In a previous study, compounds with mPRα-binding activity from secretion material of the marine algae Padina arborescens were fractionated as two peaks by HPLC steps. Compounds in both of two peaks (Peak1 and Peak2) showed competitive binding activity against human mPRα and inhibitory activity on fish oocyte maturation and ovulation. In this study, we tried to identify the chemical structure of the active compound by chemical analysis. The Peak2 was subjected to NMR analyses using 1H-NMR, 13C-NMR, DQF-COSY, TOCSY, HMQC and HMBC (Figures S1-S6). Analysis of the NMR data indicated that Peak2 was a mixture of several compounds. As a result of the analysis of the 2D NMR data, the main component was determined to have the carbon skeleton of 2-hydroxypentanoic acid (2-HPA) with assignment of chemical shift values (Fig. 1) (Table. S1).
Briefly, TOCSY and DQF-COSY indicated the proton spin system from position 2 to 5 (bold line in Fig. 1). The characteristic chemical shifts (δH 4.06, δC 64.7) of position 2 indicated the presence of a hydroxy residue. The HMBC correlation from the proton at position 2 to the carbon at position 1 suggested a carboxy residue at position 1. Although the material (Peak2) was a mixture of several compounds, we proposed that the carbon skeleton structure of 2-HPA may be essential for the activity.
Subsequently, the mPRα-binding activity of 2-HPA and its analogues was evaluated using the newly established GQD-hmPRα binding assay, which allows high through put screening of mPR-interacting compounds. Closely related 2-HPA analogues were selected as follows: hydroxy group missing version of 2-HPA; valeric acid, hydroxy group position altered version; 3-hydroxyvaleric acid, shorter fatty acid chain; 2-hydroxybutyric acid, longer fatty acid chain; 2-hydroxyhexanoic acid, missing double bonded oxygen; 1,2pentanediol (Fig. 2).
Chemical structures of two isomers of 2-HPA and its analogues used in this study. Chemical structures were drawn by the Swiss target Prediction (http://www.swisstargetprediction.ch/).
As shown in Fig. 3, the peak2 fraction isolated from Padina (Peak2) and pure (S)-2-HPA reduced the fluorescence intensity in a concentration-dependent manner as comparable to the positive control, progesterone.
Competition of the binding of P4-BSA-FITC with GQD-hmPRα by 2-HPA and its analogues. The dose-dependent effects of steroids (progesterone, estradiol-17β), (S)-type of 2-HPA, purified fraction from Padina (Padina-C) and 2-HPA analogues (see Fig. 2) were studied.
The result indicated that 2-HPA possessed hmPRα-interacting activity as the Peak 2 fraction. In contrast, no decrease in fluorescence intensity was observed for analogues of 2-HPA such as the negative control estradiol. Among the 2-HPA and its analogues tested in this study, only 2-HPA has hmPR binding properties, suggesting that the binding of 2-HPA to the hmPR is specific and that the interaction is conformationally restricted.
The physiological activities of 2-HPA and its analogues were evaluated by an in vitro oocyte maturation assay using fish oocytes. Induction of oocyte meiotic maturation has been shown to be induced by mPRa mediated signal transduction through nongenomic action as a biological process made a discovery of mPRa4. Goldfish oocytes were used for large-scale analysis. 17α,20β-dihydroxy-4-prognen-3-one (DHP), is a natural agonist for the mPRs and induces oocyte maturation by binding to the mPRs on the cell surface of the oocyte.
Although no agonistic activity to induce oocyte maturation was observed when incubated compounds alone with oocytes (data not shown), 2-HPA showed antagonistic activity against DHP-induced oocyte maturation (Fig. 4).
2-HPA and its analogues were added to indicate concentrations, and then maturation was induced by 1 µM of 17,20β-DHP (+ DHP 1). After six hours incubation, oocytes with or without germinal vesicle breakdown (GVBD) were counted and the percentage of GVBD was calculated. As a negative control, oocytes were incubated with 0.1% ethanol (EtOH) or 1 µM of DHP alone as a positive control (DHP 1). The assay was performed in triplicate and the averaged percentage of GVBD is expressed as standard deviation.
As expected, only 2-HPA showed inhibitory activity on DHP-induced oocyte maturation and its analogues did not show any activity (Fig. 4). Physiological activity of compounds was further confirmed by in vivo oocyte maturation and ovulation assay using zebrafish. Again only 2-HPA showed inhibitory activity on oocyte maturation and ovulation (Fig. 5).
The antagonistic activity of 2-HPA and its analogues against oocyte maturation and ovulation induction was analyzed by in vivo treatment in zebrafish. 2-HPA and its analogues were added into the water at 0.1 µM, and then maturation and ovulation were induced by the addition of 0.1 µM of 17,20β-DHP (DHP 0.1). After four hours of treatment with compounds by addition to water, %GVBD (closed column) and %ovulation (open column) were determined by scoring the oocytes that had become transparent and formed an egg membrane by egg activation. Fish were incubated with 0.01% ethanol (EtOH) as a negative control or with 0.1 µM DHP alone as a positive control (DHP 0.1). Three fish were used per treatment. Means of data are presented with standard deviation. Asterisks represent significant differences between DHP alone and DHP with 2-HPA treatment (** P ≤ 0.001).
Finally, the activity of 2-HPA isomers were compared. The results in Fig. 6 show that both (R)-2-HPA and (S)-2-HPA inhibit oocyte maturation and ovulation at same magnitude of activity (Fig. 6), with (R)-2 -HPA and (S)-2-HPA, strongly suggesting that both isomers act as antagonists for mPRa.