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@LiuEtAl_2021_FunctionalSelectivityBiased

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Reference

Liu, Z., Iyer, M. R., Godlewski, G., Jourdan, T., Liu, J., Coffey, N. J., Zawatsky, C. N., Puhl, H. L., Wess, J., Meister, J., Liow, J.-S., Innis, R. B., Hassan, S. A., Lee, Y. S., Kunos, G., & Cinar, R. (2021). Functional Selectivity of a Biased Cannabinoid-1 Receptor (CB1R) Antagonist. ACS Pharmacology & Translational Science, 4(3), 1175–1187. https://doi.org/10.1021/acsptsci.1c00048

Blue: Important conclusions

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we identified a compound that is highly biased toward inhibiting CB1R-agonist-induced β-arrestin-2 recruitment compared to its ability to inhibit CB1R-agonist-induced GTPγS binding. We further show that CB1R in skeletal muscle signals via β-arrestin-2 to induce insulin resistance, whereas anxiety-like behaviors elicited by CB1R blockade in the brain are mediated entirely via G protein signaling. As a result, biased antagonism of CB1R signaling via β-arrestin-2 improves obesityrelated insulin resistance without eliciting central nervous system (CNS)-mediated adverse behavioral effects.

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The CB1R antagonist/inverse agonist MRI-1891 is highly potent in suppressing CB1R-agoniststimulated βArr2 recruitment with an IC50 of 21 pM and is about 300 times less potent in inhibiting CB1R-agonist-induced activation of G protein activation (IC50: 6 nM). Importantly, this bias results in functional selectivity, as we found that CB1R modulate anxiogenic behavior, body weight, appetite, and hepatic glucose production predominantly via G protein activation, whereas CB1R modulation of muscle insulin sensitivity is predominantly via βArr2 signaling.

Yellow: Interesting

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Seven-transmembrane receptors signal via G-protein- and β-arrestindependent pathways. We describe a peripheral CB1R antagonist (MRI-1891) highly biased toward inhibiting CB1R-induced β-arrestin-2 (βArr2) recruitment over Gprotein activation.

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The two main receptors involved are CB1 receptors (CB1R) that are highly expressed in the brain but also expressed at lower yet functional levels in most peripheral tissues, and CB2R, whose expression is more limited, primarily to cells of the immune and hematopoietic systems

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In the case of the CB1R blockade, one way to achieve such separation is to limit the brain penetrance of the antagonist.

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Another approach to selectively reduce side effects relies on biased signaling, as exemplified by μ-opioid agonists that do not recruit β-arrestin-2 to the receptor and, consequently, do not induce receptor internalization and the development of tolerance

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CB1R signal mainly via Gi/o proteins, resulting in inhibition of adenylate cyclase and voltage-sensitive Ca2+ channels and activation of GIRK potassium channels and MAP kinases.

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CB1R activation results in recruitment of β-arrestins, which not only can lead to receptor desensitization and internalization12 but also could contribute to CB1R signaling, such as the activation of p42/44 MAPK, which is partially mediated by β-arrestins.

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modified the structure of the brain penetrant CB1R antagonist/inverse agonist ibipinabant16 in order to reduce its ability to cross the blood/brain barrier

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Chemical structure and physicochemical properties of (S)-MRI-1891 and its brain-penetrant parent compound SLV-319 (ibipinabant)

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retaining subnanomolar CB1R binding affinity and >2000-fold CB1R/CB2R selectivity

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In functional assays, MRI-1891 displayed very high bias toward inhibiting CB1R-agonist-induced β-arrestin-2

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  • Esto es un PET: se une ligando radiactivo al receptor CB1R. Cuanto más ligando, más señal y quiere decir que menos inhibición del receptor. Lo que vemos aquí es que hay mucho ligando que llega al receptor, por lo que el monlunabant no lo está bloqueando. Esto apoya que el fármaco no inhibe los receptores del cerebro salvo a altas dosis de forma crónica.

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Brain penetrance of (S)-MRI-1891 upon a single (acute) dose or 28 days of chronic oral dosing in lean control male C57Bl/6J mice

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Drug levels in plasma and buffer-perfused brain were measured by LC/MS/MS 1 h after the last dose (plasma Cmax). Free concentration in brain was determined by equilibrium dialysis using crude membranes from the brain of CB1R-knockout (KO) mice as described and corresponded to 0.3% of total brain levels measured.

  • cual es el tiempo de vida media del compuesto???

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recruitment (IC50: 21 pM) versus inhibiting CB1Ragonist-induced G protein activation, as monitored by GTPγS binding (IC50: 6 nM),

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acute MRI-1891 treatment at doses of 1 or 10 mg/ kg did not result in significant CB1R occupancy in the brain, as determined by CB1R positron emission tomography (PET, Figure 2b)

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id not induce anxiogenic behavior even at the high dose of 30 mg/kg. By contrast, 3 mg/kg rimonabant caused significant brain CB1R occupancy and was highly anxiogenic (Figure 2c).

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neither dose regimen was anxiogenic, whereas chronic treatment with rimonabant (3 mg/ kg/day) induced strong anxiety (Figure 2c).

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the CB1R agonist arachidonoyl chloroethylamine (ACEA) at the maximally effective intraperitoneal dose of 10 mg/kg caused a 60% inhibition of upper GI motility in lean wild-type mice. This effect was antagonized in a dose-dependent manner by MRI-1891, with 3 mg/kg causing maximal and 1 mg/kg near-maximal antagonism.

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The absence of anxiogenic behavior despite significant brain CB1R occupancy following chronic dosing with 10 mg/kg/ day MRI-1891 was puzzling, because a similar level of brain CB1R occupancy by rimonabant was associated with a strong anxiogenic response. This raised the possibility that anxiety induced by CB1R blockade results exclusively from signaling via G proteins and not via β-arrestin-2.

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β-arrestin-2 signaling is not involved in these behavioral responses to central CB1R blockade.

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We next examined the involvement of βArr2 signaling on the metabolic effects of CB1R blockade in mice with high-fat-dietinduced obesity/metabolic syndrome (DIO)

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MRI-1891 caused an acute and robust, dose-dependent decrease in food intake that returned to normal in 5−6 days, whereas a progressive decrease in body weight was maintained throughout the treatment period

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We next compared the effects of MRI-1891 in DIO wildtype and DIO βArr2-KO mice.

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Daily treatment with 3 mg/kg MRI-1891 reduced food intake and body weight nearly identically in the two strains (Figure 4a,b)

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The reversal of hyperleptinemia was also similar in the two strains (Figure 4c), which is compatible with resensitization to endogenous leptin being responsible for the appetite and weight reducing effects, as proposed earlier

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hyperinsulinemia of wild-type DIO mice was completely reversed

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wild-type DIO mice developed profound insulin resistance, which was completely reversed by MRI-1891 treatment, whereas βArr2-KO DIO mice remained insulinsensitive,

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Hepatic glucose production significantly increased in DIO versus lean wild-type mice, and this effect was attenuated by a single oral dose of 1 mg/kg MRI-1891 similarly in wild-type and βArr2-KO mice (Figure 5a). However, the obesity-induced reduction of glucose clearance was partially reversed by MRI-1891 in wildtype but not in βArr2-KO mice (Figure 5b), which was reflected by a similar differential effect of MRI-1891 on glucose infusion rate

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he tissue responsible for the differential effect of MRI-1891 on glucose clearance was skeletal (soleus) muscle,

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Crip1a Regulates CB1R Signaling via βArr2 in Skeletal Muscle. It has been recently reported that Crip1a, a CB1R distal C-terminal associated protein,18 competes with β-arrestins for binding to CB1R distal and central C-terminal domains that could affect CB1R signaling via β-arrestins

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Arms 1 and 2 of MRI-1891 (Figures 7 and 8) are well-stabilized by aromatic residues deep in the binding pocket,

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th ibipinabant and rimonabant do not interact with this residue. The S123A mutation resulted in a 4-fold reduction of inhibitory potency of MRI-1891, but not of rimonabant, toward βArr2 signaling (Figure 9c). However, the mutation did not alter either CB1R binding affinity (Figure 9a) or CB1R inhibitory potency toward G protein signaling (Figure 9b) of either MRI-1891 or rimonabant compared to that of wildtype.

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Green: Agree with the paper

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In obese wild-type and βArr2-knockout (KO) mice, MRI-1891 treatment reduces food intake and body weight without eliciting anxiety even at a high dose causing partial brain CB1R occupancy

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Activation of CB1R promotes energy conservation and inhibits energy expenditure, and an overactive ECS has been found to contribute to the development of visceral obesity and its metabolic consequences, commonly called the metabolic syndrome.2

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Crip1a is a functional antagonist of endocannabinoid/CB1R/βArr2 signaling in skeletal muscle, and loss of this function in DIO mice may contribute to the CB1R-mediated insulin resistance. We therefore measured the expression of Cnrip1, the gene encoding Crip1a, in skeletal muscle and found that Cnrip1 is robustly downregulated in wild-type DIO compared to lean control mice, without a similar diet-induced change being detectable in βArr2KO mice

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Although all the MRI-1891 conformers (cf. the Supporting Information and Figure 8a) remained stable throughout the simulations and interconversions were observed, only one conformer (conf. 1) had optimal interactions with the receptor in the orientations shown, as determined by the number and frequency of favorable contacts (cf. Methods) and was consistent with the mutation data

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The sulfonamide group of Arm 3 is stabilized by water entering the pocket, creating short chains that bridge these groups to the receptor.

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he aryl ring is well-packed against several nonpolar residues

Red: Disagree with the paper

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In contrast to the G protein signaling bias of CB1R in modulating anxiogenic behavior, the diabetogenic effect of CB1R in skeletal muscle, due to inhibition of insulin-induced glucose uptake, occurs via βArr2 signaling. First, whole-body glucose clearance and muscle 2-deoxyglucose uptake during a hyperinsulinemic clamp were significantly higher in both lean and obese βArr2-KO mice than in their respective wild-type littermates, indicating increased insulin sensitivity in the absence of βArr2. This is in agreement with a significant increase in glucose tolerance in skeletal-muscle-specific βArr2-KO mice34 but opposite an earlier report of decreased insulin sensitivity in global βArr2-KO mice.35

Purple: To learn more

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Recent findings suggest that different conformations of GPCRs mediate agonist-induced G protein activation and βarrestin recruitment25 and that a specific phosphorylation pattern at the C terminus of GPCRs, induced by GPCR kinases (GRKs), determines β-arrestin recruitment to GPCRs and regulates their intracellular functions

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Biased agonists preferentially activate conformations linked to G protein or β-arrestin signaling, respectively. Although there is no published evidence for a biased orthosteric GPCR antagonist, pregnenolone was proposed to be a biased allosteric CB1R antagonist,

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The MD simulations suggest that the presence of Arms 3 and 4 in a single molecular scaffold, with each arm playing a distinct and largely independent role (Figure 7), may be important to impart biased CB1R antagonism

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In particular, Arm 4 is absent in the unbiased inverse agonists rimonabant and taranabant and too short in ibipinabant to interact effectively with the receptor. Moreover, the structure of MRI-1891 suggests that Arm 3 spans a region common to antagonists, and Arm 4 spans a region common to agonists, as illustrated in Figure 8b. This combination may affect G protein and βArr2 signaling pathways separately

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TMH1 less flexible when compared to the wild-type CB1R, and consequently, the Nterminal segment becomes less effective in modulating the TMH1 movement in response to the action of Arm 3 or 4 (Figure 7). The mobility and adaptability of the N-terminal/ TMH1 motif appears to be important for activity, judged by the differences observed in the crystal structure of the agonistbound versus the antagonist-bound receptor.23 The X-ray structure of rimonabant-like22 and taranabant crystallized with CB1R21 show that this region changes significantly to accommodate Arm 3 of either ligand, with TMH1 moving away from the receptor core and the N-terminal tail pushed inwardly. It is plausible that the movement of TMH1 modulated by the N-terminal residues potentiates the βArr2 bias of MRI1891, particularly via Arm 4.

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