HEPN-MNT Toxin-Antitoxin System: The HEPN Ribonuclease Is Neutralized by OligoAMPylation.

Prokaryotic toxin-antitoxin (TA) systems are composed of a toxin capable of interfering with key cellular processes and its neutralizing antidote, the antitoxin. Here, we focus on the HEPN-MNT TA system encoded in the vicinity of a subtype I-D CRISPR-Cas system in the cyanobacterium Aphanizomenon flos-aquae. We show that HEPN acts as a toxic RNase, which cleaves off 4 nt from the 3' end in a subset of tRNAs, thereby interfering with translation. Surprisingly, we find that the MNT (minimal nucleotidyltransferase) antitoxin inhibits HEPN RNase through covalent di-AMPylation (diadenylylation) of a conserved tyrosine residue, Y109, in the active site loop. Furthermore, we present crystallographic snapshots of the di-AMPylation reaction at different stages that explain the mechanism of HEPN RNase inactivation. Finally, we propose that the HEPN-MNT system functions as a cellular ATP sensor that monitors ATP homeostasis and, at low ATP levels, releases active HEPN toxin.

A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model.

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.

Antidote-controlled DNA aptamer modulates human factor IXa activity.

Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.

The in silico identification of novel broad-spectrum antidotes for poisoning by organophosphate anticholinesterases.

Owing to their potential to cause serious adverse health effects, significant efforts have been made to develop antidotes for organophosphate (OP) anticholinesterases, such as nerve agents. To be optimally effective, antidotes must not only reactivate inhibited target enzymes, but also have the ability to cross the blood-brain barrier (BBB). Progress has been made toward brain-penetrating acetylcholinesterase reactivators through the development of a new group of substituted phenoxyalkyl pyridinium oximes. To help in the selection and prioritization of compounds for future synthesis and testing within this class of chemicals, and to identify candidate broad-spectrum molecules, an in silico framework was developed to systematically generate structures and screen them for reactivation efficacy and BBB penetration potential.

The Toxin-Antidote Model of Cytoplasmic Incompatibility: Genetics and Evolutionary Implications.

Wolbachia bacteria inhabit the cells of about half of all arthropod species, an unparalleled success stemming in large part from selfish invasive strategies. Cytoplasmic incompatibility (CI), whereby the symbiont makes itself essential to embryo viability, is the most common of these and constitutes a promising weapon against vector-borne diseases. After decades of theoretical and experimental struggle, major recent advances have been made toward a molecular understanding of this phenomenon. As pieces of the puzzle come together, from yeast and Drosophila fly transgenesis to CI diversity patterns in natural mosquito populations, it becomes clearer than ever that the CI induction and rescue stem from a toxin-antidote (TA) system. Further, the tight association of the CI genes with prophages provides clues to the possible evolutionary origin of this phenomenon and the levels of selection at play.

Rational design, synthesis, and evaluation of uncharged, "smart" bis-oxime antidotes of organophosphate-inhibited human acetylcholinesterase.

Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime-based therapies whose success rates are currently limited. The pyridinium cation hampers uptake of OPs into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are nonproductive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)-inhibited human AChE, here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved in vitro reactivation of diverse OP-human AChE conjugates. Oxime group reorientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Our findings provide detailed structure-activity properties of several CNS-directed, uncharged aliphatic bis-oximes holding promise for use as protonation-dependent, conformationally adaptive, "smart" accelerated antidotes against OP toxicity.

Enhancement of target toxin neutralization effect in vivo by PEGylation of multifunctionalized lipid nanoparticles.

Protein-protein (e.g., antibody-antigen) interactions comprise multiple weak interactions. We have previously reported that lipid nanoparticles (LNPs) bind to and neutralize target toxic peptides after multifunctionalization of the LNP surface (MF-LNPs) with amino acid derivatives that induce weak interactions; however, the MF-LNPs aggregated after target capture and showed short blood circulation times. Here we optimized polyethylene glycol (PEG)-modified MF-LNPs (PEG-MF-LNPs) to inhibit the aggregation and increase the blood circulation time. Melittin was used as a target toxin, and MF-LNPs were prepared with negatively charged, hydrophobic, and neutral amino-acid-derivative-conjugated functional lipids. In this study, MF-LNPs modified with only PEG5k (PEG5k-MF-LNPs) and with both PEG5k and PEG2k (PEGmix-MF-LNPs) were prepared, where PEG5k and PEG2k represent PEG with a molecular weight of 5000 and 2000, respectively. PEGylation of the MF-LNPs did not decrease the melittin neutralization ability of nonPEGylated MF-LNPs, as tested by hemolysis assay. The PEGmix-MF-LNPs showed better blood circulation characteristics than the PEG5k-MF-LNPs. Although the nonPEGylated MF-LNPs immediately aggregated when mixed with melittin, the PEGmix-MF-LNPs did not aggregate. The PEGmix-MF-LNPs dramatically increased the survival rate of melittin-treated mice, whereas the nonPEGylated MF-LNPs increased slightly. These results provide a fundamental strategy to improve the in vivo toxin neutralization ability of MF-LNPs.

Stability of Diazepam Solution for Injection Following Long-Term Storage in an Ambient Temperature of the Mediterranean Climate.

PURPOSE: Diazepam is utilized as a convulsion antidote following nerve gas attacks. As an emergency medicine, it requires storage at ambient temperatures which often doesn't meet manufacturers' requirements, leading to an early invalidation of the product. Current work investigated this issue. METHODS: Long-term stability of diazepam ampoules for injection stored in an ambient temperature of the Mediterranean climate for ~10 years vs storage at room temperature was studied. RESULTS: Diazepam assay and pH remained within pharmacopeial specifications irrespective of storage conditions. A major degradation product 2-methylamino-5-chlorobenzophenone (MACB) showed a clear trend of accumulation as a function of storage time, exceeding the permitted limit at ~2 years, irrespective of storage conditions. A strong correlation between the discoloration of the solutions and the concentration of MACB was obtained. Intravenous administration of MACB to rats at doses ~2200-fold higher than permissible specification levels caused neither mortality nor any toxicological nor post-mortem findings. CONCLUSIONS: Regarding the parameters tested: diazepam assay, MACB assay, and pH, storing ampoules of diazepam solution for injection in field conditions of high temperatures of the Mediterranean climate did not cause accelerated degradation as compared to room temperature. These findings open an option for the usage of expired ampoules in special scenarios.

Development of versatile and potent monoquaternary reactivators of acetylcholinesterase.

To date, the only treatments developed for poisoning by organophosphorus compounds, the most toxic chemical weapons of mass destruction, have exhibited limited efficacy and versatility. The available causal antidotes are based on reactivation of the enzyme acetylcholinesterase (AChE), which is rapidly and pseudo-irreversibly inhibited by these agents. In this study, we developed a novel series of monoquaternary reactivators combining permanently charged moieties tethered to position 6- of 3-hydroxypyridine-2-aldoxime reactivating subunit. Highlighted representatives (21, 24, and 27; also coded as K1371, K1374, and K1375, respectively) that contained 1-phenylisoquinolinium, 7-amino-1-phenylisoquinolinium and 4-carbamoylpyridinium moieties as peripheral anionic site ligands, respectively, showed efficacy superior or comparable to that of the clinically used standards. More importantly, these reactivators exhibited wide-spectrum efficacy and were minutely investigated via determination of their reactivation kinetics in parallel with molecular dynamics simulations to study their mechanisms of reactivation of the tabun-inhibited AChE conjugate. To further confirm the potential applicability of these candidates, a mouse in vivo assay was conducted. While K1375 had the lowest acute toxicity and the most suitable pharmacokinetic profile, the oxime K1374 with delayed elimination half-life was the most effective in ameliorating the signs of tabun toxicity. Moreover, both in vitro and in vivo, the versatility of the agents was substantially superior to that of clinically used standards. Their high efficacy and broad-spectrum capability make K1374 and K1375 promising candidates that should be further investigated for their potential as nerve agents and insecticide antidotes.

Activation of (un)regulated cell death as a new perspective for bispyridinium and imidazolium oximes.

Oximes, investigated as antidotes against organophosphates (OP) poisoning, are known to display toxic effects on a cellular level, which could be explained beyond action on acetylcholinesterase as their main target. To investigate this further, we performed an in vitro cell-based evaluation of effects of two structurally diverse oxime groups at concentrations of up to 800 µM, on several cell models: skeletal muscle, kidney, liver, and neural cells. As indicated by our results, compounds with an imidazolium core induced necrosis, unregulated cell death characterized by a cell burst, increased formation of reactive oxygen species, and activation of antioxidant scavenging. On the other hand, oximes with a pyridinium core activated apoptosis through specific caspases 3, 8, and/or 9. Interestingly, some of the compounds exhibited a synergistic effect. Moreover, we generated a pharmacophore model for each oxime series and identified ligands from public databases that map to generated pharmacophores. Several interesting hits were obtained including chemotherapeutics and specific inhibitors. We were able to define the possible structural features of tested oximes triggering toxic effects: chlorine atoms in combination with but-2(E)-en-1,4-diyl linker and adding a second benzene ring with substituents such as chlorine and/or methyl on the imidazolium core. Such oximes could not be used in further OP antidote development research, but could be introduced in other research studies on new specific targets. This could undoubtedly result in an overall improved wider use of unexplored oxime database created so far in OP antidotes field of research in a completely new perspective.

Synthetic Amphoteric Cryogels as an Antidote against Acute Heavy Metal Poisoning.

The effectiveness of an amphoteric cryogel (AAC) as an oral sorbent (enerosorbent) for the treatment of acute poisoning of small animals (rats) with heavy metals (HMs) was studied in in vivo experiments. The morphological structure of the cryogel was examined using scanning electron microscopy/energy-dispersive X-ray analysis and confocal microscopy. The use of the cryogel in the treatment of rats administered an LD50 dose of Cd(NO3)2, CsNO3, Sr(NO3)2, or HgCl2 in aqueous solution showed their high survival rate compared to the control group, which did not receive such treatment. The histological and chemical analysis of internal tissues and the biochemical analysis of the blood of the experimental animals showed the effectiveness of the cryogel in protecting the animals against the damaging effect of HMs on the organism comparable with unithiol, a chelating agent based on 2,3-dimercapto-1-propane sulfonic acid sodium salt (DMPS) approved for the treatment of acute poisoning with some heavy metals.

A Cobalt Schiff-Base Complex as a Putative Therapeutic for Azide Poisoning.

There is presently no antidote available to treat azide poisoning. Here, the Schiff-base compound Co(II)-2,12-dimethyl-3,7,11,17-tetraazabicyclo-[11.3.1]heptadeca-1(17)2,11,13,15-pentaenyl dibromide (Co(II)N4[11.3.1]) is investigated to determine if it has the capability to antagonize azide toxicity through a decorporation mechanism. The stopped-flow kinetics of azide binding to Co(II)N4[11.3.1] in the absence of oxygen exhibited three experimentally observable phases: I (fast); II (intermediate); and III (slow). The intermediate phase II accounted for ∼70% of the overall absorbance changes, representing the major process observed, with second-order rate constants of 29 (±4) M-1 s-1 at 25 °C and 70 (±10) M-1 s-1 at 37 °C. The data demonstrated pH independence of the reaction around neutrality, suggesting the unprotonated azide anion to be the attacking species. The binding of azide to Co(II)N4[11.3.1] appears to have a complicated mechanism leading to less than ideal antidotal capability; nonetheless, this cobalt complex does protect against azide intoxication. Administration of Co(II)N4[11.3.1] at 5 min post sodium azide injection (ip) to mice resulted in a substantial decrease of righting-recovery times, 12 (±4) min, compared to controls, 40 (±8) min. In addition, only two out of seven mice "knocked down" when the antidote was administered compared to the controls given toxicant only (100% knockdown).

Water-Soluble α-Amino Acid Complexes of Molybdenum as Potential Antidotes for Cyanide Poisoning: Synthesis and Catalytic Studies of Threonine, Methionine, Serine, and Leucine Complexes.

Water-soluble complexes are desirable for the aqueous detoxification of cyanide. Molybdenum complexes with α-amino acid and disulfide ligands with the formula K[(L)Mo2O2(µ-S)2(S2)] (L = leu (1), met (2), thr (3), and ser (4)) were synthesized in a reaction of [(DMF)3MoO(µ-S)2(S2)] with deprotonated α-amino acids; leu, met, thr, and ser are the carboxylate anions of l-leucine, l-methionine, l-threonine, and l-serine, respectively. Potassium salts of α-amino acids (leu (1a), met (2a), thr (3a), and ser (4a)) were prepared as precursors for complexes 1-4, respectively, by employing a nonaqueous synthesis route. The ligand exchange reaction of [Mo2O2(µ-S)2(DMF)6](I)2 with deprotonated α-amino acids afforded bis-α-amino acid complexes, [(L)2Mo2O2(µ-S)2] (6-8). A tris-α-amino acid complex, [(leu)2Mo2O2(µ-S)2(µ-leu + H)] (5; leu + H is the carboxylate anion of l-leucine with the amine protonated), formed in the reaction with leucine. 5 crystallized from methanol with a third weakly bonded leucine as a bridging bidentate carboxylate. An adduct of 8 with SCN- coordinated, 9, crystallized and was structurally characterized. Complexes 1-4 are air stable and highly water-soluble chiral molecules. Cytotoxicity studies in the A549 cell line gave IC50 values that range from 80 to 400 µM. Cyclic voltammetry traces of 1-8 show solvent-dependent irreversible electrochemical behavior. Complexes 1-4 demonstrated the ability to catalyze the reaction of thiosulfate and cyanide in vitro to exhaustively transform cyanide to thiocyanate in less than 1 h.

An Appraisal of Antidotes' Effectiveness: Evidence of the Use of Phyto-Antidotes and Biotechnological Advancements.

Poisoning is the greatest source of avoidable death in the world and can result from industrial exhausts, incessant bush burning, drug overdose, accidental toxication or snake envenomation. Since the advent of Albert Calmette's cobra venom antidote, efforts have been geared towards antidotes development for various poisons to date. While there are resources and facilities to tackle poisoning in urban areas, rural areas and developing countries are challenged with poisoning management due to either the absence of or inadequate facilities and this has paved the way for phyto-antidotes, some of which have been scientifically validated. This review presents the scope of antidotes' effectiveness in different experimental models and biotechnological advancements in antidote research for future applications. While pockets of evidence of the effectiveness of antidotes exist in vitro and in vivo with ample biotechnological developments, the utilization of analytic assays on existing and newly developed antidotes that have surpassed the proof of concept stage, as well as the inclusion of antidote's short and long-term risk assessment report, will help in providing the required scientific evidence(s) prior to regulatory authorities' approval.

An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications.

Colchicine is a toxic alkaloid prevalent in autumn crocus (Colchicum autumnale) that binds to tubulin and inhibits polymerization of microtubules. Using combinatorial and rational protein design, we have developed an artificial binding protein based on the human lipocalin 2 that binds colchicine with a dissociation constant of 120 pm, i.e. 10000-fold stronger than tubulin. Crystallographic analysis of the engineered lipocalin, dubbed Colchicalin, revealed major structural changes in the flexible loop region that forms the ligand pocket at the open end of the eight-stranded ß-barrel, resulting in a lid-like structure over the deeply buried colchicine. A cis-peptide bond between residues Phe71 and Pro72 in loop #2 constitutes a peculiar feature and allows intimate contact with the tricyclic ligand. Using directed evolution, we achieved an extraordinary dissociation half-life of more than 9 h for the Colchicalin-colchicine complex. Together with the chemical robustness of colchicine and availability of activated derivatives, this also opens applications as a general-purpose affinity reagent, including facile quantification of colchicine in biological samples. Given that engineered lipocalins, also known as Anticalin® proteins, represent a class of clinically validated biopharmaceuticals, Colchicalin may offer a therapeutic antidote to scavenge colchicine and reverse its poisoning effect in situations of acute intoxication.

Counteracting tabun inhibition by reactivation by pyridinium aldoximes that interact with active center gorge mutants of acetylcholinesterase.

Tabun represents the phosphoramidate class of organophosphates that are covalent inhibitors of acetylcholinesterase (AChE), an essential enzyme in neurotransmission. Currently used therapy in counteracting excessive cholinergic stimulation consists of a muscarinic antagonist (atropine) and an oxime reactivator of inhibited AChE, but the classical oximes are particularly ineffective in counteracting tabun exposure. In a recent publication (Kovarik et al., 2019), we showed that several oximes prepared by the Huisgen 1,3 dipolar cycloaddition and related precursors efficiently reactivate the tabun-AChE conjugate. Herein, we pursue the antidotal question further and examine a series of lead precursor molecules, along with triazole compounds, as reactivators of two AChE mutant enzymes. Such studies should reveal structural subtleties that reside within the architecture of the active center gorge of AChE and uncover intimate mechanisms of reactivation of alkylphosphate conjugates of AChE. The designated mutations appear to minimize steric constraints of the reactivating oximes within the impacted active center gorge. Indeed, after initial screening of the triazole oxime library and its precursors for the reactivation efficacy on Y337A and Y337A/F338A human AChE mutants, we found potentially active oxime-mutant enzyme pairs capable of degrading tabun in cycles of inhibition and reactivation. Surprisingly, the most sensitive ex vivo reactivation of mutant AChEs occurred with the alkylpyridinium aldoximes. Hence, although the use of mutant enzyme bio-scavengers in humans may be limited in practicality, bioscavenging and efficient neutralization of tabun itself or phosphoramidate mixtures of organophosphates might be achieved efficiently in vitro or ex vivo with these mutant AChE combinations.

Rationally designed molecules for resurgence of cyanide mitigated cytochrome c oxidase activity.

Cytochrome c oxidase (CcOX) containing binuclear heme a3-Cu B centre (BNC) mechanises the process of electron transfer in the last phase of cellular respiration. The molecular modelling based structural analysis of CcOX - heme a3-Cu B complex was performed and the disturbance to this complex under cyanide poisoning conditions was investigated. Taking into consideration the results of molecular docking studies, new chemical entities were developed for clipping cyanide from the enzyme and restoring its normal function. It was found that the molecules obtained by combining syringaldehyde, oxindole and chrysin moieties bearing propyl/butyl spacing groups occupy the BNC region and effectively remove cyanide bound to the enzyme. The binding constant of compound 2 with CN- was 2.3 × 105 M-1 and its ED50 for restoring the cyanide bound CcOX activity in 10 min was 16 µM. The compound interacted with CN- over the pH range 5-10. The comparison of the loss of enzymatic activity in the presence of CN- and resumption of enzymatic activity by compound 2 mediated removal of CN- indicated the efficacy of the compound as antidote of cyanide.

Structure of a prereaction complex between the nerve agent sarin, its biological target acetylcholinesterase, and the antidote HI-6.

Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.

Activity of silver nanoparticles on prokaryotic cells and Bothrops jararacussu snake venom.

Nanoparticle-conjugated venom-toxins of venomous animals and its therapeutic efficacy against emerging or neglecting diseases is a promising strategy. In this study, silver nanoparticles (AgNPs ∼50 nm, 0.081 mg mL-1) were studied against the neuromuscular blockade, myotoxic effects induced by Bothrops jararacussu venom (60 µg mL-1) and also against prokaryotic cells. The neurotoxicity was evaluated on ex vivo mouse phrenic nerve-diaphragm using traditional myographic technique, able to obtain functional contractile responses and to check the neurotransmission. The myotoxicity on mammalian cells was evaluated in muscles resulting from pharmacological assays using routine histological techniques and light microscopy. The toxicity to prokaryotic cells was evaluated on Salmonella typhimurium TA100 without metabolic activation. The in vitro preincubation model between AgNPs and venom was enough to abolish toxic effects of B. jararacussu venom, but mammalian cells were highly sensitive to AgNPs more than prokaryotic cells, by acting as dose-independently and dose-dependently parameters, respectively. These results allowed us to conclude that AgNPs showed promising activity as antivenom agent but for its safer use, the toxicity should be evaluated on experimental animals.

In Silico Molecular Studies of Antiophidic Properties of the Amazonian Tree Cordia nodosa Lam.

We carried out surveys on the use of Cordia nodosa Lam. in the jungles of Bobonaza (Ecuador). We documented this knowledge to prevent its loss under the Framework of the Convention on Biological Diversity and the Nagoya Protocol. We conducted bibliographic research and identified quercetrin as a significant bioactive molecule. We studied its in silico biological activity. The selected methodology was virtual docking experiments with the proteins responsible for the venomous action of snakes. The molecular structures of quercetrin and 21 selected toxins underwent corresponding tests with SwissDock and Chimera software. The results point to support its antiophidic use. They show reasonable geometries and a binding free energy of -7 to -10.03 kcal/mol. The most favorable values were obtained for the venom of the Asian snake Naja atra (5Z2G, -10.03 kcal/mol). Good results were also obtained from the venom of the Latin American Bothrops pirajai (3CYL, -9.71 kcal/mol) and that of Ecuadorian Bothrops asper snakes (5TFV, -9.47 kcal/mol) and Bothrops atrox (5TS5, -9.49 kcal/mol). In the 5Z2G and 5TS5 L-amino acid oxidases, quercetrin binds in a pocket adjacent to the FAD cofactor, while in the myotoxic homologues of PLA2, 3CYL and 5TFV, it joins in the hydrophobic channel formed when oligomerizing, in the first one similar to α-tocopherol. This study presents a case demonstration of the potential of bioinformatic tools in the validation process of ethnobotanical phytopharmaceuticals and how in silico methods are becoming increasingly useful for sustainable drug discovery.