Enzymatic and cellular study of a serotonin N-acetyltransferase phosphopantetheine-based prodrug

Abstract

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) regulates the daily rhythm in the production of melatonin and is therefore an attractive target for pharmacologic modulation of the synthesis of this hormone. Previously prepared bisubstrate analogs show potent inhibition of AANAT but have unfavorable pharmacokinetic properties due to the presence of phosphate groups which prevents transfer across the plasma membrane. Here, we examine a bis-pivaloyloxymethylene (POM)-tryptamine-phosphopantetheine prodrug (2) and its biotransformations in vitro by homogenates and pineal cells. Compound 2 is an efficient porcine liver esterase substrate for POM cleavage in vitro although cyclization of the phosphate moiety is a potential side product. Tryptamine phosphopantetheine (3) is converted to tryptamine-coenzyme A (CoA) bisubstrate analog (1) by human phosphoribosyl pyrophosphate amidotransferase (PPAT) and dephosphocoenzyme A kinase (DPCK) in vitro. Compound 2 was found to inhibit melatonin production in rat pineal cell culture. It was also found that the POM groups are readily removed to generate 3; however, further processing to tryptamine-CoA (1) is much slower in pineal extracts or cell culture. Implications for CoA prodrug development based on the strategy used here are discussed.

Figure 1

Serotonin acetyltransferase reaction catalyzed by AANAT.

Figure 2

Tryptamine-CoA (1) and its analogs.

Figure 3

In vitro reconstitution of tryptamine-CoA.

Figure 4

Intermediates generated during in vitro reconstitution.

Figure 5. HPLC analysis of in vitro reconstitution of tryptamine-CoA 1 from tryptamine-bis(POM)-phosphopantetheine 2

A. Conversion of tryptamine-bis (POM) phosphopantetheine 2 into tryptamine-mono(POM)-phosphopantetheine 6 by esterase hydrolysis. B. Conversion of tryptamine-phosphopantetheine 3 into 3′-phospho-tryptamine-CoA 5 by PPAT. C. 3′-Dephospho-tryptamine-CoA 5 into tryptamine-CoA 1 by DPCK. Products were separated on a RP-C18 column and monitored by UV absortion at 260 nm. Retention times are 20.0, 17.4, 17.0, 28.4, 26.3, 23.6 min for 2, 7, 6, 3, 5, 1, respectively.

Figure 6. Detection of compound 3 in pinealocyte extract

Compound 2 is processed by the pinealocyte extracts into compound 3. A, represents a typical elution profile of pinealocyte extract in Multiple Reaction Monitoring (MRM) mode developed to detect compounds 1, 3 and 5 simultaneously from samples as described in Experimental. B, Profile of 50 μM (30 μL injected volume) of compound 3 used as a standard. Product ions peaks, 79.1 (blue peak) and 97.0 (red peak) are generated from the target compound 3. C, control sample where Compound 2 was incubated in NH₄-acetate buffer for 60 min. D, sample where Compound 2 was incubated in pinealocyte extract for 60 min at 37°C as described in Experimental. Intensity of signal represents 10% of the compound 3 formed in the extract. Compound 3 formation in C was estimated to be about 40% of the compound 2 input in 60 min, using the peak intensity of the standard in B. Compound 1 and 5 formation could not be detected in the pinealocyte extract under the conditions used.

Figure 7. Cellular processing of compound 2. Ability of intact pinealocytes to take up and process compound 2

A. Zero time control. 50 μM of compound 2 incubated with intact pineal cells and immediately washed, lysed and deproteinated as described in Experimental. B. 20 min incubation of compound 2 in pinealocyte culture. The immediate product, compound 3 was detected in the cell extracts prepared from compound 2-treated pinealocyte culture. C. 60 min incubation of compound 2 in pinealocyte culture. D. control, represents the product ion profile of compound 3 in extracts of the untreated pinealocyte culture. The profiles shown are representatives of multiple experiments.