Melatonin (N‐acetyl‐5‐methoxytryptamine) modulates circadian rhythms and sleep‐wake cycles, primarily via activation of the MT1 and MT2 melatonin receptors which exhibit distinct pharmacological profiles. The MT1/MT2 melatonin receptor ligand, luzindole (LUZ), delays re‐entrainment of running wheel activity following an advance of dark onset via the MT1, and exerts antidepressant‐like effects via the MT2 melatonin receptor. Our goal is to design and synthesize novel analogues with at least 50‐fold selectivity for either MT1 or MT2 and a pharmacological profile on each receptor compatible with that of LUZ (ei., antagonist/inverse agonists at MT1; antagonist/partial agonists at MT2) to potentially mimic distinct receptor‐mediated behaviors. Our strategy is to assess structure‐activity relationships of indolealkyl amides to identify optimal functional groups that yield a more defined efficacy. A series of b‐methyl, b,b‐dimethyl and a‐methyl C3‐side chain functionalized indolealkyl amides, incorporating C2‐Me, C2‐Ph or C2‐H substituents were prepared. Four C5‐non‐methoxylated derivatives of melatonin, but with its side chain translocated from C3 to C2, were also synthesized. The compounds were tested in vitro for competition with 2‐[125I]‐iodomelatonin (100 pM) binding at hMT1 and hMT2 receptors stably expressed in CHO cells to assess binding affinities (Ki) as well as apparent intrinsic efficacy in the presence of GTP (100 μM). The apparent efficacy of LUZ in GTP‐shift binding assays with 2‐[125I]‐iodomelatonin shows an antagonist/inverse agonist profile at MT1 (KiGTP/Control: 0.20 ± 0.08, n=3), and an antagonist/partial agonist profile at MT2 (KiGTP/Control: 1.5 ± 0.4, n=4). The high affinity of the various analogues was dependent on the presence of b‐ and α‐substituents, and the nature of the C2‐functionality. A C2‐Ph analogue, named ATBT‐23 exhibited high affinity and selectivity for the hMT2 receptor. Indeed, ATBT‐23 showed 190‐fold selectivity for the MT2 receptor (MT1 Ki: 2,794 nM, MT2 Ki: 15 nM, KihMT1/KihMT2 Ratio: 190, n=4). This selectivity is distinct from that of LUZ which demonstrates approximately 26‐fold affinity for the MT2 receptor. However, GTP shift binding assays revealed apparent affinity ratios compatible with ATBT‐23 being a melatonin receptor antagonist/weak inverse agonist at hMT1 and an antagonist/partial agonist at hMT2 receptors, similar to non‐selective LUZ. Brain penetration determined by ex vivo binding as well as the in vivo behavioral profile for the MT2 selective analogue ATBT‐23 on circadian re‐entrainment and depressive‐like behaviors in C3H/HeN mice will be reported and compared with the behavioral profile of LUZ and other melatonin receptor type‐selective analogues. ATBT‐23 and newly synthesized analogues could signify a novel class of melatonin receptor ligands with higher affinity and efficacy yet similar pharmacology to LUZ, for the treatment of circadian sleep and depressive disorders.Support or Funding InformationSupported by the Jacobs School of Medicine and Biomedical Sciences to MLD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of melatonin receptors facilitates re‐entrainment of circadian activity rhythms following an advance of the dark onset. In an eastbound jet lag model, administration of exogenous melatonin, at the new dark onset, accelerates re‐entrainment via activation of the MT1 receptor (Dubocovich et al., 2005). Conversely, melatonin proficient mice (C3H/HeN) with a targeted deletion of the MT2 receptor (MT2KO), but not the MT1 receptor (MT1KO), experience a deceleration in re‐entrainment, probably due to impairment of the endogenous melatonin facilitated signaling mediated by activation of the MT2 receptor (Pfeffer et al., 2012). To further elucidate the functional modulatory role of melatonin receptors on the synchronization of the circadian clock, we assessed locomotor activity rhythms in C3H/HeN mice in response to pharmacological intervention to determine the ability of the MT2 selective antagonist, ATBT‐23 (hMT1/hMT2 Ki=190), to decelerate re‐entrainment by blocking the MT2 receptor, thus mimicking the MT2KO. Male C3H/HeN mice in a 12/12 L/D cycle were subjected to an eastbound jet lag paradigm in which Vehicle (30% ethanol/saline, s.c) or ATBT‐23 (1 mg/kg in Vehicle, s.c) were administered on three consecutive days at the new dark onset following an abrupt 6‐hour advance of the L/D cycle. The number of days to re‐entrain and re‐entrainment rates (hrs/day) were analyzed by two‐way ANOVA (Tukey's post hoc test) and a mixed effect two‐way repeated measure ANOVA (Sidak's post hoc test), respectively. WT (7.54±0.33 days, n=28) and MT1KO (7.36±0.54 days, n=14) mice treated with vehicle did not show a significant difference in the number of days necessary to attain re‐entrainment. However, MT2KO (11.92±0.40 days, n=13) mice, treated with vehicle, took significantly longer to re‐entrain when compared to WT and MT1KO mice (p<0.0001). This deceleration of re‐entrainment in the MT2KO was reproduced by ATBT‐23, which decelerated the rate of re‐entrainment in both WT (p<0.0001, n=25) and MT1KO (p<0.01, n=12) males, increasing the number of days to re‐entrain to 9.76±0.46 (p<0.01) and 10.00±0.63 (p<0.05) days, respectively. ATBT‐23 (n=14) did not significantly affect the number of days to re‐entrain in MT2KO mice, suggesting deceleration of re‐entrainment is not mediated by the MT1 receptor. The present study suggests that ATBT‐23 decelerates re‐entrainment of circadian activity rhythms by blocking the potent acceleration of re‐entrainment by endogenous melatonin via action on the MT2 receptor. Therefore, selective MT2 melatonin receptor antagonists can potentially be used to delay advanced circadian rhythms, as observed in depression and advance sleep phase disorders, when administered at proper circadian times.
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