CN115010788A - N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物及其应用 - Google Patents
N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物及其应用 Download PDFInfo
- Publication number
- CN115010788A CN115010788A CN202210508512.3A CN202210508512A CN115010788A CN 115010788 A CN115010788 A CN 115010788A CN 202210508512 A CN202210508512 A CN 202210508512A CN 115010788 A CN115010788 A CN 115010788A
- Authority
- CN
- China
- Prior art keywords
- antibacterial peptide
- antibacterial
- fatty acid
- terminal
- peptide analogue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 64
- 235000014113 dietary fatty acids Nutrition 0.000 title claims abstract description 21
- 229930195729 fatty acid Natural products 0.000 title claims abstract description 21
- 239000000194 fatty acid Substances 0.000 title claims abstract description 21
- 125000005313 fatty acid group Chemical group 0.000 title claims abstract description 20
- 230000003214 anti-biofilm Effects 0.000 title claims abstract description 17
- 230000000694 effects Effects 0.000 title claims abstract description 16
- 231100000053 low toxicity Toxicity 0.000 title claims abstract description 13
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims abstract description 13
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims abstract description 13
- 230000004048 modification Effects 0.000 claims abstract description 12
- 238000012986 modification Methods 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 210000004899 c-terminal region Anatomy 0.000 claims abstract description 5
- 238000007142 ring opening reaction Methods 0.000 claims abstract description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 24
- BOAHYOWLXYZJSN-UHFFFAOYSA-N 3-hydroxy-4-methylpentanoic acid Chemical compound CC(C)C(O)CC(O)=O BOAHYOWLXYZJSN-UHFFFAOYSA-N 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 5
- COLNVLDHVKWLRT-MRVPVSSYSA-N D-phenylalanine Chemical compound OC(=O)[C@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-MRVPVSSYSA-N 0.000 claims description 4
- 230000009435 amidation Effects 0.000 claims description 4
- 238000007112 amidation reaction Methods 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 230000000845 anti-microbial effect Effects 0.000 claims description 3
- 125000001433 C-terminal amino-acid group Chemical group 0.000 claims description 2
- 125000003558 D-valino group Chemical group C(=O)(O)[C@@H](C(C)C)N* 0.000 claims 2
- 241000894006 Bacteria Species 0.000 abstract description 16
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 16
- 238000002474 experimental method Methods 0.000 abstract description 12
- 206010018910 Haemolysis Diseases 0.000 abstract description 8
- 230000008588 hemolysis Effects 0.000 abstract description 8
- 230000002949 hemolytic effect Effects 0.000 abstract description 7
- 239000012528 membrane Substances 0.000 abstract description 7
- 241000192125 Firmicutes Species 0.000 abstract description 6
- 231100000419 toxicity Toxicity 0.000 abstract description 6
- 230000001988 toxicity Effects 0.000 abstract description 6
- 108010028921 Lipopeptides Proteins 0.000 abstract description 4
- 229940124350 antibacterial drug Drugs 0.000 abstract description 4
- 125000004122 cyclic group Chemical group 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- 239000011347 resin Substances 0.000 description 24
- 229920005989 resin Polymers 0.000 description 24
- 230000015572 biosynthetic process Effects 0.000 description 13
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 238000003786 synthesis reaction Methods 0.000 description 12
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 10
- 229920001184 polypeptide Polymers 0.000 description 10
- 241000588724 Escherichia coli Species 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- LIWKOFAHRLBNMG-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxycarbonylamino]butanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCNC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 LIWKOFAHRLBNMG-FQEVSTJZSA-N 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 5
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 5
- 241000191967 Staphylococcus aureus Species 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 238000006482 condensation reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 5
- 238000004949 mass spectrometry Methods 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 4
- 206010059866 Drug resistance Diseases 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 125000003275 alpha amino acid group Chemical group 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 210000000601 blood cell Anatomy 0.000 description 4
- MNWFXJYAOYHMED-UHFFFAOYSA-N heptanoic acid Chemical compound CCCCCCC(O)=O MNWFXJYAOYHMED-UHFFFAOYSA-N 0.000 description 4
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 125000006239 protecting group Chemical group 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000032770 biofilm formation Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- JAUKCFULLJFBFN-VWLOTQADSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid Chemical compound C1=CC(OC(C)(C)C)=CC=C1C[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JAUKCFULLJFBFN-VWLOTQADSA-N 0.000 description 2
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 125000003625 D-valyl group Chemical group N[C@@H](C(=O)*)C(C)C 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 108010093965 Polymyxin B Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920000024 polymyxin B Polymers 0.000 description 2
- 229960005266 polymyxin b Drugs 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 238000010267 two-fold dilution method Methods 0.000 description 2
- UGNIYGNGCNXHTR-GOSISDBHSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-GOSISDBHSA-N 0.000 description 1
- SJVFAHZPLIXNDH-JOCHJYFZSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-JOCHJYFZSA-N 0.000 description 1
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 206010061765 Chromosomal mutation Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 1
- 241000179039 Paenibacillus Species 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- JAWMENYCRQKKJY-UHFFFAOYSA-N [3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-ylmethyl)-1-oxa-2,8-diazaspiro[4.5]dec-2-en-8-yl]-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]methanone Chemical compound N1N=NC=2CN(CCC=21)CC1=NOC2(C1)CCN(CC2)C(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F JAWMENYCRQKKJY-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Oncology (AREA)
- Animal Behavior & Ethology (AREA)
- Communicable Diseases (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明设计合成了一类N‑末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物。通过对天然抗菌环脂肽PE2进行开环、N‑末端脂肪酸修饰及C‑末端Thr的替换,得到全新结构的线性抗菌肽类似物,其结构通式为:Cm‑PE2、C6‑PE2‑X9或Cn‑PE2‑Y9(其中m=6‑12;n=7、8;X=T、A、S、K、Y、F、I、W等)。体外抗菌实验表明,以上所述抗菌肽类似物对常见革兰氏阳性细菌和革兰氏阴性细菌菌株均具有较好的抗菌活性。代表性抗菌肽类似物Cn‑PE2‑Y9的溶血实验和抗生物膜实验表明,该类抗菌肽类似物溶血毒性低,安全性高,还能够抑制革兰氏阳性细菌和革兰氏阴性细菌的生物膜生成,在制备临床抗菌药物中具有良好的应用前景。
Description
技术领域
本发明属于生物化学技术领域,涉及一类全新结构的N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物,本发明同时还涉及该类抗菌肽类似物在制备临床抗菌药物中的应用。
背景技术
抗生素的使用大大降低了由细菌感染引起的发病率和死亡率。然而,由于抗生素的过度使用,使得部分细菌获得了耐药性的基因或发生染色体突变,从而产生耐药性(NatRev Microbiol.2010;8:260-271)。致病菌耐药已成为全球公共卫生危机,预计到2050年,全球将会有数百万人因耐药菌感染而死亡(BMJ.2013;346:f1493)。
抗菌肽(AMPs,Antimicrobial peptides)广泛存在于动物、植物、微生物等多种生命体中(Proteomics.2016;16:1225-1237),具有广谱抗菌活性。研究表明,阳离子抗菌肽具有通过膜裂解机制杀死细菌,或通过与DNA/RNA的相互作用,对蛋白质的合成和细胞内酶的活性产生负面影响,或抑制细胞壁/膜的形成而诱导细胞死亡(Nat Rev DrugDiscov.2012;11:37-51;Applied Microbiology&Biotechnology.2019,Vol.103(No.16):6593-6604)等多种抗菌机制,相比于抗生素,抗菌肽的使用能够显著降低耐药发生的可能性,故抗菌肽可作为抗耐药菌的新型抗菌剂(Front Cell Infect Microbiol.2016;6:194)。
天然抗菌肽稳定性差、半衰期短、生产成本高等问题限制了其临床应用和发展(Front Cell Infect Microbiol.2016;6:194)。近年来,从食品相关微生物中分离的肽类抗生素受到越来越多的关注(Appl Environ Microbiol.2012,78(9):3156-3165)。2017年,Huang等人从类芽孢杆菌B7菌株中分离出一种环脂肽类抗生素PE2,具有低毒性及广谱抗菌活性,且对耐甲氧西林金黄色葡萄球菌(MRSA)也表现出很好的抗菌活性,具有良好的发展前景(BMC Microbiology.2013,13:87-94)。但体外合成方法复杂、生产成本高等缺点限制了PE2的进一步应用。研究表明,环化对多肽的抗菌活性并不是必须的,也可通过脂肪酸修饰、疏水性氨基酸替换等方法来提高活性并增加稳定性(J Med Chem.2017,60:9630 9640;Biophys Chem.2015,199:25-33;Biochem J.2005,385:135-143)。在此基础上,可对脂肽类抗生素PE2进行结构改造,以期得到合成简便、产率高、低毒广谱的抗菌肽类似物。
发明内容
本发明的目的之一:提供一类制备成本低、产率高、抗菌活性强、溶血毒性低且具有抗生物膜活性的N-末端脂肪酸修饰的广谱抗菌肽类似物。
本发明的目的之二:提供上述抗菌肽类似物在制备临床抗菌药物中的应用。
为达到上述目的,本发明采用的技术方案为:
一、N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物的结构设计
本发明涉及的抗菌肽类似物,是对天然抗菌肽PE2进行开环改造,然后进行N-末端脂肪酸修饰和C末端酰胺化得到,其结构通式为:
Cm-D’ID’FLD’VLT-NH2,标记为Cm-PE2(1);
其中,m=6-12;D’=Dab;F=D-Phe;V=D-Val;
或:所述抗菌肽类似物是对天然抗菌肽PE2进行开环改造,然后依次进行N-末端脂肪酸修饰、C-末端氨基酸替换,最后进行C末端酰胺化得到,其结构通式为:
C6-D’ID’FLD’VLX-NH2,标记为C6-PE2-X9(2);
或Cn-D’ID’FLD’VLY-NH2,标记为Cn-PE2-Y9(3);
其中,n=7、8;D’=Dab;F=D-Phe;V=D-Val;X=T、A、S、K、Y、F、I、W。
上述结构通式(1)-(3)中,所述开环改造是指,将天然抗菌肽PE2从酯键的位置断开,得到N-末端为3-羟基-4-甲基戊酸的线性多肽类似物;结构通式(2)-(3)中,N-末端脂肪酸修饰是指,将开环后N-末端的3-羟基-4-甲基戊酸改造为正己酸、正庚酸或正辛酸。
具体地,本发明所述的一类N-末端脂肪酸修饰的广谱抗菌肽类似物,其结构式如下:
Cm-Dab-Ile-Dab-D-Phe-Leu-Dab-D-Val-Leu-Thr,标记为Cm-PE2;
或C6-Dab-Ile-Dab-D-Phe-Leu-Dab-D-Val-Leu-X,标记为C6-PE2-X9;
或Cn-Dab-Ile-Dab-D-Phe-Leu-Dab-D-Val-Leu-Tyr,标记为Cn-PE2-Y9;
其中,m=6-12;n=7、8;X=T、A、S、K、Y、F、I、W。
作为本发明技术方案的优选,上述抗菌肽类似物包括C7-PE2-Y9和C8-PE2-Y9。
上述N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物均采用经典固相合成法制备得到。
二、一类N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物在制备临床抗菌药物中的应用
1、体外抗菌实验
采用经典微量二倍稀释法测定上述抗菌肽类似物对革兰氏阳性菌株(S.aureusATCC 25923,B.subtilis ATCC 23857)和革兰氏阴性菌株(E.coli ATCC 25922,K.pneumoniae ATCC 700603,P.aeruginos ATCC 27853)的最低抑菌浓度(MIC),评估其抗菌活性。具体实验方法为:用MH肉汤培养基将生长至对数中期的实验细菌稀释成1×106CFU/mL的细菌悬浮液;将抗菌肽类似物溶解于无菌水,经二倍稀释法用培养基配制成一系列不同浓度的抗菌肽类似物溶液,再将其与等体积细菌悬浮液混合,加入96孔培养板中37℃孵育18h后观察,以无明显细菌生长的最小浓度为抗菌肽类似物的最低抑菌浓度(MIC)。上述实验以抗生素Polymyxin B作为阳性对照,平行重复三次,结果如表1。
表1结果表明,本发明所涉及的抗菌肽类似物对常见实验菌株均具有较强抗菌活性,对以金黄色葡萄球菌(S.aureus ATCC 25923)和枯草芽孢杆菌(B.subtilis ATCC23857)为代表的革兰氏阳性菌,及以大肠杆菌(E.coli ATCC 25922)、肺炎克雷伯菌(K.pneumoniae ATCC 700603)和铜绿假单胞菌(P.aeruginos ATCC 27853)为代表的革兰氏阴性菌均具有很强的抑制作用,表现为广谱抗菌活性,且对革兰氏阳性菌的抗菌活性普遍优于抗生素Polymyxin B。
表1本发明中抗菌肽类似物对抗标准菌株的最低抑菌浓度
下面以抗菌肽类似物C7-PE2-Y9和C8-PE2-Y9为代表,进行溶血实验及抗生物膜实验。
2、溶血实验
取健康小鼠的新鲜血液,1000g,4℃,离心10min,弃去上层血清,用生理盐水清洗下层血细胞后,将其配制成含有8%血细胞的悬浮液,100μL/孔加入96孔板中;将抗菌肽类似物溶解于生理盐水中,用二倍稀释法配制成不同浓度的肽溶液(256-4μg/mL),将其等体积加于已加入血细胞悬浮液的96孔板中,37℃孵育1h;共孵物1200g离心15min后,取100uL/孔上清液至新的96孔板,酶标仪检测其490nm处的吸光值。生理盐水组作阴性对照,1%TritonX-100作阳性对照,根据公式:Hemolysis rate(%)=[(OD490nm peptides-OD490nm negative control)/(OD490nm positive control-OD490nm negative control)]×100%,计算溶血率,结果如图1。
图1结果表明,本发明涉及的抗菌肽类似物具有低的溶血毒性,C7-PE2-Y9在最高检测浓度(256μg/mL)时溶血率低于10%;C8-PE2-Y9虽然在128μg/mL时溶血率超过10%,但其溶血发生浓度远大于发挥抗菌活性的有效浓度。总的来说,本发明合成的抗菌肽类似物溶血毒性低,安全性好。
3、抗生物膜实验
生物膜是由于细菌聚集后形成的粘稠状结构,这种结构可以保护细胞免受外界影响。相比于游离菌,细菌形成生物膜后对抗生素的耐受性提高10~1000倍,是造成目前细菌耐药的主要原因之一。
本发明采用结晶紫染色法测定抗菌肽类似物对金黄色葡萄球菌(S.aureus ATCC25923)和大肠杆菌(E.coli ATCC 25922)生物膜形成的抑制能力。将细菌培养在含0.5%(w/v)葡萄糖的TBS培养基中生长至对数期,稀释至5×105CFU/mL,与不同浓度的抗菌肽类似物1:10(v/v)加入96孔板,37℃孵育24h后吸除浮游细菌,PBS清洗两次,甲醇固定15分钟,0.1%(v/v)结晶紫染色15分钟,去离子水清洗残余的结晶紫染料,95%的乙醇水溶液溶解附着在生物膜上的结晶紫,测595nm处的吸光值。含0.5%(w/v)葡萄糖的TBS培养基作阴性对照,含0.5%(w/v)葡萄糖的TBS培养基加菌作阳性对照。根据公式:Biofilm inhibitionrate(%)=[1-(OD595nm peptides-OD595nm negative control)/(OD595nm positive control-OD595nm negative control)]×100%,计算抗菌肽类似物对生物膜的抑制率,结果如图2。
图2结果表明,本发明合成的抗菌肽类似物有较好的抑制生物膜生成的作用。其中,C7-PE2-Y9在1/2×MIC(4μg/mL)时几乎可完全抑制大肠杆菌(E.coli ATCC 25922)的生物膜生成,在2×MIC(16μg/mL)时几乎能够完全抑制金黄色葡萄球菌(S.aureus ATCC25923)的生物膜生成;C8-PE2-Y9在1×MIC(8μg/mL)时,对金黄色葡萄球菌(S.aureus ATCC25923)和大肠杆菌(E.coli ATCC 25922)的生物膜均具有较好的抑制能力。
基于以上所述,本发明相较于现有技术的有益效果为:
1、本发明N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物是以PE2为母肽改造而来,相较于母肽PE2而言,具有广谱抗菌活性,且对特定菌种的抗菌活性更强。代表性抗菌肽类似物Cn-PE2-Y9对革兰氏阴、阳性菌的抑菌效果均等同于或优于母肽PE2,其溶血实验和抗生物膜实验表明,该类抗菌肽类似物溶血毒性低,安全性高,还能够抑制革兰氏阳性细菌和革兰氏阴性细菌的生物膜生成,在制备临床抗菌药物中具有良好的应用前景。
2、合成过程中,本发明抗菌肽类似物用正己酸、正庚酸或正辛酸替换3-羟基-4-甲基戊酸,原料廉价易得,制备成本更低。此外,较环状母肽PE2而言,本发明的线性抗菌肽类似物合成方法简单,产率更高。
附图说明
图1为本发明抗菌肽类似物溶血毒性实验结果图;
图2为本发明抗菌肽类似物抗生物膜活性实验结果图;
图3为本发明抗菌肽类似物C7-PE2-Y9的质谱图;
图4为本发明抗菌肽类似物C8-PE2-Y9的质谱图;
图5为母肽PE2的质谱图;
图6-12为Cm-PE2的质谱图,其中,m=6,7,8,9,10,11,12;
图13-19为C6-PE2-X9的质谱图,其中,X=A、S、K、Y、F、I、W。
具体实施方式
下面通过具体实施例对本发明低毒广谱抗菌肽类似物的合成方法进行详细说明。
实施例1:抗菌肽类似物C7-PE2-Y9的合成
(1)树脂的活化及预处理
准确称取0.476g(0.2mmol)的MBHA树脂(取代值0.42mmol/g)置于合成仪中,经二氯甲烷溶液溶胀30min,DMF洗涤后,采用茚三酮显色法检验,树脂呈无色透明状,表明树脂正常。
(2)C7-PE2-Y9-resin的合成
对上述检验正常的MBHA树脂用含有20%哌啶的DMF溶液(v/v)脱去Fmoc保护基团,茚三酮显色法检验,树脂呈蓝紫色,表明Fmoc保护基已脱去;DMF洗涤后,将3倍过量(0.6mmol)的Fmoc-Tyr(tBu)-OH、3倍过量(0.6mmol)的HBTU和HOBT,及6倍过量(1.2mmol)的DIEA用重蒸DMF溶解后加入合成仪中,缩合反应1h;茚三酮显色法检验,树脂呈无色透明状,则表明缩合反应成功,得到Fmoc-Tyr(tBu)-resin。
按照上述方法,依次缩合反应后续氨基酸:Fmoc-Leu-OH、Fmoc-D-Val-OH、Fmoc-Dab(Boc)-OH、Fmoc-Leu-OH、Fmoc-D-Phe-OH、Fmoc-Dab(Boc)-OH、Fmoc-Ile-OH、Fmoc-Dab(Boc)-OH,其中氨基酸、HOBT、HBTU和DIEA用量同上,得到Fmoc-Dab(Boc)-Ile-Dab(Boc)-D-Phe-Leu-Dab(Boc)-D-Val-Leu-Tyr(tBu)-resin。
用含有20%哌啶的DMF溶液脱去末端Fmoc保护基后,将3倍过量(0.6mmol)的CH3(CH2)5COOH加入合成仪中缩合反应1h,其中HBTU、HOBT及DIEA用量同上,得到C7-Dab(Boc)-Ile-Dab(Boc)-D-Phe-Leu-Dab(Boc)-D-Val-Leu-Tyr(tBu)-resin。
(3)多肽切割
将所得C7-Dab(Boc)-Ile-Dab(Boc)-D-Phe-Leu-Dab(Boc)-D-Val-Leu-Tyr(tBu)-resin依次用二氯甲烷、甲醇洗涤,彻底压缩抽干后,加入切割试剂(三氟乙酸:三异丙基硅烷:水=9.5:0.25:0.25)反应3h进行切割,得到C7-Dab-Ile-Dab-D-Phe-Leu-Dab-D-Val-Leu-Tyr-NH2,经冰乙醚和水萃取后,冷冻干燥,得到粗肽冻干粉末。
(4)多肽纯化
将上述得到的粗肽冻干粉末经RP-HPLC分离纯化,收集流出液,再冷冻干燥,经质谱鉴定得C7-PE2-Y9,分子量为1177.76Da,质谱图见图3,氨基酸序列如SEQ ID No.1所示;其中,RP-HPLC纯化条件为:流动相A:0.1%TFA/水;流动相B:0.1%TFA/乙腈;线性梯度洗脱,收集主要吸收峰的流出液。
实施例2:抗菌肽类似物C8-PE2-Y9的合成
(1)树脂的活化及预处理
同实施例1。
(2)C8-PE2-Y9-resin的合成
同实施例1的方法依次缩合氨基酸得到Fmoc-Dab(Boc)-Ile-Dab(Boc)-D-Phe-Leu-Dab(Boc)-D-Val-Leu-Tyr(tBu)-resin,用含有20%哌啶的DMF溶液脱去末端Fmoc保护基后,将3倍过量(0.6mmol)的CH3(CH2)6COOH加入合成仪中缩合反应1h,其中HBTU、HOBT及DIEA用量同实施例1,得到C8-Dab(Boc)-Ile-Dab(Boc)-D-Phe-Leu-Dab(Boc)-D-Val-Leu-Tyr(tBu)-resin。
(3)多肽切割
同实施例1。
(4)多肽纯化
同实施例1,经质谱鉴定得C8-PE2-Y9,分子量为1191.77Da,质谱图见图4,氨基酸序列如SEQ ID No.2所示。
实施例3:母肽PE2的合成
按照文献(Org.Biomol.Chem.2017;15:7173-7180)的方法合成母肽,经质谱鉴定得PE2,分子量为1100.7Da,质谱图见图5,氨基酸序列如SEQ ID No.3所示。。
实施例4:抗菌肽类似物Cm-PE2的合成
(1)树脂的活化及预处理
同实施例1。
(2)Cm-PE2-resin的合成
同实施例1的方法依次缩合氨基酸得到Fmoc-Dab(Boc)-Ile-Dab(Boc)-D-Phe-Leu-Dab(Boc)-D-Val-Leu-Thr(tBu)-resin,用含有20%哌啶的DMF溶液脱去末端Fmoc保护基后,将3倍过量(0.6mmol)的烷基酸Cm,其中,m=6-12,得到Cm-PE2。
(3)多肽切割
同实施例1。
(4)多肽纯化
同实施例1,经质谱鉴定得Cm-PE2,质谱图分别如图6-12所示。
实施例4:抗菌肽类似物C6-PE2-X9的合成
(1)树脂的活化及预处理
同实施例1。
(2)C6-PE2-X9-resin的合成
同实施例1的方法依次缩合氨基酸得到Fmoc-Dab(Boc)-Ile-Dab(Boc)-D-Phe-Leu-Dab(Boc)-D-Val-Leu-X-resin,其中,X=Thr、Ala、Ser、Lys、Tyr、Phe、Ile、Trp等。用含有20%哌啶的DMF溶液脱去末端Fmoc保护基后,将3倍过量(0.6mmol)的CH3(CH2)4COOH,加入合成仪中缩合反应1h,其中HBTU、HOBT及DIEA用量同实施例1,得到C6-Dab(Boc)-Ile-Dab(Boc)-D-Phe-Leu-Dab(Boc)-D-Val-Leu-X-resin。
(3)多肽切割
同实施例1。
(4)多肽纯化
同实施例1,经质谱鉴定得C6-PE2-X9,质谱图分别如图13-19所示。
Claims (4)
1.N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物,其特征是,所述抗菌肽类似物是对天然抗菌肽PE2进行开环改造,然后进行N-末端脂肪酸修饰和C末端酰胺化得到,其结构通式为:
Cm-D’ID’FLD’VLT-NH2,标记为Cm-PE2;
其中,m=6-12;D’=Dab;F=D-Phe;V=D-Val;
或:所述抗菌肽类似物是对天然抗菌肽PE2进行开环改造,然后依次进行N-末端脂肪酸修饰、C-末端氨基酸替换,最后进行C末端酰胺化得到,其结构通式为:
C6-D’ID’FLD’VLX-NH2,标记为C6-PE2-X9;
或Cn-D’ID’FLD’VLY-NH2,标记为Cn-PE2-Y9;
其中,n=7、8;D’=Dab;F=D-Phe;V=D-Val;X=T、A、S、K、Y、F、I、W。
2.如权利要求1所述的N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物,其特征是,所述开环改造是指,将天然抗菌肽PE2从酯键的位置断开,得到N-末端为3-羟基-4-甲基戊酸的线性多肽类似物。
3.如权利要求1所述的N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物,其特征是,所述抗菌肽类似物包括C7-PE2-Y9和C8-PE2-Y9。
4.如权利要求1-3任一项所述的N-末端脂肪酸修饰的广谱抗菌肽类似物在制备临床抗菌药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210508512.3A CN115010788B (zh) | 2022-05-10 | 2022-05-10 | N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210508512.3A CN115010788B (zh) | 2022-05-10 | 2022-05-10 | N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115010788A true CN115010788A (zh) | 2022-09-06 |
CN115010788B CN115010788B (zh) | 2024-06-28 |
Family
ID=83068135
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210508512.3A Active CN115010788B (zh) | 2022-05-10 | 2022-05-10 | N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115010788B (zh) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2145907A1 (de) * | 2008-07-14 | 2010-01-20 | Sika Technology AG | Amorphes Polyurethanpolymer und dessen Verwendung in Heissschmelzklebstoffen |
CN102596987A (zh) * | 2009-10-30 | 2012-07-18 | 埃克斯利亚制药有限公司 | 用于纯化粘菌素的方法和纯化的粘菌素组分 |
CN109265518A (zh) * | 2018-10-10 | 2019-01-25 | 倪京满 | 具有高酶解稳定性和强抗菌活性的n-末端脂肪酸修饰抗菌肽类似物及其合成和应用 |
US20190241620A1 (en) * | 2018-02-02 | 2019-08-08 | National Pingtung University Of Science And Technology | Bacteriocin-producing Paenibacillus Ehimensis and Application thereof |
CN110563802A (zh) * | 2019-09-04 | 2019-12-13 | 倪京满 | 一组含n-甲基化氨基酸及n端脂肪酸修饰的抗菌肽类似物及其合成方法和应用 |
CN110938112A (zh) * | 2019-12-10 | 2020-03-31 | 倪京满 | 脂肪酸修饰的超短序列抗菌肽类似物及其应用 |
US20210269483A1 (en) * | 2018-08-28 | 2021-09-02 | Ultupharma Ab | Peptide compounds |
-
2022
- 2022-05-10 CN CN202210508512.3A patent/CN115010788B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2145907A1 (de) * | 2008-07-14 | 2010-01-20 | Sika Technology AG | Amorphes Polyurethanpolymer und dessen Verwendung in Heissschmelzklebstoffen |
CN102596987A (zh) * | 2009-10-30 | 2012-07-18 | 埃克斯利亚制药有限公司 | 用于纯化粘菌素的方法和纯化的粘菌素组分 |
US20190241620A1 (en) * | 2018-02-02 | 2019-08-08 | National Pingtung University Of Science And Technology | Bacteriocin-producing Paenibacillus Ehimensis and Application thereof |
US20210269483A1 (en) * | 2018-08-28 | 2021-09-02 | Ultupharma Ab | Peptide compounds |
CN109265518A (zh) * | 2018-10-10 | 2019-01-25 | 倪京满 | 具有高酶解稳定性和强抗菌活性的n-末端脂肪酸修饰抗菌肽类似物及其合成和应用 |
CN110563802A (zh) * | 2019-09-04 | 2019-12-13 | 倪京满 | 一组含n-甲基化氨基酸及n端脂肪酸修饰的抗菌肽类似物及其合成方法和应用 |
CN110938112A (zh) * | 2019-12-10 | 2020-03-31 | 倪京满 | 脂肪酸修饰的超短序列抗菌肽类似物及其应用 |
Non-Patent Citations (9)
Also Published As
Publication number | Publication date |
---|---|
CN115010788B (zh) | 2024-06-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Conlon et al. | Antimicrobial peptides from the skins of North American frogs | |
Tsubery et al. | Modulation of the hydrophobic domain of polymyxin B nonapeptide: effect on outer-membrane permeabilization and lipopolysaccharide neutralization | |
Čeřovský et al. | Lucifensin, a novel insect defensin of medicinal maggots: synthesis and structural study | |
DK2344178T3 (en) | Peptide sequences, their branched form and their use for antimicrobial applications | |
EP1853620A2 (en) | Antimicrobial hexapeptides | |
CN111363010B (zh) | 一类对称短序列抗菌肽类似物及其应用 | |
WO1992001462A1 (en) | Amphiphilic peptide compositions and analogues thereof | |
CN105566452B (zh) | 一种具有环状结构的抗菌肽及其制备方法和应用 | |
EP3434287A1 (en) | Short and ultra-short antimicrobial lipopeptides and use thereof | |
Sato et al. | Novel des-fatty acyl-polymyxin B derivatives with Pseudomonas aeruginosa-specific antimicrobial activity | |
FI92708C (fi) | Menetelmä uuden lääkeaineena käyttökelpoisen polypeptidin valmistamiseksi | |
CN115028685B (zh) | 一种阳离子双环抗菌肽及其应用 | |
TWI403330B (zh) | 低血球溶解性之抗微生物胜肽、醫藥組成物及其用途 | |
CN106749559B (zh) | 一种基于细胞穿膜肽Tat(49-57)的抗菌肽及其合成方法 | |
CN113045628B (zh) | 一种抗菌肽或其变体在制备抗菌产品中的应用 | |
CN110054664B (zh) | 含d型氨基酸的侧链脂肪酸修饰抗菌肽类似物及其合成和应用 | |
Katsuma et al. | Development of des-fatty acyl-polymyxin B decapeptide analogs with Pseudomonas aeruginosa-specific antimicrobial activity | |
JP2013523661A (ja) | 低赤血球溶解性の抗微生物ペプチド、医薬組成物およびその使用 | |
WO1998016549A1 (en) | Antimicrobial peptide analogs of gramicidin s and compositions comprising them | |
CN112625092A (zh) | 一种基于polybia-MPI的抗菌多肽化合物及其合成与应用 | |
CN112625106A (zh) | 一种抗菌多肽化合物、合成方法及其应用 | |
CN109438559B (zh) | 一种抗多重耐药鲍曼不动杆菌的多肽 | |
CN115010788B (zh) | N-末端脂肪酸修饰的具有抗生物膜活性的低毒广谱抗菌肽类似物及其应用 | |
CN113999297B (zh) | 一种抗菌肽hrNCM及其制备方法与应用 | |
CN111410681B (zh) | 一类重复小单元序列抗菌肽类似物及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |