Shoyoma et al published the chemical shifts of CBGVA in CD3OD, so by direct comparison of our NMR spectra to the published chemical shifts we conclude that we produced CBGVA. This is further supported by the work conducted by Bohlmann, which suggests that if the prenylation occurred at the C5 site, we would observe a proton with a chemical shift around 5.8 ppm, which we did not observe, Figure 2-15. We placed olivetolate in the active site of NphB in six different starting positions denoted as Olivetolate P1-6 in Table 2-2. We ran ROSETTA 5 times for each olivetolate position for a total of 30 designs. The mutations predicted in each design are listed in Table 2-2. For each olivetolate position we chose a consensus set of mutations to evaluate further: Consensus Group A through F . We then sought to evaluate the relative importance of each ROSSETTA suggested mutation. For each Consensus Group, we set the mutations back to WT residue, one at a time, and used ROSETTA to calculate the change in energy score . Those that caused the largest change in energy were deemed to be the most important mutants to include in the library for experimental testing. To model the olivetolic acid, we took the 4MX.sdf 3-D structure of olivetolate from the 5B09 crystal structure and added hydrogen atoms to the structure assuming pH 7 using Open Babel 2.3.1.39 A rotamer library was generated for olivetolic acid using the Bio Chemical Library molecule: Conformer Generator 3.5 using the PDB library.The olivetolic acid molecule was then manually placed into the co-crystal structure of NphB with GST and DHN with the DHN and crystallographic waters removed using pymol. The olivetolic acid was placed in 6 different positions in the active site with the plane of the olivetolate aromatic ring parallel to the GST alkyl tail and the desired prenylation site 3.7 angstroms away from the eventual carbocation mirroring the placement of DHN in the 1ZB6 crystal structure.
Residues 49, 162, 213, 224, 232, 233, 234, 271, 286, and 288 were allowed to be any amino acid during the Rosetta design with other sidechains held in a fixed position and the backbone fixed. The designed residues were in direct contact with the olivetolate and not in direct contact with GST. The reactions were incubated for 12 hours at room temperature, vertical grow systems then extracted 3 times with 100 µL of ethyl acetate. The organic extract was pooled for each reaction and the solvent was removed using a vacuum centrifuge. The samples were redissolved in 100 µL of methanol and subjected to HPLC analysis. The initial rate was plotted vs the concentration of substrate, and fit with the Michaelis-Menten equation to determine the kinetic parameters kcat and KM . Each Michaelis-Menten curve was performed in triplicate. The average and standard deviation of the kinetic parameters are reported. The time courses with olivetolate as the substrate were as follows: for WT, M1, M10 and M30 the time course was 3, 6, 9, and 12 minutes. For M25 the reactions were quenched at 1, 2, 4 and 8 minutes, and for M31 the reactions were quenched at 1, 2, 4 and 6 minutes. The conditions were altered slightly to characterize the constructs with divarinic acid as the substrate. For M31, the time course was 0.5, 1, 1.5 and 2 minutes. For M23, the time course was 5, 10, 15 and 20 minutes, and for WT NphB the time course was 8, 16, 24 and 32 minutes. The enzyme concentration for the mutants was ~27 µM, and the concentration of WT NphB was ~ 35 µM.Samples were dissolved in 200 µL of ethyl acetate. GC-MS measurements were carried out using an Agilent Model 7693 Autosampler, 7890B Gas Chromatograph, and 7250 Q-TOF Mass Selective Detector in the Electron Ionization mode. Sample injection was carried out in split mode with inlet temperature set to 280o C. Separation was carried out on an Agilent HP5-MS column with dimensions 30m x 250 µm x 0.25 µm. Ultra High Purity Grade He was used as carrier gas with the flow set to 1.1 mL/min in constant flow mode. The initial oven temperature was set to 120o C for 1 min followed by a 20o C/min ramp to a final temperature of 300o C which was maintained for 4 min. A 3.0 min solvent delay was used. EI energy was set to 15 eV.
The MSD was set to scan the 50 – 500 m/z range. Data collection and analysis were performed using Mass Hunter Acquisition and Qualitative Analysis software . Due to the increased temperature of the GC inlet, CBGA undergoes spontaneous decarboxylation as described by Radwan et al, resulting in an M+ ion at 316 m/z. The retention time corresponding to the 316 m/z ion for the CBGA standard was 10.48 minutes.A PyOx/PTA reaction was set up as detailed above. A 500 µL nonane overlay was added to the reaction in a 2 ml glass vial which was covered with 2 layers of breathable cell culture film. 2 18-gauge needles were inserted into a 15 mL falcon tube at the ~750 µL mark and the 3.5 mL mark. Luer locks to tubing connectors were connected to the needles and Viton tubing was connected to the other end of the luer lock. 18-gauge needles were connected to the other end of the tubing via a luer lock connector and inserted through the mesh covering so they were only touching the nonane layer and not the reaction. 2 mL of Tris buffer [pH 8.5] was added to the 15 mL conical tube, and 6 mL of nonane was added. The nonane was pumped through the system using a peristaltic pump such that the nonane flowed from the top of the reaction, through the buffered solution . The nonane pumped into the reservoir separated into the top layer of the 15 mL conical tube. The nonane from the top of the 15 mL conical tube was pumped into the top of the reaction vial . This essentially diluted the CBGA throughout the system driving the diffusion of CBGA into the nonane layer and out of the reaction. A gene block of CBDAS with the signaling peptide was ordered from IDT codon optimized for Pichia pastoris. The signal sequence was removed by PCR amplifying from the 28th residue of the protein sequence through the end of the protein, with overhangs compatible with the pPICZα vector. The PCR product was cloned into the pPICZα vector digested with EcoRI and XbaI using the Gibson cloning method. The product of the assembly reaction wastransformed into BL21 Gold cells a clone with the correct sequence isolated. The plasmid was digested with PmeI for 2 hours, and then purified using the Qiagen PCR purification protocol. The plasmid was transformed into Pichia pastoris X33 using electroporation. Immediately following electroporation, the cells were incubated in 1 mL of cold 1 M sorbitol and 1 mL of YPD media without shaking for 2 hours. The cells were plated on YPDS plates with 500 µg/mL of zeocin.
Colonies were screened using PCR for the presence of the CBDAS gene between the AOX1 promoter and terminator. For screening, pruning cannabis the colonies were re-suspended in 15 µL of sterile water and 5 µL of the resuspended colony was transferred into a PCR tube with 0.2% SDS. The samples were heated for 10 minutes at 99ºC, and then 1 µL was used as the template for PCR. Six colonies with positive colony PCR hits were screened for the expression of CBDAS. The six colonies were grown overnight at 30ºC in 25 mL of BMGY to obtain a saturated culture. The overnight cultures were used to inoculate a 25 mL culture in BMGY media and grown to an OD of ~2. The cells were harvested by centrifugation at 2,000 x g for 10 minutes. The cell pellet was re-suspended in 90 mL of BMMY media, and incubated at 30ºC for 5 days. Each day, 1 mL of the culture was removed for SDS-PAGE analysis, and 500 µL of methanol was added to the remaining culture. On day 3 the cultures were screened for CBDAS activity. The 1 mL culture samples were centrifuged to pellet the cells . 50 µL of the media was used in a subsequent activity assay, and the remainder of the media was stored at -80ºC in addition to the cell pellet. The assay conditions were as follows: 100 µL of 200 mM citrate buffer, 100 µM CBGA, 5 mM MgCl2, 5 mM KCl, 1 mM FAD and 50 µL of the expression media. in a final volume of 200 µL. The reactions were incubated overnight at room temperature and then extracted 3 times with 200 µL of ethyl acetate. The ethyl acetate extractions were pooled for each sample, and removed using a vacuum centrifuge. The samples were re-suspended in 200 µL of methanol and analyzed by HPLC. All clones produced active CBDAS. To convert the precursors CBGA and CBGVA into CBDA and CBGVA respectively, a secondary reaction was set up with cannabidiolic acid synthase . To produce CBDA, a PyOx/PTA enzymatic system was set up as detailed above to produce CBGA. After 24 hours 200 µL of the nonane overlay from the CBGA reaction was transferred to a CBDAS reaction vessel. In the aqueous layer: 50 mM Hepes [pH 7.0], 5 mM MgCl2, 5 mM KCl, 25 µM FAD, 0.1 mg/mL CBDAS concentrate. The reaction was incubated at 30ºC with gentle shaking. Reactions were quenched at 12, 24, 48, 72 and 96 hours. To produce CBDVA, HPLC purified CBGVA was converted to CBDVA using CBDAS. The final reaction volume was 200 µL, with 50 mM Hepes [pH 7.0], 5 mM MgCl2, 5 mM KCl, 25 µM FAD and 0.1 mg/mL of CBDAS concentrate. A 200 µL nonane overlay was added, and the reactions were incubated at 30 ºC with gentle shaking. The reactions were quenched at ~ 24, 48, 72 and 96 hours.Glucose is broken down to pyruvate through a modified glycolysis pathway that includes a purge valve system. The purge valve allows carbon flux to continue through the glycolysis pathway without building up excess NADPH. Pyruvate is converted to acetyl-CoA through the PDH bypass outlined in light blue. Acetyl-CoA is then converted into GPP via the mevalonate pathway . Finally, the GPP from the mevalonate pathway is used to prenylate aromaticpolyketide. Shown here is the prenylation of olivetolate to produce CBGA; however, olivetolate could be replaced with a wide range of aromatic substrates to generate various prenylated products. An example of the conversion of CBGA into CBD by the action of CBDAS and a spontaneous decarboxylation is shown.This brief review will cover potential benefits of cannabis in reducing persistent in- flammation and immune activation in virally suppressed people with HIV and the possible resulting clinical benefits. While no randomized clinical trials have been performed, both pre-clinical and clinical evidence supports these potential benefits. We discuss sources of inflammation in HIV, their clinical impact, the endocannabinoid system , effects of exogenous cannabinoids on the ECS and inflammation, particularly neuroinflammation, and potential treatment implications. This review attempts to weave together research threads from multiple areas: clinical, pre-clinical, in vivo and in vitro. The goal is to be integrative, not exhaustive. Overall, the observations reviewed here suggest a program of future basic and clinical research to explore the potential benefits of cannabinoids for the treatment of inflammation-related disorders in PWH. Additional work in this area is reviewed by Yadav-Samudrala and Fitting.The proportion of PWH who use cannabis is 2–3 times higher than in the general population < 10%, in part because many PWH use cannabis to manage symptoms such as nausea, sleep disorders, musculoskeletal and neuropathic pain, anxiety, and depression. There is, however, great heterogeneity in findings on therapeutic benefits of cannabis, likely due in part to extensive variation in the formulations being used. Components of cannabis, present in varying degrees depending on the formulation, include the principal psychoactive component,tetrahydrocannabinol , identified in the 1960s, cannabidiol , cannabinol , cannabigerol , cannabidivarin , and other compounds, such as terpenes. Each of these exerts unique pharmacological actions, with considerable evidence suggesting that the whole is greater than the sum of its parts , reflecting therapeutic synergies between all of the phytocannabinoids and phytoterpenoids.