WO2024113305A1 - Use of atazanavir for regulating gpr119 receptor activity - Google Patents

Abstract

Provided is use of atazanavir for regulating GPR119 receptor activity. Specifically, provided is use of atazanavir or a pharmaceutically acceptable salt thereof in the preparation of a reagent for regulating GPR119 receptor activity. In the process of performing high-throughput agonist screening on GPR119, it is found that atazanavir can activate a new function of GPR119, and the function is verified at the cellular level by means of a biochemical experiment. The complex structure of atazanavir and GPR119 is analyzed by means of a cryo-electron microscope, it is identified that a binding site of atazanavir and GPR119 exists at an intracellular end of a sixth helix of a receptor transmembrane region, and the site is a new allosteric regulating site of GPR119. By means of a mouse oral glucose tolerance experiment, it is found that by means of intragastric administration of atazanavir for a preventive purpose, a certain hypoglycemic effect is achieved.

Description

Use of atazanavir for modulating GPR119 receptor activity Technical Field

The present disclosure belongs to the field of biomedicine, and specifically relates to the use of atazanavir for regulating the activity of GPR119 receptor.

Background technique

Atazanavir is a widely used anti-HIV drug, approved by the U.S. Food and Drug Administration (FDA) for the treatment of AIDS in 2003. Atazanavir can inhibit the binding of HIV-1 protease to substrates, thereby inhibiting the production of new viruses. Its chemical name is 1-[4-(pyridin-2-yl)phenyl]-5(S)-2,5-di{[N-(methoxycarbonyl)-L-tert-leucine]amino}-4-(S)-hydroxy-6-phenyl-2-azahexane; the structural formula is shown in formula (I).

GPR119 (G protein-coupled receptor 119) is a G protein-coupled receptor that was first reported as an orphan receptor of the A family in 2003. In 2006, studies found that an endogenous cannabinoid, oleylethanolamide (OEA), could activate GPR119. Therefore, oleylethanolamide became a possible endogenous ligand of GPR119, and GPR119 was therefore considered to be a new type of cannabinoid receptor, but with low sequence homology to other known cannabinoid receptors. In the human body, GPR119 is mainly expressed in pancreatic β cells and the gastrointestinal tract, and plays an important role in regulating glucose homeostasis. After binding to agonists, GPR119 can activate downstream stimulating adenylate cyclase g protein (Gs), causing an increase in intracellular cyclic adenosine monophosphate (cAMP) levels. Preclinical studies have shown that GPR119 agonists can increase insulin secretion from β cells, increase the secretion of glucagon-like peptide-1 (GLP1) in primary colonic epithelial cells, and improve glucose tolerance in rodent models. GPR119's function in regulating insulin secretion and blood glucose levels makes it a potential target for the treatment of metabolic diseases such as type 2 diabetes and obesity.

Therefore, reagents and drugs that regulate the activity of the GPR119 receptor need to be further developed for the treatment of diseases with GPR119 as a therapeutic target, and as experimental reagents for related scientific research.

Summary of the invention

Problem that the invention aims to solve

G Protein-Coupled Receptors (GPCRs) regulate physiological functions such as vision, smell, taste, hormone and neurotransmitter signal transduction in the human body, and are also important targets for new drug development. GPR119 is a GPCR expressed in pancreatic islet cells and gastrointestinal cells. Activation of this receptor can promote the release of insulin and glucagon-like peptide-1, thereby regulating blood sugar. Due to its important physiological functions, GPR119 has become a potential target for the development of new diabetes drugs. In addition, the activation of GPR119 has also been reported to enhance the function of tyrosine kinase inhibitors in killing tumors. Therefore, GPR119 is also a potential target for tumor treatment.

As mentioned above, since agents and drugs for regulating the activity of the GPR119 receptor are still to be further developed, the purpose of the present disclosure is to provide the use of atazanavir for regulating the activity of the GPR119 receptor.

Solutions for solving problems

A first aspect of the present disclosure provides use of atazanavir or a pharmaceutically acceptable salt thereof in preparing an agent for regulating GPR119 receptor activity.

In some embodiments, the atazanavir or a pharmaceutically acceptable salt thereof is an agonist allosteric modulator of the GPR119 receptor.

In some specific embodiments, the atazanavir or a pharmaceutically acceptable salt thereof binds to the intracellular end of the sixth transmembrane helix of the GPR119 receptor.

In some specific embodiments, the atazanavir or a pharmaceutically acceptable salt thereof activates the GPR119 receptor via the Gs-AC-cAMP pathway.

In some embodiments, diseases for which GPR119 is a therapeutic target are treated and/or prevented by modulating GPR119 receptor activity.

A second aspect of the present disclosure provides an agent for regulating the activity of a GPR119 receptor, comprising atazanavir or a pharmaceutically acceptable salt thereof as an effective ingredient.

In some embodiments, the agent is a drug.

In some optional embodiments, the drug further comprises a pharmaceutically acceptable carrier.

A third aspect of the present disclosure provides a pharmaceutical composition for treating and/or preventing a disease with GPR119 as a therapeutic target, wherein the pharmaceutical composition comprises atazanavir or a pharmaceutically acceptable salt thereof.

In some optional embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

A fourth aspect of the present disclosure provides a method for regulating GPR119 receptor activity, comprising administering an effective amount of atazanavir or a pharmaceutically acceptable salt thereof to a system or subject in need thereof.

A fifth aspect of the present disclosure provides a method for treating and/or preventing a disease targeting GPR119, comprising administering an effective amount of atazanavir or a pharmaceutically acceptable salt thereof to a subject.

Effects of the Invention

The present disclosure discovered that Atazanavir can activate the new function of GPR119 during the high-throughput agonist screening of GPR119. This function was verified at the cellular level through biochemical experiments. The complex structure of Atazanavir and GPR119 was analyzed by cryo-electron microscopy, and the binding site of Atazanavir and GPR119 was identified to be present at the intracellular end of the sixth helix of the transmembrane region of the receptor, which is a new allosteric regulatory site of GPR119. The key amino acids that Atazanavir interacts with GPR119 at the allosteric regulatory site were verified by biochemical experiments, and the effect of Atazanavir on the GPR119 orthosteric ligand signaling pathway was identified.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A and Figure 1B show the selection and screening of GPR119 agonists using yeast survival pressure, wherein Figure 1A is a schematic diagram of the method for selecting and screening GPR119 agonists using yeast survival pressure; Figure 1B is a diagram showing the results of compound library screening using this method.

Figures 2A and 2B are biochemical validations of the activation function and allosteric regulation function of atazanavir on the GPR119 receptor, wherein Figure 2A shows the activation function of atazanavir on the GPR119 receptor in CHO cells, with AR231453, a known agonist of GPR119, as a control; Figure 2B shows the allosteric regulation of atazanavir on the GPR119 receptor.

Figure 3A and Figure 3B are schematic diagrams of the binding sites of atazanavir and GPR119, wherein Figure 3A is the orthosteric ligand binding pocket of GPR119; and Figure 3B is the allosteric binding site of atazanavir and GPR119.

FIG. 4 shows the key amino acids in the interaction between atazanavir and GPR119.

FIG5A and FIG5B show the effect of atazanavir on oral glucose tolerance in mice.

Detailed ways

The following is a detailed description of the contents of the present disclosure. The description of the technical features described below is based on representative embodiments and specific examples of the present disclosure, but the present disclosure is not limited to these embodiments and specific examples. It should be noted that:

In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values A and B.

In the present specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.

In this specification, the word "may" includes both performing a certain process and not performing a certain process.

In the present specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes cases where the event occurs and cases where it does not occur.

In this specification, the references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

The terms "including" and "having" and any variations thereof in the present disclosure are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

The term "plurality" used in this disclosure refers to two or more than two. "And/or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and/or B may represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character "/" generally indicates that the associated objects are in an "or" relationship.

According to the present disclosure, the terms "polypeptide", "protein" and "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with similar peptide backbones.

According to the present disclosure, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid molecules can be linear or circular.

According to the present disclosure, the term "G protein coupled receptor" or "GPCR" or "GPR" refers to a transmembrane receptor that is capable of transmitting signals from the outside of the cell to the inside of the cell through a G protein pathway and/or an inhibitory protein pathway. Hundreds of such receptors are known in the art; see, for example, Fredriksson et al., Mol. Pharmacol. 63: 1256-1272, 2003, and Vassilatis, D.K., Proc Natl Acad Sci USA 100: 4903-4908 (2003), each of which is incorporated herein by reference. G protein coupled receptors are polypeptides that share a common structural motif, having 7 regions of between 22 and 24 hydrophobic amino acids that form 7 alpha helices, each spanning the cell membrane. Each span is identified by numbering, i.e., transmembrane-1 (TM1), transmembrane-2 (TM2), etc., which may also be referred to as the first transmembrane helix, the second transmembrane helix, etc. in the invention. The transmembrane helices are also connected by amino acid regions between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the outside or "extracellular" side (end) of the cell membrane, which are referred to as "extracellular" regions 1, 2 and 3 (EC1, EC2 and EC3), respectively. The transmembrane helices are also connected by amino acid regions between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the inside or "intracellular" side (end) of the cell membrane, which are referred to as "intracellular" regions 1, 2 and 3 (IC1, IC2 and IC3), respectively. The "carboxyl" ("C") terminus of the receptor is located in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor is located in the extracellular space outside the cell. Any of the above regions can be easily identified by analyzing the primary amino acid sequence of the GPCR.

According to the present disclosure, the term "ligand" or "receptor ligand" means a molecule that specifically binds to a GPCR intracellularly or extracellularly. Without limiting the purpose, a ligand may be a protein, (poly)peptide, lipid, small molecule, protein scaffold, antibody, antibody fragment, nucleic acid, carbohydrate. A ligand may be synthetic or naturally occurring. The term "ligand" includes "natural ligands", which are endogenous, natural ligands of natural GPCRs. In most cases, a ligand is a "modulator" that increases or decreases an intracellular response when in contact (e.g., binding) with a GPCR expressed by a cell. Examples of ligands as modulators include agonists, partial agonists, inverse agonists, and antagonists. Among them, an "agonist" refers to a ligand that increases the signaling activity of a receptor by binding to a receptor. A full agonist can stimulate a receptor to the maximum extent; a partial agonist cannot induce full activity even at saturating concentrations. A partial agonist can also function as a "blocker" by preventing binding to a more powerful agonist. An "antagonist" refers to a ligand that binds to a receptor without stimulating any activity. "Antagonists" are also known as "blockers" because of their ability to prevent other ligands from binding and thus block agonist-induced activity. In addition, "inverse agonists" refer to antagonists that, in addition to blocking the effects of agonists, also reduce the basal or constitutive activity of a receptor below that of a receptor that does not bind a ligand.

According to the present disclosure, there are also allosteric sites on GPCRs, which do not bind to endogenous ligands, but can react specifically with allosteric modulators (AMs) to change the receptor conformation. Allosteric modulators can exhibit a certain range of activity on the target protein. Among them, positive allosteric modulators (PAMs) may not have any intrinsic efficacy, but when they bind to receptors, they enhance the binding affinity or efficacy (or both) of agonists. Negative allosteric modulators (NAMs) do not have any intrinsic efficacy, but when they bind to receptors, they inhibit the binding affinity or efficacy (or both) of agonists. Silent allosteric modulators (SAMs), also known as neutral allosteric ligands (NALs), bind to receptors but have no effect on agonist affinity or efficacy. However, SAMs can act as competitive antagonists at the same allosteric site, blocking PAM or NAM activity. Although not particularly useful from a therapeutic point of view, SAMs can be an effective tool to show that a putative PAM or NAM effect is receptor-mediated. Allosteric agonists (also referred to in this specification as agonistic allosteric modulators) can bind and produce direct agonist activation of a receptor, even in the absence of an agonist.

In this specification, the term "subject" refers to a human (i.e., a male or female of any age, for example, a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young, middle-aged, or elderly person)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog), or a bird. The non-human animal can be male or female at any stage of development. The non-human animal can be a transgenic animal or a genetically engineered animal.

The term "administering" refers to implanting, absorbing, ingesting, injecting, inhaling or otherwise introducing a drug or agent into or onto a subject.

In this specification, the term "treatment" refers to reversing, alleviating, delaying the onset or inhibiting the progression of a disease. In some embodiments, treatment can be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment can be administered in the absence of signs or symptoms of the disease. For example, treatment can be administered to susceptible subjects before the onset of symptoms (e.g., according to the medical history of symptoms). After symptom relief, treatment can also be continued, such as delaying and/or preventing the recurrence of a disease or disorder.

In this specification, the term "prevention" refers to the preventive treatment of subjects who do not have a disease now or in the past but are at risk of developing a disease or who have had a disease in the past and do not have a disease now but are at risk of disease recurrence. In certain embodiments, the subject has a higher risk of developing a disease or a higher risk of disease recurrence compared to the average healthy member of the subject population.

In this specification, "effective amount" refers to an amount sufficient to cause a desired biological response. The effective amount can vary depending on factors such as the desired biological endpoint, pharmacokinetics, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. In certain embodiments, the effective amount is a prophylactically effective amount. In certain embodiments, the effective amount is the amount of a single dose. In certain embodiments, the effective amount is the combined amount of multiple doses.

In this specification, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with a condition. A therapeutically effective amount refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall therapy; reduces or avoids symptoms, signs, or causes of a condition; and/or enhances the therapeutic efficacy of another therapeutic agent.

In this specification, a "prophylactically effective amount" is an amount sufficient to prevent a condition or one or more symptoms associated with a condition or to prevent its recurrence. A prophylactically effective amount refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a condition. The term "prophylactically effective amount" may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

The term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to animals or humans, as appropriate. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

The term "pharmaceutically acceptable salt" refers to a salt that has the potency of the parent agent and is not biologically or otherwise undesirable (e.g., is neither toxic nor otherwise harmful to the recipient thereof). Suitable salts include acid addition salts, which can be formed, for example, by mixing a solution of the parent compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid. If the drug has an acidic moiety (e.g., COOH or a phenolic group), pharmaceutically acceptable salts thereof can include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands (e.g., quaternary ammonium salts). In some exemplary embodiments, the salt form of atazanavir is atazanavir sulfate, which is disclosed in US 6,087,383.

The technical solution of the present disclosure is described in detail below.

In the process of high-throughput screening of agonists for GPR119 using a yeast survival stress screening system, it was found that atazanavir, a clinical drug approved by the FDA for the treatment of AIDS, can activate GPR119. Biochemical verification found that after the small molecule activates GPR119, it can cause the activation of Gs protein in the cell and an increase in cAMP levels. Through complex structure analysis, it was found that Atazanavir binds to the extracellular allosteric regulatory site of GPR119, and when Atazanavir is bound, the receptor exhibits an activated state. The present disclosure discovered a new function of Atazanavir as an agonist allosteric modulator of GPR119 and identified the first allosteric regulatory site of GPR119.

In some aspects of the present disclosure, there is provided a use of atazanavir or a pharmaceutically acceptable salt thereof in the preparation of an agent for modulating GPR119 receptor activity.

In some embodiments, the chemical name of atazanavir is 1-[4-(pyridin-2-yl)phenyl]-5(S)-2,5-di{[N-(methoxycarbonyl)-L-tert-leucine]amino}-4-(S)-hydroxy-6-phenyl-2-azahexane. The structural formula of atazanavir is shown in formula (I):

In some embodiments, the atazanavir or a pharmaceutically acceptable salt thereof is an agonist allosteric modulator of the GPR119 receptor.

In some specific embodiments, the atazanavir or a pharmaceutically acceptable salt thereof binds to the intracellular end of the sixth transmembrane helix of the GPR119 receptor.

In some specific embodiments, the atazanavir or a pharmaceutically acceptable salt thereof activates the GPR119 receptor via the Gs-AC-cAMP pathway.

In some embodiments, the agent that modulates the activity of the GPR119 receptor may be a drug. In some optional embodiments, the disease that uses GPR119 as a therapeutic target is treated and/or prevented by modulating the activity of the GPR119 receptor.

As mentioned above, the present disclosure discovered that atazanavir or a pharmaceutically acceptable salt thereof is an agonist allosteric modulator of the GPR119 receptor and has GPR119 receptor agonist activity, and therefore it can be used to treat and/or prevent diseases associated with GPR119 receptor activity, i.e., diseases with GPR119 as a therapeutic target.

In some embodiments, the disease associated with the GPR119 receptor is a condition that is improved by increasing incretin secretion. In some embodiments, the disease associated with the GPR119 receptor is a condition that is improved by increasing blood incretin levels. In some embodiments, the incretin is GLP-1. In some embodiments, the incretin is glucose dependent insulinotropic polyptide (GIP). In some embodiments, the incretin is peptide YY (PYY).

In some embodiments, the disease associated with the GPR119 receptor is a condition characterized by low bone mass. In some embodiments, the disease associated with the GPR119 receptor is a neurological disease. In some embodiments, the disease associated with the GPR119 receptor is a disease associated with metabolism. In some embodiments, the disease associated with the GPR119 receptor is type II diabetes. In some embodiments, the disease associated with the GPR119 receptor is obesity.

Some embodiments of the present disclosure include every combination of one or more conditions characterized by low bone mass selected from the group consisting of osteopenia, osteoporosis, rheumatoid arthritis, osteoarthritis, periodontal disease, alveolar bone loss, osteotomy bone loss, idiopathic bone loss in childhood, Paget's disease, bone loss due to metastatic cancer, osteolytic lesions, spinal curvature, and height loss.

In some embodiments, the neurological disease is selected from stroke and Parkinson's disease.

Some embodiments of the present disclosure include every combination of one or more metabolic-related diseases selected from the group consisting of type I diabetes, type II diabetes, and conditions associated therewith such as, but not limited to, coronary heart disease, ischemic stroke, restenosis after angioplasty, peripheral vascular disease, intermittent claudication, myocardial infarction (e.g., necrosis and apoptosis), dyslipidemia, postprandial lipemia, conditions of impaired glucose tolerance (IGT), conditions of impaired fasting glucose, metabolic acidosis, ketosis, arthritis, osteoporosis, hypertension, congestive heart failure, left ventricular hypertrophy, peripheral arterial disease, Diabetic retinopathy, macular degeneration, cataracts, diabetic nephropathy, glomerulosclerosis, chronic renal failure, diabetic neuropathy, metabolic syndrome, syndrome X, premenstrual syndrome, coronary heart disease, angina pectoris, thrombosis, atherosclerosis, myocardial infarction, transient ischemic attack, stroke, vascular restenosis, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertriglyceridemia, insulin resistance, impaired glucose metabolism, erectile dysfunction, skin and connective tissue diseases, foot ulcers and ulcerative colitis, endothelial dysfunction and impaired vascular compliance.

Some embodiments of the present disclosure include every combination of one or more metabolic-related diseases selected from the group consisting of diabetes, type I diabetes, type II diabetes, impaired glucose tolerance, impaired glucose tolerance, insulin resistance, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, dyslipidemia, atherosclerosis, stroke, syndrome X, hypertension, pancreatic beta-cell insufficiency, enteroendocrine cell insufficiency, glycosuria, metabolic acidosis, cataracts, diabetic nephropathy, diabetic neuropathy, peripheral neuropathy, diabetic coronary artery disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, diabetic retinopathy, metabolic syndrome, conditions associated with diabetes, myocardial infarction, learning impairment, memory impairment, neurodegenerative diseases, conditions improved by increasing blood GLP-1 levels in individuals with neurodegenerative diseases, excitotoxic brain damage due to severe epileptic seizures, Alzheimer's disease, Parkinson's disease, Huntington's disease, diseases associated with prion viruses, stroke, motor neuron disease, traumatic brain injury, spinal cord injury, and obesity.

In some embodiments, the disease is type II diabetes. In some embodiments, the disease is hyperglycemia. In some embodiments, the disease is hyperlipidemia. In some embodiments, the disease is hypertriglyceridemia. In some embodiments, the disease is type I diabetes. In some embodiments, the disease is dyslipidemia. In some embodiments, the disease is syndrome X. In some embodiments, the disease is obesity. In some embodiments, the disease is metabolic syndrome.

The term "metabolic syndrome" as used in this disclosure refers to a group of risk factors that make a patient more susceptible to developing cardiovascular disease and/or type II diabetes.

1. Overweight and/or obese (BMI ≥ 25).

2. Hyperglycemia: fasting blood glucose (FPG) ≥ 6.1mmol/L (110mg/dl) and/or 2hPG ≥ 7.8mmol/L (140mg/dl), and/or those who have been diagnosed with diabetes and are being treated.

3. Hypertension: systolic/diastolic blood pressure ≥140/90 mmHg, and/or those who have been diagnosed with hypertension and treated.

4. Dyslipidemia: fasting blood triglycerides ≥1.7mmol/L (150mg/dl), and/or fasting blood HDL-C <0.9mmol/L (35mg/dl) (male), <1.0mmol/L (39mg/dl) (female).

Those who possess three or all of the above four components can be diagnosed with metabolic syndrome.

In some embodiments, since activation of GPR119 has also been reported to enhance the tumor-killing function of tyrosine kinase inhibitors, it is shown that GPR119 is also a potential target for tumor treatment. Therefore, diseases associated with the GPR119 receptor also include tumors, such as cancer.

In other embodiments, the agent that regulates the activity of the GPR119 receptor may be a reagent for scientific research or a reagent used in other applications.

In other aspects of the present disclosure, provided is an agent for regulating the activity of a GPR119 receptor, comprising atazanavir or a pharmaceutically acceptable salt thereof as an active ingredient.

In some embodiments, the agent is a drug; in some optional embodiments, the drug further comprises a pharmaceutically acceptable carrier.

In some embodiments of the present disclosure, a pharmaceutical composition for treating and/or preventing a disease with GPR119 as a therapeutic target is provided, wherein the pharmaceutical composition comprises atazanavir or a pharmaceutically acceptable salt thereof; and, optionally, a pharmaceutically acceptable carrier.

It is understood that atazanavir or its pharmaceutically acceptable salt can be administered in a suitable dosage form with one or more pharmaceutical carriers. These dosage forms are suitable for oral, rectal, topical, oral and other parenteral administrations (e.g., subcutaneous, intramuscular, intravenous, etc.). For example, dosage forms suitable for oral administration include capsules, tablets, granules and syrups. The atazanavir contained in these preparations can be solid powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions, etc. The above dosage forms can be prepared by the active compound and one or more carriers or excipients through common pharmaceutical methods. The above carriers need to be compatible with the active compound or other excipients. For solid preparations, commonly used non-toxic carriers include but are not limited to mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, etc. Carriers for liquid preparations include water, saline, aqueous glucose solution, ethylene glycol and polyethylene glycol, etc. The active compound can form a solution or suspension with the above carriers.

The specific administration method and dosage form depend on the physical and chemical properties of the compound itself and the severity of the disease to which it is applied.

In other aspects of the present disclosure, a method of modulating GPR119 receptor activity is provided, comprising administering an effective amount of atazanavir or a pharmaceutically acceptable salt thereof to a system or subject in need thereof.

In other aspects of the present disclosure, a method for treating and/or preventing a disease with GPR119 as a therapeutic target is provided, comprising administering an effective amount of atazanavir or a pharmaceutically acceptable salt thereof to a subject. In other aspects of the present disclosure, atazanavir or a pharmaceutically acceptable salt thereof is provided for treating and/or preventing a disease with GPR119 as a therapeutic target.

The present disclosure is further illustrated by examples below in conjunction with the accompanying drawings, but is not intended to limit the present disclosure. The specific materials and their sources used in the embodiments of the present disclosure are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present disclosure, and materials identical or similar to the types, models, qualities, properties or functions of the following reagents and instruments can be used to implement the present disclosure. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

Example

Example 1. Screening of GPR119 agonists using yeast survival pressure

In this embodiment, a commercial compound library containing about 10,000 small molecules was screened using a high-throughput screening platform for GPCR agonists based on survival pressure selection established in the inventor's laboratory to obtain a new agonist for GPR119. The Saccharomyces cerevisiae strain used in the present disclosure is EBY100, and the commercial yeast plasmid pYD1 (ThermoFisher; V835-01) is used as a plasmid backbone, which is transformed into pYD-SPS-GPR119 that expresses fusion protein I and fusion protein II in EBY100. Among them, fusion protein I is formed by fusion of Trp1p ^CTD to the carboxyl terminus of GPR119; fusion protein II is formed by fusion of Trp1p ^NTD to the inside of miniGs protein. The specific construction process is as follows: the gene of GPR119 is amplified using primers 1 and 2, and inserted downstream of the promoter GAL1,10, and the gene fragment of Trp1p ^CTD is amplified using primers 3 and 4, and inserted into the carboxyl terminus of GPR119 to form fusion protein I. The gene fragment of the miniGs protein was amplified using primers 5 and 6 and inserted upstream of the promoter. The Trp1p ^NTD fragment was amplified using primers 7 and 8 and inserted into a specific position of the miniGs protein gene fragment to form fusion protein II.

Primer 1 (SEQ ID NO: 1):

Primer 2 (SEQ ID NO: 2):

Primer 3 (SEQ ID NO: 3):

Primer 4 (SEQ ID NO: 4):

Primer 5 (SEQ ID NO: 5):

Primer 6 (SEQ ID NO: 6):

Primer 7 (SEQ ID NO: 7):

Primer 8 (SEQ ID NO: 8):

Amino acid sequence of fusion protein I (SEQ ID NO: 9):

The single underlined part is the Trp1p ^CTD part; the double underlined part is the GPR119 part, and the bold part is the connecting peptide. The complete sequence of fusion protein II (SEQ ID NO: 10):

Among them, the single underlined part is the Trp1p ^NTD part; the double underlined part is the miniGs part.

The EBY100 yeast transfected with pYD-SPS-GPR119 was evenly inoculated into a 384-well plate containing small molecules, and cultured at a constant temperature and humidity of 25 degrees Celsius, and the OD600 changes of each well were continuously detected using an ELISA reader (once every day or every two days). After a period of culture and testing, the small molecule Atazanavir from the FDA-approved clinical drug library was screened (Figure 1A and Figure 1B).

Example 2. Biochemical verification of the activation function of Atazanavir on GPR119

In this example, the activation effect of Atazanavir on GPR119 was verified in a CHO cell line (purchased from ATCC) by cAMP GloSensor experiment (the kit used was purchased from Promega, item number E1291), and the known GPR119 agonist AR231453 (purchased from Cayman, item number 34898) was used as a control. CHO cells were transiently transfected with the GPR119 expression vector GPR119_pcDNA3.1 plasmid (the full-length gene of GPR119 (Gene ID: 139760; wherein, the mRNA sequence is shown in NM_178471.3; the protein amino acid sequence is shown in NP_848566.1) was amplified using primers 9 and 10, and a signal peptide and M1 tag were added, and the overlap PCR method was used to insert it into the pcDNA3.1 vector), and a luciferase plasmid that can sense cAMP by protein engineering (the kit was purchased from Promega, item number E1291). The next day, the cell culture medium was replaced with a buffer containing a luciferin substrate. After incubation for 2 hours, the test compound was added, and the changes in intracellular cAMP levels caused by the compound were observed using an ELISA instrument. The results are shown in Figures 2A and 2B. The results show that Atazanavir can effectively activate GPR119 and cause an increase in intracellular cAMP levels. It was thus identified that the activation of GPR119 by Atazanavir is achieved through the Gs-adenylate cyclase (A-cyclase, AC)-cAMP pathway. This example also verifies the allosteric regulatory effect of Atazanavir on GPR119. On the basis of adding the positive agonist AR231453 to stimulate GPR119, 5μM Atazanavir was added at the same time to observe its effect on the activation effect of the positive agonist. The results show that Atazanavir can enhance the activation of GPR119 by the positive agonist AR231453. It is thus identified that Atazanavir has the effect of a positive allosteric regulator on GPR119.

Primer 9 (SEQ ID NO: 11):

Primer 10 (SEQ ID NO: 12):

GPR119 protein sequence with added signal peptide and M1 tag (SEQ ID NO: 13):

Among them, the wavy line is the signal peptide; the double underline is the M1 tag; and the single underline is the wild-type GPR119 protein sequence.

Example 3. Determination of the binding site of Atazanavir to GPR119 by cryo-electron microscopy structure analysis

This example uses cryo-electron microscopy to analyze the complex structure of Atazanavir binding to GPR119-Gs protein. GPR119_miniG plasmid was transiently transfected in HEK293F cells (purchased from ATCC) to express a fusion protein of human GPR119 and miniGs protein. The specific construction method of GPR119_miniG plasmid is as follows. The miniGs protein was amplified using primers 11 and 12, and fused into the GPR119_pcDNA3.1 plasmid constructed in Example 2 to form the GPR119_miniG plasmid. The membrane was melted using detergents L-MNG (purchased from Anatrace, item number NG310) and CHS (purchased from Sigma, item number C6532), purified by M1 affinity column, and then purified by molecular sieve to obtain protein for frozen sample preparation. Data was collected by cryo-electron microscopy, and structural analysis was performed using Cryo-Sparc software. Through structural analysis, it can be seen that Atazanavir does not bind to the conserved extracellular endogenous ligand binding pocket of the A family GPCR, but binds to the intracellular end of the sixth transmembrane helix. This is the first identified allosteric regulatory site of GPR119 (Figure 3A and Figure 3B).

Primer 11 (SEQ ID NO: 14):

Primer 12 (SEQ ID NO: 15):

Sequence of miniGs protein (SEQ ID NO: 16):

GPR119_miniG fusion protein sequence (SEQ ID NO: 17):

Among them, the single underline part is the miniGs part; the double underline part is the GPR119 part; the wavy line part is the signal peptide; the dotted underline is the M1 tag; the italic is the protease cleavage site; the bold is the purification tag; and GS is the connecting peptide.

Example 4. Identification of key amino acids in the interaction between Atazanavir and GPR119

Based on the structure of the complex, it can be seen that the interaction between Atazanavir and GPR119 is mainly mediated by key amino acids such as S228 and R225. This example verifies the key amino acids observed in the structure for the interaction between Atazanavir and GPR119 through the cAMP GloSensor experiment of the GPR119 mutant (the experimental method is the same as in Example 2). The sequence of wild-type human GPR119 is referenced from NCBI, Gene ID: 139760 (wherein, the mRNA sequence is referred to as NM_178471.3; the protein amino acid sequence is referred to as NP_848566.1). This example uses a single-point mutation method to construct mutants of S228W and R225D (the mutation site is based on the amino acid sequence of NP_848566.1), and the primers used are as follows:

S228W-F (SEQ ID NO: 18)

S228W-R (SEQ ID NO: 19)

R225D-F (SEQ ID NO: 20)

R225D-R (SEQ ID NO: 21)

The interaction between Atazanavir (Ata for short) and wild-type and mutant GPR119 was verified by cAMP experiment. When mutations S228W and R225D were detected, the binding ability of Atazanavir to GPR119 was significantly reduced, proving that S228 and R225 are indeed the key amino acids for the interaction between Atazanavir and GPR119 (see Figure 4, where SW represents S228W; RD represents R225D; WT represents wild-type GPR119).

Example 5. Identification of the effect of atazanavir on oral glucose tolerance in mice

After the C57 mice were purchased, they were fed in a normal environment for one week; the mice were fasted but not watered overnight before the experiment. The experimental mice were randomly divided into three groups: negative control group, positive control group, and experimental group. The negative control group was gavaged with an equivalent volume of solvent at a dose of 0.1 ml/10 g; the positive control group was gavaged with a known compound AR231453 (purchased from Cayman, item number 34898) at a dose of 50 mg/kg (i.e., 0.1 ml/10 g); the experimental group was gavaged with Atazanavir (purchased from Targetmol, item number T0100) compound working solution at a dose of 100 mg/kg (i.e., 0.1 ml/10 g);

Each experimental mouse was intragastrically administered with 20% glucose solution at a dose of 2 g/kg (i.e., 0.1 ml/10 g) 30 minutes after the first intragastrial administration;

0 minute was counted after intragastric administration of glucose, and a small amount of tail vein blood was collected at 0, 30, 60, 90, and 120 minutes, and the blood glucose level was tested with a blood glucose meter (Roche); after the blood glucose test, the mice were killed by cervical dislocation.

The results are shown in Figures 5A and 5B. Compared with 0 minutes (i.e., after intragastric administration of glucose), the blood glucose of mice in the vehicle group, AR231453 group, and Atazanavir group increased significantly after 30 minutes, with increases of 184.5%, 87.6%, and 59.2%, respectively (p<0.05); after 60 minutes, the blood glucose of all animals decreased to a certain extent, and the blood glucose values of the vehicle group at 60 and 90 minutes were still statistically different from those at 0 minutes (p<0.05). Intergroup comparison at the same time showed that at 30 minutes, the blood glucose of mice in the Atazanavir group and AR231453 group was significantly lower than that of mice in the vehicle group (p<0.05), indicating that the preventive administration of Atazanavir has a certain hypoglycemic effect.

The description presented in the above exemplary embodiments is only used to illustrate the technical solution of the present disclosure, and is not intended to be exhaustive, nor is it intended to limit the present disclosure to the precise form described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described to explain the specific principles of the present disclosure and its practical application, so that other technicians in the field can easily understand, implement and use the various exemplary embodiments of the present disclosure and its various selected forms and modified forms. The scope of protection of the present disclosure is intended to be defined by the attached claims and their equivalents.

Claims (10)

Use of atazanavir or a pharmaceutically acceptable salt thereof in preparing an agent for regulating GPR119 receptor activity. The use according to claim 1, wherein the atazanavir or a pharmaceutically acceptable salt thereof is an agonist allosteric modulator of the GPR119 receptor. The use according to claim 1 or 2, wherein the atazanavir or a pharmaceutically acceptable salt thereof binds to the intracellular end of the sixth transmembrane helix of the GPR119 receptor. The use according to any one of claims 1 to 3, wherein the atazanavir or a pharmaceutically acceptable salt thereof activates the GPR119 receptor via the Gs-AC-cAMP pathway. The use according to any one of claims 1 to 4, wherein the disease with GPR119 as a therapeutic target is treated and/or prevented by regulating the activity of the GPR119 receptor. An agent for regulating the activity of a GPR119 receptor, comprising atazanavir or a pharmaceutically acceptable salt thereof as an active ingredient. The agent for regulating the activity of the GPR119 receptor according to claim 6, wherein the agent is a drug; Optionally, the drug further comprises a pharmaceutically acceptable carrier. A pharmaceutical composition for treating and/or preventing a disease with GPR119 as a therapeutic target, wherein the pharmaceutical composition comprises atazanavir or a pharmaceutically acceptable salt thereof; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. A method for modulating GPR119 receptor activity comprises administering an effective amount of atazanavir or a pharmaceutically acceptable salt thereof to a system or subject in need thereof. A method for treating and/or preventing a disease with GPR119 as a therapeutic target, comprising administering an effective amount of atazanavir or a pharmaceutically acceptable salt thereof to a subject.

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