Development of a Cannabinoid-Based Photoaffinity Probe to Determine the Δ8/9-Tetrahydrocannabinol Protein Interaction Landscape in Neuroblastoma Cells

Abstract Introduction: Δ9-Tetrahydrocannabinol (THC), the principle psychoactive ingredient in Cannabis, is widely used for its therapeutic effects in a large variety of diseases, but it also has numerous neurological side effects. The cannabinoid receptors (CBRs) are responsible to a large extent for these, but not all biological responses are mediated via the CBRs. Objectives: The identification of additional target proteins of THC to enable a better understanding of the (adverse) physiological effects of THC. Methods: In this study, a chemical proteomics approach using a two-step photoaffinity probe is applied to identify potential proteins that may interact with THC. Results: Photoaffinity probe 1, containing a diazirine as a photocrosslinker, and a terminal alkyne as a ligation handle, was synthesized in 14 steps. It demonstrated high affinity for both CBRs. Subsequently, two-step photoaffinity labeling in neuroblastoma cells led to identification of four potential novel protein targets of THC. The identification of these putative protein hits is a first step towards a better understanding of the protein interaction profile of THC, which could ultimately lead to the development of novel therapeutics based on THC.


Introduction
Preparations of the plant Cannabis sativa have been used throughout history in various cultures as medicinal concoctions or therapeutics, as well as for recreational or religious purposes. 1 In 1930, the isolation of cannabinol and cannabidiol as the first active substituents was achieved, 2 which was followed by the discovery of D 9tetrahydrocannabinol (THC) in 1964. 3 THC is the psychoactive constituent of marijuana and exists in two isomers: namely D 9 -THC and D 8 -THC, of which the latter is the most thermodynamically stable isomer. 4 THC treatment has been associated with therapeutic effects, such as analgesia, relaxation and fatigue, appetite stimulation, 5 antiemesis, 6 and reduction of nausea. 5 THC is used by patients suffering from multiple sclerosis (MS), 7 cancer, or AIDS. 8 In addition, preclinical data of THC indicate beneficial effects in several animal models of Alzheimer's, 9 Parkinson's, 10 and Huntington's disease. 11 However, THC is also associated with many undesirable side effects, including induction of psychoactivity, anxiety, memory loss, cardiac arrhythmias, and addiction. 12 Both D 9 -THC and D 8 -THC have similar affinity to the cannabinoid receptor type 1 (CB 1 R) and type 2 (CB 2 R). 13,14 The CB 1 R is the most abundant G proteincoupled receptor (GPCR) in the mammalian brain, 15 whereas the CB 2 R is predominantly present in peripheral tissues and cells of the immune system. 16 Most of the physiological effects of THC are mediated via the CB 1 R and CB 2 R as demonstrated by the use of specific CB receptor antagonists or genetically modified mice that lack the CB receptors. [17][18][19][20] It is, however, hypothesized that THC may have other non-CB receptor targets. A study, using CB 1 R and CB 2 R knockout mice, showed similar analgesia upon THC administration compared with the equivalent wild-type mice in the tail-flick test. 21 This effect was not observed in the hotplate test, which requires spinal processing of nociceptive information. These observations suggest the existence of another protein target in the brain. Previously, orphan GPCRs GPR55 and GPR18 and peroxisome proliferator-activated receptor gamma were identified to bind to THC, but it is unclear whether these targets are responsible for some of the physiological effects of THC. [22][23][24] Therefore, a more complete view of the protein interaction of THC in neuronal cells is desirable.
Photoaffinity-based protein profiling (pAfBPP) has been previously used to map the protein interaction landscape of small molecules. 25,26 Photoaffinity probes use a light-responsive element to covalently crosslink the compound with its target protein upon irradiation. To circumvent the problems associated with large reporter groups, photoaffinity probes with a bioorthogonal ligation handle (e.g., alkyne), to introduce a fluorescent or affinity tag (e.g., biotin) after crosslinking to a protein, have emerged as powerful tools to visualize small molecule-protein interactions in living systems. 27 Previously, we applied two-step pAfBPP to capture and visualize the CB 2 R on human cells. 28 Here, it was envisioned that two-step pAfBPP could be used to map the THC interaction landscape in neuroblastoma cells.
To this end, photoaffinity probe 1 (Fig. 1), a D 8 -THC analog carrying a diazirine as the photoreactive moiety and a terminal alkyne as the ligation handle, was developed. Probe 1 was synthesized in 14 steps and was found to have high affinity for both cannabinoid receptors (CBRs). The protein interaction landscape of THC was mapped in Neuro2A cells (a fast-growing neuroblastoma cell line with several neuronal properties), in which four putative novel targets of THC were identified.

Materials and Methods
Chemistry General remarks. All reactions were performed using air-or flame-dried glassware. Solvents were purchased from Sigma-Aldrich, and dry solvents were analytically dried by storing them for 24 h on activated molecular sieves. Use of dry solvents is mentioned explicitly. Reagents were purchased from Sigma-Aldrich, Acros Organics, and Merck and used without further purification. All moisture sensitive reactions performed under an Ar atmosphere are mentioned explicitly. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV 400 MHz spectrometer at 400 and 100 MHz, respectively, using CDCl 3 or CD 3 OD as solvent, unless stated otherwise. Chemical shift values are reported in ppm with TMS or solvent resonance as the internal standard (CDCl 3 / TMS, d 0.00 for 1 H [TMS], d 77.16 for 13 C [CDCl 3 ]; CD 3 OD, d 3.31 for 1 H, d 49.00 for 13 C). Data are reported as follows: chemical shifts (d) in ppm, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, ddd = doublet of doublet of doublet, dt = doublet of triplet, t = triplet, td = triplet of doublet, q = quartet, br s = broad singlet, and m = multiplet), coupling constants J (Hz), and integration. High-resolution mass spectra were recorded on a Thermo Scientific LTQ Orbitrap XL. Liquid Chromatography was performed on a Finnigan Surveyor liquid chromatography-mass spectrometry (LC/MS) system, equipped with a C18 column. Thin layer chromatography (TLC) analysis was performed on Merck silica gel 60/Kieselguhr F254, 0.25 mm TLC plates. Compounds were visualized by ultraviolet (UV) irradiation or with a KMnO 4 stain (K 2 CO 3 (40 g), KMnO 4 (6 g), and H 2 O (600 mL)). Molecules shown are drawn using the ChemDraw Professional 16.0.

Biology
General remarks. All common reagents were purchased from commercial sources and used as received. Probe 1 was synthesized as described above, D 9 -THC, D 8 -THC and CY5-N 3 were synthesized according to previously published procedures 29,30 and biotin-N 3 was purchased from Bio-Connect Life Sciences. Cannabinoid receptor ligands CP55940 and AM630 were obtained from Sigma Aldrich (St. Louis, MO), and rimonabant was obtained from F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Reagents used for the pulldown procedure are: avidin-agarose from egg white (50% glycerol suspension from Sigma Aldrich), 10· phosphate buffered saline (PBS) (proteomics grade, Sigma Aldrich) and Trypsin, sequencing grade (Promega). The CaproBoxÔ was kindly provided by Caprotec Bioanalytics GmbH, Berlin. All buffers and solutions were prepared using Millipore water (deionized using a MilliQ A10 BiocelÔ, with a 0.22 lm filter) and analytical grade reagents and solvents. Buffers are prepared at rt and stored at 4°C, unless stated otherwise.
Cell culture and membrane preparation. CHOK1hCB 1 _ bgal and CHOK1hCB 2 _bgal (source; DiscoveRx, Fremont, CA) were cultured in Ham's F12 Nutrient Mixture supplemented with 10% fetal calf serum, 1 mM glutamine, 50 lg/mL penicillin, 50 lg/mL streptomycin, 300 mg/mL hygromycin, and 800 lg/mL geneticin in a humidified atmosphere at 37°C and 5% CO 2 . Cells were subcultured twice a week at a ratio of 1:20 on 10cm ø plates by trypsinization. For membrane preparation, the cells were subcultured 1:10 and transferred to large 15 cm diameter plates. Next, the cells were detached by scraping them into 5 mL PBS and collected and centrifuged at 1000 g for 5 min. Pellets derived from 30 plates were added together and resuspended in 20 mL ice-cold buffer (50 mM Tris-HCl, 5 mM MgCl 2 , pH 7.4). An UltraThurrax homogenizer was used to homogenize the cell suspension. Membranes and the cytosolic fraction were separated by ultracentrifugation (100,000 g, with a Ti-70 rotor in a Beckham Coulter Ultracentrifuge) at 4°C for 20 min. The supernatant was discarded and the pellet was resuspended in 10 mL of the same buffer and the homogenization and centrifugation steps were repeated. Supernatant was discarded and the pellet was resuspended in 5 mL buffer. Aliquots of 200 lL were frozen at À80°C until further use. Protein concentration was determined using the BCA method. 31 [ 3 H]CP55940 displacement assay. The affinity of probe 1 on CBRs was determined on membrane fractions of CB 1 R-or CB 2 R overexpressing CHO cells, as described previously. 13 Membrane aliquots containing 5 lg (CHOK1hCB 1 _bgal) or 1 lg (CHOK1hCB 2 _bgal) of membrane protein in 100 lL assay buffer (50 mM Tris-HCl, 5 mM MgCl 2 , 0.1% BSA, pH 7.4) were incubated at 30°C for 1 h, in presence of 3.5 nM (CHOK1hCB 1 _bgal) or 1.5 nM [ 3 H]CP55940 (CHOK1hCB 2 _bgal). Nonspecific binding was determined in the presence of 10 lM SR141716A (CHOK1hCB 1 _bgal) or 10 lM AM630 (CHOK1hCB 2 _bgal). Incubation was terminated by rapid filtration performed on GF/C filters (Whatman International, Maidstone, United Kingdom), presoaked for 30 min with 0.25% polyethyleneimine (PEI), using a Brandel harvester (Brandel, Gaithersburg, MD). Filter-bound radioactivity was determined by scintillation spectrometry using a Tri-Carb 2900 TR liquid scintillation counter (Perkin Elmer, Boston, MA).

Data analysis
Graphs and statistics were performed with GraphPad Prism 7, using the results of three independent experiments performed in duplicate. The nonlinear regression analysis for one site-Fit K i (constrains: top = 100 and bottom = 0) was used to obtain logK i values, which are provided by Prism by direct application of the Cheng-Prusoff equation 32  Two-step photoaffinity labeling, gel-based analysis Wild type (WT)CHO, CB 1 R, and CB 2 R membrane aliquots were diluted to 2 lg/lL and homogenized for 20 sec with a Heidolph Silent crusher at 25,000 rpm, and benzonase was added (1:10,000 dilution from working stock of 2,500,000 U/mL, assay concentration: 250 U/mL). Eighteen microliters of protein was added per well of a 96-well flat bottom plate and 20 lM CP55940 or MilliQ water with the same% of dimethylsulfoxide (DMSO) was added, but the sample without UV was kept in an Eppendorf tube protected with alumina foil. After incubation of 30 min at rt, 2 lM LEI121 or probe 1, or MilliQ water with the same% of DMSO was added, and the protein was again incubated for 30 min at rt. The samples were then diluted with 30 lL 50 mM Hepes buffer and irradiated for 5 min with CaproBox, preset at 350 nm and cooled during irradiation. The ligation reaction was then performed with 5 lL click master mix per sample (0.455 mM CuSO 4 , 2.73 mM NaAsc, 0.09mM THPTA, 3.6lM Cy5-N 3 ). The click mix is prepared as follows: 2.5 lL 10 mM CuSO 4 and 1.5 lL 100 mM NaAsc were mixed together until the copper is fully reduced (visible by the change from the rusty brown color to bright yellow), then 0.5 lL 10 mM THPTA and 0.5 lL 0.4 mM CY5-N 3 were added. After incubation in the dark for 1 h, the protein was denatured with 18 lL 4 · Laemmli sample buffer, and the samples were resolved on a 12.5% acrylamide gel (12 lL per sample per well). Bio-Rad ImageLab was used for gel analysis and quantification.
Chemoproteomic profiling of THC protein targets Neuro2A cells were cultured at 37°C with 7% CO 2 in DMEM supplemented with 10% New Born Calf serum, 10% fetal calf serum, 1 mM glutamine, 50 lg/mL penicillin, and 50 lg/mL streptomycin and passaged twice a week. Cells were washed with PBS, then pretreated in PBS, containing 1 mM MgCl 2 and 1 mM CaCl 2 , with or without 10 lM THC, for 30 min at 37°C. Then, 1 or 10 lM probe 1 (or the same amount of DMSO for the untreated control) was added (final concentration in a total volume of 3 mL) and incubated for 30 min at 37°C. The solution was removed from the cells and replaced by 1.5 mL PBS containing 1 mM MgCl 2 and 1 mM CaCl 2 , then the plates were immediately irradiated (except the No UV control) with CaproBox (350 nm) for 5 min, and the cells were harvested by scraping.
The cells were pelleted (10 min, 1200 g, 4°C), supernatant removed, and resuspended in 250 lL 50 mM Hepes buffer. The cells were destroyed with the Heidolph Silent Crusher (20 sec, 25,000 rpm). Samples were sonicated for 10 · 2.5 sec with 0.5 sec interval (using a probe sonicator from Branson, Digital Sonifier) and 2 lL of 10% sodium dodecyl sulfate (SDS) was added. If samples were frozen at À80°C before continuation of the experiment, the samples were sonicated again for 10 · 0.5 sec with 0.5 sec interval using a probe sonicator. The protein content was quantified using Bradford 33  for 10 min. The supernatant was removed and the pellet was resuspended in 600 lL MeOH using sonication (6 · 0.5 sec, interval 0.5 sec). The protein was pelleted at 20,238 g for 10 min and the supernatant removed. The protein was then denatured in 15 min at rt with 500 lL 1% SDS containing 25 mM NH 4 HCO 3 , followed by reduction (65°C, 15 min, 700 rpm shaking) using 5 lL 1 M DTT per sample. Samples were cooled to rt before alkylation with 40 lL 0.5 M IAA per sample for 30 min at rt in the dark. One hundred forty microliters of 10% SDS was added per sample, and each sample was added to 6 mL PBS containing 50 lL avidin beads (prewashed with PBS 3 · , pelleting at 2000 g for 2 min), and incubated for 2 h at rt while rotating. Beads were pelleted (2000 g, 2 min) and washed with PBS with 0.5% SDS (1·) and with PBS (3·).
On-bead digest of peptides was performed overnight at 37°C, at 1000 rpm with digestion buffer (250 lL per sample, recipe: 300 lL 1 M Tris, 300 lL 1 M NaCl, 3 lL of 1 M CaCl 2 , 60 lL ACN, 3 lL 0.5 lg/lL Trypsin and 2334 lL MilliQ). Samples were quenched with 12.5 lL formic acid (FA) and beads were removed using a Biospin column (600 g, 2 min). Samples were added on C18 StageTips (conditioned with 50 lL MeOH, then 50 lL of 0.5% (v/v) FA in 80% (v/v) ACN/MilliQ (solution B), then 50 lL 0.5% (v/v) FA in MilliQ (solution A), each conditioning step was performed using centrifugation for 2 min at 600 g) by spinning for 15 min at 800 g, then washed with solution A for 10 min at 800 g, and eluted with solution B for 5 min at 800 g into low-binding Eppendorf tubes. Samples were evaporated using an Eppendorf SpeedVac (Eppendorf Concentrator Plus 5301) and 50 lL of LC/MS solution was added (recipe for 2 mL: 1900 lL MilliQ, 60 lL ACN, 2 lL FA, 40 lL of 1 nmol/lL yeast enolase stock). Samples were measured using a Nano-ACQUITY UPLC System coupled to a SYNAPT G2-Si high-definition mass spectrometer (Waters). The peptides were separated using an analytical column (HSS-T3 C18 1.8 lM, 75 lM · 250 mm, Waters) with a concave gradient (5-40% ACN in H 2 O with 0.1% FA). [Glu 1 ]-fibrinopeptide B was used as lock mass. Mass spectra were acquired using the UDMS e method. 34 The mass range was set from 50 to 2000 Da with a scan time of 0.6 sec in positive, resolution mode. The collision energy was set to 4 V in the trap cell for low-energy MS mode. For the elevated energy scan, the transfer cell collision energy was ramped using drift time-specific collision energies.
Raw data were processed using Progenesis QI for Proteomics (3.0, Waters), with lock mass correction (7,858,426 Da) and a database search was performed against the proteomic database of Mus musculus, with trypsin as digestion reagent, max two missed cleavages, carbamidomethyl C as a fixed modification, oxidation M as a variable modification, and FDR set to 1%. Relative quantitation using Hi-3 was performed after filtering the peptides on score (cutoff 5).

Data analysis
The average normalized abundance of proteins in sample replicates of two independent experiments was used to calculate the ratio of proteins in the probe-treated sample and the ''No UV'' sample, to determine the level of enrichment by UV-irradiation (Fig. 5A). Protein targets that were enriched >2 · by probe 1 are shown in Supplementary Table S1. Proteins that were <2-fold enriched and highly abundant (>20%) in the ''CRAPome'' database 35 www.crapome.org/, version 1.1) were excluded from further analysis. Gene ontology data of the *150 resulting putative probe targets (Fig. 5B, C) were derived using the DAVID Bioinformatics Database (https://david.ncifcrf.gov/home .jsp, version 6.8).
In THC competition experiments, the normalized abundance of proteins in sample replicates of three independent experiments was used to calculate the ratio of proteins in THC-pretreated samples over probetreated samples. The average of the mean ratios of the triplicate samples of each independent experiment was used to calculate the effect of THC (as fold change), and a Student's t-test was used to determine whether the fold change was significantly lower than 1, indicating a significant reduction of the abundance of that particular protein in the THC-treated samples (Fig. 5D). A p-value less than 0.05 was considered statistically significant. Proteins that showed <50% inhibition (Supplementary Table S2) were excluded from gene ontology analysis. This analysis yielded one putative protein target of D 9 -THC and three putative targets of D 8 -THC (Fig. 5A, B, [red dots] and E). Gene ontology and KEGG pathway analysis of the resulting putative protein targets was derived using the DAVID Bioinformatics software (https:// david.ncifcrf.gov/home.jsp, version 6.8). In addition, it was investigated whether these proteins are associated with pathophysiologies or diseases using the Online Mendelian Inheritance in Man (OMIM) database (www.omim.org/, September 2017).

Results and Discussion
Synthesis of photoaffinity probe 1 To identify the best position in THC to introduce the photoreactive group and the ligation tag, an analysis of previously reported structure-activity relationship data of THC analogs was conducted. 36 This led to the design of probe 1, which contains a diazirine and ligation handle on the alkyl side chain of THC. An advantage of this design is the di-rect coupling of the bifunctional side chain as ''minimalist linker.'' 37 The synthesis of probe 1 commenced with reduction of commercially available 3,5-dihydroxybenzoic acid 2 to corresponding benzyl alcohol 3 in near-quantitative yield, using dimethyl sulfide complex of borane, along with co-reagent trimethoxyborate (Fig. 2). 38 Benzyl alcohol 3 was oxidized to aldehyde 4 using a stoichiometric amount of Jones reagent, which prevented  D 8 -THC was synthesized in two steps from olivetol and (S)-cis-verbenol using the same procedures, in a similar yield and comparable to literature. 30 Intermediate 7 was deprotected by Ag(I) salts, using a AgNO 3 /wet EtOH system. 40 Overoxidation of the resulting aldehyde to the equivalent benzoic acid was prevented using a modified workup, comprised additional washing steps with 10 wt.% Na 2 SO 3 (aq.), on top of the sole filtration step described in the literature. 40 The resulting aldehyde was not isolated but subjected directly to phenol protection with TBS ether, to yield aldehyde 8 in excellent yield over 2 steps. Reduction of 8 to benzyl alcohol 9 with LiBH 4 proceeded with near-quantitative yield, and a subsequent Appel reaction afforded benzyl bromide 10 in excellent yield. Benzyl mercaptan 11 was obtained by substitution of the bromide by thiourea, followed by cleavage of the amidine moiety from the sulfur atom with NaOH (aq).
The synthesis of minimalist linker 17 started with the functionalization of commercially available ethyl acetoacetate 12 to propargyl ketoester 13 via generation of the dienolate under strongly basic conditions, followed by regiospecific electrophilic attack by propargyl bromide. 37 Ketoester 13 was then protected with ethylene glycol to the corresponding ketal, with azeotropic removal of water under acidic conditions, followed by direct reduction of the ester group with LiAlH 4 , afforded corresponding alcohol 14 with excellent yield over 2 steps. 41 Deprotection of ketal 14 afforded ketone 15 in a near-quantitative yield, which was next functionalized by refluxing in liquid NH 3 , followed by addition of hydroxylamine-O-sulfonic acid. The resulting crude diaziridine was subsequently oxidized to diazirine 16 using molecular iodine in mild basic conditions and was obtained in high yield over 2 steps. Sixteen then underwent a modified Appel reaction to generate minimalist linker 17 as alkyl iodide, in excellent yield.
Finally, minimalist linker 17 was coupled overnight at 30°C to resorcinol mercaptan 11 using K 2 CO 3 in a 2:1 THF/DMF solvent system and the crude sulfide underwent rapid TBS ether deprotection in the presence of TBAF, affording target probe 1 in high yield over two steps. Overall, probe 1 was synthesized from commercially available 3,5-dihydroxybenzoic acid 2 in 14 steps, with a total yield of 18%.

CBR binding affinity of probe 1
To test the affinity of probe 1 on both the CB 1 R and CB 2 R, a [ 3 H]CP55940 displacement assay was used  Two-step photoaffinity labeling of CB 1 R and CB 2 R The ability of probe 1 to label CBRs in membranes of CB 2 R-or CB 1 R-overexpressing CHO cells was tested using a two-step photoaffinity labeling assay for gelbased imaging as previously reported. 28 Probe 1 at a concentration of 2 lM, which is more than sufficient to fully occupy the binding site of the receptors, did not label either one of the CBRs (Fig. 4). Of note, positive control LEI121, a CB 2 R-selective photoaffinity probe previously reported, 28 did show profound labeling of CB 2 R. This may indicate that the diazirine of probe 1, positioned on the ''flexible'' alkylic side chain, is not in close proximity to the amino acid residues in the binding site of CB 1 R and CB 2 R to form a covalent bond with the protein. This observation is in line with previous results showing that the position of the photoreactive diazirine on the scaffold of CBR ligands is essential to capture the CBR. 28 Chemoproteomic profiling of THC protein targets using probe 1 The ability of probe 1 as a chemical tool to identify additional, non-CBR, protein targets of THC was evaluated next. Live Neuro2A cells (a fast-growing neuroblastome cell line with neuronal properties and therefore a suitable test system) were incubated with probe 1 (10 lM). Vehicle-treated and nonirradiated cells were used as control. Ligation with biotin-N 3 for affinity enrichment on avidin agarose beads enabled identification of nearly 800 proteins by mass spectrometry-based proteomics (Fig. 5A). Nearly 200 proteins were more than twofold enriched by probe 1 compared to the untreated control, of which *50 proteins were also found in the ''CRA-Pome'' database (Contaminant Repository for Affinity Purification). 35 The CRAPome database constitutes a list of frequently identified proteins (e.g., ribosomal proteins or histones) in photoaffinity labeling experiments regardless of the type of probe. These CRAPome proteins can, therefore, be considered as false positives, suggesting that nearly 150 unique probe targets were identified. Gene ontology analysis revealed that protein targets of probe 1 are mostly located in the endoplasmic reticulum, mitochondria and membranes or in the cytoplasm (Fig. 5B). The proteins are mostly associated with energy metabolism and protein transport (Fig. 5C). Probe targets that were more than twofold enriched are shown in Supplementary Table S1.
To assess which of the probe targets also interact with THC, competition experiments with probe 1 (1 lM) and D 8 -THC (10 lM) or D 9 -THC (10 lM) were performed. This resulted in one putative protein target of D 9 -THC (Cox4i1) and three for D 8 -THC (Reep5, Mtch2, Gnb1) (Fig. 5A, D [red dots]) for which the labeling of the protein by probe 1 was reduced by 40-70% (Fig. 5E, Supplementary Table S2). It should be noted that putative protein target Reep5 was enriched only 1.5-fold by probe 1, but is listed because it had the largest reduction after THC-pretreatment (69% -6%). The absence of complete inhibition by THC may be due to a low affinity to these proteins, because an inhibition between 40% and 70% indicates an IC 50 in the micromolar range. However, as this was measured in presence of 1 lM probe, the actual pK i of THC for these proteins may be a bit higher. Cox4i1 is involved in energy metabolism, whereas Reep5, Mtch2, and Gnb1 are associated with protein modification and transport, energy metabolism, apoptosis and DNA maintenance, or signal transduction, respectively (Table 1). Interestingly, these four putative protein targets are associated with various neurological diseases as reported in the KEGG and OMIM database (Table 2). 42,43 Conclusions The aim of this study was to identify unknown protein targets of THC using photoaffinity labeling and chemical proteomics. To this end, D 8 -THC-derived probe 1 was synthesized in 14 steps with a total yield of 18%. Probe 1 had nanomolar affinity for both CBRs, but was not able to undergo a covalent addition with the CBRs and therefore unable to visualize the CBRs in an established gel-based photoaffinity labeling assay. Different positioning of the photoreactive group in the probe, for example, on the more rigid tricyclic core of the scaffold to enable a stronger interaction between diazirine and amino acid residues, might allow the covalent capturing of CBRs.
Photoaffinity labeling of the proteome of live Neu-ro2A cells resulted in the identification of *150 target proteins. Competition studies with THC significantly reduced enrichment of four proteins by probe 1, which suggests that THC has a limited interaction profile in Neuro2A cells. Reep5, Mtch2, and Gnb1 were identified as putative protein targets of D 8 -THC, whereas Cox4i1 was targeted by D 9 -THC. These targets are mostly involved in protein handling, energy metabolism, apoptosis or DNA maintenance, which may suggest that long-term exposure of THC may affect a variety of (epigenetic) functions of brain cells. Of note, the affinity and functional activity of THC on these four proteins need to be further validated in orthogonal experiments using recombinant expression systems, followed by experiments to identify a mechanistic link between these proteins and physiological effects of THC.
Taken together, the identification of the putative protein hits described is a first step toward a better understanding of the protein interaction profile of THC, which could ultimately lead to the development of novel therapeutics based on THC.