Extended Data Fig. 4: CB1 selectivity with higher binding affinity.
From: A cryptic pocket in CB1 drives peripheral and functional selectivity
(a) VIP36 displays full efficacy for CB1-induced cAMP inhibition compared to CP55,940 and enhanced efficacy relative to FUB. Data is presented as a bar graph and median, maximum and minimum for each drug is: CB1-CP55,940: 100%, 103%, 97%, CB1-FUB: 85%, 85%, 78%, CB1-VIP36: 117%, 124%, 105% (CB1 n = 3 independent experiments done as quadruplets). One-way ANOVA followed by Tukey’s multiple comparison ***p = 0.0008. Data are represented as mean ± SEM. (b) Similarly, the biochemical GTP depletion assay shows VIP36 acts as a superagonist for CB1-mediated Gi turnover relative to FUB. Data is presented as a bar graph and median, maximum and minimum for each drug is: CB1 + FUB: 100%, 102%, 98%; CB1 + VIP36:108%, 110%, 107%; Gi:79%, 82%, 75%. n = 6 independent experiments, One-way ANOVA followed by Tukey’s multiple comparison ****p < 0.0001 for both. Data are represented as mean ± SEM. (c) VIP36 shows Gα-subtype efficacy bias for Gi-1 and Gi3 isoforms in comparison to both FUB and VIP2.33. Data is presented as a bar graph and median, maximum and minimum for each drug is; For Gi1 FUB: 94%, 111%, 89%; VIP36:173%, 176%, 162%; VIP2.33: 82%, 89%, 78%; Gi1 alone :39%, 40%, 37%; for Gi2 – FUB: 107%, 111%, 106%; VIP36:111%, 114%, 105%; VIP2.33:113%, 118%, 113%; Gi2 alone: 94%, 96%, 85%; for Gi3 - FUB: 128%, 133%, 122%; VIP36: 143%, 144%, 143%; VIP2.33: 128%, 128%, 127%; Gi3 alone: 96%, 98%, 88%; for Goa - FUB: 120%, 122%, 117%; VIP36: 118%, 118%, 116%; VIP2.33: 112%, 113%, 111%; Goa alone: 87%, 97%, 85%; for Gz - FUB: 105%, 110%, 103%; VIP36: 103%, 106%, 99%; VIP2.33: 106%, 110%, 103%; Gz alone: 21%, 47%, 9%. Data are represented as mean ± SEM. n = 3 independent experiments. One-way ANOVA followed by Tukey’s multiple comparison ****p < 0.0001 for all, VIP2.33 vs. Gi1 alone ***p = 0.0004, FUB vs. Gi2 alone **p = 0.0097, VIP36 vs. Gi2 alone **p = 0.0048, VIP2.33 vs. Gi2 alone ***p = 0.0006, FUB vs. Gi2 alone **p = 0.0097, VIP36 vs. Gi2 alone **p = 0.0048, VIP2.33 vs. Gi2 alone ***p = 0.0006, FUB vs. VIP36 *p = 0.0137, VIP36 vs. VIP2.33 *p = 0.0157, VIP2.33 vs. Goa alone ***p = 0.0003. (d). β-arrestin-1 profiling of lead bitopics VIP36 (EC50 = n.d, Emax < 20%) and VIP2.33 (EC50 = 4.2 nM, pEC50 ± SEM = 8.38 ± 0.2, EMax = 110 ± 10) compared with FUB (EC50 = 3.74 nM, pEC50 ± SEM = 8.43 ± 0.2, EMax = 96 ± 8), and CP55,940 (EC50 = n.d, Emax < 20%) controls. Note: Since CP55,940 did not signal through β-arrestin1 the data is normalized against FUB.(e) VIP36 (IC50 = 22.5 nM, CI 95% = 5.481e-008 to 1.046e-007) shows similar (even elevated) potency in a radioligand (3H-SR141716) displacement assay relative to the parent FUB (IC50 = 75.7 nM, CI 95% = 1.646e-008 to 3.068e-008). CI = Confidence interval. n values and primary statistics for all panels are provided in Supplementary Table S4.
