Complete biosynthesis of cannabinoids and their unnatural analogues in yeast

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“Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.”

https://www.nature.com/articles/s41586-019-0978-9

“Yeast can produce THC, CBD, novel cannabinoids”  https://www.upi.com/Science_News/2019/02/28/Yeast-can-produce-THC-CBD-novel-cannabinoids/4411551303863/

“Yeast produce low-cost, high-quality cannabinoids”  https://www.eurekalert.org/pub_releases/2019-02/uoc–ypl022419.php

“Engineered yeast can brew up the active ingredients in cannabis plants”  https://www.newscientist.com/article/2195103-engineered-yeast-can-brew-up-the-active-ingredients-in-cannabis-plants/

“High grade cannabis chemicals produced using brewing yeast”  https://www.independent.co.uk/news/science/cannabis-drug-produced-yeast-marijuana-thc-cbd-medicine-california-a8799576.html

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Crystal Structure of the Human Cannabinoid Receptor CB2

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“The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257’s unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.”
“Study reveals the structure of the 2nd human cannabinoid receptor”   HTTPS://MIPT.RU/ENGLISH/NEWS/STUDY_REVEALS_THE_STRUCTURE_OF_THE_2ND_HUMAN_CANNABINOID_RECEPTOR
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CBN: The cancer fighting Cannabinoid

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“CBN, cannabinol, is a mildly psychoactive cannabinoid found within the cannabis plant. We examine the very complex mechanisms that give allowance for this cannabinoids entrance into the cell membrane and its effect on cannabinoid receptors and the inhibition of the enzyme adenylate cyclase that is responsible for phosphate production. Prior study bears weight accordingly; we examine this phosphate as a potent energy source, the enzymes responsible for cell replication cycle and inhibition thereof. Moreover, how IL-2, (Interleukin-2), a type of cytokine signaling molecule in the immune system stops being produced when immune T cells are exposed to cannabinoids. How IL-2 stimulates the cell cycle via promotion of the c-Fos protein and is responsible for modulation of the immune response. This is shown by Faubert and Kaminski, that administration of CBN can slow cell replication and endure cell death (apoptosis).”

http://www.imedpub.com/proceedings/cbn-the-cancer-fighting-cannabinoid-5528.html

“Programmed Cell Death (Apoptosis)” http://www.ncbi.nlm.nih.gov/books/NBK26873/

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Lung alveolar tissue destruction and protein citrullination in diesel exhaust exposed mouse lungs.

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“Humanity faces an increasing impact of air pollution worldwide, including threats to human health. Air pollutants prompt and promote chronic inflammation, tumourigenesis, autoimmune and other destructive processes in the human body.

Post-translational modification of proteins, e.g. citrullination, results from damaging attacks of pollutants, including smoking, air pollution and others, rendering host tissues immunogenic. Citrullinated proteins and citrullinating enzymes, deiminases, are more prevalent in patients with COPD and correlate with ongoing inflammation and oxidative stress.

In this study, we installed an in-house-designed diesel exhaust delivery and cannabidiol vaporization system where mice were exposed to relevant, urban traffic-related levels of diesel exhaust for 14 days and assessed integrity of alveolar tissue, gene expression shifts and changes in protein content in the lungs and other tissues of exposed mice. Systemic presence of modified proteins was also tested.

The protective effect of phytocannabinoids was investigated as well.

Data obtained in our study show subacute effects of diesel exhaust on mouse lung integrity and protein content. Emphysematous changes are documented in exposed mouse lungs. In parallel, increased levels of citrulline were detected in the alveolar lung tissue and peripheral blood of exposed mice.

Pretreatment with vaporized cannabidiol ameliorated some damaging effects.

Results reported hereby provide new insights into subacute lung tissue changes that follow diesel exhaust exposure and suggest possible dietary and/or other therapeutic interventions for maintaining lung health and healthy ageing.”

https://www.ncbi.nlm.nih.gov/pubmed/30801928

https://onlinelibrary.wiley.com/doi/abs/10.1111/bcpt.13213

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Intractable Generalized Epilepsy: Therapeutic Approaches.

 

“PURPOSE OF REVIEW:

To summarize recent developments in therapeutic options, both medical and surgical, for patients with drug-resistant generalized epilepsy syndromes, which continue to be a multifaceted challenge for patients and physicians.

RECENT FINDINGS:

Newer generation pharmaceutical options are now available, such as brivaracetam, rufinamide, lacosamide, perampanel, and cannabidiol. Less restrictive dietary options appear to be nearly as effective as classic ketogenic diet for amelioration of seizures. The latest implantable devices include responsive neurostimulation and deep brain stimulation. Corpus callosotomy is an effective treatment for some seizure types, and newer and less invasive approaches are being explored. Resective surgical options have demonstrated success in carefully selected patients despite generalized electrographic findings on electroencephalogram. The current literature reflects a widening range of clinical experience with newer anticonvulsant medications including cannabinoids, dietary therapies, surgical approaches, and neurostimulation devices for patients with intractable generalized epilepsy.”

https://www.ncbi.nlm.nih.gov/pubmed/30806817

https://link.springer.com/article/10.1007%2Fs11910-019-0933-z

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Cannabis Oil Use by Adolescents and Young Adults With Inflammatory Bowel Disease.

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“The aim of the study was to describe use of oral or sublingual cannabis oil (CO) by adolescent and young adult patients with inflammatory bowel disease (IBD).

METHODS:

A descriptive study of IBD patients 13 to 23 years of age seen between January 2015 through December 2017 at Children’s Hospital Colorado. Information obtained included chart abstraction, electronic and interview self-report, and serum cannabinoid levels. We compared CO users and cannabis non-users for clinical characteristics and perceptions of risk. Users of CO provided information on routes, patterns, motivations, and perceived benefits and problems with use.

RESULTS:

The 15 users and 67 non-users were similar for clinical characteristics and pain and appetite scores. 9 of 15 (60%) CO users had used in the past 30 days, an average of 22 ± 9 times; and 4 used daily. A variety of strengths and CBD:THC ratios were reported. Most common perceived effect of use was on sleep quality, nausea, and increase in appetite. Of the 15 users, 6 used only CO and no additional forms of cannabis. Of these 6 CO only users, 5 reported a medical reason for use, most commonly to relieve pain.

CONCLUSIONS:

Adolescent and young adults with IBD used oral CO and many used other cannabis products as well. Users perceived some medical benefit. Care teams should strive for open communication about use until further information on safety and efficacy becomes available.”

https://www.ncbi.nlm.nih.gov/pubmed/30801394

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Decreased Expression of Cannabinoid Receptors in the Eutopic and Ectopic Endometrium of Patients with Adenomyosis.

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“Adenomyosis is a common gynecologic benign disease that may have a life-long negative impact on women.

Previous studies have indicated that the endocannabinoid system may participate in the progress of endometriosis.

Our research aims to analyze the expression patterns of the typical cannabinoid receptors (CB1 and CB2), the main constituents of the endocannabinoid system, in endometrial samples derived from patients diagnosed as adenomyosis or not.

RESULTS:

In either the proliferative or the secretory phase, CB1 and CB2 protein and mRNA levels were both significantly lower in the eutopic and ectopic endometrium of adenomyosis when compared with normal endometrium. For women with adenomyosis, CB1 and CB2 protein and mRNA levels were much lower in the ectopic endometrium than the eutopic in both phases of the cycle. Both CB1 and CB2 protein and mRNA levels were increased during the secretory phase in normal endometrium, while CB1 lost its cyclic variation in the eutopic and ectopic endometrium from patients diagnosed as adenomyosis.

CONCLUSION:

The decreased expression of CB1 and CB2 in the eutopic and ectopic endometrium from patients diagnosed as adenomyosis suggests that cannabinoid receptors may participate in the pathogenesis of adenomyosis.”

https://www.ncbi.nlm.nih.gov/pubmed/30800671

“In conclusion, we found a significant decrease in the cannabinoid receptors CB1 and CB2 in the eutopic and ectopic endometrium of patients with adenomyosis, regardless of the menstrual phase, suggesting that CB1 and CB2 participate in the pathogenesis of this condition.”

https://www.hindawi.com/journals/bmri/2019/5468954/

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Cannabinoid receptor 2 deficiency exacerbates inflammation and neutrophil recruitment.

“Cannabinoid receptor (CB)2 is an immune cell-localized GPCR that has been hypothesized to regulate the magnitude of inflammatory responses.

However, there is currently no consensus as to the mechanism by which CB2 mediates its anti-inflammatory effects in vivo. To address this question, we employed a murine dorsal air pouch model with wild-type and CB2-/- 8-12-wk-old female and male C57BL/6 mice and found that acute neutrophil and lymphocyte antigen 6 complex, locus Chi monocyte recruitment in response to Zymosan was significantly enhanced in CB2-/- mice.

Additionally, levels of matrix metalloproteinase 9 and the chemokines C-C motif chemokine ligand (CCL)2, CCL4, and C-X-C motif chemokine ligand 10 in CB2-/- pouch exudates were elevated at earlier time points. Importantly, using mixed bone marrow chimeras, we revealed that the proinflammatory phenotype in CB2-/- mice is neutrophil-intrinsic rather than stromal cell-dependent. Indeed, neutrophils isolated from CB2-/- mice exhibited an enhanced migration-related transcriptional profile and increased adhesive phenotype, and treatment of human neutrophils with a CB2 agonist blocked their endothelial transmigration.

Overall, we have demonstrated that CB2 plays a nonredundant role during acute neutrophil mobilization to sites of inflammation and, as such, it could represent a therapeutic target for the development of novel anti-inflammatory compounds to treat inflammatory human diseases.”

https://www.ncbi.nlm.nih.gov/pubmed/30799631

https://www.fasebj.org/doi/10.1096/fj.201802524R

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Effect of cannabis on weight and metabolism in first-episode non-affective psychosis: Results from a three-year longitudinal study.

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“Recent evidence indicates a protective effect of cannabis on weight gain and related metabolic alterations. However, there are no previous studies on the long-term longitudinal effects of cannabis on first-episode drug-naïve patients, which would thereby avoid the confounding effects of chronicity and previous treatment exposure.

We aimed to explore the effect of cannabis smoking on weight and lipid/glycaemic metabolic measures in a sample of first-episode non-affective psychosis patients.

RESULTS::

Cannabis users at baseline presented a lower weight ( F=14.85, p<0.001), body mass index ( F=13.14, p<0.001), total cholesterol ( F=4.85, p=0.028) and low-density lipoprotein-cholesterol ( F=6.26, p=0.013) compared to non-users. These differences were also observed after three years: weight ( F=8.07, p=0.005), body mass index ( F=4.66, p=0.032) and low-density lipoprotein-cholesterol ( F=3.91, p=0.049). Moreover, those patients discontinuing cannabis use presented a higher increase in weight ( F=2.98, p=0.052), body mass index ( F=2.73, p=0.067) and triglyceride-high-density lipoprotein ratio ( F=2.72, p=0.067) than the ‘non-users’ and ‘continuers’.

CONCLUSIONS::

The study suggests that cannabis use may produce a protective effect against weight gain and related metabolic alterations in psychosis.”

https://www.ncbi.nlm.nih.gov/pubmed/30702972

https://doi.org/10.1177/0269881118822173

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Palmitoylethanolamide and Cannabidiol Prevent Inflammation-induced Hyperpermeability of the Human Gut In Vitro and In Vivo—A Randomized, Placebo-controlled, Double-blind Controlled Trial

Inflammatory Bowel Diseases

“We aimed to examine, for the first time, the effect of cannabidiol (CBD) and palmitoylethanolamide (PEA) on the permeability of the human gastrointestinal tract in vitro, ex vivo, and in vivo.

Results
In vitro, PEA, and CBD decreased the inflammation-induced flux of dextrans (P< 0.0001), sensitive to PPARα and CB1 antagonism, respectively. Both PEA and CBD were prevented by PKA, MEK/ERK, and adenylyl cyclase inhibition (P < 0.001). In human mucosa, inflammation decreased claudin-5 mRNA, which was prevented by CBD (P < 0.05). Palmitoylethanolamide and cannabidiol prevented an inflammation-induced fall in TRPV1 and increase in PPARα transcription (P< 0.0001). In vivo, aspirin caused an increase in the absorption of lactulose and mannitol, which were reduced by PEA or CBD (P < 0.001).

Conclusion

Cannabidiol and palmitoylethanolamide reduce permeability in the human colon. These findings have implications in disorders associated with increased gut permeability, such as inflammatory bowel disease.”

https://academic.oup.com/ibdjournal/advance-article-abstract/doi/10.1093/ibd/izz017/5341970?redirectedFrom=fulltext

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