Psychedelic indolethylamines with therapeutic potential are naturally produced in plants, fungi, and animals. Here, we elucidated the complete N,N-dimethyltryptamine (DMT) biosynthetic pathway in hallucinogenic plant species traditionally used in shamanic rituals for spiritual healing. Leveraging the similarities in their chemical structures, we reconstructed in one plant assay the full biosynthetic pathways of five renowned natural psychedelics; psilocin and psilocybin found in mushrooms, DMT from plants, and bufotenin and 5-methoxy-DMT secreted by the Sonoran Desert toad. We further engineered halogenated analogs of these molecules, which do not naturally occur in plants and exhibit prospective therapeutic potential for psychiatric conditions. Blending catalytic functions across the tree of life, coupled with metabolic engineering guided by rational protein design of mutant enzymes, enabled substantially more efficient in planta production of the indolethylamine components. This work establishes a versatile platform for concurrent biosynthesis and diversification of psychoactive indolethylamines, paving the way for their production in plants.

Despite extensive study of its chemical composition and long history of medicinal use, the occurrence of glycosylated cannabinoid derivatives in Cannabis sativa has not been documented to date. Here, we identified glycosylated cannabinoids and their common intermediate olivetolic acid (OA) in various C. sativa tissues and cultivars. We moreover identified four UDP-glycosyltransferases (UGTs) from C. sativa with OA glycosylating activity. Enhancing the water solubility of cannabinoids through glycosylation holds potential for pharmaceutical development and cosmetic applications. However, glycosylation of pathway intermediates such as OA may divert metabolic flux away from cannabinoid production, complicating efforts to engineer glycosylated forms. To resolve this, we applied FuncLib design to an AlphaFold-predicted structure of one of the identified enzymes CsUGT14, generating active-site variants. Through functional screening, we identified mutants that display increased specificity toward cannabinoid end products over the OA intermediate. Moreover, we recognized a single point mutation that dictates OA positioning within the active site, thereby altering isomer formation. These findings expand the known repertoire of natural cannabinoids and provide a rare example of crystallography-free enzyme design to improve stability, substrate selectivity, and isomer specificity. Furthermore, this work lays the foundation for the tailored biosynthesis of soluble glycosylated cannabinoids in heterologous systems.

Despite extensive study of its chemical composition and long history of medicinal use, the occurrence of glycosylated cannabinoid derivatives in Cannabis sativa has not been documented to date. Here, we identified glycosylated cannabinoids and their common intermediate olivetolic acid (OA) in various C. sativa tissues and cultivars. We moreover identified four UDP-glycosyltransferases (UGTs) from C. sativa with OA glycosylating activity. Enhancing the water solubility of cannabinoids through glycosylation holds potential for pharmaceutical development and cosmetic applications. However, glycosylation of pathway intermediates such as OA may divert metabolic flux away from cannabinoid production, complicating efforts to engineer glycosylated forms. To resolve this, we applied FuncLib design to an AlphaFold-predicted structure of one of the identified enzymes CsUGT14, generating active-site variants. Through functional screening, we identified mutants that display increased specificity toward cannabinoid end products over the OA intermediate. Moreover, we recognized a single point mutation that dictates OA positioning within the active site, thereby altering isomer formation. These findings expand the known repertoire of natural cannabinoids and provide a rare example of crystallography-free enzyme design to improve stability, substrate selectivity, and isomer specificity. Furthermore, this work lays the foundation for the tailored biosynthesis of soluble glycosylated cannabinoids in heterologous systems.

Endocannabinoids (eCBs) and related lipids play crucial roles in brain function, including the regulation of circadian rhythms and sleep. To comprehensively map these molecules, we employed liquid chromatography high-resolution tandem mass spectrometry (LC/HRMS/MS) to quantify 78 lipids across 14 families in seven brain areas of male mice at four time points throughout the day (every six hours), and during sleep initiation. We found that most eCBs from the fatty acids (FAs) family, particularly arachidonic acid (AA), were highly abundant in the mouse brain in all brain areas and during the circadian rhythm. High eCBs abundance was shown in deeper brain areas, while temporal differences using the Cosinor analysis revealed 26 eCBs behaving in a circadian rhythm response, with linolenic acid (LnA) being the only lipid to show rhythmicity across all brain areas. Sleep initiation (ZT1) was associated with increased N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) activity and N-acylethanolamide (NAE) levels in the cortex and hippocampus, while wake extension (WEx) altered 2-monoacylglycerol (2-MAG) metabolism and increased cannabinoid receptor 1 (CB1) expression. These findings provide a detailed lipidomic map of eCBs and related lipids in the male mouse brain, highlighting their area-specific distribution, circadian regulation, and involvement in sleep/wake transitions. Given the link between sleep disruption and neurodegeneration, future studies should investigate whether the observed eCB dysregulation contributes to sleep disturbances in these conditions, and if targeting these pathways offers novel therapeutic strategies. Significant Statement This comprehensive study provides a high-dimensional map of eCBs and related lipids in the male mouse brain, revealing intricate spatial and temporal dynamics highlighting the role of these lipids in regulating fundamental physiological processes such as circadian rhythm and sleep. ### Competing Interest Statement The authors have declared no competing interest.

In T-cell acute lymphoblastic leukemia (T-ALL) more than 50% of cases display autoactivation of Notch1 signaling, leading to oncogenic transformation. We have previously identified a specific chemovar of Cannabis that induces apoptosis by preventing Notch1 maturation in leukemia cells. Here, we isolated three cannabinoids from this chemovar that synergistically mimic the effects of the whole extract. Two were previously known, Cannabidiol (CBD) and Cannabidivarin (CBDV); whereas the third cannabinoid, which we termed 331-18A, was identified and fully characterized in this study. We demonstrated that these cannabinoids act through Cannabinoid receptor type 2 and TRPV1 to activate the integrated stress response pathway by depleting intracellular Ca 2+ . This is followed by increased mRNA and protein expression of ATF4, CHOP and CHAC1, which is hindered by inhibiting the upstream initiation factor eIF2α. The increased abundance of CHAC1 prevents Notch1 maturation, thereby reducing the levels of the active Notch1 intracellular domain, and consequently decreasing cell viability and increasing apoptosis. Treatment with the three isolated molecules resulted in reduced tumor size and weight in-vivo and slowed leukemia progression in mice models. Altogether, this study elucidated the mechanism of action of three distinct cannabinoids in modulating the Notch1 pathway, and constitutes an important step in the establishment of a new therapy for treating NOTCH1 -mutated diseases and cancers such as T-ALL.

Mescaline, among the earliest identified natural hallucinogens, holds great potential in psychotherapy treatment. Nonetheless, despite the existence of a postulated biosynthetic pathway for more than half a century, the specific enzymes involved in this process are yet to be identified. In this study, we investigated the cactus Lophophora williamsii (Peyote), the largest known natural producer of the phenethylamine mescaline. We employed a multi-faceted approach, combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling, enzymatic assays, molecular modeling, and pathway engineering for pathway elucidation. We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway, and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates, likely modulating mescaline levels in Peyote. Finally, we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells, providing novel insights into several challenges hindering complete heterologous mescaline production. Taken together, our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline, safeguarding this valuable natural resource for future generations.

EDITORIAL article Front. Plant Sci., 08 November 2023Sec. Plant Metabolism and Chemodiversity Volume 14 - 2023 | https://doi.org/10.3389/fpls.2023.1327345

Dear Editor, In T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematologic cancer with poor clinical outcomes, more than 50% of cases show NOTCH1-driven transformation [1]. The NOTCH1 receptor signaling pathway is activated through a series of proteolytic cleavages, ultimately causing the release of the active intracellular domain (NICD), which translocates to the nucleus where it promotes transcription of target genes involved in cell growth. The importance of NOTCH1 mutations in T-ALL has generated great interest in the development of anti-NOTCH1 targeted therapies. A new and promising emerging field in cancer treatment is medical cannabis. Accumulating evidence suggests the direct effects of cannabis on tumor progression in cell lines and animal models [2]. Cannabis, and its unique secondary metabolites, known as phytocannabinoids, directly affect the propagation of cancer cells by modulating key cellsignaling pathways [3].Wehave previously demonstrated that different cannabis extracts, each containing a unique composition ofmetabolites, selectively impaired the survival of cancer cell lines depending on amatch between the chemical composition of the extract and the characteristics of the specific cancer cell line [4]. In the present work, we set out to investigate whether cannabis extracts with unique phytocannabinoid profiles can selectively facilitate antitumor effects in T-ALL cells that harbor a Notch1mutation. We first performed a preliminary screening in which the viability of four distinct T-ALL cell lines was tested when

Modulation of the endocannabinoid system is projected to have therapeutic potential in almost all human diseases. Accordingly, the high demand for novel cannabinoids stimulates the discovery of untapped sources and efficient manufacturing technologies. Here we explored Helichrysum umbraculigerum, an Asteraceae species unrelated to Cannabis sativa that produces Cannabis-type cannabinoids (for example, 4.3% cannabigerolic acid). In contrast to Cannabis, cannabinoids in H. umbraculigerum accumulate in leaves' glandular trichomes rather than in flowers. The integration of de novo whole-genome sequencing data with unambiguous chemical structure annotation, enzymatic assays and pathway reconstitution in Nicotiana benthamiana and in Saccharomyces cerevisiae has uncovered the molecular and chemical features of this plant. Apart from core biosynthetic enzymes, we reveal tailoring ones producing previously unknown cannabinoid metabolites. Orthology analyses demonstrate that cannabinoid synthesis evolved in parallel in H. umbraculigerum and Cannabis. Our discovery provides a currently unexploited source of cannabinoids and tools for engineering in heterologous hosts.

Studies have shown that women are more susceptible to adverse effects (AEs) from conventional drugs. This study aimed to investigate the differences of medical cannabis (MC)-related AEs between women and men in patients with chronic noncancer pain (CNCP). This is a cross-sectional study of adult patients licensed for MC treatment who were also diagnosed as patients with CNCP by a physician. Data included self-reported questionnaires and comprehensive MC treatment information. Simultaneously, identification and quantification of phytocannabinoids and terpenoids from the MC cultivars were performed. Comparative statistics were used to evaluate differences between men and women. Four hundred twenty-nine patients with CNCP (64% males) reported fully on their MC treatment. Subgrouping by sex demonstrated that the weight-adjusted doses were similar between men and women (0.48 [0.33-0.6] gr for men and 0.47 [0.34-0.66] gr for women). Nonetheless, women reported more than men on MC-related AEs. Further analysis revealed that women consumed different MC cultivar combinations than men, with significantly higher monthly doses of the phytocannabinoids CBD and CBC and significantly lower monthly doses of the phytocannabinoid 373-15c and the terpenoid linalool. Our findings demonstrate sex differences in MC-related AEs among patients with CNCP. Women are more susceptible to MC-related AEs, presumably because of both the inherent sex effect and the consumption of specific phytocannabinoid compositions in the MC cultivar(s). The understanding of these differences may be crucial for planning MC treatments with safer phytocannabinoid and terpenoid compositions and to better inform patients of expected AEs.

Medical Cannabis and its major cannabinoids (−)-trans-Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are gaining momentum for various medical purposes as their therapeutic qualities are becoming better established. However, studies regarding their efficacy are oftentimes inconclusive. This is chiefly because Cannabis is a versatile plant rather than a single drug and its effects do not depend only on the amount of THC and CBD. Hundreds of Cannabis cultivars and hybrids exist worldwide, each with a unique and distinct chemical profile. Most studies focus on THC and CBD, but these are just two of over 140 phytocannabinoids found in the plant in addition to a milieu of terpenoids, flavonoids and other compounds with potential therapeutic activities. Different plants contain a very different array of these metabolites in varying relative ratios, and it is the interplay between these molecules from the plant and the endocannabinoid system in the body that determines the ultimate therapeutic response and associated adverse effects. Here, we discuss how phytocannabinoid profiles differ between plants depending on the chemovar types, review the major factors that affect secondary metabolite accumulation in the plant including the genotype, growth conditions, processing, storage and the delivery route; and highlight how these factors make Cannabis treatment highly complex.

Introduction: Chronic non-cancer pain (CNCP) is one of the most prevalent indications for medical cannabis (MC) treatment globally. In this study, we investigated CNCP parameters in patients during prolonged MC treatment, and assessed the interrelation between CNCP parameters and the chemical composition of MC chemovar used. Methods: A cross-sectional questionnaire-based study was performed in one-month intervals for the duration of six months. Subjects were adult patients licensed for MC treatment who also reported a diagnosis of CNCP by a physician. Data included self-reported questionnaires. MC treatment features included administration route, cultivator, cultivar name and monthly dose. Comparison statistics were used to evaluate differences between the abovementioned parameters and the monthly MC chemovar doses at each time point. Results: 429, 150, 98, 71, 77 and 82 patients reported fully on their MC treatment regimens at six one-month intervals, respectively. Although pain intensities did not change during the study period, analgesic medication consumption rates decreased from 46 to 28% (p < 0.005) and good Quality of Life (QoL) rates increased from 49 to 62% (p < 0.05). These changes overlapped with increase in rates of (-)-Δ9-trans-tetrahydrocannabinol (THC) and α-pinene high dose consumption. Conclusion: Even though we observed that pain intensities did not improve during the study, QoL did improve and the rate of analgesic medication consumption decreased alongside with increasing rates of high dose THC and α-pinene consumption. Understanding MC treatment composition may shed light on its long-term effects.

In the last decades, growing evidence showed the therapeutic capabilities of Cannabis plants. These capabilities were attributed to the specialized secondary metabolites stored in the glandular trichomes of female inflorescences, mainly phytocannabinoids and terpenoids. The accumulation of the metabolites in the flower is versatile and influenced by a largely unknown regulation system, attributed to genetic, developmental and environmental factors. As Cannabis is a dioecious plant, one main factor is fertilization after successful pollination. Fertilized flowers are considerably less potent, likely due to changes in the contents of phytocannabinoids and terpenoids; therefore, this study examined the effect of fertilization on metabolite composition by crossbreeding (-)-Δ9-trans-tetrahydrocannabinol (THC)- or cannabidiol (CBD)-rich female plants with different male plants: THC-rich, CBD-rich, or the original female plant induced to develop male pollen sacs. We used advanced analytical methods to assess the phytocannabinoids and terpenoids content, including a newly developed semi-quantitative analysis for terpenoids without analytical standards. We found that fertilization significantly decreased phytocannabinoids content. For terpenoids, the subgroup of monoterpenoids had similar trends to the phytocannabinoids, proposing both are commonly regulated in the plant. The sesquiterpenoids remained unchanged in the THC-rich female and had a trend of decrease in the CBD-rich female. Additionally, specific phytocannabinoids and terpenoids showed an uncommon increase in concentration followed by fertilization with particular male plants. Our results demonstrate that although the profile of phytocannabinoids and their relative ratios were kept, fertilization substantially decreased the concentration of nearly all phytocannabinoids in the plant regardless of the type of fertilizing male. Our findings may point to the functional roles of secondary metabolites in Cannabis.

Medical cannabis (MC) treatment for chronic pain is increasing, but evidence regarding short- and long-term efficacy and associated adverse effects (AEs) of the different cannabis plant components is limited. Most reports focus on two phytocannabinoids, (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). This study, aimed to identify patterns of phytocannabinoid compositions associated with MC treatment response and with related AEs. Participants in this multicenter prospective cohort were patients with chronic non-cancer pain that were prescribed MC by physicians. Data was collected before MC treatment, at one month (short-term) and at 12 months (long-term). Simultaneously, liquid chromatography mass spectrometry identification and quantification of phytocannabinoids from the cultivars were performed. The monthly dose of each phytocannabinoid for each patient was z-scaled and clustered into ten groups to assess the difference in analgesic treatment response (≥30%/50% pain intensity reduction) and AEs rates. We identified ten clusters that had similar analgesic treatment response rates. However, there were significant differences in AEs rates both at short- and long-term. We identified specific phytocannabinoid compositions that were associated with overall AEs rates (5% compared to 53% at short-term and 44% at long-term) and with specific AEs rates such as MC related central nervous system, gastrointestinal and psychological AEs. To conclude, Evaluating only Δ9-THC or CBD is insufficient to find associations with MC related AEs. Therefore, comprehensive profiling of phytocannabinoids is needed to discover associations to related AEs and help physicians prescribe safer cannabis with less AEs while still relieving pain.

Cannabis or its derivatives are widely used by patients with cancer to help with cancer symptoms and treatment side effects. However, cannabis has potent immunomodulatory properties. To determine if cannabis consumption during immunotherapy affects therapy outcomes, we conducted a prospective observatory study including 102 (68 immunotherapy and 34 immunotherapy plus cannabis) consecutive patients with advanced cancers who initiated immunotherapy. Cannabis consumption correlated with a significant decrease in time to tumor progression and overall survival. On the other hand, the use of cannabis reduced therapy-related immune-related adverse events. We also tested the possibility that cannabis may affect the immune system or the tumor microenvironment through the alteration of the endocannabinoid system. We analyzed a panel of serum endocannabinoids (eCBs) and eCB-like lipids, measuring their levels before and after immunotherapy in both groups. Levels of serum eCBs and eCB-like lipids, before immunotherapy, showed no significant differences between cannabis users to nonusers. Nevertheless, the levels of four eCB and eCB-like compounds were associated with patients’ overall survival time. Collectively, cannabis consumption has considerable immunomodulatory effects, and its use among cancer patients needs to be carefully considered due to its potential effects on the immune system, especially during treatment with immunotherapy.

Therapeutic use of medical cannabis among cancer patients has become highly prevalent, while its overall effects on the immune system are unclear. This study aims to determine if cannabis consumption during immunotherapy affects therapy outcome for patients with advanced malignancies. The study was conducted at single Oncology center, in Israel between 01 Sep 2016 and 25 Sep 2018; included 102 [68 immunotherapy alone (I-G) and 34 immunotherapy plus cannabis (IC-G)] consecutive patients with advanced cancers who initiated one of the checkpoint inhibitors. Blood samples were taken before immunotherapy treatment. Endocannabinoid (eCB) levels from various lipid families, were evaluated in a subgroup of 36 patients. Safety and effectivity of cannabis treatment in advanced cancers commencing treatment with immune checkpoint blockers was evaluated with time to tumor progression (TTP) used as a post hoc primary endpoint and overall survival (OS) and eCB concentrations as secondary endpoints with a minimum follow-up time of 7 months. Kaplan Maier curve showed a significant difference in TTP [I-G 13.1m (95%CI 6.0-NAm) vs. IC-G 3.4m (95%CI 1.8-6.0m), p=0.0025] and OS [IG 28.5m (95%CI 15.6-NAm) vs. IC-G 6.4m (95%CI 3.2-9.7m), p=0.0009]. After adjusting for the line of treatment, Cox regression analysis showed that cannabis consumption decreases OS (HR= 2.18, 95%CI 1.241-3.819. p=0.007) and TTP (HR= 1.95, 95%CI 1.17-3.26. p=0.011). The use of cannabis reduced grade ≥2 immune-related adverse events (iAE) (I-G 39% vs. IC-G 21%, p=0.057). Further analysis of baseline levels of circulating eCB from various lipid families showed no significant changes in their overall concentrations. However, analyzing a cohort comparing patients with progressive disease to those with complete remission correlates baseline eCB levels and expected OS, suggesting that the eCB system may play a role in immunotherapy outcomes. Initiating immunotherapy with cannabis use negatively affects OS and TTP of cancer patients treated with immunotherapy.

Background: Medical cannabis (MC) treatment for migraine is practically emerging, although sufficient clinical data are not available for this indication. This cross-sectional questionnaire-based study aimed to investigate the associations between phytocannabinoid treatment and migraine frequency. Methods: Participants were migraine patients licensed for MC treatment. Data included self-reported questionnaires and MC treatment features. Patients were retrospectively classified as responders vs. non-responders (≥50% vs. <50% decrease in monthly migraine attacks frequency following MC treatment initiation, respectively). Comparative statistics evaluated differences between these two subgroups. Results: A total of 145 patients (97 females, 67%) with a median MC treatment duration of three years were analyzed. Compared to non-responders, responders (n = 89, 61%) reported lower current migraine disability and lower negative impact, and lower rates of opioid and triptan consumption. Subgroup analysis demonstrated that responders consumed higher doses of the phytocannabinoid ms_373_15c and lower doses of the phytocannabinoid ms_331_18d (3.40 95% CI (1.10 to 12.00); p < 0.01 and 0.22 95% CI (0.05–0.72); p < 0.05, respectively). Conclusions: These findings indicate that MC results in long-term reduction of migraine frequency in >60% of treated patients and is associated with less disability and lower antimigraine medication intake. They also point to the MC composition, which may be potentially efficacious in migraine patients.

The therapeutic use of medical Cannabis is growing, and so is the need for standardized and therapeutically stable Cannabis products for patients. The therapeutic effects of Cannabis largely depend on the content of its pharmacologically active secondary metabolites and their interactions, mainly terpenoids and phytocannabinoids. Once harvested and during storage, these natural compounds may decarboxylate, oxidize, isomerize, react photochemically, evaporate and more. Despite its widespread and increasing use, however, data on the stability of most of the plant's terpenoids and phytocannabinoids during storage is scarce. In this study, we therefore aimed to determine postharvest optimal storage conditions for preserving the composition of naturally biosynthesized secondary metabolites in Cannabis inflorescences and Cannabis extracts. To this end, Cannabis inflorescences (whole versus ground samples) and Cannabis extracts (dissolved in different solvents) from (-)-Δ9-trans-tetrahydrocannabinol- or cannabidiol-rich chemovars, were stored in the dark at various temperatures (25, 4, -30 and -80°C), and their phytocannabinoid and terpenoid profiles were analyzed over the course of 1 year. We found that in both Cannabis inflorescences and extracts, a storage temperature of 25°C led to the largest changes in the concentrations of the natural phytocannabinoids over time, making this the most unfavorable temperature compared with all others examined here. Olive oil was found to be the best vehicle for preserving the natural phytocannabinoid composition of the extracts. Terpenoid concentrations were found to decrease rapidly under all storage conditions, but temperatures lower than -20°C and grinding of the inflorescences were the least favorable conditions. Overall, our conclusions point that storage of whole inflorescences and extracts dissolved in olive oil, at 4°C, were the optimal postharvest conditions for Cannabis.

Increasing evidence for the therapeutic potential of Cannabis in numerous pathological and physiological conditions has led to a surge of studies investigating the active compounds in different chemovars and their mechanisms of action, as well as their efficacy and safety. The biological effects of Cannabis have been attributed to phytocannabinoid modulation of the endocannabinoid system. In-vitro and in-vivo studies have shown that pure phytocannabinoids can alter the levels of endocannabinoids and other cannabimimetic lipids. However, it is not yet understood whether whole Cannabis extracts exert variable effects on the endocannabinoid metabolome, and whether these effects vary between tissues. To address these challenges, we have developed and validated a novel analytical approach, termed "cannabinoidomics," for the simultaneous extraction and analysis of both endogenous and plant cannabinoids from different biological matrices. In the methodological development liquid chromatography high resolution tandem mass spectrometry (LC/HRMS/MS) was used to identify 57 phytocannabinoids, 15 major phytocannabinoid metabolites, and 78 endocannabinoids and cannabimimetic lipids in different biological matrices, most of which have no analytical standards. In the validation process, spiked cannabinoids were quantified with acceptable selectivity, repeatability, reproducibility, sensitivity, and accuracy. The power of this analytical method is demonstrated by analysis of serum and four different sections of mouse brains challenged with three different cannabidiol (CBD)-rich extracts. The results demonstrate that variations in the minor phytocannabinoid contents of the different extracts may lead to varied effects on endocannabinoid concentrations, and on the CBD metabolite profile in the peripheral and central systems. We also show that the Cannabis challenge significantly decreases the levels of several endocannabinoids in specific brain sections compared to the control group. This effect is extract-specific and suggests the importance of minor, other-than CBD, phytocannabinoid or non-phytocannabinoid compounds.