Aralkylamine N-acetyltransferase

The AANAT mRNA transcript is mainly expressed in the central nervous system (CNS). It is detectable at low levels in several brain regions including the pituitary gland as well as in the retina. It is most highly abundant in the pineal gland which is the site of melatonin synthesis. Brain and pituitary AANAT may be involved in the modulation of serotonin-dependent aspects of human behavior and pituitary function.[3]

In the pinealocyte cells of the pineal gland, aralkylamine N-acetyltransferase is involved in the conversion of serotonin to melatonin. It is the penultimate enzyme in the melatonin synthesis controlling the night/day rhythm in melatonin production in the vertebrate pineal gland. Melatonin is essential for seasonal reproduction, modulates the function of the circadian clock in the suprachiasmatic nucleus, and influences activity and sleep. Due to its important role in circadian rhythm, AANAT is subjected to extensive regulation that is responsive to light exposure (see Regulation). It may contribute to multifactorial genetic diseases such as altered behavior in sleep/wake cycle and mood disorders.[2]

The primary chemical reaction that is catalyzed by aralkylamine N-acetyltransferase uses two substrates, acetyl-CoA and serotonin. AANAT catalyzes the transfer of the acetyl group of Acetyl-CoA to the primary amine of serotonin, thereby producing CoA and N-acetylserotonin. In humans, other endogenous substrates of the enzyme include specific trace amine neuromodulators, namely phenethylamine, tyramine, and tryptamine, in turn forming N-acetylphenethylamine, N-acetyltyramine, and N-acetyltryptamine.[5]

In the biosynthesis of melatonin, N-acetylserotonin is further methylated by another enzyme, N-acetylserotonin O-methyltransferase (ASMT) to generate melatonin. The N-acetyltransferase reaction has been suggested to be the rate-determining step, and thus Serotonin N-acetyltransferase has emerged as a target for inhibitor design (see below).[6]

AANAT obeys an ordered ternary-complex mechanism. The substrates bind sequentially (ordered) with acetyl-CoA binding to the free enzyme followed by the binding of serotonin to form the ternary complex. After the transfer of the acetyl group has occurred, the products are orderly released with N-acetyl-serotonin first and CoA last.[7]

Aralkylamine N-acetyltransferase belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily which consists 10,000 acetyltransferases, named so because of their sequence homology to a class of eukaryotic transcription factors, therein the yeast GCN5. Other well-studied members of the superfamily are glucosamine-6-phosphate N-acetyltransferase and histone acetyltransferases.

All members of this superfamily has a structurally conserved fold consisting of an N-terminal strand followed by two helices, three antiparallel β-strands, followed by a ‘‘signature’’ central helix, a fifth β-strand, a fourth α-helix and a final β-strand. These elements are nearly universally conserved in spite of poor pairwise identity in sequence alignments.[12]

Since it was reported that melatonin is a competitive inhibitor of AANAT, this neurotransmitter seems to exert an autoregulatory control on its own biosynthesis. Thus, loose structural analogues of the indolamine hormone were evaluated on AANAT, and moderate inhibitors were discovered.[23]

Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were screened as potential sources of inhibitors, to see if it serves as either pure or mixed competitive inhibitor for the hAANAT enzyme. Molecular modeling and structure-activity relationship studies made it possible to pinpoint the amino acid residue of the pentapeptide inhibitor S 34461 that interacts with the cosubstrate-binding site.[24]

It is suggested that AANAT catalyzes the transfer of an acetyl group from acetyl-CoA to serotonin, with the involvement of an intermediate ternary complex, to produce N-acetylserotonin. Based on this mechanism, it might be expected that a bisubstrate analog inhibitor, derived from the tethering of indole and CoASH parts, could potentially mimic the ternary complex and exert strong inhibition of AANAT.[25] The first bisubstrate analog (1), which links tryptamine and CoA via an acetyl bridge, was synthesized by Khalil and Cole, and shown to be a very potent and specific AANAT inhibitor.[26]

AANAT has shown that it also has a secondary alkyltransferase activity as well as acetyltransferase activity.[27] N-Haloacetyltryptamines were developed and serve as substrates of AANAT alkyltransferase and are also potent (low micromolar) in vitro inhibitors against AANAT acetyltransferase activity. AANAT catalyzes reaction between N-bromoacetyltryptamine (BAT) and reduced CoA, resulting a tight-binding bisubstrate analog inhibitor.[27][28] The first synthesized cell-permeable inhibitor of AANAT N-bromoacetyltryptamine was studied further on melatonin secretion from rat and pig pineal glands.[29] New N-halogenoacetyl derivatives leading to a strong in situ inhibition of AANAT. The concept behind the mechanism of action of these precursors was studied by following the biosynthesis of the inhibitor from tritiated-BAT in a living cell.[20]

The first druglike and selective inhibitors of AANAT has been identified. Lawrence M. Szewczuk et al. have virtually screened more than a million compounds by 3D high-throughput docking into the active site of X-ray structure for AANAT, and then tested 241 compounds as inhibitors. One compound class which containing a rhodanine scaffold has shown low micromolar competitive inhibition against acetyl-CoA and proved to be effective in blocking melatonin production in pineal cells.[19]

The recent study about inhibitor of AANAT has described the discovery of a new class of nonpeptidic AANAT inhibitors based on a 2,2′-bithienyl scaffold.[22]