Anxiolytic effects of N-(4,5-dihydro-5-oxo-1,2-dithiolo-[4,3,b]-pyrrole-6-yl)-N-methylformamide, a pyrroloformamide isolated from a marine Streptomyces sp., in adult zebrafish by the 5-HT system

Abstract

General anxiety disorders are among the most prevalent mental health problems worldwide. The emergence and development of anxiety disorders can be due to genetic (30–50%) or non-genetic (50–70%) factors. Despite medical progress, available pharmacotherapies are sometimes ineffective or can cause undesirable side effects. Thus, it becomes necessary to discover new safe and effective drugs against anxiety. This study evaluated the anxiolytic effect in adult zebrafish (Danio rerio) of a natural pyrroloformamide (PFD), N-(4,5-dihydro-5-oxo-1,2-dithiolo-[4,3,b]-pyrrole-6-yl)-N-methylformamide, isolated from a Streptomyces sp. bacterium strain recovered from the ascidian Eudistoma vannamei. The complete structure of PFD was determined by a detailed NMR analysis, including ¹H–¹³C and ¹H–¹⁵N-HBMC data. In addition, conformational and DFT computational studies also were performed. A group of fishes (n = 6) was treated orally with PFD (0.1, 0.5 and 1.0 mg/mL; 20 μL) and subjected to locomotor activity and light/dark tests, as well as, acute toxicity 96 h. The involvement of the GABAergic and serotonergic (5-HT) systems was investigated using flumazenil (a silent modulator of GABA receptor) and 5-HT₁, 5-HT_2A/2C and 5-HTR_3A/3B receptors antagonists, known as pizotifen, granisetron and cyproheptadine, respectively. PFD was nontoxic, reduced locomotor activity and promoted the anxiolytic effect in zebrafish. Flumazenil did not inhibit the anxiolytic effect of the PFD via the GABAergic system. This effect was reduced by a pretreatment with pizotifen and granisetron, and was not reversed after treatment with cyproheptadine. Molecular docking and dynamics studies confirmed the interaction of PFD with the 5-HT receptor.

Communicated by Ramaswamy H. Sarma

Highlights

• Pyrroloformamide (PFD), isolated from the marine Streptomyces sp. associated ascidian Eudistoma vannamei, showed no toxicity in adult zebrafish but reduced its locomotor activity.

• The structural elucidation of PFD was determined by the analysis of 1D and 2D NMR and HRESIMS data.

• The density functional theory (DFT) study confirmed the existence of two conformers as determined by NMR spectra.

• The serotonergic system modulated the anxiolytic effect of PFD via the 5-HT receptor in adult zebrafish.

• Molecular docking and dynamics studies confirmed the interaction of PFD with the 5-HT receptor.

Keywords:

• Pyrroloformamide
• Eudistoma vannamei
• central nervous system
• serotonergic system
• anxiety

Disclosure statement

The authors declare no conflicts of interest.

Additional information

Funding

The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico – ​​CNPq (n° 420454/2016-0 and 310183/2020-0) for the financial support and grants granted to carry out this work. The authors also thank CENAPAD-SP for using the Gaussian09 software package and the computational support provided through the “proj373” project.