Peptide compound for regulating function of human lysyl-tRNA synthetase

Abstract

The invention provides a polypeptide compound capable of regulating human lysyl-tRNA (transfer ribonucleic acid) synthetase. Specifically, the invention provides a compound as shown in a formula (I), or a solvate thereof, or a pharmaceutically acceptable salt thereof, the compound can target the human lysyl-tRNA synthetase, is used for adjusting the new function of the human lysyl-tRNA synthetase, and participates in treating diseases or symptoms related to the new function of the human lysyl-tRNA synthetase. The # imgabs0 # (Peptide) 1 is an amino acid sequence, and the amino acid sequence is as follows: X-X-X1-Tyr-X-X2-X-X3-X4-X5, and the amino acid sequence is as shown in the specification; and the (Peptide) 2 is a cell-penetrating peptide, and the sequence of the cell-penetrating peptide is Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Gln. The invention further discloses a preparation method of the cell-penetrating peptide.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a class of peptide compounds for regulating the function of human lysyl-tRNA synthetase. Background Art

[0002] Lysyl-tRNA synthetase (LysRS) is a highly conserved enzyme in the aminoacyl-tRNA synthetase (aaRSs) family and plays an important role in protein translation. Its classic function is to catalyze a two-step reaction to attach lysine (Lys) to tRNA Lys The 3' end of the mitochondria generates Lys-tRNA, one of the raw materials for protein translation Lys .

[0003] With the evolution of organisms, multiple aminoacyl-tRNA synthetase complexes (MSCs) gradually formed in cells. The complex is composed of nine aminoacyl-tRNA synthetases (LysRS, ArgRS, GlnRS, AspRS, MetRS, IleRS, LeuRS, GluRS and GluProRS formed by the natural fusion of ProRS) and three scaffold proteins (AIMP1, AIMP2 and AIMP3).

[0004] LysRS has been reported to have a variety of non-classical functions after dissociation from MSCs and is involved in a variety of diseases. In activated mast cells, LysRS is phosphorylated at serine 207 and then dissociated from MSCs, translocated to the nucleus, and interacted with microphthalmia-associated transcription factor (MITF). At the same time, phosphorylated LysRS catalyzes the production of the second messenger AP 4 A, A.P. 4 A binds to the tumor suppressor protein HINT1, inhibiting HINT1's inhibition of MITF transcription. Both promote the transcription of MITF target genes and participate in regulating immune activation responses. LysRS can synthesize AP 4 A, can further participate in the regulation of the STING signaling pathway, and plays an important role in the inflammatory response dependent on the STING signaling pathway. In HIV-1 virus-infected cells, it was also observed that the serine 207 of LysRS was phosphorylated and then dissociated from MSC. Free LysRS interacts with the viral precursor protein Gag, promoting the regeneration of the virus. In addition, when laminin is present in the cell matrix, the threonine 52 of LysRS is phosphorylated and released from MSC. The released LysRS interacts with the 67LR on the cell membrane, promoting the migration of cancer cells.

[0005] Therefore, it is of great significance to dissociate LysRS from MSCs through other treatments and discover new functions of lysyl-tRNA synthetase. Summary of the invention

[0006] The purpose of the present invention is to provide a class of peptide compounds for regulating the function of human lysyl-tRNA synthetase.

[0007] In the first aspect of the present invention, there is provided a use of a peptide compound represented by general formula (I) or a salt thereof in the preparation of a pharmaceutical composition for binding to human lysyl-tRNA synthetase and regulating its function;

[0008] H-(Peptide) 1 -Linker-(Peptide) 2 -OH(I)

[0009] In the general formula (I),

[0010] (Peptide) 1 is an amino acid sequence, and the amino acid sequence is: XXX 1 -Tyr-XX 2 -XX 3 -X 4 -X 5 ;

[0011] in:

[0012] X is selected from the group consisting of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), tryptophan (Trp), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), or a combination thereof;

[0013] X 1 Selected from the group consisting of methionine (Met), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), histidine (His), or a combination thereof;

[0014] X 2Selected from the group consisting of valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), or a combination thereof;

[0015] X 3 Selected from the group consisting of proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), methionine (Met), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), tyrosine (Tyr), histidine (His), or a combination thereof;

[0016] X 4 Selected from the group consisting of tyrosine (Tyr), phenylalanine (Phe), valine (Val), leucine (Leu), isoleucine (Ile), glutamine (Gln), lysine (Lys), histidine (His), or a combination thereof;

[0017] X 5 Selected from the group consisting of histidine (His), tyrosine (Tyr), glutamine (Gln), glutamic acid (Glu), lysine (Lys), arginine (Arg), or a combination thereof;

[0018] Linker is selected from the following group: Gly-Gly-Gly, Gly-Gly-Gly-Z-(peptide) 3 -Gly-Gly-Gly, or a combination thereof;

[0019] Among them, the sequence of Z is Ala-Pro-Leu-Arg-Val-Glu-Leu-Pro;

[0020] (Peptide) 2 It is a cell-penetrating peptide, and the cell-penetrating peptide sequence is: Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Gln;

[0021] (Peptide) 3 Selected from (Peptide) 1 , sequence and (Peptide) 1 Same or different.

[0022] In another preferred embodiment, all amino acids in the polypeptide compound are in L-type configuration.

[0023] In another preferred embodiment, the salt of the compound represented by the general formula (I) includes a salt formed by the compound represented by the general formula (I) and an inorganic acid or an organic acid, or a salt formed by the compound represented by the general formula (I) and an organic base.

[0024] In another preferred embodiment, the organic acid comprises an acid selected from the group consisting of formic acid, acetic acid, propionic acid, citric acid, lactic acid, oxalic acid, malonic acid, maleic acid, tartaric acid, benzoic acid, succinic acid, aspartic acid, glutamic acid, methanesulfonic acid, toluenesulfonic acid, or a combination thereof;

[0025] The inorganic acid includes an acid selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, carbonic acid, or a combination thereof;

[0026] The organic base includes a base selected from the group consisting of ethylamine, ethylenediamine, diethylamine, ethylamino alcohol, diethylamino alcohol, arginine, lysine, histidine, piperazine, or a combination thereof.

[0027] In another preferred embodiment, the peptide compound is selected from the following group: M1 (SEQ ID NO.1), M2 (SEQ ID NO.2), M3 (SEQ ID NO.3), M4 (SEQ ID NO.4), M5 (SEQ ID NO.5), M6 (SEQ ID NO.6), or a combination thereof.

[0028] In another preferred embodiment, the human lysyl-tRNA synthetase is selected from the following group: lysyl-tRNA synthetase bound to a polyaminoacyl-tRNA synthetase complex, free lysyl-tRNA synthetase, or a combination thereof.

[0029] In another preferred embodiment, the pharmaceutical composition is used for:

[0030] (a) promoting the dissociation of human lysyl-tRNA synthetase from the polyaminoacyl-tRNA synthetase complex;

[0031] (b) Combined with human lysyl-tRNA synthetase to form a co-crystal;

[0032] (c) regulating human lysyl-tRNA synthetase;

[0033] (d) Promote cellular fat metabolism.

[0034] In another preferred embodiment, the regulation of the function of human lysyl-tRNA synthetase comprises regulating one or more functions selected from the following group:

[0035] (a) Human lysyl-tRNA synthetase participates in HIV virus replication;

[0036] (b) Immune response involving human lysyl-tRNA synthetase;

[0037] (c) Human lysyl-tRNA synthetase participates in cellular autophagy.

[0038] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for promoting fat metabolism in hepatocytes.

[0039] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical preparation selected from the following group: intravenous injection, oral preparation, topical preparation, or a combination thereof.

[0040] In the second aspect of the present invention, a peptide compound or a salt thereof is provided as shown in the following formula I:

[0041] H-(Peptide) 1 -Linker-(Peptide) 2 -OH(I)

[0042] In the general formula (I),

[0043] (Peptide) 1 is an amino acid sequence, and the amino acid sequence is: XXX 1 -Tyr-XX 2 -XX 3 -X 4 -X 5 ;

[0044] in:

[0045] X is selected from the group consisting of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), tryptophan (Trp), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), or a combination thereof;

[0046] X 1 Selected from the group consisting of methionine (Met), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), histidine (His), or a combination thereof;

[0047] X 2Selected from the group consisting of valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), or a combination thereof;

[0048] X 3 Selected from the group consisting of proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), methionine (Met), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), tyrosine (Tyr), histidine (His), or a combination thereof;

[0049] X 4 Selected from the group consisting of tyrosine (Tyr), phenylalanine (Phe), valine (Val), leucine (Leu), isoleucine (Ile), glutamine (Gln), lysine (Lys), histidine (His), or a combination thereof;

[0050] X 5 Selected from the group consisting of histidine (His), tyrosine (Tyr), glutamine (Gln), glutamic acid (Glu), lysine (Lys), arginine (Arg), or a combination thereof;

[0051] Linker is selected from the following group: Gly-Gly-Gly, Gly-Gly-Gly-Z-(peptide) 3 -Gly-Gly-Gly, or a combination thereof;

[0052] Among them, the sequence of Z is Ala-Pro-Leu-Arg-Val-Glu-Leu-Pro;

[0053] (Peptide) 2 It is a cell-penetrating peptide, and the cell-penetrating peptide sequence is: Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Gln;

[0054] (Peptide) 3 Selected from (Peptide) 1 , sequence and (Peptide) 1 Same or different.

[0055] In another preferred embodiment, the peptide compound is selected from the following group: M1 (SEQ ID NO.1), M2 (SEQ ID NO.2), M3 (SEQ ID NO.3), M4 (SEQ ID NO.4), M5 (SEQ ID NO.5), M6 (SEQ ID NO.6), or a combination thereof.

[0056] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The binding curve of the peptide compound of the present invention and human lysyl-tRNA synthetase is shown.

[0058] Figure 2 The co-crystal structure of the peptide compound M3 of the present invention and human lysyl-tRNA synthetase is shown.

[0059] Figure 3 The structure of the LysRS-M3 complex is aligned with the structure of the LysRS-M1 complex.

[0060] Figure 4 The structure of the LysRS-M3 complex is aligned with the structure of the LysRS-M2 complex.

[0061] Figure 5 It was shown that the peptide compounds of the present invention promote the dissociation of human lysyl-tRNA synthetase from the multi-enzyme complex.

[0062] Figure 6 The peptide compounds of the present invention promote the expression of the autophagy marker LC3 II

[0063] Figure 7 It was shown that the peptide compounds of the present invention promote the formation of GFP-LC3 bright spots.

[0064] Figure 8 It is shown that the peptide compounds of the present invention promote the fat metabolism of liver cancer cells HepG2. DETAILED DESCRIPTION

[0065] After extensive and in-depth research, the inventors unexpectedly discovered the use of a polypeptide compound of general formula (I) or a salt thereof in binding to human lysyl-tRNA synthetase and regulating its new function. The experiments of the present invention show that the polypeptide compound of the present invention can promote the dissociation of LysRS from MSC, and can form a eutectic structure with LysRS, and can even regulate LysRS to achieve upregulation of autophagy and promote fat metabolism. The present invention was completed on this basis.

[0066] Compounds of the present invention

[0067] As used herein, "the peptide compound represented by the general formula (I) of the present invention", "the compound of the present invention" and "the polypeptide compound of the present invention" are used interchangeably and all refer to the peptide compound represented by the general formula (I) or its salt.

[0068] In another preferred embodiment, the pharmaceutically acceptable salts include salts formed by combining with inorganic acids, organic acids, or organic bases that can provide physiologically acceptable cations, and ammonium salts.

[0069] In another preferred embodiment, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, boric acid, phosphoric acid or carbonic acid; the organic acid is selected from formic acid, acetic acid, propionic acid, citric acid, lactic acid, oxalic acid, malonic acid, maleic acid, tartaric acid, benzoic acid, succinic acid, aspartic acid, glutamic acid, methanesulfonic acid or toluenesulfonic acid; the alkali metal ion is selected from lithium ion, sodium ion, potassium ion; the organic base is selected from ethylamine, ethylenediamine, diethylamine, ethylamino alcohol, diethylamino alcohol, arginine, lysine, histidine or piperazine.

[0070] All such salts within the scope of the present invention may be prepared by conventional methods.

[0071] Pharmaceutical compositions and methods of administration

[0072] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0073] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0074] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration or subcutaneous administration).

[0075] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0076] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0077] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0078] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0079] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0080] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0081] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds (eg, other therapeutic agents).

[0082] When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds can be used simultaneously, separately or sequentially with the compound of the present invention to prevent and / or treat ClpP-related diseases.

[0083] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0084] The main advantages of the present invention include:

[0085] 1. The polypeptide compounds of the present invention have strong binding to human lysyl-tRNA synthetase, and the binding force is in the nanomolar to micromolar range.

[0086] 2. The polypeptide compounds of the present invention target human lysyl-tRNA synthetase with a clear mechanism of action, promote the dissociation of human lysyl-tRNA synthetase from the MSC complex or regulate the conformational changes of human lysyl-tRNA synthetase.

[0087] 3. The polypeptide compounds of the present invention can regulate new functions of human lysyl-tRNA synthetase (such as autophagy).

[0088] 4. The polypeptide compounds of the present invention have a significant effect on the fat metabolism of hepatocytes and are expected to be developed into new drugs for treating autophagy-related diseases (such as obesity, fatty liver, neurodegenerative diseases, etc.).

[0089] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0090] Example 1

[0091] Small peptide binding experiment with human lysyl-tRNA synthetase

[0092] 1.1 Experimental methods

[0093] A binding experiment is performed using the polypeptide M1 (SEQ ID NO.1), M2 (SEQ ID NO.2), M3 (SEQ ID NO.3), M4 (SEQ ID NO.4), M5 (SEQ ID NO.5), or M6 (SEQ ID NO.6) of the present invention and human lysyl-tRNA synthetase, comprising the following steps:

[0094] (1) 120 μM LysRS was diluted in half with a buffer containing 10 mM 4-hydroxyethylpiperazineethanesulfonic acid (pH adjusted to 7.5 with hydrochloric acid), 200 mM sodium chloride, 2.5% glycerol, and 1 mM magnesium chloride for a total of 11 dilutions. A blank control group without LysRS was kept at 120 μL for each concentration.

[0095] (2) Different concentrations of LysRS were dispensed into low-adsorption, opaque, half-well, black, 96-well plates, 25 μL per well, for a total of 4 replicates.

[0096] (3) Prepare 10 nM FITC-labeled peptide compound using the same buffer and dispense into EP tubes, 120 μL per tube, for a total of 12 tubes.

[0097] (4) Using a 12-channel gun, add 25 μL of 10 nM FITC-labeled polypeptide compound to different concentrations of LysRS, that is, the final concentration of the polypeptide compound is 5 nM, and the final concentration of LysRS is 60 μM, 30 μM, 15 μM, ... 0 μM.

[0098] (5) The completely mixed samples were reacted at room temperature for 3 h and the fluorescence anisotropy value was read using an ELISA reader (excitation light: 480 nm, emission light: 535 nm).

[0099] Binding strength K d Calculated using the following formula:

[0100]

[0101] in,

[0102] A: experimentally measured anisotropy value;

[0103] A f : anisotropy value of free ligand;

[0104] A b : anisotropy value of the fully bound ligand;

[0105] L T : total concentration of added ligand;

[0106] R T : Total concentration of added receptor.

[0107] The amino acid sequences of M1-M6 are shown in Table 1.

[0108] Table 1

[0109]

[0110] 1.2 Experimental Results

[0111] The experimental results are as follows Figure 1 As shown, the polypeptide compound of the present invention has a good binding effect with human lysyl-tRNA synthetase.

[0112] Among them, the binding affinity of polypeptide compound M1 to human lysyl-tRNA synthetase is 18.4±1.09μM, the binding affinity of polypeptide compound M2 to human lysyl-tRNA synthetase is 7.35±1.24μM, the binding affinity of polypeptide compound M3 to human lysyl-tRNA synthetase is 6.01±0.29μM, the binding affinity of polypeptide compound M4 to human lysyl-tRNA synthetase is 98.08±12.38nM, the binding affinity of polypeptide compound M5 to human lysyl-tRNA synthetase is 233.8±26.88nM, and the binding affinity of polypeptide compound M6 to human lysyl-tRNA synthetase is 15.43±5.90nM.

[0113] Example 2

[0114] Co-crystal experiment of small peptide and human lysyl-tRNA synthetase

[0115] 2.1 Experimental methods

[0116] Human lysyl-tRNA synthetase protein and polypeptide compounds M1, M2, and M3 were mixed at a ratio of 1:1.1, and crystallization screening was performed using the sitting drop method. The crystallization conditions of the complex and the protective solution conditions are shown in Table 2:

[0117] Table 2

[0118]

[0119] 2.2 Experimental Results

[0120] The experimental results are as follows Figure 2-4 shown. Figure 2 The crystal structure of the complex between LysRS and the peptide compound M3 is shown. M3 binds in the groove formed by the anticodon binding domain of one LysRS and the catalytic domain of another LysRS, stabilizing the conformation of LysRS and playing an important role in the new function of LysRS.

[0121] Figure 3 The structure of the LysRS-M3 complex is aligned with that of the LysRS-M1 complex. When the residue at position 8 in the peptide compound is serine, it interacts more strongly with D259 and E260 of LysRS than with proline at position 8.

[0122] Figure 4The structure of the LysRS-M3 complex is compared with that of the LysRS-M2 complex. When the residue at position 8 in the peptide compound is serine, the interaction with D259 of LysRS is enhanced compared with asparagine at position 8, but the interaction with E260 is weakened. When the residue at position 9 in the peptide compound is tyrosine, the interaction with G262 and R314 of LysRS is enhanced compared with valine at position 9.

[0123] Example 3

[0124] Small peptide promotes the dissociation of human lysyl-tRNA synthetase from polyaminoacyl-tRNA synthetase complex

[0125] 3.1 Experimental methods

[0126] The culture medium was 90% DMEM + 10% FBS at 37°C and 5% CO 2 HeLa cells were cultured in an incubator.

[0127] After the cell state is stable and no mycoplasma contamination is confirmed, HeLa cells are treated with 20 μM of the polypeptide compound. After 24 hours, the culture medium is removed, the cells are gently washed with PBS, and the cells are lysed with 300 μL of lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40). In order to fully lyse the cells, the cells are repeatedly blown with a 1 mL syringe after adding the lysis buffer, and then the lysate is transferred to a 1.5 mL centrifuge tube and centrifuged at 4 ° C, 12000 rpm for 10 minutes.

[0128] The supernatant was centrifuged again for 10 min in the same manner. The supernatant was separated using a gel exclusion chromatography column S6, and the elution buffer was 150 mM NaCl, 20 mM Tris-HCl pH 8.0.

[0129] The eluted samples were taken at equal intervals and separated by SDS-PAGE gel, and the signal intensity of LysRS in different elution volumes was detected by WB.

[0130] Anti-LysRS antibody was diluted 1:5000 in TBST containing 5% BSA. When the gel exclusion chromatography column is used to separate proteins, proteins with large molecular weight cannot enter the gel pores and will be eluted quickly. Proteins with small molecular weight enter the gel pores and are eluted later.

[0131] Therefore, it is possible to determine whether LysRS is dissociated from MSCs based on the signal distribution of LysRS in the high molecular weight region (11 mL, about 970 KDa) and the low molecular weight region (16 mL, about 66 KDa).

[0132] 3.2 Experimental Results

[0133] The experimental results are as follows Figure 5 As shown, in the control group, the signal of LysRS was mainly concentrated in the high molecular weight, indicating that LysRS existed in the form of MSC complex. After treatment with polypeptide compounds M1, M2, and M3, the signal of LysRS was detected at the low molecular weight, indicating that the polypeptide compounds promoted the dissociation of LysRS from MSC.

[0134] Example 4

[0135] Peptide compounds induce upregulation of autophagy

[0136] 4.1 Experimental methods

[0137] Treatment 1: HeLa cells were treated with 20 μM of the peptide compound for 24 h. The positive control group was starved with EBSS for 2 h. The cells were gently washed with cold PBS to remove the residual culture medium, and then lysed with 300 μL of lysis buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol). The lysate was separated by SDS-PAGE, and the expression of the autophagy marker LC3 II was detected by WB.

[0138] Treatment 2: HeLa cells were transiently transfected with GFP-LC3 plasmid for 24 hours, and then treated with 20μM peptide compound for 24 hours. The positive control group starved cells with EBSS 2 hours before preparation. After that, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, stained with DAPI, and sealed, and the bright spots of GFP-LC3 formed in the cells were observed using a fluorescent confocal microscope.

[0139] 4.2 Experimental Results

[0140] The experimental results are as follows Figure 6-7 shown.

[0141] Figure 6 The results showed that the peptide compounds M1, M2, and M3 performed consistently with the positive control group. After treatment, the expression of the autophagy marker LC3 II increased significantly, indicating that the peptide compounds caused upregulation of autophagy.

[0142] Figure 7 It was shown that the performance of polypeptide compounds M1, M2, M3 and the positive control group was consistent. After treatment, the number of GFP-LC3 bright spots formed in the cells increased significantly, indicating that the polypeptide compounds caused upregulation of autophagy.

[0143] Example 5

[0144] Peptide compounds promote fat metabolism in liver cancer cells HepG2

[0145] 5.1 Experimental Methods

[0146] HepG2 cells were cultured in a complete medium containing 87% MEM + 10% FBS + 1% Glutamax + 1% NEAA + 1% Sodium Pyruvate. After confirming that the cells were growing stably and without contamination, HepG2 cells were cultured at 4 × 10 5 cells / mL were inoculated into a 96-well cell culture plate. After 24 h, in order to reduce the influence of lipid droplets in serum on the experiment, the FBS in the complete culture medium was replaced with 2% BSA.

[0147] HepG2 cells were treated with 1 mM Oleic acid with or without 20 μM peptide compounds and 500 nM Digoxin (DG, positive compound) for 24 h.

[0148] The treated cells were fixed with 4% paraformaldehyde and stained with Oil Red O, and the formed lipid droplets were observed and photographed under a bright field microscope.

[0149] 5.2 Experimental Results

[0150] The experimental results are as follows Figure 8 As shown, there are fewer lipid droplets in HepG2 cells in the medium containing only 2% BSA.

[0151] After treatment with oleic acid, the number of lipid droplets in HepG2 cells increased significantly. When oleic acid was added, the peptide compounds M1, M2, M3 and the positive compound Digoxin were added at the same time, and the number of lipid droplets in HepG2 cells decreased significantly, indicating that small peptide compounds promoted the fat metabolism of liver cancer cells HepG2.

[0152] discuss

[0153] In the study of the present invention, it was found that LysRS dissociated from MSCs after starvation or drug treatment, and then participated in the regulation of autophagy.

[0154] Therefore, intervening in the new function of lysyl-tRNA synthetase has potential value in anti-allergy, anti-inflammatory, anti-HIV, anti-cancer, and autophagy-related diseases (such as neurodegenerative diseases, obesity, fatty liver, etc.), and has medicinal value in promoting fat metabolism.

[0155] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a peptide compound represented by general formula (I) or a salt thereof in the preparation of a pharmaceutical composition for binding to human lysyl-tRNA synthetase and regulating its function; H-(Peptide)1-Linker-(Peptide)2-OH(I) In the general formula (I), (Peptide) 1 is an amino acid sequence, wherein the amino acid sequence is: XX-X1-Tyr-X-X2-X-X3-X4-X5; in: X is selected from the group consisting of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), tryptophan (Trp), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), or a combination thereof; X1 is selected from the group consisting of methionine (Met), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), histidine (His), or a combination thereof; X2 is selected from the group consisting of valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), or a combination thereof; X3 is selected from the group consisting of proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), methionine (Met), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), tyrosine (Tyr), histidine (His), or a combination thereof; X4 is selected from the group consisting of tyrosine (Tyr), phenylalanine (Phe), valine (Val), leucine (Leu), isoleucine (Ile), glutamine (Gln), lysine (Lys), histidine (His), or a combination thereof; X5 is selected from the group consisting of histidine (His), tyrosine (Tyr), glutamine (Gln), glutamic acid (Glu), lysine (Lys), arginine (Arg), or a combination thereof; Linker is selected from the group consisting of Gly-Gly-Gly, Gly-Gly-Gly-Z-(peptide)3-Gly-Gly-Gly, or a combination thereof; Among them, the sequence of Z is Ala-Pro-Leu-Arg-Val-Glu-Leu-Pro; (Peptide) 2 is a cell-penetrating peptide, and the cell-penetrating peptide sequence is: Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Gln; (Peptide) 3 is an amino acid sequence, and the amino acid sequence is: XX-X1-Tyr-X-X2-X-X3-X4-X5; and the sequence is the same as or different from (Peptide) 1.

2. The use according to claim 1, characterized in that All amino acids in the polypeptide compound are in L-type configuration.

3. The use according to claim 1, characterized in that The salt of the compound represented by the general formula (I) includes a salt formed by the compound represented by the general formula (I) and an inorganic acid or an organic acid, or a salt formed by the compound represented by the general formula (I) and an organic base.

4. The use according to claim 3, characterized in that The organic acid comprises an acid selected from the group consisting of formic acid, acetic acid, propionic acid, citric acid, lactic acid, oxalic acid, malonic acid, maleic acid, tartaric acid, benzoic acid, succinic acid, aspartic acid, glutamic acid, methanesulfonic acid, benzenesulfonic acid, or a combination thereof; The inorganic acid includes an acid selected from the group consisting of hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, carbonic acid, or a combination thereof; The organic base includes a base selected from the group consisting of ethylamine, ethylenediamine, diethylamine, ethylamino alcohol, diethylamino alcohol, arginine, lysine, histidine, piperazine, or a combination thereof.

5. The use according to claim 1, characterized in that The peptide compound is selected from the following group: M1 (SEQ ID NO.1), M2 (SEQ ID NO.2), M3 (SEQ ID NO.3), M4 (SEQ ID NO.4), M5 (SEQ ID NO.5), M6 (SEQID NO.6), or a combination thereof.

6. The use according to claim 1, characterized in that The human lysyl-tRNA synthetase is selected from the group consisting of a lysyl-tRNA synthetase bound to a polyaminoacyl-tRNA synthetase complex, a free lysyl-tRNA synthetase, or a combination thereof.

7. The use according to claim 1, characterized in that The pharmaceutical composition is used for: (a) promoting the dissociation of human lysyl-tRNA synthetase from the polyaminoacyl-tRNA synthetase complex; (b) Combined with human lysyl-tRNA synthetase to form a co-crystal; (c) regulating human lysyl-tRNA synthetase; (d) Promote cellular fat metabolism.

8. The use according to claim 7, characterized in that The regulating the function of human lysyl-tRNA synthetase comprises regulating one or more functions selected from the following group: (a) Human lysyl-tRNA synthetase participates in HIV virus replication; (b) Immune response involving human lysyl-tRNA synthetase; (c) Human lysyl-tRNA synthetase participates in cellular autophagy.

9. A peptide compound or a salt thereof as shown in the following formula I: H-(Peptide)1-Linker-(Peptide)2-OH(I) In the general formula (I), (Peptide) 1 is an amino acid sequence, wherein the amino acid sequence is: XX-X1-Tyr-X-X2-X-X3-X4-X5; in: X is selected from the group consisting of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), tryptophan (Trp), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), or a combination thereof; X1 is selected from the group consisting of methionine (Met), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), histidine (His), or a combination thereof; X2 is selected from the group consisting of valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), or a combination thereof; X3 is selected from the group consisting of proline (Pro), serine (Ser), threonine (Thr), cysteine ​​(Cys), methionine (Met), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), tyrosine (Tyr), histidine (His), or a combination thereof; X4 is selected from the group consisting of tyrosine (Tyr), phenylalanine (Phe), valine (Val), leucine (Leu), isoleucine (Ile), glutamine (Gln), lysine (Lys), histidine (His), or a combination thereof; X5 is selected from the group consisting of histidine (His), tyrosine (Tyr), glutamine (Gln), glutamic acid (Glu), lysine (Lys), arginine (Arg), or a combination thereof; Linker is selected from the group consisting of Gly-Gly-Gly, Gly-Gly-Gly-Z-(peptide)3-Gly-Gly-Gly, or a combination thereof; Among them, the sequence of Z is Ala-Pro-Leu-Arg-Val-Glu-Leu-Pro; (Peptide) 2 is a cell-penetrating peptide, and the cell-penetrating peptide sequence is: Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Gln; (Peptide)3 is selected from (Peptide)1, and its sequence is the same as or different from that of (Peptide)1.

10. The peptide compound or salt thereof according to claim 9, characterized in that: The peptide compound is selected from the following group: M1 (SEQ ID NO.1), M2 (SEQ ID NO.2), M3 (SEQ ID NO.3), M4 (SEQ ID NO.4), M5 (SEQ ID NO.5), M6 (SEQ ID NO.6), or a combination thereof.

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