Molecular Mechanisms of Nicergoline from Ergot Fungus in Blocking Human 5-HT3A Receptor

Abstract

This study investigates the modulatory effects of nicergoline, a major bioactive compound derived from ergot fungus, on the 5-hydroxytryptamine 3A (5-HT3A) receptor. Utilizing a two-electrode voltage-clamp technique, we evaluated the impact of nicergoline on the 5-HT-induced inward current (I_5-HT) in 5-HT3A receptors. Our findings reveal that nicergoline inhibits I_5-HT in a reversible and concentration-dependent manner. Additionally, the observed voltage-dependent and use-dependent inhibition indicates that nicergoline acts as an open channel blocker of the 5-HT3A receptor. To further elucidate the interaction between nicergoline and the 5-HT3A receptor, we conducted molecular docking studies. Overactivation of the 5-HT3A receptor can enhance excitatory neurotransmission, potentially leading to heightened anxiety and stress responses. It may also interfere with hippocampal functions, adversely affecting learning and memory. Additionally, exceed activation of these receptors is a primary mechanism underlying nausea and vomiting, commonly observed during chemotherapy or in response to certain toxins. Collectively, our results suggest that nicergoline has the potential to inhibit 5-HT3A receptor activity by interacting with binding residues L260 and V264. This inhibition may enhance cognitive function by stabilizing neural circuits involved in cognitive processes and can improve cognitive symptoms in patients with dementia. Additionally, the anxiolytic effects resulting from 5-HT3A receptor inhibition could promote overall psychological well-being in affected individuals. Thus, the role of nicergoline as a 5-HT3A receptor antagonist not only highlights its therapeutic potential but also warrants further exploration into its mechanisms and broader implications for managing neurodegenerative diseases.

Keywords: 5-HT3A receptor; Ergot fungus; nicergoline; open channel blocker.

Fig. 1. Inhibitory effects of nicergoline on 5-HT3A receptor.

(A) The chemical structures of nicergoline. (B) A comparative analysis of the inhibitory effects observed when 100 μM 5-HT was co-incubated or pre-incubated with 30 μM of nicergoline on 5-HT3A receptor. Pre-incubation was performed with 30 μM nicergoline for 1 min prior to simultaneous treatment with 5-HT. To sustain the environment for each antagonist, the concentration was maintained for 1 min after 5-HT treatment. The ionic current induced by nicergoline was recorded through a two-electrode voltage clamp. Notably, the inhibitory action of nicergoline on 5-HT3A receptor-mediated currents was determined to be reversible. (C) The concentrationresponse relationship reflecting the co-treatment of 5-HT alongside varying concentrations of nicergoline on 5-HT3A receptor. The graph illustrates that the I_5-HT of 5-HT3A receptor experiences a progressive inhibition with increasing nicergoline concentrations. (D) The percentage of inhibition induced by nicergoline on the 5-HT inward current of 5-HT3A receptor was calculated based on the average current generated by 5-HT alone across the 5-HT3A receptor. All experiments were performed at a holding potential of −80 mV, with data presented as mean ± SEM (n = 9–12, derived from four distinct frogs).

Fig. 2. Inhibition mechanism of nicergoline on the 5-HT3A receptor.

(A) The I_5-HT resulting from co-treatment with nicergoline and 100 μM 5-HT was reversible at a holding potential of −80 mV. Representative voltage-current relationship curves were obtained at holding potentials ranging from −80 to +50 mV. Voltage steps were applied to oocytes following the addition of 100 μM 5-HT, with or without 30 μM nicergoline on 5-HT3A receptor. (B) The I_5-HT was evaluated in oocytes at a holding potential of −80 mV during the co-administration of 3 or 30 μM nicergoline with varying concentrations of 5-HT on 5- HT3A receptor. The control (Con) condition involves varying concentrations of 5-HT acting on the 5-HT3A receptor without the presence of nicergoline. The curves indicated that nicergoline inhibited 5-HT3A receptor in a non-competitive fashion (n = 8–12 from four distinct frogs). (C and D) Recovery Times for nicergoline Inhibition. Representative traces of nicergoline application and recovery times for the 5-HT3A receptor (C, top left). In two-electrode voltage-clamp experiments, oocytes expressing the 5-HT3A receptor exhibited a stable response to repeated 100 μM 5-HT applications, with a washout period of approximately 3 min. The initial 5-HT-induced response served as the control current for recovery analysis. Nicergoline (30 μM) was administered for 2 min, followed by 5-HT application. Recovery times after nicergoline administration were 0, 10, and 30 sec (C, top right, bottom left, and bottom right). (D) A quantitative fitted curve representing the current amplitudes from tested traces shows that the activity of the 5-HT3A receptor was reduced to 56.2 ± 10.9% of the baseline current when 5- HT was applied immediately following nicergoline, with a subsequent stepwise recovery. Full recovery of the currents occurred after approximately 30 sec washout (n = 5–7, derived from four distinct frogs).

Fig. 3. Molecular study and docking model confirmation of nicergoline interaction with the 5-HT3A receptor.

(A) Front view of the interaction between nicergoline and the 5-HT3A receptor. (B) Up view of the interaction of nicergoline with the wild-type 5-HT3A receptor. The interaction is depicted with the 5-HT3A receptor represented as a tertiary protein structure, while nicergoline is modeled as a ball-and-stick representation. (C) Confirmation of the interaction pocket site within the 5-HT3A receptor. (D) The chemical interaction structure between nicergoline and the residues of 5-HT3A receptor. (E) Visualization of the interaction distances and involved residues between nicergoline and the wild-type 5-HT3A receptor. (F) Visualization of the interaction between nicergoline and the mutant 5-HT3A receptor, indicating modifications in interaction distances and involved residues.

Fig. 4. Inhibitory effects of nicergoline on double mutant 5-HT3A receptor.

(A–C) The I_5-HT mediated by 5-HT3A receptor at a holding potential of −80 mV. Each mutant is revealing differences in the inhibitory effect of 30, 100 and 300 μM nicergoline when treated with 100 μM 5-HT. (D) The concentration-response graph of nicergoline on the mutant subunits, measuring 5-HT-induced inward current with 100 μM 5-HT in the presence of various nicergoline concentrations. [Wild (■), L260A (●), V264A (▲), L260A + V264A (▼)]. Data points are expressed as mean ± SEM (n = 7–11 from four distinct frogs). Additional parameters such as maximum inhibition, half maximal inhibitory concentration, and Hill coefficient values are detailed in Table 3. One-way ANOVA test was used for comparison comparisons between among the groups. (**p < 0.001 and ***p < 0.0001, compared with 300 μM nicergoline of the wild type. (E) Fast inactivation of the peak amplitude current in the double mutant (L260A/V264A) was analyzed. To investigate the rapid activation and inactivation observed in the mutant, the currents induced by serotonin in both the wild type (black line) and the mutant (blue line) were normalized and compared (E, insert). The current from peak to complete inactivation was fitted using exponential decay curve (red line) to determine the half-maximum and the maximum inactivation time. (F) To analyze the changes in antagonist activity of the double mutant (L260A/V264A), the well-known 5-HT receptor blocker MDL72222 was applied to confirm its inhibitory effect. In the double mutant (L260A/V264A), 100 μM nicergoline’s inhibitory effect was abolished, while the inhibition induced by 0.5 μM MDL72222 remained unaffected, indicating that the mutation selectively impaired nicergoline's antagonistic activity without altering the response to MDL72222.