De novo discovery of serotonin N-acetyltransferase inhibitors

Abstract

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) is a member of the GCN5 N-acetyltransferase (GNAT) superfamily and catalyzes the penultimate step in the biosynthesis of melatonin; a large daily rhythm in AANAT activity drives the daily rhythm in circulating melatonin. We have used a structure-based computational approach to identify the first druglike and selective inhibitors of AANAT. Approximately 1.2 million compounds were virtually screened by 3D high-throughput docking into the active site of X-ray structures for AANAT, and in total 241 compounds were tested as inhibitors. One compound class, containing a rhodanine scaffold, exhibited low micromolar competitive inhibition against acetyl-CoA (AcCoA) and proved to be effective in blocking melatonin production in pineal cells. Compounds from this class are predicted to bind as bisubstrate inhibitors through interactions with the AcCoA and serotonin binding sites. Overall, this study demonstrates the feasibility of using virtual screening to identify small molecules that are selective inhibitors of AANAT.

Figure 1

Structure of the bisubstrate inhibitor CoA-S-acetyltryptamine.

Figure 2

Verification of hits via direct [¹⁴C]-acetyltransferase assay. Potential hits from the primary and secondary screens were confirmed in a TLC-based assay that directly measures acetylation of TrpNH₂ by transfer of the [¹⁴C]-acetyl group from AcCoA. The 10 min reactions were carried out in 0.1 M ammonium acetate (pH 6.8), contained 0.1 mM TrpNH₂, 0.1 mM [¹⁴C]-AcCoA, and were initiated with 3.5 nM oAANAT. [¹⁴C]-product (N-acetyltryptamine) was extracted with EtOAc, resolved via TLC, and quantified by PhosphorImage analysis. The product inhibitor N-acetyltryptamine (1 mM) was used as a positive control.

Figure 3

Structures of verified AANAT inhibitors identified by VS.

Figure 4

Structures of false positives.

Figure 5

Cell-based screen of AANAT inhibitors. (A) Representative compounds from each class of inhibitors were evaluated for their ability to inhibit melatonin biosynthesis in rat pinealocytes. All cells received 100 μM drug for 1 hr followed by either 1 μM NE + drug or 1 μM NE alone for 5 hrs. Secreted melatonin was quantified via liquid chromatography-quadrupole linear ion trap mass spectrometry. Melatonin values were calculated from the area of the intensity peak of the melatonin daughter ion, expressed as counts per second. (B) Compound 2B was selected for dose response analysis in the rat pinealocyte assay. All cells received 25-200 μM 2B for 1 hr followed by 25-200 μM 2B + NE for 5 hrs. Secreted melatonin was quantified as described for A. (C) Western blot analysis of resultant cell pellets from the dose response experiment with compound 2B. Protein concentrations remain constant, indicating that the compound is not likely to be toxic to the cells over the course of the experiment.

Figure 6

Mechanistic analysis of compound 2B. IC₅₀s were used to probe the mechanism of inhibition for compound 2B. In this analysis, the effect of raising substrate concentration (either TrpNH₂ or AcCoA) from K_m to 5× K_m on IC₅₀ of 2B was determined using the αKD-coupled spectrophotometric assay. (A) IC₅₀ curves under varying substrate conditions: (●) K_m TrpNH₂ and K_m AcCoA, (○) K_m TrpNH₂ and 5× K_m AcCoA, and (□) 5× K_m TrpNH₂ and K_m AcCoA. (B) Replot of IC₅₀ values, showing dependence on AcCoA concentration but not TrpNH₂ concentration.

Figure 7

Proposed mode of binding of compound 2B to oAANAT. (A) Compound 2B docked to oAANAT. oAANAT is depicted in ribbon form and colored green. 2B is positioned as a bisubstrate inhibitor shown as a stick model colored as follows: Carbon is yellow, oxygen is red, nitrogen is blue, and sulfur is orange. The p-fluorophenyl (R₁) group is docked in the serotonin binding pocket and the remainder of this hydrophobic pocket is filled by the indolinone moiety. The thiazolidone carbonyl is hydrogen bonded to the side chain of Y168. The 6-carbon aliphatic linker spans the AcCoA binding site where the carboxylate is anchored by hydrogen bonding. Hydrogen bonds are represented by dashed gray lines. (B) Crystal structure of AANAT bound to CoA-S-acetyltryptamine (PDB code 1CJW). AANAT is represented as a green ribbon, CoA-S-acetyltryptamine is colored as follows: Carbon is gray oxygen is red, nitrogen is blue, sulfur is yellow, and phosphorus is orange.

Figure 8

Evaluation of compound 2B as a PCAF HAT inhibitor. Assays were carried out at 30 °C with reaction volumes of 30 μL that contained 10 μM substrate (H3-20), 10 nM purified PCAF HAT domain in 50 mM Tris-HCl (pH 8.0). Reactions were initiated with 20 μM [¹⁴C]-AcCoA after the other components were equilibrated at 30 °C and quenched after 5 min with 6× Tris-tricine gel loading buffer. Mixtures were separated on 16% SDS Tris-tricine polyacrylamide gels and dried, and radioactivity was quantified by PhosphorImage analysis.

Figure 9

IC₅₀ of compounds 2B and 4B in the direct acetyltransferase assay. In this TLC-based assay, acetylation of TrpNH₂ by transfer of [¹⁴C]-acetyl group from AcCoA is directly monitored. The 10 min reactions were carried out in 0.1 M ammonium acetate (pH 6.8), contained 0.1 mM TrpNH₂, 0.1 mM [¹⁴C]-AcCoA, and were initiated with 3.5 nM oAANAT. [¹⁴C]-product (N-acetyltryptamine) was extracted with EtOAc, resolved via TLC, and quantified by PhosphorImage analysis. (A) Raw data from the PhosphorImage analysis showing the dose response with compound 2B. (B) IC₅₀ determination of compound 2B and 4B from resultant dose response data.