Novel, small molecules targeting the 5-HT4 receptor protect against stress-induced maladaptive behavior with efficacy across age

Abstract

Background: Stress is a risk factor for developing psychiatric disorders, including major depressive disorder (MDD). Compounds targeting the serotonin type 4 receptor (5-HT₄R) hold promise as novel rapid-acting treatments of mood disorders. However, a lack of selectivity and numerous side effects have been limiting factors for their clinical use. Here, we developed and characterized novel-composition 5-HT₄R compounds in mouse models of stress.

Methods: Three 5-HT₄R-targeting compounds were designed and synthesized based on PF-04995274, a high-affinity 5-HT₄R ligand reported to be a partial agonist. G-protein assays were utilized to characterize molecular activity. Saline, PF-04995274, or a novel compound were administered before or after stress in male and female mice. Drug effects were assayed using behavioral paradigms. Patch clamp electrophysiology was used to determine the effect of drug on glutamatergic activity in hippocampal Cornu Ammonis 3 (CA3).

Results: Prophylactic administration of DL5, DL7, or DL8 was effective at reducing stress-induced maladaptive behaviors in male and female mice; DL7 and DL8 were effective when administered after stress. When administered following learned helplessness (LH), DL7 reduced behavioral despair and increased c-Fos in the dentate gyrus (DG) and CA3. All novel compounds attenuated large-amplitude AMPA receptor-mediated bursts in ventral CA3 (vCA3). In aged male mice, prophylactic DL7 reduced behavioral despair.

Conclusions: These results characterize novel 5-HT₄R-targeting compounds for stress-induced psychiatric disease with the potential to address unmet needs in adult and aged patients with stress-induced psychiatric illness. Future work will characterize their mechanism of action with the goal of clinical development.

Keywords: anxiety; fear; hippocampus; hyponeophagia; memory; serotonin.

Figure 1.. Activity of novel 5-HT₄R-targeting compounds in direct miniG and a cAMP accumulation competition assay

(A) Schematic of the miniGs direct recruitment assay, showing the 5-HT₄R fused to nanoluciferase at its C-terminus and miniGs fused to the acceptor mVenus. Receptor activation due to ligand binding results in recruitment of miniG proteins from the cytoplasm to the receptor at the plasma membrane leading to an increase in BRET between the donor and acceptor molecules. (B) Dose response curves for the miniGs assays with standards serotonin, prucalopride, partial agonist RS-67,333, and parental compound PF-04995274, which acts as an inverse agonist. Compounds DL5, DL7, and DL8 have minimal effects in this system. (C) Schematic of the cAMP assay, showing activation of the Gα subunit of the G_s heterotrimeric complex in response to receptor activation, resulting in an increase in intracellular cAMP. Changes in intracellular cAMP are monitored using the cAMP sensor CAMYEL, an EPAC protein that is fused to both a donor luciferase and acceptor fluorescent protein. As cAMP is produced, it binds to EPAC and causes conformational changes that lead to a decrease in the BRET signal. The experiments in (D) are competition assays against an EC80 concentration of serotonin (35 nM). RS-67,333 decreases residual cAMP levels in a concentration dependent manner, replacing serotonin but acting as a partial agonist. PF-04995274 also leads to concentration dependent decrease in cAMP levels but to a value below baseline, consistent with its actions as an inverse agonist. Compounds DL5 and DL7 lead to concentration dependent decreases in residual cAMP to near baseline values. Compound DL8 lead to decreases in residual cAMP below the vehicle baseline similar to PF-04995274. Data represented as a percentage normalized to E_max of serotonin (D) or EC80 serotonin (D). Dose response curves were fit using a 3-parameter model and values represent the mean of 3 – 7 individual replicates. Each replicate was performed with triplicate determinations. Error bars represent ± SEM; cAMP, cyclic adenosine monophosphate; EC80, effective concentration; 80% nluc, nanoluciferase.

Figure 2.. A single prophylactic injection of a novel 5-HT₄R compound protects against stress-induced maladaptive behavior in male mice.

(A) Experimental protocol. (B-C) Freezing was comparable across all groups during CFC training (B) across time and (C) averaged across the entire test session. (D-E) During context re-exposure, PF-04995274 (10 mg/kg) and DL7 (3 mg/kg) decreased fear expression when compared with saline mice. (F-G) On day 1 of the FST, immobility time was comparable across groups. (H-I) On day 2 of the FST, PF-04995274, DL5 (3 and 10 mg/kg), DL7 (3 mg/kg), and DL8 (10 mg/kg) decreased immobility time when compared with saline. (J) In the MB task, DL5 (10 mg/kg) and DL7 (3 and 10 mg/kg) decreased the number of marbles buried when compared to saline. (K) Prior to administration of NSF, mice lost a comparable amount of weight during food restriction across groups. (L-M) In the NSF, PF-04995274 (10 mg/kg), DL5 (3 mg/kg), and DL8 (3 mg/kg) reduced the latency to feed in the OF. In the home cage, groups exhibited comparable (N) latency to feed and (O) amount of food consumed. Sal n = 11, PF n = 11, DL5 (3 mg/kg) n = 5, DL5 (10 mg/kg) n = 11, DL7 (3 mg/kg) n = 7, DL7 (10 mg/kg) n = 10, DL8 (3 mg/kg) n = 5, DL8 (10 mg/kg) n = 13, male mice per group; error bars represent ± SEM; *p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; HC, home cage; MB, marble burying; NSF, novelty-suppressed feeding; OF, open field; PF, PF-04995274; Sal, saline. Behavioral timeline created with BioRender.com.

Figure 3.. A single injection of a novel 5-HT₄R compound decreases behavioral despair when administered following stress in male mice.

(A) Experimental design. (B-C) Freezing was comparable across all groups during CFC exposure. (D-E) On day 1 of the FST, DL5, DL7, and DL8 (all 3 mg/kg) increased immobility time. (F-G) On day 2 of the FST, PF-04995274 (10 mg/kg), DL7 (10 mg/kg), and DL8 (10 mg/kg) reduced immobility time when compared with saline. (H) In the MB task, all groups buried a comparable number of marbles. (I) In the NSF, all groups lost a similar amount of weight when compared directly to saline although there was an overall effect of Drug. (J-K) During the NSF, there was an overall effect of Drug, but no drug decreased the latency to feed in the OF when compared to saline. In the home cage, (L) latency to feed and (M) food consumed was comparable across all groups. Sal n = 14, PF n = 9, DL5 (3 mg/kg) n = 5, DL5 (10 mg/kg) n = 14, DL7 (3 mg/kg) n = 5, DL7 (10 mg/kg) n = 13, DL8 (3 mg/kg) n = 5, DL8 (10 mg/kg) n = 14 male mice per group; error bars represent ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; HC, home cage; Sal, saline; PF, PF-04995274; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.

Figure 4.. Prophylactic administration of novel 5-HT₄R compounds reduce large AMPA-driven synaptic bursts in CA3.

(A) Experimental design. Mice were administered saline, DL5 (3 mg/kg), DL7 (3 mg/kg), or DL8 (3 mg/kg) 1 week before whole-cell voltage clamp electrophysiology. Representative EPSCs following administration of (B) saline, (C) DL5, (D) DL7, and (E) DL8. (F) The mean amplitude was attenuated in DL5, DL7, and DL8-administered mice when compared to saline (G) The mean burst amplitude was significantly reduced by DL5, DL7, and DL8 when compared with saline. (H) The mean frequency of all AMPAR-mediated EPSCs within a 20-second recording period was significantly decreased by DL5 and DL7, but not by DL8, when compared with saline. Sal n = 4, DL5 n = 6, DL7 n = 5, DL8 n = 5 cells per group; error bars represent ± SEM; *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. AMPAR, AMPA receptor; CA3, cornu ammonis area 3; EPSC, excitatory postsynaptic current; Sal, saline; ms, millisecond; pA, picoampere. Behavioral timeline created with BioRender.com.

Figure 5.. DL7 reduces behavioral despair when administered after learned helplessness in male mice.

(A) Experimental design. (B) Total activity during habituation period was comparable across all groups. (C) Total session latencies were comparable across all groups. (D) Mean latency to escape the shock was comparable across all groups. (E) Mean latency to escape in trials 21 – 30 was comparable across all groups. (F-G) On day 1 of the FST, all groups exhibited comparable immobility time compared to saline. (H-I) On day 2 of the FST, PF-04995274 and DL7 reduced immobility time compared with saline. (J) Representative images of c-Fos expression (green) in the HPC; scale bar = 500 μm. (K) PF-04995274, DL5, DL7, and DL8 increased c-Fos expression in the DG when compared with saline. (L) PF-04995274, DL5, and DL7 increased c-Fos expression in CA3 when compared with saline. (M) c-Fos expression was comparable across groups in CA1. Sal n = 10, PF n = 4, DL5 n = 10, DL7 n = 11, DL8 n = 5 male mice per group; error bars represent SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; ; CA3, cornu ammonis area 3; CA1, cornu ammonis area 1; DG, dentate gyrus; FST, forced swim test; HC, home cage; HPC, hippocampus; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; PF, PF-04995274; Sal, saline; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.

Figure 6.. Prophylactic DL7 exerts therapeutic potential in aged male mice.

(A) Experimental timeline for aged (~17 month) male mice. (B-C) Freezing during CFC training was comparable across groups. (D-E) During CFC exposure, freezing behavior was comparable across groups. (F) On day 1 of the FST, RS-67,333 reduced immobility across the entire experimental session compared to saline, but (G) average immobility time for 3 – 6 min was comparable across groups. (H-I) On day 2 of the FST, RS-67,333 (10 mg/kg), PF-04995274 (10 mg/kg), and DL7 (3 mg/kg) reduced immobility compared to saline. (J) In the MB task, all groups buried a similar number of marbles compared to saline. (K) During food restriction phase of the NSF assay, drug groups lost a comparable amount of weight when compared directly to saline although there was a main effect of Drug. (L-M) Latency to feed in the OF in the NSF was comparable across all groups. In the home cage, (N) latency to feed and (O) the amount of food consumed was comparable between the groups. Sal n = 11, RS n = 7, Pruc n = 7, PF n = 11, DL7 n = 9, male mice per group; error bars represent ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001. CFC, contextual fear conditioning; FST, forced swim test; MB, marble burying; NSF, novelty suppressed feeding; OF, open field; HC, home cage; Sal, saline; PF, PF-04995274; P, prucalopride; RS, RS-67,333; min, minutes; sec, seconds; mg, milligram; kg, kilogram. Behavioral timeline created with BioRender.com.