CN115554403B - Use of the steroid hormone DHEA as receptor ADGRG2 agonist ligand - Google Patents
Use of the steroid hormone DHEA as receptor ADGRG2 agonist ligand Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
- A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
- A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
Abstract
The invention provides application of steroid hormone DHEA as a receptor ADGRG2 agonist ligand, belonging to the technical fields of bioengineering and biological medicine. According to the invention, the steroid hormone DHEA can be used as an endogenous ligand of an orphan receptor ADGRG2, so that the ADGRG2 is effectively activated to generate Gs signals, and the steroid hormone DHEA can form the basis for developing diagnostic and/or therapeutic drugs for male reproductive system diseases, so that the invention has good practical application value.
Description
Technical Field
The invention belongs to the technical fields of bioengineering and biological medicine, and particularly relates to application of steroid hormone DHEA as an ADGRG2 receptor agonist ligand.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
G protein-coupled receptors are the largest family of membrane receptors in the human genome and drug targets, and about 30% of approved drugs act by binding GPCRs and modulating intracellular signaling. Among them, the adhesion class G protein-coupled receptors (aGPCRs) are the second largest family of GPCRs, with a total of 33 members. All aGPCRs have a classical seven-transmembrane structure, and have several unique features, namely a very long N-terminus with adhesive potential, which includes an induced self-hydrolysable membrane-proximal domain (Hamann et al 2015). Mutations in the adhesion-type receptor are associated with many human diseases, such as adhesion-type receptors ADGRV1 and LPHN2/3, which are critical for cochlear hair cell survival and excitatory synapse formation, respectively, ADGRF5 mediates alveolar surfactant homeostasis through Gq signaling, ADGRG6 modulates skeletal development through downstream Gs signaling, etc. The agcr has been linked to a variety of different diseases and regulates many important physiological processes throughout the body, so the agcr family has great potential for drug discovery.
ADGRG2 (adhesion G-like protein-coupled receptor G2), also known as GPR64 or human epididymal specific protein 6 (HE 6), ADGRG2 is highly expressed in epididymis and output tubules of humans and mice, and plays an important role in regulating male reproductive function. The literature reports that knockout of ADGRG2 can lead to deregulation of fluid reabsorption in the output tubules, leading to male sterility (Davies et al 2004). Zhang Daolai further demonstrates that ADGRG2 and Gq co-localize with the anion channel CFTR on the specific structural top membrane of the output tubule with the aid of β -arestin-1, forming a regional signal transduction complex and constituting a rapid GPCR-ion channel coupling, thereby precisely regulating the chloride current of non-ciliated cells and the process of tubule reabsorption. Overexpression of ADGRG2 in vitro can lead to constitutive viability of Gs and Gq. However, endogenous ligands for ADGRG2 are not currently found.
Dehydroepiandrosterone (DHEA) is a naturally occurring C-19 adrenal steroid (5-androstane-3β -ol-17-1) derived from cholesterol (Webb et al, 2006). Dehydroepiandrosterone is secreted primarily by the reticulum of the adrenal cortex of humans and other primates. The adrenal cortex excretes 75-90% of dehydroepiandrosterone daily, the remainder being produced by the testes and ovaries. DHEA 3 beta-dehydroepiandrosterone sulfate (DHEA-S) is a hydrophilic storage form that circulates in the blood (baulie, 2002). DHEAS can be interconverted with dehydroepiandrosterone by dehydroepiandrosterone sulfotransferase and hydroxysteroid sulfate (baulie, 2002). DHEA and DHEAs are the most abundant steroid hormones in humans, and are a potential androgen at higher blood concentrations than other steroids. DHEA is currently increasingly used in the treatment of women with low ovarian function In Vitro Fertilisation (IVF), and is reported to be effective in improving pregnancy rates and embryo quality, while reducing miscarriage rates (Arlt et al, 1999; shohatt-tal et al, 2015; poplar et al, 2020 a). However, the physiological activity of DHEA is still to be studied further.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides application of steroid hormone DHEA as a receptor ADGRG2 agonist ligand. According to the invention, the steroid hormone DHEA can be used as an endogenous ligand of an orphan receptor ADGRG2, so that the ADGRG2 is effectively activated to generate Gs signals, and the steroid hormone DHEA can form the basis for developing diagnostic and/or therapeutic drugs for male reproductive system diseases, so that the invention has good practical application value.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention there is provided the use of an ADGRG2 modulating compound in the manufacture of a medicament for the diagnosis and/or treatment of a disorder associated with the male reproductive system.
Such male reproductive system related disorders include, but are not limited to, oligospermia, necrospermia, epididymal stasis, associated testicular dysspermia, and male infertility.
Wherein the ADGRG2 modulating compound is a receptor ADGRG2 agonist or a receptor ADGRG2 inducible expression agent.
Further, the ADGRG2 agonist includes a ligand, polypeptide, antibody, or small molecule compound that activates ADGRG 2.
The ligand includes the steroid hormone DHEA.
In a second aspect of the invention there is provided the use of dehydroepiandrosterone as a ligand for the receptor ADGRG2 agonist.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the dehydroepiandrosterone, or a solvate or pharmaceutically acceptable salt thereof, as described above.
And a pharmaceutical preparation comprising the dehydroepiandrosterone or a solvate or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable adjuvant and/or carrier.
In a fourth aspect of the invention there is provided the use of the above DHEA or a solvate or pharmaceutically acceptable salt thereof or the above pharmaceutical composition or formulation in the manufacture of an ADGRG2 activator medicament or agent.
In a fifth aspect, the present invention provides the use of the DHEA or a solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition or formulation described above, in the manufacture of a medicament or agent for use in the treatment of a disease associated with aberrant expression of ADGRG 2.
In a sixth aspect of the invention, there is provided a method of treating a disease associated with aberrant expression of ADGRG2 comprising administering to a subject a therapeutically effective amount of DHEA or the pharmaceutical composition or pharmaceutical formulation.
The beneficial technical effects of one or more of the technical schemes are as follows:
1. the prior research provides the adhesion receptor ADGRG2 for reabsorption of the output small tube liquid of the regulatory male reproductive system, and provides a potential drug target for infertility treatment. The research results of the technical proposal reveal the relation between the adhesion receptor ADGRG2 and the male reproductive system diseases in the mechanism level, and provide reliable basis for the application of the adhesion receptor as a therapeutic target of the male reproductive system diseases.
2. The technical proposal provides the name and the compound structural formula of the endogenous ligand steroid hormone of the adhesion receptor ADGRG2, mediates Gs signal path through the specific combination with the ADGRG2, provides a relatively clear regulation and control mechanism, provides corresponding guidance for the application of the steroid hormone in male reproductive system diseases, and has good practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a graph showing the effect of the steroid hormone DHEA-induced extracellular end configuration change dose of ADGRG2 as an adhesion type receptor in example 1 of the present invention;
FIG. 2 is a graph showing the effect of the steroid hormone DHEA-induced change in extracellular end configuration of the adhesive receptor ADGRG2 in example 1 of the present invention;
FIG. 3 is a graph showing the effect of the steroid hormone DHEA on activating Gs signaling pathway of murine and human adhesion receptor ADGRG2 in example 1 of the present invention;
FIG. 4 is a graph showing the rise in CFTR current in primary cells of output tubules of steroid hormone DHEA-induced adhesion receptor ADGRG 2-mediated wild-type and ADGRG2 knockout mice in example 1 of the present invention.
FIG. 5 is a graph of the rise in CFTR current of steroid hormone DHEA-induced HEK293 cells overexpressing the adhesion-type receptors ADGRG2 and CFTR or CFTR alone in example 1 of the present invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology within the skill of the art, and are fully explained in the literature. See, e.g., sambrook et al, molecular cloning: the techniques and conditions described in the handbook, or as recommended by the manufacturer.
As described above, ADGRG2 is specifically and highly expressed in the output tubule and epididymis, and has important regulatory effect on male reproductive system function. Previous studies by the inventors showed that the adhesion receptor ADGRG2 was coupled to CFTR on the mouse output tubule, regulating fluid reabsorption in the tubule.
In view of the above, the present invention provides a steroid hormone DHEA which acts as an endogenous ligand for the adhesion receptor ADGRG2 and is capable of effectively activating ADGRG2 to generate Gs signals, thereby being expected to be a therapeutic agent for male reproductive system diseases.
In one embodiment of the invention, there is provided the use of an ADGRG2 modulating compound in the manufacture of a medicament for the diagnosis and/or treatment of a disorder associated with the male reproductive system.
In yet another embodiment of the present invention, the male reproductive system related diseases include, but are not limited to, oligospermia, necrospermia, epididymal congestion, associated testicular seminification dysfunction and male infertility.
In yet another embodiment of the invention, the ADGRG2 modulating compound is a receptor ADGRG2 agonist or a receptor ADGRG2 inducible expression agent.
In yet another embodiment of the invention, the ADGRG2 agonist comprises a ligand, polypeptide, antibody, or small molecule compound that activates ADGRG 2.
In yet another embodiment of the present invention, the ligand comprises the steroid hormone DHEA.
In yet another embodiment of the invention, there is provided the use of dehydroepiandrosterone as a ligand for the receptor ADGRG2 agonist.
In yet another embodiment of the present invention, there is provided a pharmaceutical composition comprising the dehydroepiandrosterone, or a solvate or a pharmaceutically acceptable salt thereof, as described above.
And a pharmaceutical preparation comprising the dehydroepiandrosterone or a solvate or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable adjuvant and/or carrier.
The auxiliary materials refer to components except active ingredients in the pharmaceutical composition or the pharmaceutical preparation, and the auxiliary materials are nontoxic to a subject. Excipients commonly used in the art such as buffers, stabilizers, preservatives or excipients, commonly used excipients such as binders, fillers, wetting agents, disintegrants and the like.
As an example, excipients that may be used in the formulation of the present invention include, but are not limited to: the excipient is selected from calcium phosphate, magnesium stearate, talcum powder, dextrin, starch, gel cellulose, methyl cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidone.
The pharmaceutical carriers of the present invention may be pharmaceutically acceptable solvents, suspending agents, vesicles, nanomaterials, and the like, for delivery into an animal or human. The carrier may be liquid or solid and is selected according to the intended mode of administration, while the proteins and liposomes are also pharmaceutical carriers.
The compounds of the present invention may be formulated into pharmaceutical compositions or formulations by those skilled in the art using well known techniques. For example, the DHEA of the present invention as described above is mixed with a pharmaceutical excipient and then, if necessary, the resulting mixture is formed into a desired shape. And, in addition to the ones mentioned in the present invention, suitable pharmaceutical excipients are known in the art, for example, see the pharmaceutical excipients handbook of 2005 edition (original fourth edition), authors (english) r.c. ro (RaymondCRowe) (m) p.j. schenski (PaulJSheskey).
In yet another embodiment of the present invention, there is provided the use of the above-described DHEA or a solvate or pharmaceutically acceptable salt thereof, or the above-described pharmaceutical composition or pharmaceutical formulation, in the manufacture of an ADGRG2 activator drug or agent.
In yet another embodiment of the present invention, there is provided the use of the above DHEA or a solvate or pharmaceutically acceptable salt thereof or the above pharmaceutical composition or pharmaceutical formulation for the manufacture of a medicament or agent for use in the treatment of a disease associated with aberrant expression of ADGRG 2.
In yet another embodiment of the present invention, the diseases associated with abnormal expression of ADGRG2 include diseases associated with male reproductive system, further including but not limited to oligospermia, necrospermia, epididymal stasis, associated testicular seminiferous dysfunction, and male infertility.
In yet another embodiment of the invention, a method of treating a disease associated with aberrant expression of ADGRG2 is provided that includes administering to a subject a therapeutically effective amount of DHEA or the pharmaceutical composition or pharmaceutical formulation.
The subject of the present invention is an animal, preferably a mammal, most preferably a human, which has been the subject of treatment, observation or experiment, and is male (male).
The therapeutically effective amount of the present invention refers to that amount of the active compound or pharmaceutical formulation, including the compound of the present invention, which results in a biological or medical response of the tissue system, animal or human being sought by the researcher, veterinarian, medical doctor or other medical personnel, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.
The range of therapeutically effective amounts that can be used is known to researchers, veterinarians, doctors, or other medical personnel in the art from clinical trials or other means known in the art.
The diseases related to abnormal expression of ADGRG2 comprise diseases related to male reproductive system, and further comprise diseases including but not limited to oligospermia, necrospermia, epididymal stasis, testicular seminification dysfunction and male infertility.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1 steroid hormone DHEA specifically binds ADGRG2 and activates the ADGRG2 downstream signaling pathway and modulates the functional experimental steps of ADGRG2 in the output tubule:
1. commercial steroid hormone DHEA was purchased from the ara Ding Shiji official network.
2. By adopting a molecular biological method, constructing a recombinant murine ADGRG2 plasmid, over-expressing the murine ADGRG2 by HEK293 cells, stimulating by DHEA, and detecting the self-configuration change degree of the ADGRG2 induced by the DHEA by using a bioluminescence resonance energy transfer method of an Nluc group at the N end of the ADGRG2 and a Flash group on an extracellular ring, wherein the specific binding of the DHEA and the ADGRG2 is reflected by the energy transfer degree of a bioluminescence function.
3. The principle that the second messenger cAMP can be detected by using the Glosensor method by over-expressing human or murine ADGRG2 in HEK293 cells, and the condition that ADGRG2 activates Gs signal pathway is characterized by that the agonist DHEA stimulates cells to detect the concentration of intracellular cAMP to the extent that the concentration of intracellular cAMP is increased.
4. Previous studies demonstrated that the adhesion-type receptor ADGRG2 and cystic fibrosis transmembrane conductance regulator (CFTR) co-localize at the output tubule non-ciliated cell apical membrane and that ADGRG2 mediates the current flow of CFTR. Output tubule single cells of wild mice and knockout mice were isolated and infected with adenovirus ADGRG 2-promoter-RFP-expanded virus. The primary cells were stimulated with different concentrations of DHEA and electrophysiological means recorded the current of ADGRG 2-promoter-RFP-expanded cells.
Hek293 cells overexpress ADGRG2 of human or murine origin with CFTR or alone with DHEA at different concentrations, and electrophysiological means record the whole cell currents of both cells.
DHEA was formulated with PBS to a working concentration of 10 -8 M、10 -7 M、10 -6 M、10 -5.5 M、10 -5 M、10 -4.5 M、10 -4 M, in cells over-expressing Nluc-ADGRG2-ECL1/2/3-Flash, incubating for 40 minutes at a constant temperature of 37 ℃ by using a substrate Flash-EDT2 ℃, washing twice by using PBS, then stimulating by using DHEA with different concentrations, recording fluorescence values in real time by using a multifunctional enzyme-labeled instrument, and carrying out statistical analysis and mapping on the results, wherein the results are shown in figure 1.
DHEA was formulated with PBS to a working concentration of 10 -8 M、10 -7 M、10 -6 M、10 -5.5 M、10 -5 M、10 -4.5 M、10 -4 M, ligand stimulation with different concentrations is given to HEK293 cells over-expressing human or mouse ADGRG2 and Glosensor, negative control is HEK293 cells expressing empty vector pcDNA and Glosensor, fluorescence values are recorded in real time by using a multifunctional enzyme-labeled instrument, and the result is obtainedStatistical analysis was performed and the results are shown in FIG. 2.
Wild mice and ADGRG2 whole-body knockout mice are subjected to neck removal and sacrifice, output tubules are separated, collagenase is digested for half an hour and scattered into single cells, adenovirus ADGRG 2-precursor-RFP-labeled virus is used for infection, primary cells are digested to a 24mm multiplied by 24mm slide after 48 hours, after 12 hours primary cells are attached to the wall, the cell slide is taken to a stage of a fluorescence microscope, DHEA stimulation with different concentrations is given, and the primary cells which emit red fluorescence are selected and recorded into whole cell currents by a HEKA EPC10 double-probe amplifier, and the results are shown in figures 3 and 4.
In vitro HEK293 cells were tested for DHEA-induced changes in CFTR current, and DHEA was formulated with PBS to a working concentration of 10 -8 M、10 -7 M、10 -6 M、10 -5.5 M、10 -5 M、10 -4.5 M、10 -4 M, ligand stimulation at different concentrations was given to HEK293 cells co-expressing human or murine ADGRG2 with CFTR or over-expressing CFTR alone, whole cell currents were recorded with HEKA EPC10 dual probe amplifier, and the results are shown in fig. 5.
Experimental results show that DHEA is able to specifically bind to the adhesion receptor ADGRG2 and that the half-effective concentration that induces a change in extracellular loop ECL2 is 25uM; DHEA stimulates the adhesion receptor ADGRG2 to produce Gs pathways, where half-effective concentrations of human and murine are 11uM and 2uM, respectively; DHEA can induce CFTR current increase in wild mice and HEK293 cells co-expressing human or murine ADGRG2 and CFTR, and has no effect on primary cells of fully knockout ADGRG2 mice and HEK293 cells over-expressing CFTR alone, half-effective concentration of DHEA-induced ADGRG 2-mediated increase in CFTR current in primary cells is 14.2uM, half-effective concentration of DHEA-induced increase in CFTR current in human or murine ADGRG 2-mediated increase in overexpression system in vitro is 19.6uM and 2.1uM, respectively.
The above experimental results indicate that the steroid compound DHEA provided in this example is capable of targeting ADGRG2 to its specific ligand and is capable of effectively activating ADGRG2, resulting in a Gs signaling pathway. Thus, the invention provides a promising compound which can form the basis for intervention of medicines for diagnosis and treatment of male reproductive diseases.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. Use of dehydroepiandrosterone to stimulate the adhesion receptor ADGRG2 to produce Gs pathways in HEK293 cells in vitro;
the above applications are performed in an in vitro environment and do not include disease diagnosis and therapeutic uses.
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