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Home , Caryophyllene, JZL195, Reuptake inhibitor

Chemical probes to potently and selectively inhibit PNAS PLUS endocannabinoid cellular

Andrea Chiccaa,1, Simon Nicolussia,1, Ruben Bartholomäusb, Martina Blunderc,d, Alejandro Aparisi Reye, Vanessa Petruccia, Ines del Carmen Reynoso-Morenoa,f, Juan Manuel Viveros-Paredesf, Marianela Dalghi Gensa, Beat Lutze, Helgi B. Schiöthc, Michael Soeberdtg, Christoph Abelsg, Roch-Philippe Charlesa, Karl-Heinz Altmannb, and Jürg Gertscha,2

aInstitute of Biochemistry and Molecular Medicine, National Centre of Competence in Research NCCR TransCure, University of Bern, 3012 Bern, Switzerland; bDepartment of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, 8093 Zurich, Switzerland; cDepartment of Neuroscience, Biomedical Center, Uppsala University, 751 24 Uppsala, Sweden; dBrain Institute, Universidade Federal do Rio Grande do Norte, Natal 59056- 450, Brazil; eInstitute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, D-55099 Mainz, Germany; fCentro Universitario de Ciencias Exactas e Ingenierías, University of Guadalajara, 44430 Guadalajara, Mexico; and gDr. August Wolff GmbH & Co. KG Arzneimittel, 33611 Bielefeld, Germany

Edited by Benjamin F. Cravatt, The Scripps Research Institute, La Jolla, CA, and approved May 10, 2017 (received for review March 14, 2017) The extracellular effects of the endocannabinoids and ECs over arachidonate and other N-acylethanolamines (NAEs) 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after (15–19). However, although suitable inhibitors are available for crossing cellular membranes by facilitated diffusion. The lack of potent most targets within the ECS (20), the existing AEA uptake in- and selective inhibitors for endocannabinoid transport has prevented hibitors lack potency and show poor selectivity over the other the molecular characterization of this process, thus hindering its components of the ECS, in particular FAAH (21, 22). biochemical investigation and pharmacological exploitation. Here, Given the lack of appropriate inhibitors, it is not unexpected we report the design, chemical synthesis, and biological profiling of that the process of EC cellular uptake has remained largely natural product-derived N-substituted 2,4-dodecadienamides as a selec- uncharacterized at the biochemical level and, therefore, is also = tive endocannabinoid uptake inhibitor. The highly potent (IC50 10 nM) controversially discussed (21). Here, building on previous work on inhibitor N-(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pro- N-alkyl-2,4-dodecadienamides from Echinacea purpurea (L.) nounced -dependent anxiolytic, antiinflammatory, Moench, which have been shown to interact with the ECS (22, 23), and analgesic effects in mice by increasing endocannabinoid levels. a series of derivatives and analogs of these natural unsaturated A tailored WOBE437-derived diazirine-containing photoaffinity fatty acid amides were synthesized, and their effects on EC probe (RX-055) irreversibly blocked membrane transport of transport were investigated. This work has resulted in the both endocannabinoids, providing mechanistic insights into this com- identification of (2E,4E)-N-[2-(3,4-dimethoxyphenyl)ethyl] plex process. Moreover, RX-055 exerted site-specific anxiolytic effects dodeca-2,4-dienamide (WOBE437; 1) as a highly potent and on in situ photoactivation in the brain. This study describes suitable in- selective EC uptake inhibitor, which was extensively profiled. In hibitors to target endocannabinoid membrane trafficking and uncovers addition, we have designed and synthesized the WOBE437- an alternative endocannabinoid pharmacology. derived photoaffinity probe RX-055 (2) as a potent and irre- endocannabinoid reuptake | 2-AG | inhibitor | | versible EC uptake inhibitor, which has enabled unambiguous lipid transport insights into the uptake process.

Significance he endocannabinoid system (ECS) is a pan-organ lipid sig- Tnaling network that modulates numerous biological pro- cesses, including and immune function (1, 2). Suitable chemical tools have been instrumental in the discov- The major endogenous [i.e., endocannabinoids (ECs)] ery and characterization of the endocannabinoid system. However, the lack of potent and selective inhibitors for endo- for cannabinoid receptors CB1 and CB2 are the arachidonic acid (AA)-derived lipids 2-arachidonoyl glycerol (2-AG) and cannabinoid transport has prevented the molecular character- N-arachidonoylethanolamine [anandamide (AEA)]. Altered EC ization of this process. Current uptake inhibitors are poorly signaling in the brain has been implicated in nociception (3), bioavailable to the central nervous system (CNS) and weakly learning and memory (4), anxiety (5), and depression (6). The selective because they also inhibit fatty acid amide hydro- indirect modulation of EC levels may lead to fewer side effects lase (FAAH), the major anandamide-degrading . Few studies have addressed the uptake inhibition of 2-arachidonoyl than the direct activation of CB1 receptors in terms of neuro- transmission, , and immunomodulation (7). glycerol (2-AG), which is the major endocannabinoid. Here, we report a highly potent and selective endocannabinoid reuptake CB1 receptor agonists are intrinsically associated with strong central side effects that are far less pronounced for increasing inhibitor. Our data indicate that endocannabinoid transport EC levels upon blockage of the main EC hydrolytic fatty across the membrane can be targeted, leading to general acid amide hydrolase (FAAH) and antiinflammatory and anxiolytic effects in mice. (MAGL). In addition to general antiinflammatory and analgesic Author contributions: A.C., S.N., M.B., B.L., K.-H.A., and J.G. designed research; A.C., S.N., effects, the modulation of EC tissue concentrations is a prom- R.B., M.B., A.A.R., V.P., I.d.C.R.-M., J.M.V.-P., and M.D.G. performed research; M.S., C.A., ising therapeutic approach to treat diseases related to the central and R.-P.C. contributed new reagents/analytic tools; A.C., S.N., R.B., M.B., A.A.R., V.P., nervous system (CNS) (8, 9). Pharmacological strategies to treat I.d.C.R.-M., J.M.V.-P., B.L., H.B.S., M.S., and J.G. analyzed data; and A.C., S.N., M.S., neuropsychiatric disorders currently focus on the inhibition of K.-H.A., and J.G. wrote the paper. EC degradation (10). FAAH and MAGL inhibitors such as The authors declare no conflict of interest. URB597 (11) and JZL184 (12), respectively, have been in- This article is a PNAS Direct Submission. strumental to elucidate the role of AEA and 2-AG in rodent 1A.C. and S.N. contributed equally to this work. models of anxiety and depression (6, 12–14). Although AEA and 2To whom correspondence should be addressed. Email: [email protected]. 2-AG have different intracellular fates, they may share a com- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mon mechanism of membrane trafficking that is selective for 1073/pnas.1704065114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1704065114 PNAS Early Edition | 1of10 Downloaded by guest on September 30, 2021 Results 2,691; and 1,242 nM, respectively (Fig. 1A and SI Appendix,Table Discovery of WOBE437 as a Highly Potent AEA Uptake Inhibitor. Based on S1). The most potent compounds were those with an N-phenethyl the natural product (2E,4E)-N-isobutylamidedodeca-2,4-dienamide head group, which showed IC50 values in the low nanomolar range SI Appendix (3)(SI Appendix,Fig.S1andTableS1) from the medicinal plant ( ,TableS1). Compared with the unsubstituted parent 8 = E. purpurea as a starting point, we synthesized a library of 634 ana- compound (IC50 1,142 nM), the presence of a single methoxy group at different positions of the aryl system (20, 21,and22) was logs and derivatives with varying alkyl chain lengths and structure of = SI Appendix SI Experimental associated with approximately fourfold increased potency (IC50 the head group moiety ( ,Fig.S1and – A B SI Appendix Procedures). All new derivatives were tested for AEA uptake in- 198 271 nM) (Fig. 1 and and ,TableS1). Potency SI Appendix SI could be increased dramatically by 3,4-dimethoxylation, providing the hibition in U937 cells by using a screening assay ( , 1 Experimental Procedures highly potent and selective inhibitor (WOBE437)withanIC50 value ). From a total of 348 analogs, we identified ± 2E,4E N of 10 8 nM for AEA uptake inhibition (using 100 nM total AEA) the dodeca- -dienoyl -alkylamide scaffold as the most prom- and an outstanding 1,000-fold selectivity over FAAH (Fig. 1 A and B ising framework for the development of EC transport inhibitors SI Appendix > A and ,TableS1). The structurally related benzodioxole being highly selective over FAAH ( 100-fold selectivity; Fig. 1 and (24) and dihydrobenzodioxine (25)analogswere∼13timeslesspo- B N N ). For instance, -(pyridin-3-yl)ethyl, -(2-methoxyphenyl)ethyl, tent (Fig. 1 A and B and SI Appendix,Fig.S2). Modifications in the N E E and -(2,3-dihydro-1,4-benzodioxin-6-yl)ethyl dodeca-2 ,4 - acyl part of WOBE437 (chain length and degree of unsaturation) led 14 20 25 dienamide ( , ,and , respectively) exhibited IC50 values in the to less potent analogs (SI Appendix,Fig.S3). The similarity between nanomolar range for AEA uptake and 60- to 105-fold selectivity over the head groups in WOBE437 and the minor EC N-arachidonoyl AEA hydrolysis (Fig. 1 A and B and SI Appendix,TableS1). The (24) prompted us to also prepare (2E,4E)-dodecadienoyl ethyl linker connecting the amide group with the aromatic system dopamine (32) and the AA-based analog of WOBE437 (i.e., 34). provided the optimal length for effective uptake inhibition, as ex- Interestingly, 32 was ∼100-fold less potent than WOBE437, whereas emplified by the comparison between the 14, pyridin-3-ylmethyl (15), 34 retained significant potency and was only sixfold less potent and pyridin-3-yl (16) head groups, which led to IC50 values of 101; (SI Appendix, Table S1). Similarly, the (2E,4E)-dodecadienoyl

O ABCDR N H Neuro2a cells U937 cells )lortno AEA 2-AG -4 Guineensine 15 120 120 20 N N c N ] (M)) OMDM-2 14 fo%(ekatpuGA-2roAE 50 -5 UCM707 100 100 WOBE437 1142 nM (8) 101 nM (14) 2691 nM (15) 1242 nM (16) 16 21 80 O -6 AM404 80 25 24 VDM11 60 O -7 60 22 O -8 40 40 198 nM (20) 271 nM (21) 221 nM (22) URB597 O O O -9 LY2183240 20 20

O O O -10 AEA uptake (% of control) 0 0 142 nM (24) 129 nM (25) 10 nM (1; WOBE437) [IC (log hydrolysis AEA -10 -9 -8 -7 -6 -5 -4 -11-10 -9 -8 -7 -6 -5 -4 -12-11-10-9 -8 -7 -6 -5 -4 A AEA uptake (log [IC50] (M)) log [WOBE437] (M) log [WOBE437] (M)

EGNeuro2a F Cortical Neuro2a H Vehicle WOBE437 Vehicle WOBE437 Vehicle WOBE437 )sllecnoill )slle )sllecnoill uptake hydrolysis 12 12 160 cn 10 10 o il l i 120 im/lomp i m/lomp(ekatpuGA-2

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Fig. 1. Design and in vitro pharmacological characterization of the highly potent and selective EC transport inhibitor WOBE437. (A) Chemical structures of a

selection of dodeca-2E,4E-dienoyl N-alkylamides and IC50 values for AEA uptake. WOBE437 is highlighted in red. (B) Correlation of IC50 values for AEA uptake and hydrolysis inhibition of the most potent N-alkylamides and reference compounds (FAAH inhibitors are in green; AEA uptake inhibitors are in blue). No significant correlation between these two processes (indicated with the gray dotted line) was observed [Pearson’s ρ = 0.258, P = 0.214, not significant (α = 0.05)]. WOBE437 (red) shows exceptional potency for AEA uptake inhibition and selectivity over FAAH inhibition. (C) Inhibition of AEA uptake by WOBE437 in Neuro2a cells (after 10 min) showing normalized data. (D) Inhibition of AEA and 2-AG uptake induced by WOBE437 in U937 cells (after 5 min) showing normalized data. Time course of AEA uptake in Neuro2a cells (E; 100 nM WOBE437 vs. vehicle) and primary rat cortical neurons (F;1μM WOBE437 vs. vehicle). (G) Time course of 2-AG uptake in Neuro2a cells in the presence of WOBE437 (5 μM) or vehicle. (H) WOBE437 does not inhibit any of the major hy- drolases in membranes obtained from mouse brain (ABPP experiment). URB597, JZL184, JZL195, and WWL70 were used as positive controls for FAAH, MAGL, dual FAAH/MAGL, and ABHD6 inhibition, respectively. Data show mean values ± SD from at least three independent experiments, each performed in triplicate. For D, data show mean values ± SEM from 10 independent experiments, each performed in triplicate. Statistical significance was calculated with two-tailed unpaired t test. *P < 0.05; **P < 0.01 vs. vehicle. CTRL, control.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1704065114 Chicca et al. Downloaded by guest on September 30, 2021 ethanolamine (33) generated by linking the acyl chain of WOBE437 S4). These data suggest a saturable membrane target for WOBE437 PNAS PLUS with the head group of AEA significantly lost potency (IC50 = 3.47 μM) (Fig. 2 A–D and SI Appendix, Table S4 and Fig. S8). To further (SI Appendix,TableS1). investigate this point, 14C-WOBE437 was applied to membrane preparations generated from mouse brain and U937 cells. The data Biochemical and Pharmacological Profiling of WOBE437 Shows Functional showed a clear saturation binding kinetics with similar Kd values Specificity. In addition to their effects on AEA uptake, the inhibition (15.7 ± 2.8 nM and 11.3 ± 2.4 nM for membrane preparations of FAAH was thoroughly investigated for all nanomolar inhibitors derived from mouse brain and U937 cells, respectively) and Bmax (SI Appendix,TableS1). Several N-alkylamides exhibited dramati- values (18.2 ± 1.5 fmol/mg protein and 15.6 ± 1.4 fmol/mg protein cally improved selectivity for AEA uptake reduction over FAAH for mouse brain and U937 membranes, respectively) (Fig. 2 E and inhibition compared with any previously described nonselective and F). The Kd values are in line with the IC50 values shown in the -permeable AEA uptake inhibitors (Fig. 1B and SI Appendix, functional AEA uptake assays in Neuro2a and U937 cells (Fig. 1 C Table S2). The lack of biologically significant FAAH inhibition by and D). In further experiments, 10 nM 14C-WOBE437 was coin- WOBE437 was established in different assay systems by using hu- cubated with different concentrations of AEA and U937 mem- man recombinant enzyme, cell, and brain homogenates. The com- branes. Increasing amounts of AEA induced a right-down shift of 14 pound did not inhibit AEA hydrolysis in all biological matrices the C-WOBE437 binding curve, leading to an increase of Kd ≥ investigated at pharmacologically relevant concentrations (IC50 values, while not significantly modifying Bmax values (Fig. 2F and 10 μM) (SI Appendix, Fig. S4 A and B). Conversely, WOBE437 SI Appendix,TableS5). Furthermore, the EC AEA and noladin showed low nanomolar potency for the inhibition of AEA uptake ether and the AEA uptake inhibitor OMDM-2 could compete with in different cell lines and assay formats, including the FAAH- 14C-WOBE437 binding in brain and U937 membranes (SI Appen- = ± deficient HMC-1 human mast cells (25) (IC50 137 31 nM, dix,Fig.S9). Together, these data indicate a saturable membrane Hill slope = −0.934; SI Appendix,Fig.S5A and Table S3), Neuro2a for WOBE437 that is competitive with ECs and the = ± = mouse neuroblastoma cells (IC50 55 18 nM, Hill slope membrane transport inhibitor OMDM-2. In intact U937 cells, − C = ± = 0.705) (Fig. 1 ), and U937 cells (IC50 10 8 nM, Hill slope Michaelis–Menten analysis of AEA transport kinetics displayed a −0.715) (Fig. 1D). Independent time-course experiments showed competitive type of inhibition (Fig. 2G and SI Appendix,TableS6). that in Neuro2a cells, WOBE437 (100 nM) inhibited the overall Secondary plots of the slope (Kmapp/Vmaxapp vs. [WOBE437]) and ∼ – AEA uptake by 35% (nonnormalized data) after 2 10 min of intercept (1/Vmaxapp vs. [WOBE437]) further confirmed the com- incubation, reducing the effect to 20% inhibition after 15– petitive inhibition of AEA uptake (SI Appendix,Fig.S10). In sub- E

20 min of incubation (Fig. 1 ). Importantly, in a comparable sequent experiments, the mechanism of AEA uptake inhibition by PHARMACOLOGY assay, WOBE437 (1 μM) also inhibited AEA uptake in primary rat WOBE437 was shown to be reversible (rapid dilution assay; SI cortical neurons by 50% after 2–7 min of incubation with a reduced Appendix, Fig. S11 A and B) and independent of the pre- inhibition (20–35%) after 10–20minofincubation(Fig.1F). We incubation time (SI Appendix,Fig.S11andC). Incubation of and others have suggested that AEA and 2-AG compete for cellular 14C-WOBE437 (100 nM) with U937 cells over time (0–30 min) did uptake despite their differing intracellular fates (15, 17). In line with not show any increase of the cell-associated radioactive signal this concept, WOBE437 inhibited 2-AG uptake (using 1 μMtotal (∼10% of total signal; SI Appendix,Fig.S11D). The same result was ± = 14 2-AG) in U937 cells with an IC50 of 283 121 nM (Hill slope obtained by coincubating C-WOBE437with50timeshigher −0.973; Fig. 1D). In Neuro2a cells, incubation with WOBE437 (5 concentration of AEA, thus suggesting that WOBE437 does not act μM) at 2 and 5 min reduced overall 2-AG uptake by 40% (Fig. 1G). as a substrate of EC transport (SI Appendix,Fig.S11D). Interestingly, the α/β-hydrolase domain-6 (ABHD6) inhibitor We further investigated cell penetration by quantifying in- WWL70 prevented 2-AG uptake and hydrolysis occurring at later tracellular levels of WOBE437 by LC-MS/MS and measuring its time points, unlike JZL184, which specifically inhibited hydrolysis distribution in the parallel artificial membrane permeability as- (SI Appendix,Fig.S5C and D). Thus, WOBE437 inhibited 2-AG say (PAMPA) (SI Appendix, Fig. S12 and Table S7). Both results uptake, but not hydrolysis (Fig. 1G). In agreement with the hy- confirmed that WOBE437 did not significantly penetrate cell drolase activity-based protein profiling (ABPP) in mouse brain membranes up to 10 μM. homogenate (Fig. 1H) and classical radioactivity-based hydrolytic It is noteworthy that in a CEREP screen, WOBE437 did not assays in other biological matrices (SI Appendix,Fig.S4C and D), exhibit any significant interaction with 45 CNS-related receptors, WOBE437 did not inhibit the 2-AG hydrolyzing enzymes MAGL, including cannabinoid receptors (SI Appendix,Fig.S13A), high- ABHD6, and ABHD12. Importantly, WOBE437 was completely lighting its selectivity toward EC membrane transport. A full as- SI Ap- stable in the presence of the main EC-degrading enzymes ( sessment of the binding of WOBE437 to CB1 and CB2 receptors pendix A ,Fig.S6 ). Furthermore, it did not inhibit COX-2 activity revealed only negligible binding interactions (Ki values of 17 and and exhibited no binding to fatty acid binding protein 5 (FABP5) 48 μM, respectively) (SI Appendix,Fig.S13B and Table S8). (SI Appendix,Fig.S6C–E). Because AEA and 2-AG transport was previously suggested to be bidirectional (15, 21), we also explored Pharmacological Effects of WOBE437 in Mice. WOBE437 was initially the effect of WOBE437 on EC efflux and release in U937 cells. assessed in a battery of four individual tests typically associated WOBE437 moderately, but significantly, inhibited the efflux of with CB1 receptor activation in mice (nociception, locomotion, AEA from preloaded cells (SI Appendix,Fig.S7A and B)and body temperature, and cataleptic behavior), collectively referred blocked the release of both ECs, while increasing their intracellular to as the “tetrad” (26). Dose–response experiments identified levels in cells stimulated with ATP, thapsigargin, and ionomycin (SI 10 mg/kg as the lowest dose to elicit a moderate, but complete, Appendix,Fig.S7C and D). We then treated cells with radiolabeled tetrad in BALB/c mice upon i.p. administration (Fig. 3 A–D). 14 C-WOBE437 to investigate its cellular penetration. In U937 cells, Compared with the CB1 receptor (R)-WIN55,212-2, the the vast majority of the radioactive signal was collected in the ex- observed hypothermia, catalepsy, and antinociception were sig- tracellular and membrane-bound fractions (75–90%), with negligi- nificantly less pronounced, whereas the reduction in locomotion ble amounts (5–9%) detected in the intracellular fraction (Fig. 2 A– was comparable (SI Appendix,Fig.S14A–D). To assess whether D and SI Appendix,TableS4). Interestingly, the highest membrane- the WOBE437-induced effects were CB1 receptor-mediated, mice associated signal was shown at the concentration of 10 nM (9.8% of were pretreated with the selective antagonist/ total signal), whereas at higher 14C-WOBE437 concentrations, the (SR1). As in the case of (R)-WIN55,212-2, the membrane-associated radioactivity significantly dropped to 1.7%, WOBE437-induced hypothermia, catalepsy, and antinociception 0.7%, and 0.7% of total signal for the concentration of 100 nM, were completely blocked by SR1, whereas hypolocomotion was 1 μM, and 10 μM, respectively (Fig. 2 A–D and SI Appendix,Table partially reversed (Fig. 3 A–D). The tetrad was fully replicated in

Chicca et al. PNAS Early Edition | 3of10 Downloaded by guest on September 30, 2021 AB C D )sl ) )s ) sl s llecoi llecoiM/lomp(noitubirtsid734E l l eco eco 5 pmol (10 nM) 4 30 50 pmol (100 nM) 250 500 pmol (1 M) 2500 5000 pmol (10 M) iM i M/lomp(n M / /lo lo 200 2000 mp(n 3 m p(noitu 20 o o it i 150 1500 t u ubir b 2 bi i r rt t ts sid s 100 1000 id734E 10 id734EB 7

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Fig. 2. In vitro characterization of WOBE437. (A–D) Different initial amounts of 14C-WOBE437 were incubated for 15 min with 2 × 106 U937 cells at 37 °C. Afterward, cells were centrifuged, and the 14C-signal was measured from the extracellular (light gray), membrane-bound (dark gray), and intracellular (white) fractions. The results are reported in absolute amounts and as percentage of the initial amount (SI Appendix, Table S4). (E and F) Binding kinetics of 14C-WOBE437 using membrane preparations of mouse brain (E)andU937cells(F). In U937 cell membranes, 14C-WOBE437 was coincubated with vehicle or

different concentrations of AEA. (G) of AEA cellular uptake in U937 cells as shown by Michaelis–Menten analysis. Apparent Km and Vmax values are shown in SI Appendix,TableS6. Data shown are mean values ± SD from at least four independent experiments each performed in triplicate.

wild-type C57BL6/N mice and completely absent in CB1 receptor- significantly increased at 1 h after injection, thus reconfirming the deficient littermates (SI Appendix,Fig.S14E–H). These experi- anxiolytic effect of WOBE437 (Fig. 3J). ments provided a proof-of-concept for the mechanism of action of WOBE437 as an indirect CB1 receptor agonist. In subsequent WOBE437 Selectively Modulates 2-AG and AEA Concentrations in Mice. studies, we evaluated the effects of WOBE437 in pain, in- To investigate whether there was a link between the in vivo flammatory, and anxiety models. At doses of 5 and 10 mg/kg, pharmacological effects of WOBE437 and the inhibition of EC WOBE437 elicited significant analgesic and antiinflammatory ef- uptake in vitro, the levels of WOBE437, AEA, 2-AG, and related fects, as indicated by the reduced number of abdominal stretches lipids in different tissues were quantified by LC-MS/MS. After i.p. in the acetic writhing test, comparable with indomethacin (5 mg/kg), injection of 10 mg/kg WOBE437, the peak plasma concentration of and by protective effects in lipopolysaccharide (LPS)-induced 492 ± 103 pmol/mL was reached after 15 min and decreased with a endotoxemia in BALB/c mice (Fig. 3 E and F and SI Appendix, half-life of 203 min (Fig. 4A and SI Appendix,Fig.S17A). In the Fig. S15 A and B). Both effects were reversed by SR1. In the brain, the highest concentration was measured after 30 min (919 ± formalin test, WOBE437 reduced inflammatory pain and paw 314 pmol/g brain), remaining unchanged up to 1 h (652 ± thickness comparable with indomethacin (5 mg/kg) (Fig. 3 G and 165 pmol/g brain). The Cbrain/Cplasma ratio (Kp)ofWOBE437was H and SI Appendix,Fig.S15C). At the lower dose of 3 mg/kg, 0.65 after 5 min, 1.64 after 15 min, and reached 1.9 after 30 and WOBE437 still showed significant anxiolytic effects in C57BL6/N 60 min (Fig. 4 A and E). The peak concentration of WOBE437 in mice in the elevated plus maze (EPM) and in the holeboard (HB) the brain was 780 ± 267 nM, thus being in line with the bioactive test (Fig. 3 I and J). The number of entries and the time spent in concentrations determined in cellular assays. WOBE437 did not the open arms of the EPM were both significantly increased inhibit any of the major serine hydrolases in vivo (SI Appendix,Fig. compared with vehicle-treated animals (Fig. 3I and SI Appendix, S17 B–D). After single and repeated injection(s), AEA and 2-AG Fig. S16 A and B). The anxiolytic effect was completely blocked by levels in the plasma were not significantly affected (Fig. 4 B and C). SR1, indicating an indirect CB1 receptor-mediated mechanism for Only at 15 min after injection, WOBE437 prevented the moderate WOBE437. The same dose of 3 mg/kg was ineffective in the tetrad increase of 2-AG induced by DMSO (Fig. 4C). Interestingly, al- test and did not alter locomotion in the EPM assay (SI Appendix, though WOBE437 did not alter corticosterone levels after a single Fig. S16 C and D). In the HB test, the head-dipping frequency was injection, it doubled the circulating concentration of corticosterone

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Fig. 3. Pharmacological profile of WOBE437 in vivo. (A–D) Concentration-dependent WOBE437-induced hypothermia, hypolocomotion, catalepsy, and analgesia (tetrad) in BALB/c mice. The tetrad was assessed after 1 h from WOBE437 injection (i.p.). Hypothermia, catalepsy, and analgesia were fully blocked by pretreating the animals for 30 min with SR1, whereas hypolocomotion was only partially reversed. (E) WOBE437 (10 mg/kg) reduced the number of abdominal stretches in the acetic acid-induced writhing test. This analgesic effect was prevented by pretreatment with SR1. (F) WOBE437 (5 mg/kg) exerted protective effects in the LPS-induced drop of rectal temperature, which is a consequence of endotoxin challenge. The protective effect was inhibitedby pretreatment with SR1. (G and H) Analgesic and antiinflammatory effects of WOBE437 during the second phase of formalin-induced pain and inflammatory responses. For E, G, and H, indomethacin (Indometh.) (5 mg/kg) is shown as reference analgesic . (I and J) Anxiolytic effects of WOBE437 in the EPM (I)and the HB (J) tests performed in C57BL6/N mice. The data show mean values ± SEM. Groups were compared with the vehicle treated control group or as indicated by arcs using a one-way (A–E and G–I) or two-way (F) ANOVA after Bonferroni’s post hoc test or unpaired t test (G–J). n = 5–20 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant. All doses are indicated in mg/kg, i.p. Indometh., indomethacin.

after 7 d (Fig. 4D). In the total brain, AEA levels did not change measured in adipose tissue, followed by the spleen, liver, and compared with basal level after a single injection with WOBE437 kidney (SI Appendix,Fig.S24). The LC-MS/MS and 14C isotope (Fig. 4F). On the contrary, 2-AG levels increased 1.7-fold com- quantifications provided a comparable tissue distribution of pared with vehicle at 1 h after injection (Fig. 4G). After 7 d of daily WOBE437, indicating its CNS bioavailability. treatment, both AEA and 2-AG levels were significantly raised by 1.5-fold compared with DMSO. The moderate, but significant, Development of the Irreversible EC Transport Inhibitor RX-055. Based increase of both ECs in total brain did not affect the number of on the WOBE437 chemical scaffold, we investigated the possibility functional CB1 receptors (SI Appendix,Fig.S18). Repeated ad- of designing a tailored functional photoaffinity probe. Analogs ministrations of WOBE437 led to a significant threefold increase were prepared by replacing either of the two methoxy groups on of corticosterone in the brain, which mirrors the changes observed the phenyl ring with a hexyloxy chain bearing a photoactivatable in plasma (Fig. 4H). In peripheral organs, WOBE437 reached peak trifluoromethylphenyl diazirine moiety (SI Appendix,Fig.S25). concentrations of 5–20 nmol/g tissue, without any signs of accu- Intriguingly, the ability of these probes to inhibit AEA uptake was mulation over 7 d (SI Appendix,Figs.S21–S23). After a single in- strongly dependent on the positioning of the linker between the jection, the AEA level did not change in liver, whereas in kidney phenyl moiety of the WOBE scaffold and the photoactivatable and spleen, it moderately diminished (SI Appendix,Figs.S21–S23). head group. Thus, 2 (RX-055), which has the hexyloxy linker in the Similarly, 2-AG levels were not altered in the spleen, but dropped position meta to the amide nitrogen, retained the same potency as significantly after 60–360 min in liver and kidney (SI Appendix, WOBE437 (IC50 = 14 ± 3 nM, Hill slope = −0.791; Fig. 5 A and B Figs. S21–S23). However, EC concentrations were not affected by and SI Appendix,TableS9), whereas the parasubstituted derivative repeated administrations of WOBE437 in peripheral tissues. Again, 48 was a significantly weaker inhibitor of AEA uptake, with an IC50 corticosterone levels were increased in spleen, liver, and kidney (SI value of 1,979 nM (SI Appendix,Fig.S26A and B). Similar to Appendix,Figs.S21–S23). Importantly, the concentrations of other WOBE437, RX-055 (500 nM) inhibited AEA uptake in U937 cells lipids (NAEs, AA, prostaglandins, and progesterone) were not independently of preincubation time (SI Appendix,Fig.S11C). In significantly altered upon WOBE437 treatment in brain, plasma, Neuro2a cells, RX-055 (1 μM) inhibited the overall AEA uptake and peripheral organs (SI Appendix,Figs.S19–S23). We also de- by 30–40% in the time range 2–20 min (Fig. 5C). termined the tissue distribution of 14C-WOBE437 (10 mg/kg, i.p.) RX-055 also inhibited 2-AG uptake with low nanomolar po- 14 in C57BL6/N mice after 1 h, and the highest Csignalwas tency (IC50 = 32 ± 7 nM, Hill slope = −0.694; Fig. 5B). In

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Fig. 4. LC-MS/MS quantification of WOBE437, AEA, 2-AG and corticosterone in brain and plasma of C57BL6/N mice treated with 10 mg/kg of WOBE437 for different times. Plasma (A–D) and brain (E–H) concentrations of WOBE437, AEA, 2-AG, and corticosterone measured at different time points after treatment with 10 mg/kg (i.p., single injection and repeated administrations, once daily for 7 d) in C57BL6/N mice. Data represent means ± SD; n = 5 mice per group. Statistical analysis was performed by using one-way ANOVA to compare distinct groups of animals injected with DMSO or WOBE437 and killed at different time points. At every time point, DMSO- and WOBE437-treated animals were compared by using the two-tailed unpaired t test. *P < 0.05; **P < 0.01; ***P < + ++ 0.001, WOBE437 vs. vehicle; #P < 0.05; ##P < 0.01 vs. baseline (time 0); P < 0.05; P < 0.01 repeated administration vs. single administration (at 60 min after injection).

washout experiments, the inhibition of AEA and 2-AG uptake RX-055, but with a lower efficacy (Fig. 5 E and F). It is noteworthy was maintained for RX-055 as opposed to WOBE437, for which that both inhibitors did not affect the plasma concentrations of AEA inhibition was significantly reduced (SI Appendix, Fig. S26 other NAEs (SI Appendix,Fig.S29). To explore the potential C–E). RX-055 (0.5 μM) was also more efficient than WOBE437 utility of this probe in vivo, RX-055 was assessed in an animal in inhibiting 2-AG uptake in adherent Neuro2a cells (Fig. 5D). model of anxiety behavior. Because of limited systemic bio- These data clearly indicate that, upon photoactivation, RX-055 availability, RX-055 was injected into specific brain regions and covalently binds to a membrane target involved in AEA and 2-AG UV-photoactivated in situ by using cannula implantations tem- uptake. The probe was profiled on the main ECS targets showing porarily inserted by a fiber-optic cable (Fig. 6A). At 90 min after no significant binding to CB1 and CB2 receptors (Ki > 100 μM) treatment, the EPM test revealed that RX-055–injected mice and negligible effects on MAGL, ABHD6, and ABHD12 up showed significantly increased numbers of entries and time spent to 10 μMandanIC50 value for FAAH of 4 μM(SI Appendix, in the open arms compared with vehicle-injected and UV- Fig. S27), corresponding to a 250-fold selectivity for AEA up- irradiated littermate control mice (Fig. 6 B and C). The anxio- take inhibition. Similarly to WOBE437, RX-055 moderately in- lytic effects were evident at all tested concentrations injected bi- hibited the efflux of AEA and 2-AG from preloaded U937 cells laterally into the basolateral amygdala (BLA), whereas they were and blocked the release of both ECs in stimulated cells completely absent upon injection into the lateral ventricle (LV) (SI Appendix, Fig. S28). (Fig. 6 B and C). RX-055 did not affect locomotion in the animals Next, we investigated the effects of RX-055 (1 μM) on EC levels (SI Appendix,Fig.S30). in isolated human whole blood challenged with LPS for 1 h. As shown in Fig. 5 E and F, the incubation with LPS raised the Discussion plasma concentrations of AEA and 2-AG, and pretreatment with The development of tool compounds that potently and selectively RX-055 further increased the levels of both ECs by 30–40%. In modulate the different components of the ECS has been fun- the same experiments, 1 μM WOBE437 qualitatively behaved like damental in the exploration of the biochemical basis and

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Fig. 5. In vitro pharmacological characterization of the tailored irreversible EC transport inhibitor RX-055. (A) UV-induced mechanism of photoactivation of the diazirine group. (B) Inhibition curve (normalized) of AEA and 2-AG uptake in U937 cells for RX-055. (C and D) Time course of AEA (C) and 2-AG (D) uptake in adherent Neuro2a cells in presence of RX-055 (0.5 μM) or vehicle. (E and F) AEA (E) and 2-AG (F) levels measured by LC-MS/MS in isolated human whole blood after 1 h stimulation with LPS (2 μg/mL) and 15 min pretreatment with WOBE437 (1 μM), RX-055 (1 μM), or vehicle. The open circles are basal control (not LPS-challenged) and the closed circles are LPS-challenged. Data show mean values ± SEM (A–D) calculated from at least three independent experiments each performed in triplicate. (E and F) Whole blood was collected from 10–12 different human donors. Statistical analysis was carried out by using unpaired two-tailed t test (B–D) and a one-way ANOVA following Dunnett’s post hoc test (E and F). *P < 0.05; **P < 0.01; ***P < 0.001.

pharmacological potential of this complex signaling network (11, further indicate that WOBE437 binds to a membrane site with high 12, 27, 28). However, the lack of selective inhibitors of EC mem- affinity (10–20 nM). However, a second, low-affinity binding site brane transport has prevented an unambiguous biochemical and and/or nonspecific binding to phospholipids cannot be excluded. pharmacological characterization of this process. Hence, the exact WOBE437 is hydrolytically stable, and after i.p. administration at EC transport mechanism remains elusive (21). Here, we report the 10 mg/kg, it rapidly and efficiently accumulates in the brain (Kp > design and generation of the highly potent and selective EC 1 after 15 min). To obtain biological insights into the potential role reuptake inhibitors WOBE437 (1) and RX-055 (2). WOBE437 of EC uptake in vivo, we thoroughly characterized the pharmaco- inhibits AEA and 2-AG cellular uptake at substoichiometric logical and biochemical effects of WOBE437. In mice, 10 mg/kg concentrations (relative to the ECs) without altering the levels of WOBE437 elicited a full tetrad response [although less pro- other NAEs in cell lines and human whole blood, thus serving as a nouncedthan(R)-WIN55,212-], which is a hallmark of either direct selective EC uptake inhibitor (for comparison with known AEA CB1 receptor activation or the simultaneous elevation of AEA and uptake inhibitors, see SI Appendix,TableS2). Intriguingly, despite 2-AG levels in the brain (Fig. 3 A–D) (26, 29). In contrast, FAAH the fact that WOBE437 can inhibit both EC release and reuptake, inhibitors only trigger analgesia, whereas MAGL inhibitors induce the net effect observed in complex systems (i.e., whole blood and hypolocomotion, hypothermia, and analgesia, but not catalepsy (11, mice) showed an increase of EC levels. A possible explanation for 12). The LC-MS/MS data showed that, in total brain tissue, acute this finding may derive from a higher affinity of WOBE437 for WOBE437 treatment significantly increased 2-AG levels without reuptake inhibition, which could be due to its limited cell pene- affecting AEA (Fig. 4 F and G). Although the CB1 receptor- tration. Indeed, residual AEA and 2-AG reuptake upon WOBE437 mediated WOBE437 induced behavioral changes might suggest treatment is in the range of 20–50% (depending on the cell line), that the significant increase in 2-AG was likely accompanied by a whereas it can inhibit only 20% of EC release (i.e., 80% residual mild and/or region-specific rise of AEA levels that was not de- release) (SI Appendix,Fig.S7). Moreover, in tissues, the catabolic tectable, it cannot be ruled out that 2-AG uptake inhibition might and metabolic EC enzymes driving the release and facilitated up- differ pharmacologically from MAGL inhibition. At 5–10 mg/kg, take may be expressed in different cell types. Binding kinetics WOBE437 elicited significant analgesic and antiinflammatory

Chicca et al. PNAS Early Edition | 7of10 Downloaded by guest on September 30, 2021 functional antagonism (32, 33). Similarly, a reversible MAGL in- A UV light RX-055 hibitor showed protective effects in a mouse model of multiple sclerosis after 21 d of treatment, which was associated with a two- UV light fold increase of 2-AG levels in the brain (34). Our data show that a selective competitive EC reuptake inhibitor modulates the ho- meostasis of AEA and 2-AG levels in a time- and space-restricted LV manner, without leading to an EC overflow or altering the levels of RX-055 BLA other lipids. Intriguingly, after repeated administrations, WOBE437 (10 mg/kg) did not significantly alter the levels of AEA, 2-AG, and NAEs in kidney and liver (SI Appendix, Figs. S21 and S22). These data represent another pharmacological difference be- tween the inhibition of EC reuptake and the blockage of EC In vivo RX-055 injection followed by in-situ UV irradiation degradation. The possibility to tissue-specifically increase EC levels provides a targeted approach without leading to chronic activation of liver and kidney CB1 receptors that exacerbate in- flammation, promoting liver and renal fibrosis, insulin resistance, BCLateral ventricle (LV) Lateral ventricle (LV) steatosis, and nephropathy (35–38). Basolateral amygdala (BLA) Basolateral amygdala (BLA) Unexpectedly, upon repeated administration, WOBE437 led to a 60 20 ** significant twofold to fourfold increase of the corticosterone levels in ** **** brain, plasma, and peripheral organs (Fig. 4 D and H and SI Ap- 50 * pendix –

seirtnemra-nepo% ,Figs.S19S23). Although this observation requires further

e 15

m investigations, the modulation of corticosterone (the rodent equiv- 40 itmr alent of cortisol) tissue levels is interesting. A potential correlation 9 30 ns a- 10 between Δ -THC–induced suppression of neuroinflammation and

ns nepo% ns activation of the hypothalamic–pituitary–adrenal (HPA) axis in ns 20 ns ns multiple sclerosis was recently discussed (39). Although stress con- 5 10 ditions are usually characterized by high cortisol levels, burnout patients have an impaired response of the HPA axis, which leads to 0 0 hypocortisolism (40, 41). In these patients, the chronic low level of DMSO 1.5 3 6 DMSO 1.5 3 6 cortisol has been correlated with the severity of clinical and non- RX-055 (nmol/g brain) RX-055 (nmol/g brain) clinical symptoms (42, 43). To further study the mechanism of EC reuptake inhibition, we UV-irradiation in situ UV-irradiation in situ designed the suitable photoaffinity probe RX-055. This probe irre- Fig. 6. Anxiolytic effects of RX-055 upon injection and in situ photo- versibly blocked the uptake of both AEA and 2-AG with essentially activation in the BLA in C57BL6/J mice. (A) Schematic representation of in- the same potency as WOBE437, thus offering opportunities to elu- tracranial injection of RX-055 followed by in situ UV photoactivation in the cidate the mechanism of EC reuptake and to investigate the phar- BLA. (B and C) At all doses tested, RX-055 increased the open arm entries macology of its irreversible blockage. Administration of both (B) and the time spent in open arms (C) upon injection and photoactivation WOBE437 and RX-055 to human whole blood and subsequent LPS in the BLA. In contrast, no significant changes of the same parameters were stimulation showed a specific increase of ECs (Fig. 5 E and F), in line observed upon injection in the LV. In B and C, the control group was injected with the in vitro effects of these compounds and the cannabimimetic with solvent (DMSO) and UV-irradiated similarly to the RX-055 injected mice. in vivo pharmacology observed with WOBE437. Generally, RX- ± The data show mean values SEM. Groups were compared with the vehicle- 055 was more potent and produced longer-lasting effects than treated control group or as indicated by arcs using a one-way ANOVA fol- lowing Bonferroni’s post hoc test. n = 12–36 mice per group. *P < 0.05; **P < WOBE437 on 2-AG uptake in vitro. These data are in agreement 0.01. ns, not significant. with a competitive reversible mechanismofaction(WOBE437)thatis dependent on the amount of substrate (100 nM AEA vs. 1 μM2-AG) vs. an irreversible blockage (RX-055). When RX-055 was injected in effects in different animal models; at the subtetrad-inducing dose the BLA, followed by in situ UV irradiation, it showed evident an- of 3 mg/kg, the inhibitor exhibited anxiolytic effects in two mouse xiolytic effects, whereas no effects were observed upon injection into models of anxiety behavior (Fig. 3 I and J), similar to FAAH and the LV (Fig. 6). The role of BLA in cannabinoid-mediated anxiolytic MAGL inhibitors (11, 14, 29, 30). It is noteworthy that, after 7 d of effects has been shown (5, 9, 44). The difference between BLA and treatment, both AEA and 2-AG levels had significantly increased LV may reflect a distinct distribution of the probe and/or its inability in total brain by a factor of ∼1.5 compared with vehicle. Similarly, to diffuse out from the LV into other brain regions. WOBE437 accumulated in the brain, reaching an estimated con- Overall, these data show that EC membrane trafficking and centration of 926 nM after 7 d of treatment (10 mg/kg i.p., daily) cellular reuptake can be targeted. The potent and selective blockage of EC membrane transport by WOBE437 and RX- compared with 555 nM 1 h after a single injection (Fig. 4 A and E). 055 thus represents an alternative type of pharmacological mod- The levels of other NAEs remained essentially unchanged, sug- ulation of the ECS. As shown, the currently known cytoplasmic gesting that FAAH activity was not affected, which is also in AEA shuttling proteins (cytoplasmic transporters) like FABP5, agreement with the minor cell penetration of WOBE437 (Fig. 2 hsp70, and FLAT1, or TRPV1, which was postulated to mediate A–D SI Appendix and ,Fig.S12). The moderate increase of AEA AEA uptake in endothelial cells (21, 45), are not the target of and 2-AG concentrations induced by WOBE437 did not alter the WOBE437. Therefore, the present study strongly indicates that SI Appendix number of functional CB1 receptors in the brain ( ,Fig. EC membrane transport mechanisms independent from cyto- S20). In contrast, the prolonged 2-AG “overflow” (10–12 times plasmic binding proteins or degrading enzymes play a crucial role basal levels) resulting from repeated administrations of JZL184 has in the brain, as previously suggested from data with less selective been shown to desensitize CB1 receptors (31). However, repeated and potent AEA reuptake inhibitors (21). Being selective low low doses of JZL184, in combination with FAAH inhibition, pro- nanomolar EC transport inhibitors, WOBE437 and RX-055 are duce antinociceptive and anxiolytic effects without causing CB1 not only suitable tool compounds to study lipid membrane

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1704065114 Chicca et al. Downloaded by guest on September 30, 2021 transport mechanisms, which is highly challenging, but also ad- Binding Assay, CB1 Functional Assays and in Vitro Pharmacological Screening. PNAS PLUS 35 γ dress the need for high-quality chemical probes for research (46). CB1 and CB2 receptor binding assays and [ S]GTP S assays were performed Moreover, RX-055 is a unique covalent probe to unravel the as described in ref. 49 and reported in SI Appendix, SI Experimental Proce- biochemistry of EC trafficking and could be instrumental for the dures. WOBE437 was profiled in more detail in an extended CNS adverse identification of the membrane proteins involved in this process. drug reaction screening panel (CEREP) (50). The list of targets is reported in SI Appendix, SI Experimental Procedures. Materials and Methods Localization and Quantification of 14C-WOBE437 in Cellular Fractions of U937 Animals. An extended section is provided in SI Appendix, SI Experimental Cells and Mouse Tissues. The cell penetration and tissue distribution of Procedures. Animal experiments were approved by the Local Animal Welfare 14C-WOBE437 was assessed in U937 cells and C57BL6/N mice as described in Committees, in strict compliance with the ethical guidelines of the European SI Appendix, SI Experimental Procedures. Union (University of Mainz, University of Bern, and Uppsala University) and Mexican Federal Regulations for the Care and Use of Laboratory Animals (University of Guadalajara). In Vivo Experiments in BALB/c, C57BL6 and Phenotypic CB1-KO Mice. WOBE437 and RX-055 were tested in independent laboratories, in differ- Chemical Synthesis of N-Alkylamides and WOBE437. The synthesis of com- ent mouse strains, and in multiple (behavioral) models for cannabimimetic, pounds is described in the SI Appendix, SI Experimental Procedures. analgesic, antiinflammatory, and anxiolytic effects in wild-type and pheno- typic CB1-KO mice as described in SI Appendix, SI Experimental Procedures. Inhibition of AEA and 2-AG Uptake and Release in Different Cell Types. All radioactivity-based AEA and 2-AG uptake and release assays performed in LC-MS/MS Quantification of WOBE437, ECs, and Other Lipids. WOBE437, AEA, U937 and HMC-1 cells were carried out by using two different assay formats as 2-AG, and other lipids and hormones were quantified by LC-MS/MS in dif- reported (15, 25, 47). AEA and 2-AG uptake assay in Neuro2a and primary rat ferent biological matrices (cells, human whole blood, and rodent tissues) as cortical neurons were performed with a different method described in SI reported in SI Appendix, SI Experimental Procedures. Appendix, SI Experimental Procedures. ACKNOWLEDGMENTS. We thank M. Salome Gachet for measuring the ABPP. ABPP experiments were performed by using membrane preparations UCM707 and WOBE437 distribution in U937 cells and Mark Rau, Sabine Rihs, obtained from mouse brains as described in ref. 25 and in SI Appendix, SI Patricia Schenker, and Tatiana Hofer for performing some replicate measure- ments. This work was supported by the Swiss National Science Foundation Experimental Procedures. NCCR TransCure and the EIN Roche grant. F. Hoffmann-La Roche provided support for the PAMPA assay and in vitro profiling and confirmation of CB Enzyme Activity and Metabolic Stability Assays. FAAH, ABHDs, MAGL, and COX-2 receptor binding data. M.B. was supported by the Brazilian Research Council

activity assays were performed by using cell homogenates, human recombinant and the Fredrik, Ingrid Thurings Stiftelse and Stiftelse Lars Hiertas Minne and PHARMACOLOGY enzymes, and mouse, rat, and pig brain homogenates as described in refs. 15, Åhlén-stiftelse. M.S. and C.A. were supported by Bundesministerium für Wirt- 25, 47, and 48 and reported in SI Appendix, SI Experimental Procedures. schaft und Energie (ZIM KOOP) Grant KF2611301MD0.

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