome-inactivating protein (RIP type 2) consisting of two subunits, ricin toxin A (RTA) and ricin toxin B (RTB). Because of its cytotoxicity, ricin has worried world authorities for its potential use as a chemical weapon; therefore, its inhibition is of great biotechnological interest. RTA is the target for inhibitor synthesis, and pterin derivatives are promising candidates to inhibit it. In this study, we used a combination of the molecular docking approach and fast steered molecular dynamics (SMD) to assess the correlation between nonequilibrium work, ⟨ W⟩, and the IC50 for six RTA inhibitors. The results showed that molecular docking is a powerful tool to predict good bioactive poses of RTA inhibitors, and ⟨ W⟩ presented a strong correlation with IC50 ( R2 = 0.961). Such a profile ranked the RTA inhibitors better than the molecular docking approach. Therefore, the combination of docking and fast SMD simulation was shown to be a promising tool to distinguish RTA-active inhibitors from inactive ones and could be used as postdocking filtering approach.

Evidence for thyroid hormone as a positive regulator of serum thyrotropin bioactivity.

CONTEXT: The regulation of TSH bioactivity in humans is not completely understood. OBJECTIVE: The aim of the study was to investigate the role of serum thyroid hormones in regulating the bioactivity of TSH. DESIGN: We determined in vitro TSH bioactivity and glycosylation in nine patients (six females and three males, age 41.3 yr) with primary hypothyroidism before and after L-T(4) replacement, in 11 age- and sex-comparable controls (seven females and four males, age 37.6 yr), and in two thyroidectomized patients with TSH-secreting adenomas during and after L-T(4) withdrawal. METHODS: In vitro TSH bioactivity was measured by a sensitive and specific bioassay based on cAMP generation by Chinese hamster ovary cells transfected with human TSH receptor. TSH glycosylation was assessed by concanavalin A lectin and ricin column affinity chromatography. RESULTS: In vitro TSH bioactivity in hypothyroid patients was low as compared with controls (0.48 +/- 0.1 vs. 1.1 +/- 0.2; P = 0.004) and increased during L-T(4) (0.48 +/- 0.1 vs. 0.8 +/- 0.1; P = 0.01). A strong significant correlation (r = +0.80; P = 0.004, Spearman) was observed between the absolute increments of serum TSH bioactivity and T(3) during L-T(4) replacement. The degree of sialylation was elevated in hypothyroid patients before treatment (47 +/- 2.4% vs. 29 +/- 4.3%; P = 0.002) and decreased significantly after L-T(4) (47 +/- 2.4% vs. 33 +/- 4.3%; P = 0.02). The mannose content of serum TSH in hypothyroid patients was similar to controls and did not change during L-T(4). In vitro TSH bioactivity also decreased in patients with TSH-secreting adenomas during L-T(4) withdrawal. CONCLUSION: These data indicate that serum thyroid hormone level is a positive regulator of TSH bioactivity.

Acetone powder from dormant seeds of Ricinus communis L: lipase activity and presence of toxic and allergenic compounds.

The influence of several factors on the hydrolytic activity of lipase, present in the acetone powder from dormant castor seeds (Ricinus communis) was evaluated. The enzyme showed a marked specificity for short-chain substrates. The best reaction conditions were an acid medium, Triton X-100 as the emulsifying agent and a temperature of 30 degrees C. The lipase activity of the acetone powder of different castor oil genotypes showed great variability and storage stability of up to 90%. The toxicology analysis of the acetone powder from genotype Nordestina BRS 149 showed a higher ricin (toxic component) content, a lower 2S albumin (allergenic compound) content, and similar allergenic potential compared with untreated seeds.