Lipopeptide HIV membrane fusion inhibitors and their drug uses

Abstract

This invention discloses a lipopeptide HIV membrane fusion inhibitor and its pharmaceutical use. The lipopeptide of this invention is of formula I: X1-EMTWEEWEK KVEELEKKIEELLK-X2-X3-X4-X5; or formula II: X1-ELTWEEWEKKVEELEKKIEELLK-X6-X7-X4-X5; wherein, X1 is an amino-terminal protecting group; X2 is a polypeptide sequence (EAAAK)n, A[(EAAAK)n]A, or (EP)n, where n represents the number of repetitions; X3 is Lys, Cys, Dap, Orn, Dab, Dah, or absent; X4 is a lipophilic compound group used to modify the C-terminus; X5 is a carboxyl-terminal protecting group; X6 is a polypeptide sequence KAEEQQKKNE; and X7 is Lys, Cys, Dap, Orn, Dab, or Dah. The lipopeptide of this invention has higher antiviral activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to lipopeptide-based HIV membrane fusion inhibitors and their drug uses. Background Technology

[0002] Acquired Immune Deficiency Syndrome (AIDS) is a chronic disease caused by infection with the Human Immunodeficiency Virus (HIV). HIV primarily infects CD4+ T cells and dendritic cells, monocytes, and macrophages that express the CD4 molecule, attacking the body's immune system and causing opportunistic infections and tumors. It seriously threatens the lives and health of patients. To date, there is neither an effective preventative vaccine nor a cure, and AIDS remains a major global public health problem.

[0003] Currently approved anti-HIV drugs primarily target the four stages of the viral life cycle: viral invasion, reverse transcription, integration, and maturation. The process of HIV invading target cells is mediated by its surface envelope glycoprotein (Env), which consists of the surface subunit gp120 and the transmembrane subunit gp41 linked by non-covalent bonds. When the virus infects a target cell, the surface subunit gp120 first binds to the CD4 receptor on the target cell membrane, causing a conformational change in gp120 that exposes its binding site for co-receptors. Subsequently, gp120 binds to co-receptors (CCR5 or CXCR4) on the target cell surface, leading to its separation from the transmembrane subunit gp41. Finally, gp41 undergoes a conformational change, initiating the fusion of the viral envelope with the target cell membrane, completing the process of viral entry into the host cell. In terms of mechanism of action, HIV fusion inhibitors competitively bind to the N- or C-terminal heptapeptide repeat region (NHR or CHR) of gp41, thereby blocking the formation of the six-hexagonal bundle (6-HB) structure and inhibiting viral entry into target cells. T20 is the first and currently the only HIV fusion inhibitor drug approved by the US FDA. It is derived from amino acids 638-673 of the gp41 CHR and consists of 36 amino acid residues. However, its relatively low antiviral activity requires high-dose injections (90 mg twice daily), and it suffers from drawbacks such as a short half-life, easy induction of drug resistance, and high treatment costs, which significantly limit its clinical application. Therefore, developing novel HIV fusion inhibitors with improved drug properties is crucial.

[0004] Previous research by the inventors has shown that the MT hook structure can significantly enhance the binding and antiviral activity of CHR-derived peptide inhibitors. Based on this, they designed a short peptide membrane fusion inhibitor, 2P23, containing the MT hook structure and the PBD region sequence. 2P23 exhibits potent and broad-spectrum antiviral activity against HIV-1, HIV-2, and SIV. To further improve the stability and antiviral activity of 2P23, palmitic acid (C16) modified lipopeptide LP-19 and stearic acid (C18) modified lipopeptide LP-25 were subsequently prepared. Both lipopeptides showed highly efficient anti-HIV activity both in vivo and in vitro. Multiple studies have shown that cholesterol modification is also an important strategy for developing lipopeptide-based viral fusion inhibitors, such as the HIV fusion inhibitory lipopeptides C34-Chol and LP-98. During the COVID-19 pandemic, a series of SARS-CoV-2 fusion inhibitory lipopeptides were rapidly developed through cholesterol modification, including SARS-CoV-2HR2-derived lipopeptides IPB02, IPB24, IPB29, SARSHRC-PEG4-chol, P40-LP, and P315V3, as well as HCoV-OC43HR2-derived lipopeptide EK1C4. In lipopeptide-based design strategies, a linker is typically added between the lipid molecule and the peptide sequence to act as a connecting arm. Flexible linker combinations of small molecule polyethylene glycol (PEG)n and glycine (G) and serine (S), such as (GGGGS)n or (GSGSG)n, have been widely used. Summary of the Invention

[0005] In order to improve the antiviral activity of HIV membrane fusion inhibitors, this invention aims to provide lipopeptide HIV membrane fusion inhibitors and their pharmaceutical uses.

[0006] The specific technical solution of this invention is as follows:

[0007] This invention provides a lipopeptide or a pharmaceutically acceptable salt thereof, characterized in that the lipopeptide is represented by formula I or formula II as follows:

[0008] Formula I: X1-EMTWEEWEKKVEELEKKIEELLK-X2-X3-X4-X5;

[0009] Formula II: X1-ELTWEEWEKKVEELEKKIEELLK-X6-X7-X4-X5;

[0010] Wherein, X1 is an amino-terminal protecting group, X4 is a lipophilic compound group used to modify the C-terminus, and X5 is a carboxyl-terminal protecting group;

[0011] X2 and X3 are either one of 1) and 2) below:

[0012] 1) X2 is a polypeptide sequence (EAAAK)n, which acts as a rigid linker and serves as a connecting arm, where n represents the number of repeats of EAAAK, and n takes the value 1, 2, 3, 4, 5 or 6; X3 is lysine (Lys), cysteine ​​(Cys), 2,3-diaminopropionic acid (Dap), ornithine (Orn), 2,4-diaminobutyric acid (Dab), 2,7-diaminoheptanoic acid (Dah) or is absent;

[0013] 2) X2 is a polypeptide sequence A[(EAAAK)n]A or (EP)n, which acts as a rigid linker and serves as a connecting arm, where n represents the number of repetitions of EAAAK or EP, and n takes the value 1, 2, 3, 4, 5 or 6; X3 is lysine (Lys), cysteine ​​(Cys), 2,3-diaminopropionic acid (Dap), ornithine (Orn), 2,4-diaminobutyric acid (Dab) or 2,7-diaminoheptanoic acid (Dah);

[0014] X6 is the polypeptide sequence KAEEQQKKNE, and X7 is lysine (Lys), cysteine ​​(Cys), 2,3-diaminopropionic acid (Dap), ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,7-diaminoheptanoic acid (Dah).

[0015] Furthermore, the amino-terminal protecting group is any one of acetyl (Ac), amino (NH2), maleyl, succinyl, tert-butoxycarbonyl, benzyloxy, or other hydrophobic groups or macromolecular carrier groups.

[0016] Furthermore, the carboxyl-terminal protecting group is any one of amino (NH2), carboxyl, hydroxyl, amide, tert-butoxycarbonyl, or other hydrophobic groups or macromolecular carrier groups.

[0017] Further, the lipophilic compound is any one of cholesterol succinate monoester, 2-cholesterol acetic acid, 2-cholesterol propionic acid, 3-cholesterol propionic acid, 2-cholesterol butyric acid, 2-cholesterol isobutyric acid, 3-cholesterol butyric acid, 3-cholesterol isobutyric acid, 4-cholesterol butyric acid, 2-cholesterol valerate, 2-cholesterol isovaleric acid, 3-cholesterol valerate, 5-cholesterol valerate, 2-cholesterol hexanoic acid, 6-cholesterol hexanoic acid, 2-cholesterol heptanoic acid, 7-cholesterol heptanoic acid, 2-cholesterol octanoic acid, 8-cholesterol octanoic acid, bromoacetate cholesterol ester, fatty acids containing 8 to 20 carbon atoms (such as octadecanoic acid), dihydrosphingosine, and vitamin E, preferably cholesterol succinate monoester.

[0018] Furthermore, the amino-terminal protecting group is an acetyl group; and / or, the carboxyl-terminal protecting group is an amino group.

[0019] Furthermore, X2 and X3 are either 1) or 2) below:

[0020] 1) X2 is a polypeptide sequence (EAAAK)n, where n represents the number of repeats, and n is 1, 2, 3, 4, 5 or 6; X3 is lysine or is absent.

[0021] 2) X2 is a polypeptide sequence A[(EAAAK)n]A or (EP)n, where n represents the number of repeats, and n is 1, 2, 3, 4, 5 or 6; X3 is a lysine residue.

[0022] X7 is lysine.

[0023] Furthermore, the compound of Formula I is LP-35, X1 is Ac, X2 is EAAAK (n=1), X3 is lysine, X4 is cholesterol succinate monoester, and X5 is NH2.

[0024] Furthermore, the compound of Formula I is LP-36, X1 is Ac, X2 is EAAAKEAAAK (n=2), X3 is lysine, X4 is cholesterol succinate monoester, and X5 is NH2.

[0025] Furthermore, the compound of Formula I is LP-37, X1 is Ac, X2 is AEAAAAKEAAAKA (n=2), X3 is lysine, X4 is cholesterol succinate monoester, and X5 is NH2.

[0026] Furthermore, the compound of Formula I is LP-38, X1 is Ac, X2 is EAAAKEAAAKEAAAK (n=3), X3 is not present, X4 is cholesterol succinate monoester, and X5 is NH2.

[0027] Furthermore, the compound of Formula I is LP-39, X1 is Ac, X2 is EPEPEEPEPEPEPEP (n=6), X3 is lysine, X4 is cholesterol succinate monoester, and X5 is NH2.

[0028] Furthermore, the compound of Formula II is LP-32, X1 is Ac, X6 is KAEEQQKKNE, X7 is lysine, X4 is cholesterol succinate monoester, and X5 is NH2.

[0029] The abbreviations of amino acids in Formula I and Formula II have meanings known in the art, such as E for glutamic acid, M for methionine, T for threonine, W for tryptophan, K for lysine, V for valine, L for leucine, I for isoleucine, and P for proline.

[0030] The present invention also provides a pharmaceutical composition comprising the lipopeptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0031] The present invention also provides the use of the lipopeptide or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in the preparation of a lipopeptide HIV membrane fusion inhibitor.

[0032] This invention also provides the use of the lipopeptide or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in the preparation of any of the following:

[0033] 1) Antiviral drugs;

[0034] 2) Medications for the prevention and / or treatment of diseases caused by viral infections;

[0035] 3) Drugs that inhibit viral cell fusion;

[0036] 4) Drugs that inhibit viral invasion of cells;

[0037] 5) Drugs that inhibit viral replication;

[0038] The virus is one or more of HIV-1, HIV-2, and SIV.

[0039] Furthermore, the disease caused by the viral infection is AIDS.

[0040] Further details of this invention are described in detail below, or some of them may be reflected in the embodiments of this invention.

[0041] Unless otherwise specified, the quantities of different components and reaction conditions used herein are to be interpreted as "approximate" or "about". Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under their respective experimental conditions due to different standard errors.

[0042] In this document, when there is disagreement or ambiguity regarding the chemical structure and chemical name of a compound, the compound is defined precisely by its chemical structure. The compounds described in this invention may contain one or more chiral centers, and / or double bonds and similar structures, and may also exist as stereoisomers, including isomers of the double bonds (e.g., geometric isomers), optical enantiomers, or diastereomers. Accordingly, any chemical structure within the scope of this description, whether partially or entirely containing similar structures, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (e.g., a single geometric isomer, a single enantiomer, or a single diastereomer) and any mixture of these isomers. These mixtures of racemic and stereoisomers can be further separated into enantiomers or stereoisomers of their constituent components by those skilled in the art using various separation techniques or methods of chiral molecule synthesis.

[0043] Compounds of Formula I and II include, but are not limited to, optical isomers, racemates, and / or other mixtures of these compounds. In the above cases, the single enantiomer or diastereomer, such as the optically active isomer, can be obtained by asymmetric synthesis or by racemic resolution. Racemic resolution can be achieved by various methods, such as conventional recrystallization with a resolving agent or by chromatographic methods. Additionally, compounds of Formula I and II also include cis and / or trans isomers with double bonds.

[0044] The compounds described in this invention include, but are not limited to, the compounds shown in Formula I and Formula II, and all their pharmaceutically available forms. These pharmaceutically available forms include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, drug prodrugs based on the above substances, and any mixtures of these forms.

[0045] In practical applications, the drug of this invention can be administered directly to patients or mixed with a suitable carrier or excipient before administration to achieve the purpose of treating and / or preventing HIV infection. The carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Various dosage forms can be formulated using these materials, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. Various carriers known in the art can be widely used to formulate unit dosage forms into suppositories. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.To formulate unit-dose dosage forms for injection, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all commonly used diluents in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Additionally, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injection formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. The above dosage forms can be administered via injection, including subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; or via cavities, such as rectal and vaginal administration. Injection is the preferred route of administration.

[0046] The dosage of the drug of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight and individual response of the patient or animal, the specific active ingredient used, the route of administration and the frequency of administration, etc. The above dosage can be administered in a single dose form or in several, such as two, three or four dose forms.

[0047] The present invention also provides the use of the pharmaceutical composition described above in the prevention and treatment of HIV infection.

[0048] The drug of this invention can be used alone for the treatment and prevention of HIV infection, or it can be used in combination with one or more other antiviral drugs to improve the overall treatment effect. These anti-HIV drugs include, but are not limited to, reverse transcriptase inhibitors, protease inhibitors, invasion inhibitors, integration inhibitors, and maturation inhibitors. The aforementioned reverse transcriptase inhibitors may be one or more of AZT, 3TC, ddI, d4T, ddT, TDF, Abacavir, Nevirapine, Efavirenz, Delaviridine, Azvudine, and Enavirin; the aforementioned protease inhibitors may be one or more of Saquinavir mesylate, Idinavir, Ritonavir, Amprenavir, Kaletra, and Nelfinavir mesylate; the aforementioned invasion inhibitors may be one or more of Maraviroc, TAK-779, T20, T2635, Sifviride, and Eboviride; and the aforementioned integration inhibitors may be one or more of Raltegravir, Dolutegravir, and Elvitegravir.

[0049] For any given patient, the specific effective therapeutic dose level must be determined based on a number of factors, including the disorder being treated and its severity; the activity of the specific active ingredient used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific active ingredient; the duration of treatment; medications used in combination with or concurrently with the specific active ingredient; and similar factors known in the medical field. For example, it is practiced in the art to start the dose of the active ingredient below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0050] The beneficial effects of this invention are as follows:

[0051] The lipopeptide or its pharmaceutically acceptable salt form provided by this invention is stable and is a highly effective, novel anti-HIV membrane fusion inhibitor with higher antiviral activity. It can be used to prepare pharmaceutical compositions for the prevention and / or treatment of diseases caused by HIV infection. Attached Figure Description

[0052] Figure 1 Anti-HIV-1 activity of a newly designed lipopeptide membrane fusion inhibitor;

[0053] Figure 2 The inhibitory activities of LP-37 and LP-39 against different HIV-1 subtypes were evaluated.

[0054] Figure 3 The inhibitory activity of LP-37 and LP-39 on HIV-1Env-mediated cell fusion;

[0055] Figure 4 The inhibitory activities of LP-37 and LP-39 against HIV-2 and SIV strains were evaluated.

[0056] Figure 5 The inhibitory activities of LP-37 and LP-39 against T20 and HP23 resistant mutants were evaluated.

[0057] Figure 6 The secondary structure and thermal stability of LP-37 and LP-39;

[0058] Figure 7 Results of in vitro metabolic stability analysis of LP-37 and LP-39. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The embodiments provided below can serve as a guide for those skilled in the art to make further improvements and do not constitute a limitation on the present invention in any way. Those skilled in the art can refer to the content of this document and appropriately improve the relevant parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The method of the present invention has been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] Example 1: Design and synthesis of lipopeptides

[0062] LP-25 and LP-26 are lipopeptides modified with stearic acid and cholesterol, respectively, based on the 2P23 short peptide and modified with a PEG8 linker, developed by the inventors in the past. To develop a more effective HIV fusion inhibitor, this embodiment further designed cholesterol-modified lipopeptides with different linker arms. First, the second amino acid "M" at the N-terminus of LP-26 was replaced with "L," and amino acid sequences of different lengths were introduced at its C-terminus to obtain lipopeptides LP-30, LP-31, and LP-32, respectively. The introduced amino acid sequences may serve as natural linkers between the 2P23 peptide sequence and cholesterol. Next, lipopeptides LP-33 and LP-34 were obtained by introducing a linker consisting of the common combination of glycine (G) and serine (S). Finally, repeating rigid linkers "EAAAK" and "EP" were introduced between the peptide sequence and cholesterol to obtain lipopeptides LP-35, LP-36, LP-37, LP-38, and LP-39.

[0063] All lipopeptides involved in this embodiment were synthesized using the standard solid-phase 9-fluorenylmethoxycarbonyl (FMOC) method on rinkamide 4-methylphenylhydroxylamine (MBHA) resin. All peptides were acetylated at the N-terminus and aminoized at the C-terminus. For cholesterol conjugation, the template peptide contained a lysine residue at the carboxyl terminus with a 1-(4,4-dimethyl-2,6-dioxycyclohexylethylene)ethyl side-chain protecting group, and underwent a specific deprotection step in 2% hydrazine hydrate-n,n-dimethylformamide (DMF) solution for fatty acid conjugation. Finally, the peptides were purified by reversed-phase high-performance liquid chromatography (HPLC) to a purity >95%. The specific peptide names and sequence structures are as follows:

[0064] Lipopeptide name sequence structure LP-25 <![CDATA[Ac-EMTWEEWEKKVEELEKKIEELLK-PEG8-K(C18)-NH2]]> LP-26 <![CDATA[Ac-EMTWEEWEKKVEELEKKIEELLK-PEG8-C(chol)-NH2]]> LP-30 <![CDATA[Ac-ELTWEEWEKKVEELEKKIEELLKKAEEQQKK(chol)-NH2]]> LP-31 <![CDATA[Ac-ELTWEEWEKKVEELEKKIEELLKAAQEQQEK(chol)-NH2]]> LP-32 <![CDATA[Ac-ELTWEEWEKKVEELEKKIEELLKKAEEQQKKNEK(chol)-NH2]]> LP-33 <![CDATA[Ac-ELTWEEWEKKVEELEKKIEELLKGSGGSGGK(chol)-NH2]]> LP-34 <![CDATA[Ac-EMTWEEWEKKVEELEKKIEELLKGGGGSGGGGSK(chol)-NH2]]> LP-35 <![CDATA[Ac-EMTWEEWEKKVEELEKKIEELLKEAAAKK(chol)-NH2]]> LP-36 <![CDATA[Ac-EMTWEEWEKKVEELEKKIEELLKEAAAKEAAAKK(chol)-NH2]]> LP-37 <![CDATA[Ac-EMTWEEWEKKVEELEKKIEELLKAEAAAKEAAAKAK(chol)-NH2]]> LP-38 <![CDATA[Ac-EMTWEEWEKKVEELEKKIEELLKEAAAKEAAAKEAAAK(chol)-NH2]]> LP-39 <![CDATA[Ac-EMTWEEWEKKVEELEKKIEELLKEPEPEPEPEPEPK(chol)-NH2]]>

[0065] Example 2: Discovery and Identification of Highly Active Lipopeptides Based on Rigid Linkers

[0066] 1. Experimental Materials and Methods

[0067] The test lipopeptides were LP-30, LP-31, LP-32, LP-33, LP-34, LP-35, LP-36, LP-37, LP-38, and LP-39 synthesized in Example 1, and the control lipopeptides LP-25 and LP-26. The viruses used were HIV-1 strain NL4-3 and JRFL pseudovirus, prepared in our laboratory; the target cells, TZM-bl, were a product of the National Experimental Cell Resource Sharing Service Platform. The specific experimental steps are as follows:

[0068] ①Preparation of HIV-1 pseudovirus: plasmids expressing NL4-3 or JRFL envelope proteins (Env) were co-transfected with HIV-1 backbone plasmid pSG3Δenv into HEK293T cells; transfected cells were cultured in a 37°C, 5% CO2 cell culture incubator for 48 hours, and then the supernatant was collected, filtered and the filtrate was collected, which is the pseudovirus solution containing NL4-3 pseudovirus or JRFL or SF162. After titration, it was stored at -80°C for later use.

[0069] ② Dissolve the test lipopeptides in deionized water or dimethyl sulfoxide (DMSO), then dilute the lipopeptides to the initial concentration using DMEM medium. Next, serially dilute the lipopeptides 3-fold in a 96-well cell culture plate to obtain the lipopeptide dilutions. Nine dilutions are set for each test lipopeptide.

[0070] ③ In a 96-well cell culture plate, add lipopeptide dilution buffer (50 μL / well) to the drug wells and DMEM medium (50 μL / well) to the control wells, with 3 replicates for each well. Then add 1000 TCID50. 50 The viral solution (adjusted to 50 μL / well) was incubated at room temperature for 30 minutes.

[0071] ④ Resuspend the pre-cultured target cells TZM-bl in DMEM medium and adjust the cell concentration to 10 × 10⁻⁶. 4 Cells were added at a concentration of 15 μg / mL, followed by the addition of DEAE-dextran to a final concentration of 15 μg / mL. Finally, cells (100 μL / well) were added to 96-well plates containing the virus & lipopeptide complex and cultured at 37°C in a 5% CO2 cell incubator for 48 hours.

[0072] ⑤ Discard the cell culture supernatant, add 30 μL of cell lysis buffer (Promega, catalog number E1531) to each well, lyse at room temperature for 15 minutes, then add luciferase detection substrate reagent (Promega, catalog number E1501), measure the relative fluorescence units (RLU) using a microplate photometer, construct an inhibition rate curve, and calculate the half-maximal inhibitory concentration (IC50) of the drug. 50 ).

[0073] 2. Experimental Results and Analysis

[0074] The results are as follows Figure 1 As shown, compared with LP-26, which has a PEG8 flexible linker, the anti-HIV activity of lipopeptides LP-30 and LP-31, which have a C-terminal extended amino acid "KAEEQQK" and "AAQEQQE" respectively, was not improved; while the lipopeptide LP-32, which has a C-terminal extended amino acid "KAEEQQKKNE", inhibited CXCR4-tropic HIV. NL4-3The activity of the strains was significantly enhanced. Furthermore, LP-33, with a shorter "GSGGSGG" flexible linker, exhibited lower antiviral activity, while LP-34, with a longer "GGGGSGGGGS" flexible linker, showed slightly increased antiviral activity. These results indicate that relatively longer linkers can enhance the antiviral activity of the inhibitors. Among the lipopeptide inhibitors with rigid linkers, LP-35, with a single "EAAAK" linker, showed no increased antiviral activity, while LP-36 and LP-37, with two repeating "EAAAK" sequence linkers, showed significantly increased antiviral activity. Clearly, the two helical-promoted amino acids (alanine) on either side of the linker in LP-37 can further enhance the inhibitory activity. LP-38, with three repeating "EAAAK" sequence linkers, and LP-39, with six repeating "EP" sequence linkers, showed antiviral activity comparable to LP-37. The above results indicate that rigid linkers with repeating “EAAAK” or “EP” amino acid sequences are superior to PEG8 or repeating “GGGGS” amino acid sequences. In contrast, LP-37 and LP-39, with 12 amino acid rigid linkers “AEAAAKEAAAKA” and “EPEPEPEPEPEPEP” respectively, showed the greatest optimization and exhibited the highest antiviral activity compared to LP-26, which contains a flexible linker. LP-37 and LP-39 inhibited HIV-1. NL4-3 IC 50 The values ​​were 8.08 and 8.17 pM, respectively, indicating inhibition of HIV-1. JRFL IC 50 The values ​​were 76.85 and 56.96 pM, respectively.

[0075] Example 3: Broad-spectrum inhibitory activity of LP-37 and LP-39 against HIV-1, HIV-2 and SIV

[0076] AIDS is primarily caused by HIV, which is further divided into HIV-1 and HIV-2. HIV-1 is more prevalent, highly infectious, and widely distributed, accounting for 95% of infections. HIV-2 is mainly distributed in Africa, showing a localized epidemic trend. HIV-1 is highly variable, having evolved into many subtypes and recombinant viruses. HIV-1 subtypes A, B, and C are the main viruses causing the global AIDS epidemic, while in China, A / E and B / C recombinant viruses are predominant. This embodiment further evaluates the antiviral activity of the aforementioned dominant lipopeptides LP-37 and LP-39 against a group of 36 HIV-1, HIV-2, and SIV strains through pseudovirus inhibition experiments, viral replication inhibition experiments, and fusion inhibitor experiments based on a DSP system.

[0077] 1. Experimental Materials and Methods

[0078] (1) Virus inhibition experiment

[0079] The test lipopeptides were LP-37 and LP-39 synthesized in Example 1. The HIV-1 pseudovirus used was... Figure 2 As shown, the SIV pseudovirus used is SIV. 239 and SIV PBJ The HIV-2 replicating virus used is HIV-2. ROD and HIV-2 ST The target cells, TZM-bl, are a product of the National Experimental Cell Resource Sharing Service Platform. The specific experimental steps are as follows:

[0080] ① Virus preparation: plasmids expressing HIV-1 or SIV envelope protein (Env) were co-transfected with HIV-1 backbone plasmid pSG3Δenv into HEK293T cells; molecular cloning plasmids encoding HIV-2 virus strain ROD or ST were transfected into HEK293T cells; transfected cells were cultured in a 37℃, 5% CO2 cell culture incubator for 48 hours, and then the supernatant was collected, filtered and the filtrate was collected as the virus solution, titrated and stored at -80℃ for later use.

[0081] After obtaining the viral fluid, the experimental steps are the same as steps ②-⑤ in Example 2.

[0082] (2) Fusion suppression experiment based on DSP system

[0083] The tested lipopeptides were LP-37 and LP-39 synthesized in Example 1. Plasmids expressing different HIV-1 Env subtypes were preserved in our laboratory; the pDSP8-11 fluorescent reporter plasmid and plasmids stably expressing CXCR4 / CCR5 and DSP were also used. 8-11 The target cells, 293FT, were provided by Professor Zene Matsuda of the University of Tokyo, Japan, and are routinely used and preserved in our laboratory; 293T cells were purchased from the American Type Culture Collection. The specific experimental procedures are as follows:

[0084] ① Seed 293T cells (effective cells) into 96-well plates for culture (approximately 1.5 × 10⁻⁶ cells). 4 (each well / hole) will simultaneously stably express CXCR4 / CCR5 and DSP. 8-11 Target cells 293FT (approximately 1.5 × 10⁻⁶) 4 The cells (number per mL) were seeded into a 10 cm cell culture dish and cultured at 37°C with 5% CO2.

[0085] ② After culturing for 16 hours, the HIV-1Env expression plasmid was combined with DSP. 1-7 The plasmid was co-transfected 1:1 into 293T effector cells.

[0086] ③ Twenty-four hours after transfection, the lipopeptide membrane was dissolved in deionized water, and the inhibitor was diluted to the initial concentration in DMEM medium. This was then serially diluted 3-fold and added to the effector cells in a 96-well plate (50 μL / well), with 9 dilution gradients and 3 replicates. DMEM medium was used as a control for cells without inhibitor. The culture plates were incubated at 37°C in a 5% CO2 cell culture incubator for 1 hour.

[0087] ④ Resuspend the 293FT cells and adjust the cell concentration to approximately 30 × 10⁻⁶. 4 Add EnduRen live cell substrate (Promega) at a ratio of 1:4000 to mL, mix well, and incubate at 37°C and 5% CO2 for 30 minutes.

[0088] ⑤ Add 100 μL of 293FT cells to each well of HIV-1 effector cells, centrifuge at 300g for 1 minute to ensure sufficient contact between effector cells and target cells, incubate at 37°C for 1 hour, then measure luciferase activity (relative fluorescence unit, RLU), construct an inhibition rate curve, and calculate the half-maximal inhibitory concentration (IC50) of the drug. 50 ).

[0089] 2. Experimental Results and Analysis

[0090] The inventors previously reported that LP-25 can effectively inhibit a group of different HIV-1 pseudoviruses. This embodiment uses the same group of HIV-1 pseudoviruses to evaluate the antiviral activity of LP-37 and LP-39 in a TZM-bl cell single-cycle infection assay. The results are as follows... Figure 2 As shown, consistent with previous results, LP-25 effectively inhibited 36 HIV-1 pseudoviruses, with an average IC50 value of 1. 50 The value was 196.89 pM; while the antiviral activity of LP-37 and LP-39 was significantly improved, with an average IC50 value of 196.89 pM. 50 The values ​​were 30.72 and 29.86 pM, respectively.

[0091] LP-37 and LP-39 exhibit inhibitory activities against seven representative HIV-1 Env-mediated intercellular fusions, such as... Figure 3 As shown, the average IC50 of the two lipopeptides 50 The values ​​were 74.59 and 72.09 pM, respectively, indicating that LP-37 and LP-39 can also efficiently inhibit HIV-1Env-mediated intercellular fusion.

[0092] The inhibitory activities of LP-37 and LP-39 against HIV-2 and SIV strains are as follows: Figure 4 As shown, LP-37 and LP-39 inhibit HIV-2. ROD Infected IC 50 The values ​​were 215.13 and 371.5 pM, respectively; inhibition of HIV-2ST Infected IC 50 The values ​​were 89.57 and 92.99 pM, respectively. LP-37 and LP-39 inhibited SIV. 239 IC 50 The values ​​were 17.14 and 25.2 pM, respectively; SIV inhibition PBJ IC 50 The values ​​are 38.8 and 41.97 pM.

[0093] Example 4: LP-37 and LP-39 maintain effective inhibitory activity against drug-resistant HIV-1 strains

[0094] Another important biological characteristic of HIV is its susceptibility to drug resistance, leading to treatment failure and posing a key challenge for drug development. This embodiment uses two groups of HIV-1 drug-resistant strains: the first group consists of T20-resistant mutant strains carrying single-point or double-point mutations (I37T, V38A, V38M, Q40H, N43K, G36S / V38M, I37T / N43K, V38A / N42T), which exhibit high resistance to T20; the second group consists of HP23-resistant mutant strains carrying single-point, double-point, or triple-point mutations (E49A). The mutant strains (E49K, Q52R, L57R, N126K, E136G, N43K / E49A, E49K / N126K, L57R / E136G, Q39R / N43K / N126K, N43K / E49A / N126K) showed mild to high resistance to 2P23; the antiviral activity of LP-37 and LP-39 was evaluated using the pseudovirus inhibition experiment described in Example 2 above.

[0095] The results are as follows Figure 5 As shown, LP-37 and LP-39 both exhibited significant inhibitory activity against drug-resistant mutations. Their average IC50 values ​​against eight T20 drug-resistant mutants were [data missing]. 50 The mean values ​​were 9.16 and 8.61 pM, respectively, while the IC50 for wild-type NL4-3 was... 50 The values ​​were 7.28 and 7.45 pM, respectively. Based on the resistance fold calculation results, the IC50 values ​​for the eight resistance mutations were... 50 The fold changes were all less than 3-fold, indicating that LP-37 and LP-39 were sensitive to all T20 resistance mutations and maintained high activity levels. Regarding HP23 resistance mutants, LP-37 and LP-39, besides showing mild resistance to a few mutants, also exhibited highly efficient inhibitory activity against other mutants, with an average IC50 value of less than 3%. 50The values ​​were 22.13 and 41.91 pM, respectively. Interestingly, LP-37 showed no significant resistance to the L57R mutant, while LP-39 exhibited a 6.73-fold resistance to the L57R mutant; LP-37 showed a 7.4-fold resistance to the L57R / E136G mutant, while LP-39 showed a 37.72-fold resistance to the L57R / E136G mutant; compared with the lipopeptide membrane fusion inhibitors such as LP-19 and LP-25 previously reported by our team, LP-37 showed a significantly enhanced activity level, and in comparison, LP-37 is the most effective inhibitor against HP23 resistant mutants.

[0096] Example 5: Structural features of LP-37 and LP-39 and their interaction with the target sequence

[0097] 1. Experimental Materials and Methods

[0098] Circular dichroism (CD) chromatography was used to determine the secondary structure (α-helix) and thermal stability of the lipopeptide inhibitor, as well as the interaction between the lipopeptide and the target sequence mimic peptide. The target sequence mimic peptide N36 (Ac-SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL-NH2) was derived from the NHR sequence of HIV-1 gp41. The test lipopeptides were LP-37 and LP-39 synthesized in Example 1, and the test complex was a mixture of the lipopeptide and N36. The specific experimental steps were as follows:

[0099] ① Prepare a 10 μM solution of the test peptide (or test complex) using phosphate-buffered saline (PBS, pH 7.2). (For the test complex, 10 μM means that the concentration of both the test lipopeptide and N36 is 10 μM.) and place the solution in a 37°C water bath for 30 minutes.

[0100] ② Transfer the solution obtained in step 1 to the corresponding cuvette and use a Jasco spectropolarimeter (model J-815) to scan the change of the molar ellipticity [θ]λ of the solution in the wavelength range of 195-270nm. The typical α-helix structure can show the maximum negative peaks at 208nm and 222nm. Subtract the PBS blank control to correct the spectral value. In the calculation process, the peak value of -33000degree.cm2.dmol-1 is used as the standard of 100% α-helix content. The percentage of α-helix content is calculated based on the molar ellipticity of the solution at 222nm.

[0101] ③ Add the solution obtained in step 1 to the thermal stability testing cuvette, and adjust the CD temperature control module to scan the change of [θ]222 of the solution with temperature from 2 to 98℃ at a rate of 2℃ per minute. Smooth the melting curve, and use Origin software to calculate the midpoint temperature (Tm) of the thermal dissociation transition to reflect the degree of helical thermal stability.

[0102] 2. Experimental Results and Analysis

[0103] The results are as follows Figure 6 As shown in Figure A, LP-37 and LP-39 alone exhibit typical double negative peaks at 208 and 222 nm, indicating that LP-37 and LP-39 form typical α-helix structures, with α-helix contents of 96% and 44%, respectively. However, the melting curves cannot accurately calculate their Tm values, but the thermal stability of LP-37 is significantly higher than that of LP-39. In contrast, LP-37, with its rigid linker featuring a repeating "EAAAK" sequence, significantly increases the α-helix structure and thermal stability of the lipopeptide. The relatively high self-α-helix content can effectively reduce the binding entropy between the lipopeptide and the target site, increase the binding constant, and improve activity; the relatively stable secondary structure can also improve the stability of the lipopeptide in vivo, which is beneficial for antiviral activity.

[0104] like Figure 6 As shown in Figure B, both LP-37 and LP-39 can interact with the target sequence mimicking peptide N36 to form a stable α-helix structure; the N36 / LP-37 complex has an α-helix content of 93% and a Tm value of 93℃; the N36 / LP-39 complex has an α-helix content of 65% and a Tm value of 88℃. In contrast, LP-37, with its rigid linker featuring a repeating "EAAAK" sequence, significantly enhances its ability to bind to the viral target sequence while maintaining stable binding to the target.

[0105] Example 6: Stability Study of LP-37 and LP-39

[0106] To further verify the drug-likeness of the membrane fusion inhibitor disclosed herein, in this embodiment, LP-37 and LP-39 were placed at 4°C or 37°C for an extended period, incubated with human serum, and digested with proteases. Their stability was then determined by detecting changes in the antiviral activity of the lipopeptides.

[0107] 1. Experimental Materials and Methods

[0108] Temperature stability test: Lipopeptides were prepared into 500 μM aqueous solutions and stored at 4℃ and 37℃ for 0, 3, 7, 14, 21, 28, 35, 49, or 63 days, respectively. Samples were then collected and diluted to the corresponding IC50 value of the lipopeptide. 90 The concentration (i.e., the concentration at which the lipopeptide achieves a 90% inhibitory effect on the virus) was used in the sham virus inhibition experiment in Example 2 to detect the effect of different samples at this concentration on HIV-1. NL4-3 The inhibitory effect of TZM-bl infection on cells was investigated, and the inhibition rate was calculated.

[0109] Human serum stability test: The lipopeptide was prepared into a 500 μM aqueous solution, and human serum was added to it to a final concentration of 20%. After mixing, the solution was incubated at 37°C for 0, 5, 30, 60, 120, 180, or 240 min, and the samples were collected. The sample concentration was then diluted to the corresponding IC50 value of the lipopeptide. 90 The concentration (i.e., the concentration at which the lipopeptide achieves a 90% inhibitory effect on the virus) was used in the sham virus inhibition experiment in Example 2 to detect the effect of different samples at this concentration on HIV-1. NL4-3 The inhibitory effect of TZM-bl infection on cells was investigated, and the inhibition rate was calculated.

[0110] Digestion of proteases: LP-127 or LP-128 was mixed with proteinase K, trypsin, or α-chymotrypsin (Sigma-Aldrich products, catalog numbers P2308, T4799, and C4129, respectively) at a ratio of 20:1 (final concentrations of 2 mg / mL and 0.1 mg / mL, respectively). The mixture was incubated at 37°C for 0, 30, 60, 120, 180, or 240 minutes, and samples were collected. The sample concentration was then diluted to the corresponding IC50 value of the lipopeptide. 90 The concentration (i.e., the concentration at which the lipopeptide achieves a 90% inhibitory effect on the virus) was used in the sham virus inhibition experiment in Example 2 to detect the effect of different samples at this concentration on HIV-1. NL4-3 The inhibitory effect of TZM-bl infection on cells was investigated, and the inhibition rate was calculated.

[0111] Digestion by proteases: Lipopeptides were mixed with proteinase K, trypsin, or α-chymotrypsin (Sigma-Aldrich products, catalog numbers P2308, T4799, and C4129, respectively) at a ratio of 20:1 (final concentrations of 2 mg / mL and 0.1 mg / mL, respectively). Samples were collected after incubation at 37°C for 0, 30, 60, 120, 180, or 240 minutes, and the sample concentrations were diluted to the corresponding IC50 values ​​of the lipopeptides. 90 The concentration (i.e., the concentration at which the lipopeptide achieves a 90% inhibitory effect on the virus) was used in the sham virus inhibition experiment in Example 2 to detect the effect of different samples at this concentration on HIV-1. NL4-3 The inhibitory effect of TZM-bl infection on cells was investigated, and the inhibition rate was calculated.

[0112] 2. Experimental Results and Analysis

[0113] like Figure 7As shown, at 4℃, LP-37 was highly stable, and its antiviral activity remained essentially unchanged after 63 days. LP-39 showed a decreasing trend in activity, maintaining approximately 77% of its original level after 63 days. At 37℃, LP-37 remained highly stable and maintained its original antiviral activity after 49 days, and its activity remained approximately 80% of its original level after 63 days. LP-39, however, showed a significant decrease in activity, almost losing its inhibitory activity after 49 days. In contrast, LP-37, with its rigid linker containing the repeating "EAAAK" sequence, was more stable than LP-39 in terms of temperature. After treatment with proteinase K, trypsin, and α-chymotrypsin, LP-37 exhibited high resistance to digestion by all three proteases, with no significant change in antiviral activity. LP-39 was relatively stable with trypsin and α-chymotrypsin, but it was highly sensitive to proteinase K, and its antiviral activity decreased significantly after proteinase K treatment. Furthermore, both lipopeptides maintained stable antiviral activity after incubation with human serum samples at 37°C. In contrast, LP-37, with its rigid linker featuring a repeating "EAAAK" sequence, exhibited significant superiority in stability.

[0114] The present disclosure has been described in detail above. Those skilled in the art will recognize that the present disclosure can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experimentation. While specific embodiments are given in this disclosure, it should be understood that further modifications can be made to the present disclosure. In summary, in accordance with the principles of this disclosure, this application is intended to include any changes, uses, or improvements to the present disclosure, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A lipopeptide or a pharmaceutically acceptable salt thereof, characterized in that, The lipopeptide is shown in Formula I or Formula II as follows: Formula I: Ac-EMTWEEWEKKVEELEKKIEELLKAEAAAKEAAAKAK(chol)-NH2; Formula II: Ac-EMTWEEWEKKVEELEKKIEELLKEPEPEPEPEPEPK(chol)-NH2.

2. A pharmaceutical composition, characterized by, It comprises the lipopeptide or its pharmaceutically acceptable salt as claimed in claim 1, and a pharmaceutically acceptable carrier or excipient.

3. Use of the lipopeptide or its pharmaceutically acceptable salt as claimed in claim 1, or the pharmaceutical composition as claimed in claim 2, in the preparation of a lipopeptide HIV membrane fusion inhibitor.

4. Use of the lipopeptide or its pharmaceutically acceptable salt as claimed in claim 1, or the pharmaceutical composition as claimed in claim 2, in the preparation of any one of the following drugs: 1) an antiviral drug; 2) a drug for preventing and / or treating a disease caused by a viral infection; 3) a drug for inhibiting cell fusion of a virus; 4) a drug for inhibiting invasion of a cell by a virus; 5) a drug for inhibiting replication of a virus; wherein, The virus is one or more of HIV1, HIV-2 and SIV.

5. Use according to claim 4, characterized in that, The disease caused by the viral infection is AIDS.

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