Evaluation of Rhodanine Indolinones as AANAT Inhibitors

Abstract

Circadian rhythm (CR) dysregulation negatively impacts health and contributes to mental disorders. The role of melatonin, a hormone intricately linked to CR, is still a subject of active study. The enzyme arylalkylamine N-acetyltransferase (AANAT) is responsible for melatonin synthesis, and it is a potential target for disorders that involve abnormally high melatonin levels, such as seasonal affective disorder (SAD). Current AANAT inhibitors suffer from poor cell permeability, selectivity, and/or potency. To address the latter, we have employed an X-ray crystal-based model to guide the modification of a previously described AANAT inhibitor, containing a rhodanine-indolinone core. We made various structural modifications to the core structure, including testing the importance of a carboxylic acid group thought to bind in the CoA site, and we evaluated these changes using MD simulations in conjunction with enzymatic assay data. Additionally, we tested three AANAT inhibitors in a zebrafish locomotion model to determine their effects in vivo. Key discoveries were that potency could be modestly improved by replacing a 5-carbon alkyl chain with rings and that the central rhodanine ring could be replaced by other heterocycles and maintain potency.

Keywords: acetyl transferase; circadian rhythm; enzyme models; heterocycles; melatonin.

Figure 1.

Previously published AANAT inhibitors^[–,–27] that we evaluated in our new enzymatic assay before beginning work on modifying 17. IC₅₀ values reported for sheep AANAT enzyme, unless indicated otherwise. Compound 1’s activity has been ascribed to the formation of compound 2 in situ.^[25] Compound 4 had no inhibition at 40 μM, so the IC₅₀ value is estimated to be at least 2-fold greater.

Figure 2.

A) Docking pose of compound 17 in the AANAT active site with key residues highlighted. Figure made using PyMOL version 2.4.1. B) Sections of molecule 17 that we are modifying in the current work.

Figure 3.. Molecular dynamics simulation of compound 17/ AANAT complex.

A) Diagram showing the percentage of the MD trajectory that AANAT residues and water molecules interact with compound 17. B) RMSD of AANAT α-carbons and compound 17 relative to AANAT over the course of the MD trajectory. C) Type of interaction of AANAT residues with compound 17 and their interaction fraction: hydrogen bonds; hydrophobic; ionic; water bridges.

Figure 4.. Molecular dynamics simulation of compound 30/ AANAT complex.

A) Diagram showing the percentage of the MD trajectory that AANAT residues and water molecules interact with compound 30. B) RMSD of AANAT α-carbons and compound 30 relative to AANAT over the course of the MD trajectory. C) Type of interaction of AANAT residues with compound 30 and their interaction fraction: hydrogen bonds; hydrophobic; ionic; water bridges.

Figure 5.. Molecular dynamics simulation of compound 37/ AANAT complex.

A) Diagram showing the percentage of the MD trajectory that AANAT residues and water molecules interact with compound 37. B) RMSD of AANAT α-Carbons and compound 37 relative to AANAT over the course of the MD trajectory. C) Type of Interaction of AANAT residues with compound 37 and their interaction fraction: hydrogen bonds; hydrophobic; ionic; water bridges.

Figure 6.

Evaluation of aqueous stability for 17 and 33 (both at 10 μM) in pH 7 phosphate buffer (50 mM in phosphate) over 48 h. Concentrations were monitored by HPLC (254 nm). The peak area at the initial timepoint, taken within 1 minute of adding compound to buffer, was set equal to 10 μM and subsequent peak areas were normalized to that initial peak area. Each timepoint (0, 1.5h, 4h, 24h, 48h) was analyzed in triplicate.

Figure 7.

Results of toxicity tests of compound 17 at five different concentrations and E3 media control data for 135 ZF larvae over 24 h. No concentration of 17 was found to produce mortality rates exceeding that which occurred in the control.

Figure 8.

Comparison of the overnight activity levels of ZF larvae exposed to three different concentrations of rhodanine 17, two known AANAT inhibitors, myricetin 3 (50 μM) and bromoacetyltryptamine (BAT) 1 (10 μM), and E3 media control (media without test compounds). (○): E3 medium control (n=55); (σ): rhodanine 17, 50 μM (n=56); (□): rhodanine 17, 20 μM (n=36); (◊): rhodanine 17, 10 μM (n=40); (■): BAT 1, 10 μM (n=58); (●): myricetin 3, 50 μM (n=67). Compound 17 did not alter overnight behavior in a manner expected for a compound that would influence melatonin synthesis in vivo.

Scheme 1.

Synthesis of carboxylic acids with alternative heterocycles 37–39.