Discovery of specific activity of 2-HPA acting on the membrane progestin receptor alpha (paqr7) by purification of natural products from the marine algae Padina

Discovery of specific activity of 2-HPA acting on the membrane progestin receptor alpha (paqr7) by purification of natural products from the marine algae Padina | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Discovery of specific activity of 2-HPA acting on the membrane progestin receptor alpha (paqr7) by purification of natural products from the marine algae Padina Mohammad Tohidul Amin, Mrityunjoy Acharjee, Md. Maisum Jyoti, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3360164/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Membrane progestin receptors (mPRs) are members of the progestin and adipoQ (PAQR) receptor family that are stimulated by endogenous steroids to initiate rapid intracellular signalling through a nongenomic pathway. Previously, water-soluble compounds with mPRα-binding activity from the marine algae Padina arborescens were fractionated by HPLC steps. In this study, the structure of one of the major compounds in the fraction was identified as 2-hydroxypentanoic acid (2-HPA) using Nuclear Magnetic Resonance spectroscopy. 2-HPA showed a substantial competitive binding affinity for hmPRα in the GQD-hmPRα binding assay. In contrast, synthetic structural analogues of 2-HPA showed no competitive binding activity. The physiological activity of 2-HPA and its analogues was then investigated using in vitro goldfish and in vivo zebrafish oocyte maturation and ovulation assays. As with the hmPRα binding assay, only 2-HPA showed inhibitory activity on oocyte maturation and ovulation of fish oocytes. Furthermore, the inhibitory activity of 2-HPA was compared between S- and R-type 2-HPA. The results showed that both types had the same level of activity. These results indicate that 2-HPA, found as a secreted compound from Padina arborescens , is a novel mPRα antagonist and its chemical structure is highly restricted to show its activity. Biological sciences/Biochemistry Biological sciences/Chemical biology Biological sciences/Developmental biology Biological sciences/Drug discovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Membrane progestin receptors (mPRs) are seven- or eight-transmembrane plasma membrane receptors for progesterone or its analogues. mPRs are members of a novel family of progestin and adipoQ receptors (PAQRs) consisting of 11 genes that show homology to the adipoQ receptors 1 . The mPR molecule has five types, α, β, γ, δ and ε, corresponding to PAQR7, PAQR8, PAQR5, PAQR6 and PAQR9, respectively 2 , 3 . It is also known that mPRs are conserved in a wide range of vertebrate cells, from humans to fish, and are expressed in a variety of tissues, including brain and kidney 2 , 4 . To date, mPRs have been implicated in biological regulation, including oocyte maturation in fish and amphibians, induction of reproductive behaviour in mammals 5 – 8 . Recently, it has attracted particular attention for its involvement in brain development as a target for neurosteroids 9 . In addition, mPRs have long been shown to be highly expressed in cancer cells and are thought to mediate the pathways of cancer growth and invasiveness 10 , 11 . This group of mPR molecules is known to be involved in the regulation of many cellular activities, and the search for mPR reactive substances that respond to them is ongoing 12 , 13 . In the initial studies of mPR discovery, Org OD 02 was already discovered as an mPR-specific agonist and has been used for research purposes 11 , 14 . We have also shown that Org has the ability to induce oocyte maturation in fish 15 . The experimental systems for in vitro oocyte maturation in fish and amphibians have long been shown to be induced by nongenomic actions of progesterone, and were have been used as a test for nongenomic actions, leading to the discovery of mPR 5 . This assay has been used exclusively as a method for testing mPR reactive substances 16 , 17 . We have developed in vivo zebrafish oocyte maturation and ovulation induction methods as screening methods for mPR-responsive substances, as well as a test method using the modified luciferase gene (Glosensor) to measure intracellular cAMP concentration 18 , 19 . We have also succeeded in expressing and purifying mPRa molecules using yeast, and have developed a high-throughput screening method for mPR-responsive substances using GQD-mPRa, which is a nanoparticle-associated mPRa 20 , 21 . On the other hand, we are also trying to isolate and purify mPR-reactive natural products using these screening methods 22 . We have discovered the presence of mPR-reactive substances in the seawater of coral reefs in Mauritius and recently succeeded in separating them into two peaks 23 , 24 . In this study, the chemical structure of the main component of one of these two peaks was successfully determined. The results of the physiological activity of the resulting organic component showed that it is an inhibitor of oocyte maturation, i.e., a novel antagonist of mPRa. mPRa-specific antagonists have never been reported before and are expected to be useful research tools and novel drug candidates in mPR research in the future. It is expected to be a useful research tool in mPR research and a new drug candidate in the future. Results 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 1 H-NMR, 13 C-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 mPRa 4 . 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. Discussion In this study, a novel natural compound from Padina arborescence was identified that interacts with mPRa. In GQD-mPRa binding assay, the compound in peak 2 showed higher affinity than progesterone. Synthetic compounds identified as a major component of Peak2, 2-HPA, showed more higher affinity than Peak2 (Fig. 3 ). Corresponding to this high affinity, 2-HPA showed inhibitory activity against fish oocyte maturation and ovulation at the same concentration of progestin (Figs. 5 and 6 ). We investigated the structure-activity relationship of 2-HPA analogues to identify the key residues that interact with mPRa. Interestingly, none of the analogues showed activity. The results showed that the hydroxide at position 2 is essential for interaction with mPRa. The length of the carbon chain is also important for binding to mPRa. Thus, it is suggested that the relatively simple structure of 2-HPA is a strict fit for the steroid binding site of mPRa. Conversely, the structural isomers of 2-HPA, (S)- 2-HPA and (R)- 2-HPA, showed no difference in activity (Fig. 6 ). The results suggest that hydrogens binding to carbon 5 do not contribute to any binding with mPRa. Treatment with 2-HPA resulted in the prevention of oocyte maturation and ovulation in fish. It can be concluded that the prevention of fish oocyte maturation is due to the binding of 2-HPA to mPRa. Although we cannot exclude the possibility that the inhibition of ovulation is due to the binding of 2-HPA to the nuclear progesterone receptor, which induces ovulation 25 . We believe that this inhibition is due to inhibition of the induction of oocyte maturation, which is the first response in the sequential induction of oocyte maturation and ovulation. In this study, we have succeeded in identifying the first antagonist of mPRa, the first molecule discovered to mediate a steroid nongenomic action. mPR has five subtypes, and in addition to its function in the reproductive system, which is the subject of this study, mPRa has been implicated in mediating steroid nongenomic actions in the brain and other parts of the body, and in regulating various biological processes. Antagonists of mPRa may be useful for in validating these functions and may also be candidates for new drugs. The identification of novel substances from seaweeds, particularly water-soluble substances, is currently underway. It is highly likely that new antagonists and agonists of mPR will be discovered among these substances. Further elucidation of novel compounds should be encouraged in the future. Methods Materials Chemicals were purchased from companies accordingly: (S)-2-HPA (Combi-Blocks, San Diego, CA); (R)-2-HPA (AMATEK CHEMISTRY, Hong Kong); 2-hydroxybutyric acid (Tokyo Kasei, Japan), valeric acid, 2-hydroxyhexanoic acid, progesterone and 17b-estradiol (Sigma-Aldrich); 1,2-pentanediol, and 3-hydroxyvaleric acid (AdooQ BioScience, Irvine, CA). Experimental Fishes Goldfish were reared and maintained under standard laboratory conditions. The fish used in the experiments were kept in a flow-through aquarium at 20–25°C under a 14-hour light/10-hour dark cycle. Zebrafish were reared and maintained under standard laboratory conditions. The fish used in the experiments were maintained in a flow-through culture system at 28.5°C under a 14-hour light/10-hour dark cycle. All fish experiments were approved by the Institutional Ethics Committee of Shizuoka University, Japan (approval numbers 2022F-3 and 2023F-9), and the guidelines for the use of animals established by this committee were strictly adhered to. The study was carried out in compliance with the ARRIVE guidelines 26 . Separation of components in Padina secretions by HPLC Sampling and HPLC purification were carried out as previously described 24 . Two peaks (Peak1 and 2) were obtained in the final purification step. The Peak2 fraction was dried using a freeze dryer (FDU-810, EYELA). The dried compounds were dissolved in ethanol, which was used for the GQD-hmPRa binding assay and for assays of physiological activities. Identification of the major component in the Peak2 from Padina secretions by NMR experiment The Peak2 obtained in the HPLC separation was analyzed by NMR. The fraction of Peak2 was lyophilized and then dissolved in 500 µL of CDCl 3 . The NMR spectra including 1 H, 13 C, DQF-COSY, TOCSY, HMQC and HMBC were measured using a JNM-ECZ500R spectrometer (JEOL, Tokyo, Japan), according to the manufacturer's instructions. Evaluation of the specific binding of 2-HPA and its analogues to mPRa The binding properties of 2-HPA and its analogues to mPRa were evaluated by GQD-hmPRa according to the method described previously 21 . In vitro goldfish oocyte maturation assay An in vitro oocyte maturation assay was performed in goldfish as described previously 16 . In vitro and in vivo oocyte maturation assay in zebrafish. An in vitro and in vivo zebrafish oocyte maturation and ovulation assay was performed as described previously 15 , 18 . Statistical Analysis Results were represented as Mean ± SD (Standard Deviation). Data significance values were calculated by using paired t-test. All analytical data and graphs were prepared on GraphPad Prism software version 9.4.0 for Mac OS (GraphPad Software, San Diego, California, USA) Declarations Data availability The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments We thank Mr. Daijiro Tokumoto for cultivation of goldfish. This study was supported by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and JSPS KAKENHI Grant Number 20K06719 and 23K05830 (to TT). We also acknowledge student scholarship from Japanese Government (MEXT) Scholarship for Md. F. Hossain and S. Hossain; Honda Benjiro to Md. M. S. Jyoti; Bangabandhu Science and Technology Fellowship Trust, Ministry of Science and Technology, Government of the People’s Republic of Bangladesh for S. Ahamed (memo no:08//04-01-2021). Author contributions statement MTA and MA performed the purification. MTA and SA performed and the in vitro and in vivo assay. MTA analyzed the data and drafted the manuscript. MSJ conducted GQD-mPR binding assay. MR, MH and MFH collected and cultured Padina . SK performed NMR analysis. TT participated in the study design, supervised the study and wrote the paper. All authors read and approved the final manuscript. Additional information Competing interests: The authors declare no competing interests. References Tang, Y. T. et al. PAQR proteins: a novel membrane receptor family defined by an ancient 7-transmembrane pass motif. J Mol Evol 61, 372–380 (2005). Thomas, P. et al. Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins. Endocrinology 148, 705–718, doi: 10.1210/en.2006-0974 (2007). Pang, Y., Dong, J. & Thomas, P. 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Plos Biol 18, doi:ARTN e3000411 10.1371/journal.pbio.3000411 (2020). Additional Declarations No competing interests reported. Supplementary Files SupplementalFigures.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3360164","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":235703901,"identity":"5211d33c-c7da-4ba6-acd2-d010cc8c4b09","order_by":0,"name":"Mohammad Tohidul Amin","email":"","orcid":"","institution":"National University Corporation, Shizuoka University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Tohidul","lastName":"Amin","suffix":""},{"id":235703902,"identity":"6f5b9800-57e9-4b9f-ab9d-983045039a3a","order_by":1,"name":"Mrityunjoy Acharjee","email":"","orcid":"","institution":"National University Corporation, Shizuoka University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mrityunjoy","middleName":"","lastName":"Acharjee","suffix":""},{"id":235703903,"identity":"86d77ba6-f3e7-4832-999d-0ff8ea46b9d8","order_by":2,"name":"Md. 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Maksudul Hassan","email":"","orcid":"","institution":"National University Corporation, Shizuoka University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Maksudul","lastName":"Hassan","suffix":""},{"id":235703906,"identity":"8120ff30-b7a2-4f9c-8dc2-8b6b95e3852c","order_by":5,"name":"Md. Forhad Hossain","email":"","orcid":"","institution":"National University Corporation, Shizuoka University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Forhad","lastName":"Hossain","suffix":""},{"id":235703907,"identity":"ee748d65-c25c-42bc-bf2b-0100138b569a","order_by":6,"name":"Saokat Ahamed","email":"","orcid":"","institution":"National University Corporation, Shizuoka University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Saokat","middleName":"","lastName":"Ahamed","suffix":""},{"id":235703908,"identity":"0360ae1c-4ad6-4fdf-afe4-416be5beb41f","order_by":7,"name":"Shinya Kodani","email":"","orcid":"","institution":"National University Corporation, Shizuoka University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shinya","middleName":"","lastName":"Kodani","suffix":""},{"id":235703909,"identity":"e4dc9a84-c4db-4ea7-ad33-14d7445f27ac","order_by":8,"name":"Toshinobu Tokumoto","email":"data:image/png;base64,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","orcid":"","institution":"National University Corporation, Shizuoka University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Toshinobu","middleName":"","lastName":"Tokumoto","suffix":""}],"badges":[],"createdAt":"2023-09-16 02:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3360164/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3360164/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":43908974,"identity":"57929c0e-eeee-494a-ac62-e48aa53a7aff","added_by":"auto","created_at":"2023-09-29 22:11:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKey correlations of 2D NMR spectra of 2-hydroxypentanoic acid.\u003c/strong\u003e Numbers indicate carbon position. One-sided arrow indicates HMBC correlation. Bold line indicates TOCSY correlation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3360164/v1/16907a5e32d56ffac9b0d7b0.png"},{"id":43910304,"identity":"ab74052c-2934-4f7e-afb8-3af677a582d2","added_by":"auto","created_at":"2023-09-29 22:19:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure of 2-HPA isomers and its analogues. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChemical 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/).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3360164/v1/2e525587b9aa15880c015029.png"},{"id":43910305,"identity":"c2ef5f8b-8b69-4e47-a201-ab64ef25b675","added_by":"auto","created_at":"2023-09-29 22:19:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":347812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGQD-hmPRa binding assay of 2-HPA and its analogues. \u003c/strong\u003e(A)\u003c/p\u003e\n\u003cp\u003eCompetition of the binding of P4-BSA-FITC with GQD-hmPRa by 2-HPA and its analogues. The dose-dependent effects of steroids (progesterone, estradiol-17b), (S)-type of 2-HPA, purified fraction from Padina (Padina-C) and 2-HPA analogues (see Figure 2) were studied.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3360164/v1/c6e38a82fe51380be6641a74.jpg"},{"id":43908977,"identity":"f6621671-503f-46a7-90e9-081283526f24","added_by":"auto","created_at":"2023-09-29 22:11:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":621779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e oocyte maturation assay of 2-HPA and its analogues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2-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.\u003c/p\u003e","description":"","filename":"Figure.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3360164/v1/cd090f2ce317a3068bffd962.jpg"},{"id":43908976,"identity":"15babd8f-4664-4667-b307-0cf9ee1b962b","added_by":"auto","created_at":"2023-09-29 22:11:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":345409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoocyte maturation and ovulation assay of 2-HPA and its analogues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antagonistic activity of 2-HPA and its analogues against oocyte maturation and ovulation induction was analyzed by \u003cem\u003ein vivo\u003c/em\u003etreatment 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. \u0026nbsp;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).\u003c/p\u003e","description":"","filename":"Figure.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3360164/v1/7f697410dafc671a0ffb2885.jpg"},{"id":43908978,"identity":"a114dcc8-81ab-402f-aa22-8d20edd9b80d","added_by":"auto","created_at":"2023-09-29 22:11:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":83121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eassay of (S) and (R) types of 2-HPA.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The \u003cem\u003ein vitro \u003c/em\u003eassay results for the (S) and (R) types of 2-HPA are shown. Zebrafish oocytes were used for this low number assay. \u0026nbsp;Each compound was added to the zebrafish Ringer’s solution at a final concentration of 1 μM with 1 μM DHP. After two hours of incubation, the percentage of germinal vesicle breakdown (%GVBD: indicated by the closed column) was determined by scoring the oocytes that became transparent. The assay was performed in triplicate on three fish. Mean of triplicate data is presented with standard deviation. Asterisk indicate significant differences between DHP alone and DHP with 2-HPA treatments (* P≤ 0.05).\u003c/p\u003e\n\u003cp\u003e(B) The \u003cem\u003ein vivo \u003c/em\u003eassay results for the (S) and (R) types of 2-HPA are shown. Each compound was added to the water at a final concentration of 0.1 μM with 0.1 μM DHP. After four hours of incubation, %GVBD (closed column) and %ovulation (open column) were determined by scoring the oocytes as they became transparent and formed an egg membrane. Three fish per treatment were used. Means of data are presented with standard deviation. Asterisk indicate significant differences between DHP alone and DHP with 2-HPA treatments (** P≤ 0.001).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3360164/v1/66c45b3f2684fe71a8dbeae1.png"},{"id":46325212,"identity":"b7ed864a-ff27-4c1a-b252-14cc5d33d397","added_by":"auto","created_at":"2023-11-13 07:53:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":856479,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3360164/v1/a428d9b8-fffd-40bb-9b7d-442c5341b371.pdf"},{"id":43908980,"identity":"2d784c3e-6161-471b-b71b-de1701eb1c4e","added_by":"auto","created_at":"2023-09-29 22:11:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":253423,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3360164/v1/d684b8b4194f980deabe5e93.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discovery of specific activity of 2-HPA acting on the membrane progestin receptor alpha (paqr7) by purification of natural products from the marine algae Padina","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMembrane progestin receptors (mPRs) are seven- or eight-transmembrane plasma membrane receptors for progesterone or its analogues. mPRs are members of a novel family of progestin and adipoQ receptors (PAQRs) consisting of 11 genes that show homology to the adipoQ receptors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The mPR molecule has five types, α, β, γ, δ and ε, corresponding to PAQR7, PAQR8, PAQR5, PAQR6 and PAQR9, respectively\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. It is also known that mPRs are conserved in a wide range of vertebrate cells, from humans to fish, and are expressed in a variety of tissues, including brain and kidney\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. To date, mPRs have been implicated in biological regulation, including oocyte maturation in fish and amphibians, induction of reproductive behaviour in mammals\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Recently, it has attracted particular attention for its involvement in brain development as a target for neurosteroids\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In addition, mPRs have long been shown to be highly expressed in cancer cells and are thought to mediate the pathways of cancer growth and invasiveness\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This group of mPR molecules is known to be involved in the regulation of many cellular activities, and the search for mPR reactive substances that respond to them is ongoing\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In the initial studies of mPR discovery, Org OD 02 was already discovered as an mPR-specific agonist and has been used for research purposes\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We have also shown that Org has the ability to induce oocyte maturation in fish\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The experimental systems for \u003cem\u003ein vitro\u003c/em\u003e oocyte maturation in fish and amphibians have long been shown to be induced by nongenomic actions of progesterone, and were have been used as a test for nongenomic actions, leading to the discovery of mPR\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This assay has been used exclusively as a method for testing mPR reactive substances\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe have developed \u003cem\u003ein vivo\u003c/em\u003e zebrafish oocyte maturation and ovulation induction methods as screening methods for mPR-responsive substances, as well as a test method using the modified luciferase gene (Glosensor) to measure intracellular cAMP concentration\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We have also succeeded in expressing and purifying mPRa molecules using yeast, and have developed a high-throughput screening method for mPR-responsive substances using GQD-mPRa, which is a nanoparticle-associated mPRa\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. On the other hand, we are also trying to isolate and purify mPR-reactive natural products using these screening methods\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We have discovered the presence of mPR-reactive substances in the seawater of coral reefs in Mauritius and recently succeeded in separating them into two peaks\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In this study, the chemical structure of the main component of one of these two peaks was successfully determined. The results of the physiological activity of the resulting organic component showed that it is an inhibitor of oocyte maturation, i.e., a novel antagonist of mPRa. mPRa-specific antagonists have never been reported before and are expected to be useful research tools and novel drug candidates in mPR research in the future. It is expected to be a useful research tool in mPR research and a new drug candidate in the future.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn a previous study, compounds with mPR\u0026alpha;-binding activity from secretion material of the marine algae \u003cem\u003ePadina arborescens\u003c/em\u003e were fractionated as two peaks by HPLC steps. Compounds in both of two peaks (Peak1 and Peak2) showed competitive binding activity against human mPR\u0026alpha; 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 \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR, \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC-NMR, DQF-COSY, TOCSY, HMQC and HMBC (Figures \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e-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. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) (Table. S1).\u003c/p\u003e\n\u003cp\u003eBriefly, TOCSY and DQF-COSY indicated the proton spin system from position 2 to 5 (bold line in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The characteristic chemical shifts (\u0026delta;H 4.06, \u0026delta;C 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.\u003c/p\u003e\n\u003cp\u003eSubsequently, the mPR\u0026alpha;-binding activity of 2-HPA and its analogues was evaluated using the newly established GQD-hmPR\u0026alpha; 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. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eChemical structures of two isomers of 2-HPA and its analogues used in this study. Chemical structures were drawn by the Swiss target Prediction (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swisstargetprediction.ch/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the peak2 fraction isolated from \u003cem\u003ePadina\u003c/em\u003e (Peak2) and pure (S)-2-HPA reduced the fluorescence intensity in a concentration-dependent manner as comparable to the positive control, progesterone.\u003c/p\u003e\n\u003cp\u003eCompetition of the binding of P4-BSA-FITC with GQD-hmPR\u0026alpha; by 2-HPA and its analogues. The dose-dependent effects of steroids (progesterone, estradiol-17\u0026beta;), (S)-type of 2-HPA, purified fraction from Padina (Padina-C) and 2-HPA analogues (see Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) were studied.\u003c/p\u003e\n\u003cp\u003eThe result indicated that 2-HPA possessed hmPR\u0026alpha;-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.\u003c/p\u003e\n\u003cp\u003eThe physiological activities of 2-HPA and its analogues were evaluated by an \u003cem\u003ein vitro\u003c/em\u003e 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 mPRa\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Goldfish oocytes were used for large-scale analysis. 17\u0026alpha;,20\u0026beta;-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.\u003c/p\u003e\n\u003cp\u003eAlthough 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. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e2-HPA and its analogues were added to indicate concentrations, and then maturation was induced by 1 \u0026micro;M of 17,20\u0026beta;-DHP (+\u0026thinsp;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 \u0026micro;M 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.\u003c/p\u003e\n\u003cp\u003eAs expected, only 2-HPA showed inhibitory activity on DHP-induced oocyte maturation and its analogues did not show any activity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Physiological activity of compounds was further confirmed by \u003cem\u003ein vivo\u003c/em\u003e oocyte maturation and ovulation assay using zebrafish. Again only 2-HPA showed inhibitory activity on oocyte maturation and ovulation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe antagonistic activity of 2-HPA and its analogues against oocyte maturation and ovulation induction was analyzed by \u003cem\u003ein vivo\u003c/em\u003e treatment in zebrafish. 2-HPA and its analogues were added into the water at 0.1 \u0026micro;M, and then maturation and ovulation were induced by the addition of 0.1 \u0026micro;M of 17,20\u0026beta;-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 \u0026micro;M 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\u0026thinsp;\u0026le;\u0026thinsp;0.001).\u003c/p\u003e\n\u003cp\u003eFinally, the activity of 2-HPA isomers were compared. The results in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e show that both (R)-2-HPA and (S)-2-HPA inhibit oocyte maturation and ovulation at same magnitude of activity (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), with (R)-2 -HPA and (S)-2-HPA, strongly suggesting that both isomers act as antagonists for mPRa.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, a novel natural compound from \u003cem\u003ePadina arborescence\u003c/em\u003e was identified that interacts with mPRa. In GQD-mPRa binding assay, the compound in peak 2 showed higher affinity than progesterone. Synthetic compounds identified as a major component of Peak2, 2-HPA, showed more higher affinity than Peak2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Corresponding to this high affinity, 2-HPA showed inhibitory activity against fish oocyte maturation and ovulation at the same concentration of progestin (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe investigated the structure-activity relationship of 2-HPA analogues to identify the key residues that interact with mPRa. Interestingly, none of the analogues showed activity. The results showed that the hydroxide at position 2 is essential for interaction with mPRa. The length of the carbon chain is also important for binding to mPRa. Thus, it is suggested that the relatively simple structure of 2-HPA is a strict fit for the steroid binding site of mPRa. Conversely, the structural isomers of 2-HPA, (S)- 2-HPA and (R)- 2-HPA, showed no difference in activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results suggest that hydrogens binding to carbon 5 do not contribute to any binding with mPRa.\u003c/p\u003e \u003cp\u003eTreatment with 2-HPA resulted in the prevention of oocyte maturation and ovulation in fish. It can be concluded that the prevention of fish oocyte maturation is due to the binding of 2-HPA to mPRa. Although we cannot exclude the possibility that the inhibition of ovulation is due to the binding of 2-HPA to the nuclear progesterone receptor, which induces ovulation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. We believe that this inhibition is due to inhibition of the induction of oocyte maturation, which is the first response in the sequential induction of oocyte maturation and ovulation.\u003c/p\u003e \u003cp\u003eIn this study, we have succeeded in identifying the first antagonist of mPRa, the first molecule discovered to mediate a steroid nongenomic action. mPR has five subtypes, and in addition to its function in the reproductive system, which is the subject of this study, mPRa has been implicated in mediating steroid nongenomic actions in the brain and other parts of the body, and in regulating various biological processes. Antagonists of mPRa may be useful for in validating these functions and may also be candidates for new drugs. The identification of novel substances from seaweeds, particularly water-soluble substances, is currently underway. It is highly likely that new antagonists and agonists of mPR will be discovered among these substances. Further elucidation of novel compounds should be encouraged in the future.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eChemicals were purchased from companies accordingly: (S)-2-HPA (Combi-Blocks, San Diego, CA); (R)-2-HPA (AMATEK CHEMISTRY, Hong Kong); 2-hydroxybutyric acid (Tokyo Kasei, Japan), valeric acid, 2-hydroxyhexanoic acid, progesterone and 17b-estradiol (Sigma-Aldrich); 1,2-pentanediol, and 3-hydroxyvaleric acid (AdooQ BioScience, Irvine, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Fishes\u003c/h2\u003e \u003cp\u003eGoldfish were reared and maintained under standard laboratory conditions. The fish used in the experiments were kept in a flow-through aquarium at 20\u0026ndash;25\u0026deg;C under a 14-hour light/10-hour dark cycle.\u003c/p\u003e \u003cp\u003eZebrafish were reared and maintained under standard laboratory conditions. The fish used in the experiments were maintained in a flow-through culture system at 28.5\u0026deg;C under a 14-hour light/10-hour dark cycle.\u003c/p\u003e \u003cp\u003eAll fish experiments were approved by the Institutional Ethics Committee of Shizuoka University, Japan (approval numbers 2022F-3 and 2023F-9), and the guidelines for the use of animals established by this committee were strictly adhered to. The study was carried out in compliance with the ARRIVE guidelines\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSeparation of components in\u003c/b\u003e \u003cb\u003ePadina\u003c/b\u003e \u003cb\u003esecretions by HPLC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSampling and HPLC purification were carried out as previously described \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Two peaks (Peak1 and 2) were obtained in the final purification step. The Peak2 fraction was dried using a freeze dryer (FDU-810, EYELA). The dried compounds were dissolved in ethanol, which was used for the GQD-hmPRa binding assay and for assays of physiological activities.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of the major component in the Peak2 from\u003c/b\u003e \u003cb\u003ePadina\u003c/b\u003e \u003cb\u003esecretions by NMR experiment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Peak2 obtained in the HPLC separation was analyzed by NMR. The fraction of Peak2 was lyophilized and then dissolved in 500 \u0026micro;L of CDCl\u003csub\u003e3\u003c/sub\u003e. The NMR spectra including \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC, DQF-COSY, TOCSY, HMQC and HMBC were measured using a JNM-ECZ500R spectrometer (JEOL, Tokyo, Japan), according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of the specific binding of 2-HPA and its analogues to mPRa\u003c/h2\u003e \u003cp\u003eThe binding properties of 2-HPA and its analogues to mPRa were evaluated by GQD-hmPRa according to the method described previously\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003egoldfish oocyte maturation assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAn \u003cem\u003ein vitro\u003c/em\u003e oocyte maturation assay was performed in goldfish as described previously\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro and in vivo\u003c/b\u003e \u003cb\u003eoocyte maturation assay in zebrafish.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAn \u003cem\u003ein vitro and in vivo\u003c/em\u003e zebrafish oocyte maturation and ovulation assay was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eResults were represented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (Standard Deviation). Data significance values were calculated by using paired t-test. All analytical data and graphs were prepared on GraphPad Prism software version 9.4.0 for Mac OS (GraphPad Software, San Diego, California, USA)\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mr. Daijiro Tokumoto for cultivation of goldfish. This study was supported by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and JSPS KAKENHI Grant Number 20K06719 and 23K05830 (to TT). We also acknowledge student scholarship from Japanese Government (MEXT) Scholarship for Md. F. Hossain\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand S. Hossain; Honda Benjiro to Md. M. S. Jyoti; Bangabandhu Science and Technology Fellowship Trust, Ministry of Science and Technology, Government of the People\u0026rsquo;s Republic of Bangladesh for S. Ahamed (memo no:08//04-01-2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTA and MA performed the purification. MTA and SA performed and the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assay. MTA analyzed the data and drafted the manuscript. MSJ conducted GQD-mPR binding assay. MR, MH and MFH collected and cultured \u003cem\u003ePadina\u003c/em\u003e. SK performed NMR analysis. TT participated in the study design, supervised the study and wrote the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTang, Y. T. \u003cem\u003eet al.\u003c/em\u003e PAQR proteins: a novel membrane receptor family defined by an ancient 7-transmembrane pass motif. J Mol Evol 61, 372\u0026ndash;380 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThomas, P. \u003cem\u003eet al.\u003c/em\u003e Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins. 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Plos Biol 18, doi:ARTN e3000411 \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pbio.3000411\u003c/span\u003e\u003cspan address=\"10.1371/journal.pbio.3000411\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3360164/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3360164/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMembrane progestin receptors (mPRs) are members of the progestin and adipoQ (PAQR) receptor family that are stimulated by endogenous steroids to initiate rapid intracellular signalling through a nongenomic pathway. Previously, water-soluble compounds with mPRα-binding activity from the marine algae \u003cem\u003ePadina arborescens\u003c/em\u003e were fractionated by HPLC steps.\u003c/p\u003e \u003cp\u003eIn this study, the structure of one of the major compounds in the fraction was identified as 2-hydroxypentanoic acid (2-HPA) using Nuclear Magnetic Resonance spectroscopy. 2-HPA showed a substantial competitive binding affinity for hmPRα in the GQD-hmPRα binding assay. In contrast, synthetic structural analogues of 2-HPA showed no competitive binding activity. The physiological activity of 2-HPA and its analogues was then investigated using \u003cem\u003ein vitro\u003c/em\u003e goldfish and \u003cem\u003ein vivo\u003c/em\u003e zebrafish oocyte maturation and ovulation assays. As with the hmPRα binding assay, only 2-HPA showed inhibitory activity on oocyte maturation and ovulation of fish oocytes. Furthermore, the inhibitory activity of 2-HPA was compared between S- and R-type 2-HPA. The results showed that both types had the same level of activity. These results indicate that 2-HPA, found as a secreted compound from \u003cem\u003ePadina arborescens\u003c/em\u003e, is a novel mPRα antagonist and its chemical structure is highly restricted to show its activity.\u003c/p\u003e","manuscriptTitle":"Discovery of specific activity of 2-HPA acting on the membrane progestin receptor alpha (paqr7) by purification of natural products from the marine algae Padina","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-29 22:11:23","doi":"10.21203/rs.3.rs-3360164/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ef908a5-30a0-404a-90e2-27b4fbe7e608","owner":[],"postedDate":"September 29th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":24963880,"name":"Biological sciences/Biochemistry"},{"id":24963881,"name":"Biological sciences/Chemical biology"},{"id":24963882,"name":"Biological sciences/Developmental biology"},{"id":24963883,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2023-11-13T07:45:14+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-29 22:11:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3360164","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3360164","identity":"rs-3360164","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}