Methyl transfer in psilocybin biosynthesis.

Psilocybin, the natural hallucinogen produced by Psilocybe ("magic") mushrooms, holds great promise for the treatment of depression and several other mental health conditions. The final step in the psilocybin biosynthetic pathway, dimethylation of the tryptophan-derived intermediate norbaeocystin, is catalysed by PsiM. Here we present atomic resolution (0.9 Å) crystal structures of PsiM trapped at various stages of its reaction cycle, providing detailed insight into the SAM-dependent methylation mechanism. Structural and phylogenetic analyses suggest that PsiM derives from epitranscriptomic N6-methyladenosine writers of the METTL16 family, which is further supported by the observation that bound substrates physicochemically mimic RNA. Inherent limitations of the ancestral monomethyltransferase scaffold hamper the efficiency of psilocybin assembly and leave PsiM incapable of catalysing trimethylation to aeruginascin. The results of our study will support bioengineering efforts aiming to create novel variants of psilocybin with improved therapeutic properties.

Determination of psilocybin and psilocin content in multiple Psilocybe cubensis mushroom strains using liquid chromatography - tandem mass spectrometry.

A method for clinical potency determination of psilocybin and psilocin in hallucinogenic mushroom species Psilocybe cubensis was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Five strains of dried, intact mushrooms were obtained and analyzed: Blue Meanie, Creeper, B-Plus, Texas Yellow, and Thai Cubensis. An extraction protocol was developed; this included an evaluation of sample milling technique, extraction solvents, and recovery/stability. Reversed phase chromatography on fused-core particle phases was developed for the determination of the two analytes using internal standard calibration with deuterated isotopologues of each analyte. The separation takes less than 5 min. Matrix effects were investigated by comparing signal response of calibration samples in neat solution and several mushroom matrices; no significant matrix effects were observed. The limit of detection for psilocybin was 1.5 ng/mL (1.5 pg on-column; 300 ng/g mushroom) and for psilocin was 0.15 ng/mL (0.15 pg on-column; 30 ng/g mushroom) using a Shimadzu LCMS-8050 triple quadrupole mass spectrometer. Assessment of the accuracy and precision of the method indicated percent error and RSD were <6 % at all concentration levels. Three whole, intact mushrooms from each strain were analyzed individually to obtain average content differences both between strains and between mushrooms of the same strain. From most to least potent, the study found that the average total psilocybin and psilocin concentrations for the Creeper, Blue Meanie, B+, Texas Yellow, and Thai Cubensis strains were 1.36, 1.221, 1.134, 1.103, and 0.879 % (w/w), respectively. A subset of these mushrooms was also tested in a separate non-affiliated laboratory, and the results were comparable between the two laboratories. Results from the secondary laboratory showed improved precision when multiple mushrooms were homogenized together, prior to extraction.

Psychedelics promote plasticity by directly binding to BDNF receptor TrkB.

Psychedelics produce fast and persistent antidepressant effects and induce neuroplasticity resembling the effects of clinically approved antidepressants. We recently reported that pharmacologically diverse antidepressants, including fluoxetine and ketamine, act by binding to TrkB, the receptor for BDNF. Here we show that lysergic acid diethylamide (LSD) and psilocin directly bind to TrkB with affinities 1,000-fold higher than those for other antidepressants, and that psychedelics and antidepressants bind to distinct but partially overlapping sites within the transmembrane domain of TrkB dimers. The effects of psychedelics on neurotrophic signaling, plasticity and antidepressant-like behavior in mice depend on TrkB binding and promotion of endogenous BDNF signaling but are independent of serotonin 2A receptor (5-HT2A) activation, whereas LSD-induced head twitching is dependent on 5-HT2A and independent of TrkB binding. Our data confirm TrkB as a common primary target for antidepressants and suggest that high-affinity TrkB positive allosteric modulators lacking 5-HT2A activity may retain the antidepressant potential of psychedelics without hallucinogenic effects.

The evolution and ecology of psilocybin in nature.

Fungi produce diverse metabolites that can have antimicrobial, antifungal, antifeedant, or psychoactive properties. Among these metabolites are the tryptamine-derived compounds psilocybin, its precursors, and natural derivatives (collectively referred to as psiloids), which have played significant roles in human society and culture. The high allocation of nitrogen to psiloids in mushrooms, along with evidence of convergent evolution and horizontal transfer of psilocybin genes, suggest they provide a selective benefit to some fungi. However, no precise ecological roles of psilocybin have been experimentally determined. The structural and functional similarities of psiloids to serotonin, an essential neurotransmitter in animals, suggest that they may enhance the fitness of fungi through interference with serotonergic processes. However, other ecological mechanisms of psiloids have been proposed. Here, we review the literature pertinent to psilocybin ecology and propose potential adaptive advantages psiloids may confer to fungi.

In silico characterization of the psilocybin biosynthesis pathway.

Nearly all mushrooms of the Psilocybe genus contain the natural product psilocybin, which is a psychoactive alkaloid derived from l-tryptophan. Considering their use in ancient times, as well as their psychedelic properties, these mushrooms have re-emerged with psychotherapeutic potential for treating depression, which has triggered increased pharmaceutical interest. However, the psilocybin biosynthesis pathway was only recently defined and, as such, little exists in the way of structural data. Accordingly, the aim of this study was to structurally characterize this pathway by generating homology models for the four Psilocybe cubensis enzymes involved in psilocybin biosynthesis (PsiD, a decarboxylase; PsiH, a monooxygenase; PsiK, a phosphotransferase; PsiM, a methyltransferase). Following initial model generation and alignment with the identified structural templates, repeated refinement of the models was carried out using secondary structure prediction, geometry evaluation, energy minimization, and molecular dynamics simulations in water. The final models were then evaluated using molecular docking interactions with their substrates, i.e., psilocybin precursors (l-tryptophan, tryptamine, 4-hydroxytryptamine, and norbaeocystin/baeocystin), all of which generated feasible binding modes for the expected biotransformation. Further plausibility of the psilocybin → aeruginascin, 4-hydroxytryptamine → norpsilocin, and tryptamine → N,N-dimethyltryptamine conversions, all mediated by the generated model for PsiM, suggests valid routes of formation for these key secondary metabolites. The structural characterization of these enzymes and their binding modes which emerged from this study can lead to a better understanding of psilocybin synthesis, thereby paving the way for the development of novel substrates and selective inhibitors, as well as improved biotechnological manipulation and production of psilocybin in vitro.

Structure-based discovery of nonhallucinogenic psychedelic analogs.

Drugs that target the human serotonin 2A receptor (5-HT2AR) are used to treat neuropsychiatric diseases; however, many have hallucinogenic effects, hampering their use. Here, we present structures of 5-HT2AR complexed with the psychedelic drugs psilocin (the active metabolite of psilocybin) and d-lysergic acid diethylamide (LSD), as well as the endogenous neurotransmitter serotonin and the nonhallucinogenic psychedelic analog lisuride. Serotonin and psilocin display a second binding mode in addition to the canonical mode, which enabled the design of the psychedelic IHCH-7113 (a substructure of antipsychotic lumateperone) and several 5-HT2AR ß-arrestin-biased agonists that displayed antidepressant-like activity in mice but without hallucinogenic effects. The 5-HT2AR complex structures presented herein and the resulting insights provide a solid foundation for the structure-based design of safe and effective nonhallucinogenic psychedelic analogs with therapeutic effects.

The Therapeutic Potential of Psilocybin.

The psychedelic effects of some plants and fungi have been known and deliberately exploited by humans for thousands of years. Fungi, particularly mushrooms, are the principal source of naturally occurring psychedelics. The mushroom extract, psilocybin has historically been used as a psychedelic agent for religious and spiritual ceremonies, as well as a therapeutic option for neuropsychiatric conditions. Psychedelic use was largely associated with the "hippie" counterculture movement, which, in turn, resulted in a growing, and still lingering, negative stigmatization for psychedelics. As a result, in 1970, the U.S. government rescheduled psychedelics as Schedule 1 drugs, ultimately ending scientific research on psychedelics. This prohibition on psychedelic drug research significantly delayed advances in medical knowledge on the therapeutic uses of agents such as psilocybin. A 2004 pilot study from the University of California, Los Angeles, exploring the potential of psilocybin treatment in patients with advanced-stage cancer managed to reignite interest and significantly renewed efforts in psilocybin research, heralding a new age in exploration for psychedelic therapy. Since then, significant advances have been made in characterizing the chemical properties of psilocybin as well as its therapeutic uses. This review will explore the potential of psilocybin in the treatment of neuropsychiatry-related conditions, examining recent advances as well as current research. This is not a systematic review.

Chemistry and Toxicology of Major Bioactive Substances in Inocybe Mushrooms.

Mushroom poisoning has always been a threat to human health. There are a large number of reports about ingestion of poisonous mushrooms every year around the world. It attracts the attention of researchers, especially in the aspects of toxin composition, toxic mechanism and toxin application in poisonous mushroom. Inocybe is a large genus of mushrooms and contains toxic substances including muscarine, psilocybin, psilocin, aeruginascin, lectins and baeocystin. In order to prevent and remedy mushroom poisoning, it is significant to clarify the toxic effects and mechanisms of these bioactive substances. In this review article, we summarize the chemistry, most known toxic effects and mechanisms of major toxic substances in Inocybe mushrooms, especially muscarine, psilocybin and psilocin. Their available toxicity data (different species, different administration routes) published formerly are also summarized. In addition, the treatment and medical application of these toxic substances in Inocybe mushrooms are also discussed. We hope that this review will help understanding of the chemistry and toxicology of Inocybe mushrooms as well as the potential clinical application of its bioactive substances to benefit human beings.

Development and validation of an LC-MS/MS method for the bioanalysis of psilocybin's main metabolites, psilocin and 4-hydroxyindole-3-acetic acid, in human plasma.

Psilocin is the active metabolite of psilocybin, a serotonergic psychedelic substance. It is used recreationally and investigated in substance-assisted psychotherapy. The pharmacokinetic properties of psilocin are only partially characterized. Therefore, we developed and validated a rapid LC-MS/MS method to quantify psilocin and its metabolite 4-hydroxyindole-3-acetic acid (4-HIAA) in human plasma. Plasma samples were processed by protein precipitation using methanol. The injected sample was mixed with water in front of a C18 analytical column to increase retention of the analytes. Psilocin and 4-HIAA were detected by multiple reaction monitoring (MRM) in positive and negative electrospray ionisation mode, respectively. An inter-assay accuracy of 100-109% and precision of ≤8.7% was recorded over three validation runs. The recovery was near to complete (≥94.7%) and importantly, consistent over different concentration levels and plasma batches (CV%: ≤4.1%). The plasma matrix caused negligible ion suppression and endogenous interferences could be separated from the analytes. Psilocin and 4-HIAA plasma samples could be thawed and re-frozen for three cycles, kept at room temperature for 8 h or 1 month at -20 °C without showing degradation (≤10%). The linear range (R ≥ 0.998) of the method covered plasma concentrations observed in humans following a common therapeutic oral dose of 25 mg psilocybin and was therefore able to assess the pharmacokinetics of psilocin and 4-HIAA. The LC-MS/MS method was convenient and reliable for measuring psilocin and 4-HIAA in plasma and will facilitate the clinical development of psilocybin.

Effects and safety of Psilocybe cubensis and Panaeolus cyanescens magic mushroom extracts on endothelin-1-induced hypertrophy and cell injury in cardiomyocytes.

Prevalence of major depression in people with chronic heart failure is higher than in normal populations. Depression in heart failure has become a major issue. Psilocybin-containing mushrooms commonly known as magic mushrooms, have been used since ancient times for their mind healing properties. Their safety in cardiovascular disease conditions is not fully known and may pose as a risk for users suffering from these illnesses. Study investigates the effects and safety of Psilocybe cubensis and Panaeolus cyanescens magic mushrooms use from genus Psilocybe and Panaeolus respectively, in a pathological hypertrophy conditions in which endothelin-1 disorder is a contributor to pathogenesis. We examined the effects of the mushrooms extracts on endothelin-1-induced hypertrophy and tumor necrosis factor-α (TNF- α)-induced cell injury in H9C2 cardiomyocytes. Mushrooms were oven dried and extracted with cold and boiling-hot water. H9C2 cardiomyocytes were induced with endothelin-1 prior to treatment with extracts over 48 h. Cell injury was stimulated with TNF-α. Results proposed that the water extracts of Panaeolus cyanescens and Psilocybe cubensis did not aggravate the pathological hypertrophy induced by endothelin-1 and also protected against the TNF-α-induced injury and cell death in concentrations used. Results support medicinal safe use of mushrooms under controlled conditions and cautioned use of higher concentrations.

The Psilocin (4-hydroxy-N,N-dimethyltryptamine) and Bufotenine (5-hydroxy-N,N-dimethyltryptamine) Case: Ensuring the Correct Isomer has Been Identified.

Psilocin (4-hydroxy-N,N-dimethyltryptamine, 4-HO-DMT) and bufotenine (5-hydroxy-N,N-dimethyltryptamine, 5-HO-DMT), which are both naturally occurring compounds, are classified as controlled substances in numerous countries due to their pharmacological activities and recreational usage. There are two other benzene ring regioisomers, 6-hydroxy-N,N-dimethyltryptamine (6-HO-DMT) and 7-hydroxy-N,N-dimethyltryptamine (7-HO-DMT), which are not classified by name as controlled substances, and which were synthesized for this current work. The four isomers were analyzed using routine methodologies employed by the Israel's Police Division of Identification and Forensic Science (DIFS) Laboratory, namely thin layer chromatography (TLC), Fourier transform infrared spectroscopy (FTIR), and gas chromatography mass spectroscopy (GC-MS). It was found possible to differentiate the four isomers. Forensic specimens that were suspected to be psilocybe mushrooms were examined, confirming that it is now possible to unequivocally identify the presence of psilocin and rule out the presence of its other isomers.

Self-reported negative outcomes of psilocybin users: A quantitative textual analysis.

Psilocybin, a substance mainly found in mushrooms of the genus psilocybe, has been historically used for ritualistic, recreational and, more recently, medicinal purposes. The scientific literature suggests low toxicity, low risk of addiction, overdose, or other causes of injury commonly caused by substances of abuse, with growing interest in the use of this substance for conditions such as treatment-resistant depression. However, the presence of negative outcomes linked to psilocybin use is not clear yet. The objective of this study is to investigate the negative effects of psilocybin consumption, according to the users' own perception through self-reports extracted from an online platform. 346 reports were analyzed with the assistance of the IRAMUTEQ textual analysis software, adopting the procedures of Descending Hierarchical Classification, Correspondence Factor Analysis and Specificities Analysis. The text segments were grouped in 4 main clusters, describing thinking distortions, emergencies, perceptual alterations and the administration of the substance. Bad trips were more frequent in female users, being associated with thinking distortions. The use of multiple doses of psilocybin in the same session or its combination with other substances was linked to the occurrence of long-term negative outcomes, while the use of mushrooms in single high doses was linked to medical emergencies. These results can be useful for a better understanding of the effects of psilocybin use, guiding harm-reduction initiatives.

Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin.

Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment-resistant depression and major depressive disorder. The psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot-scale protocol for recombinant PsiK that yielded 150 mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost.

Synthesis and Biological Evaluation of Tryptamines Found in Hallucinogenic Mushrooms: Norbaeocystin, Baeocystin, Norpsilocin, and Aeruginascin.

A general synthetic method was developed to access known tryptamine natural products present in psilocybin-producing mushrooms. In vitro and in vivo experiments were then conducted to inform speculations on the psychoactive properties, or lack thereof, of the natural products. In animal models, psychedelic activity by baeocystin alone was not evident using the mouse head twitch response assay, despite its putative dephosphorylated metabolite, norpsilocin, possessing potent agonist activity at the 5-HT2A receptor.

Simultaneous Production of Psilocybin and a Cocktail of ß-Carboline Monoamine Oxidase Inhibitors in "Magic" Mushrooms.

The psychotropic effects of Psilocybe "magic" mushrooms are caused by the l-tryptophan-derived alkaloid psilocybin. Despite their significance, the secondary metabolome of these fungi is poorly understood in general. Our analysis of four Psilocybe species identified harmane, harmine, and a range of other l-tryptophan-derived ß-carbolines as their natural products, which was confirmed by 1D and 2D NMR spectroscopy. Stable-isotope labeling with 13 C11 -l-tryptophan verified the ß-carbolines as biosynthetic products of these fungi. In addition, MALDI-MS imaging showed that ß-carbolines accumulate toward the hyphal apices. As potent inhibitors of monoamine oxidases, ß-carbolines are neuroactive compounds and interfere with psilocybin degradation. Therefore, our findings represent an unprecedented scenario of natural product pathways that diverge from the same building block and produce dissimilar compounds, yet contribute directly or indirectly to the same pharmacological effects.

DARK Classics in Chemical Neuroscience: Psilocybin.

Psilocybin is found in a family of mushrooms commonly known as "magic mushrooms" that have been used throughout history to induce hallucinations. In the late 1950s Albert Hofmann, of Sandoz Laboratories, identified and synthesized the psychoactive compounds psilocybin and psilocin which are found in psilocybe mushrooms. Psilocybin was marketed by Sandoz as Indocybin for basic psychopharmacological and therapeutic clinical research. Psilocybin saw a rapid rise in popularity during the 1960s and was classed as a Schedule I drug in 1970. This led to a significant decrease in psilocybin research. Recently, however, preliminary studies with psilocybin have shown promise as potential for the treatment of obsessive compulsive disorder, alcohol addiction, tobacco addiction, and major depressive disorder, and the treatment of depression in terminally ill cancer patients. This review describes in detail the synthesis, metabolism, pharmacology, adverse drug reactions, and importance of psilocybin to neuroscience in the past and present.

Statistical comparison of mass spectra for identification of amphetamine-type stimulants.

A method for the statistical comparison of mass spectral data is demonstrated for applications in controlled substance analysis. The method uses an unequal variance t-test at each mass-to-charge ratio in the scan range to determine if two spectra are statistically associated or discriminated. If the two spectra are associated, a random-match probability is calculated to estimate the likelihood that the mass spectral fragmentation pattern in question occurs by random chance alone. If the two spectra are discriminated, the fragment ions responsible for the discrimination are determined. In this work, mass spectral data from case samples containing amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), phentermine, and psilocin were investigated. All spectra were collected in an accredited forensic laboratory using routine methods for controlled substance analysis. Using the statistical method, spectra of case samples were statistically associated to the corresponding reference standard at the 99.9% confidence level. In these instances, random-match probabilities ranged from 10-39 to 10-29, indicating the probability that the characteristic fragmentation pattern occurred by random chance is extremely small. Further, spectra of case samples were discriminated from other reference standards at the 99.9% or 99.0% confidence level, with 1-26 ions responsible for discrimination in each comparison.

[Psilocybin - public available psychodysleptic]. / Psylocybina ­ ogólnodostepny psychodysleptyk.

Substances of plant origin have been used to induce hallucinations for a long time, in religious ceremonies and rituals as well as in pain relief. Psilocybin and psilocin naturally occur in the fungal genus Psilocybe. Due to the psychedelic effects and relative harmlessness of these substances and the fact that they do not cause physical addiction, psilocybin and psilocin recently have been increasingly replacing synthetic psychodysleptics, such as diethylamide D-lysergic acid. Both compounds as psychoactive substances are illegal, but psilocybin, in addition to psychotropic action, also shows positive effects, which from a medical point of view indicate its therapeutic potential and capacity for use in therapy. However, poisoning by psilocin and its derivatives is still a major clinical and social problem, mainly among young people, which is why quick and reliable identification of these substances is very important. Traditional ways of assigning the sample to a particular taxon, such as morphological and biochemical analysis or palynological and sporological studies, are not very universal and often do not provide clear results. Credibility, high speed and lower cost of DNA analysis make genetic methods more often used to determine the species of fungi. These methods are random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and high resolution melting (HRM). Moreover, analysis of the regions ITS1 and nLSU was suggested as a valid method for application in the molecular taxonomy of fungi for forensic purposes. Modern methods of identifying psilocybin and psilocin in fungi and biological material are: zone capillary electrophoresis, high performance liquid chromatography, gas chromatography and liquid chromatography coupled with mass spectrometry. The mentioned methods are successfully used for the identification of psychoactive substances in fungi as well as in blood and urine samples.

Synthesis, hydrolysis and stability of psilocin glucuronide.

A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for ß-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples.

Multiple receptors contribute to the behavioral effects of indoleamine hallucinogens.

Serotonergic hallucinogens produce profound changes in perception, mood, and cognition. These drugs include phenylalkylamines such as mescaline and 2,5-dimethoxy-4-methylamphetamine (DOM), and indoleamines such as (+)-lysergic acid diethylamide (LSD) and psilocybin. Despite their differences in chemical structure, the two classes of hallucinogens produce remarkably similar subjective effects in humans, and induce cross-tolerance. The phenylalkylamine hallucinogens are selective 5-HT(2) receptor agonists, whereas the indoleamines are relatively non-selective for serotonin (5-HT) receptors. There is extensive evidence, from both animal and human studies, that the characteristic effects of hallucinogens are mediated by interactions with the 5-HT(2A) receptor. Nevertheless, there is also evidence that interactions with other receptor sites contribute to the psychopharmacological and behavioral effects of the indoleamine hallucinogens. This article reviews the evidence demonstrating that the effects of indoleamine hallucinogens in a variety of animal behavioral paradigms are mediated by both 5-HT(2) and non-5-HT(2) receptors.