CN104892744B - 一种具有拮抗趋化因子受体cxcr4的活性多肽的及其设计制备和生物医学应用 - Google Patents
一种具有拮抗趋化因子受体cxcr4的活性多肽的及其设计制备和生物医学应用 Download PDFInfo
- Publication number
- CN104892744B CN104892744B CN201510037065.8A CN201510037065A CN104892744B CN 104892744 B CN104892744 B CN 104892744B CN 201510037065 A CN201510037065 A CN 201510037065A CN 104892744 B CN104892744 B CN 104892744B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- amino acid
- lys
- branched
- cxcr4
- 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.)
- Expired - Fee Related
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 112
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 111
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 110
- 230000003042 antagnostic effect Effects 0.000 title claims abstract description 7
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 title claims description 41
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 title claims description 41
- 238000002360 preparation method Methods 0.000 title claims description 9
- 238000013461 design Methods 0.000 title abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 15
- 238000000329 molecular dynamics simulation Methods 0.000 claims abstract description 8
- 238000012360 testing method Methods 0.000 claims abstract description 7
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 4
- 230000004071 biological effect Effects 0.000 claims abstract 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 14
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 9
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 208000031261 Acute myeloid leukaemia Diseases 0.000 claims description 5
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 230000003185 calcium uptake Effects 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 210000003958 hematopoietic stem cell Anatomy 0.000 claims description 4
- 102000005962 receptors Human genes 0.000 claims description 4
- 108020003175 receptors Proteins 0.000 claims description 4
- -1 salt compounds Chemical class 0.000 claims description 4
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 230000003834 intracellular effect Effects 0.000 claims description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 230000035605 chemotaxis Effects 0.000 claims description 2
- 239000002576 chemokine receptor CXCR4 antagonist Substances 0.000 claims 5
- 238000012216 screening Methods 0.000 claims 2
- 108010061299 CXCR4 Receptors Proteins 0.000 claims 1
- 102000012000 CXCR4 Receptors Human genes 0.000 claims 1
- PLQWGQUNUPMNOD-KKUMJFAQSA-N Ser-Tyr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O PLQWGQUNUPMNOD-KKUMJFAQSA-N 0.000 claims 1
- 238000009650 gentamicin protection assay Methods 0.000 claims 1
- 230000005764 inhibitory process Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000002464 receptor antagonist Substances 0.000 claims 1
- 229940044551 receptor antagonist Drugs 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 13
- 239000007790 solid phase Substances 0.000 abstract description 8
- 241000713772 Human immunodeficiency virus 1 Species 0.000 abstract description 3
- 239000005482 chemotactic factor Substances 0.000 abstract 2
- 150000008574 D-amino acids Chemical class 0.000 description 118
- 210000004027 cell Anatomy 0.000 description 42
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 23
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 15
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 13
- OVQJAKFLFTZDNC-GUBZILKMSA-N Arg-Pro-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O OVQJAKFLFTZDNC-GUBZILKMSA-N 0.000 description 10
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 description 10
- OXRLYTYUXAQTHP-YUMQZZPRSA-N Leu-Gly-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(O)=O OXRLYTYUXAQTHP-YUMQZZPRSA-N 0.000 description 10
- XTWXRUWACCXBMU-XIRDDKMYSA-N Ser-Trp-His Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)NC(=O)[C@H](CO)N XTWXRUWACCXBMU-XIRDDKMYSA-N 0.000 description 10
- 108010092854 aspartyllysine Proteins 0.000 description 10
- 239000012043 crude product Substances 0.000 description 9
- 239000003112 inhibitor Substances 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 239000011347 resin Substances 0.000 description 8
- 229920005989 resin Polymers 0.000 description 8
- 206010027476 Metastases Diseases 0.000 description 6
- 206010028980 Neoplasm Diseases 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 6
- 125000003277 amino group Chemical group 0.000 description 6
- 230000009401 metastasis Effects 0.000 description 6
- RMVRSNDYEFQCLF-UHFFFAOYSA-N thiophenol Chemical compound SC1=CC=CC=C1 RMVRSNDYEFQCLF-UHFFFAOYSA-N 0.000 description 6
- JOEHPBQVSCDCHE-BKGQOYFSSA-N (4r,7s,10s,13s,19s,22s,25s,28s,31s,34r)-34-amino-22-(4-aminobutyl)-10-(3-amino-3-oxopropyl)-31-benzyl-13,19-bis[3-(diaminomethylideneamino)propyl]-25-[(1r)-1-hydroxyethyl]-28-(2-methylpropyl)-6,9,12,15,18,21,24,27,30,33-decaoxo-7-propan-2-yl-1,2-dithia-5, Chemical compound N1C(=O)[C@@H](N)CSSC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CC1=CC=CC=C1 JOEHPBQVSCDCHE-BKGQOYFSSA-N 0.000 description 5
- UCDHVOALNXENLC-KBPBESRZSA-N Leu-Gly-Tyr Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 UCDHVOALNXENLC-KBPBESRZSA-N 0.000 description 5
- YRRCOJOXAJNSAX-IHRRRGAJSA-N Leu-Pro-Lys Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N YRRCOJOXAJNSAX-IHRRRGAJSA-N 0.000 description 5
- FUKDBQGFSJUXGX-RWMBFGLXSA-N Lys-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCCN)N)C(=O)O FUKDBQGFSJUXGX-RWMBFGLXSA-N 0.000 description 5
- ZAWOJFFMBANLGE-CIUDSAMLSA-N Lys-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCCN)N ZAWOJFFMBANLGE-CIUDSAMLSA-N 0.000 description 5
- YSPZCHGIWAQVKQ-AVGNSLFASA-N Lys-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN YSPZCHGIWAQVKQ-AVGNSLFASA-N 0.000 description 5
- 108010060534 MSH (11-13) Proteins 0.000 description 5
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 5
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 108010054167 vMIP-II Proteins 0.000 description 5
- 102000009410 Chemokine receptor Human genes 0.000 description 4
- 108050000299 Chemokine receptor Proteins 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000003399 chemotactic effect Effects 0.000 description 4
- 238000010511 deprotection reaction Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 3
- 101710088580 Stromal cell-derived factor 1 Proteins 0.000 description 3
- 239000012190 activator Substances 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- 230000012292 cell migration Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000009545 invasion Effects 0.000 description 3
- DZNKOAWEHDKBEP-UHFFFAOYSA-N methyl 2-[6-[bis(2-methoxy-2-oxoethyl)amino]-5-[2-[2-[bis(2-methoxy-2-oxoethyl)amino]-5-methylphenoxy]ethoxy]-1-benzofuran-2-yl]-1,3-oxazole-5-carboxylate Chemical compound COC(=O)CN(CC(=O)OC)C1=CC=C(C)C=C1OCCOC(C(=C1)N(CC(=O)OC)CC(=O)OC)=CC2=C1OC(C=1OC(=CN=1)C(=O)OC)=C2 DZNKOAWEHDKBEP-UHFFFAOYSA-N 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 2
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- IIJWXEUNETVJPV-IHRRRGAJSA-N Tyr-Arg-Ser Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N)O IIJWXEUNETVJPV-IHRRRGAJSA-N 0.000 description 2
- 238000002820 assay format Methods 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 208000026278 immune system disease Diseases 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000001869 matrix assisted laser desorption--ionisation mass spectrum Methods 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- DBABZHXKTCFAPX-UHFFFAOYSA-N probenecid Chemical compound CCCN(CCC)S(=O)(=O)C1=CC=C(C(O)=O)C=C1 DBABZHXKTCFAPX-UHFFFAOYSA-N 0.000 description 2
- 229960003081 probenecid Drugs 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FVBZXNSRIDVYJS-AVGNSLFASA-N Arg-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N FVBZXNSRIDVYJS-AVGNSLFASA-N 0.000 description 1
- 102100024167 C-C chemokine receptor type 3 Human genes 0.000 description 1
- 101710149862 C-C chemokine receptor type 3 Proteins 0.000 description 1
- 108091008928 CXC chemokine receptors Proteins 0.000 description 1
- 102000054900 CXCR Receptors Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical group [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010008951 Chemokine CXCL12 Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241001502974 Human gammaherpesvirus 8 Species 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- SEFNTZYRPGBDCY-IHRRRGAJSA-N Tyr-Arg-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N)O SEFNTZYRPGBDCY-IHRRRGAJSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000006315 carbonylation Effects 0.000 description 1
- 238000005810 carbonylation reaction Methods 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000006203 ethylation Effects 0.000 description 1
- 238000006200 ethylation reaction Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- VPSRLGDRGCKUTK-UHFFFAOYSA-N fura-2-acetoxymethyl ester Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=C(C)C=C1OCCOC(C(=C1)N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=CC2=C1OC(C=1OC(=CN=1)C(=O)OCOC(C)=O)=C2 VPSRLGDRGCKUTK-UHFFFAOYSA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940126181 ion channel inhibitor Drugs 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 210000004224 pleura Anatomy 0.000 description 1
- 210000000414 pleural mesothelial cell Anatomy 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002922 simulated annealing Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种设计具有天然趋化因子N端区域的活性多肽。这类活性多肽通过拮抗趋化因子受体的活性,从而抑制HIV‑1侵入细胞并感染。本发明还公开了这类活性多肽的设计方法,通过分子动力学模拟,设计合适的连接桥将两个多肽片段连接起来,确定多肽合成序列,固相合成多肽,并最终测试活性多肽的生物学活性。本发明的活性多肽可作为CXCR4受体的拮抗剂,用于治疗艾滋病的先导药物,干细胞动员剂,急性髓系白血病,以及与CXCR4相关的多种疾病的治疗。
Description
技术领域
本发明涉及一种具有拮抗趋化因子受体CXCR4的活性多肽及其制备方法和应用。
背景技术
CXCR4是CXC趋化因子受体家族的一员,总共有356个氨基酸残基。CXCR4是趋化因子基质细胞衍生因子-1(SDF-1,CXCL12)的特异性受体,与SDF-1结合后使细胞产生趋化作用。SDF-1a和CXCR4广泛地表达于多种细胞和组织中,包括免疫细胞、脑、心脏、肾、肝、肺和脾,在免疫系统、循环系统及中枢神经系统的发育中起着至关重要的作用。
CXCR4蛋白被发现与多种疾病密切相关。上世纪90年代,研究人员发现了HIV病毒感染的共受体,即CXCR4和CCR5。这两个穿膜蛋白均属于趋化因子受体家族,且与HIV病毒的入侵有着很大的联系。CXCR4是研究最多的趋化因子受体之一,主要是它可作为HIV-1入侵的辅助受体。因此,CXCR4蛋白也被认为是研究抗HIV药物的重要靶点蛋白。
CXCR4与SDF-1a轴可使造血干细胞回溯到骨髓。因此,阻断CXCR4与SDF-1a的相互作用可以使得造血干细胞从骨髓中迁移出来,用于治疗非霍杰金淋巴瘤和多发性骨髓瘤化疗之后的造血功能损伤。
CXCR4在很多肿瘤组织中被发现过量表达,并且在肿瘤细胞的增殖,侵润,血管增生,以及转移中起到非常重要的作用。CXCR4在23种不同类型肿瘤中均有表达,是肿瘤细胞表达最为普遍的趋化因子受体,并且和患者的预后相关。乳腺癌患者中,CXCR4表达阳性与患者淋巴结、远处转移相关,而高表达CXCR4的乳房癌患者预后较差。关于结直肠癌的研究表明,CXCR4高表达与肿瘤的复发、肝脏转移相关,并且相应患者生存率也较低。另外,在卵巢癌、黑素瘤、前列腺癌、神经母细胞瘤等其他肿瘤的研究中也得到了相似的结果。非小细胞肺癌患者恶性胸水中肿瘤细胞CXCR4表达阳性,而胸膜间皮细胞则表达SDF-1a,CXCR4/SDF-1a信号轴可能在非小细胞肺癌胸膜播散过程中起到了一定的作用。因此,CXCR4也是靶向治疗肿瘤及肿瘤转移的研究新热点。
发明内容
本发明提供了一种分支多肽及其衍生物的设计、合成和应用,目的之一是提供一种具有拮抗CXCR4活性的抗艾滋病和相关免疫疾病的先导化合物。
本发明所提供的分支多肽结构式为Leu-Gly-Ala-Ser-Trp-His-Arg-Pro-Asp-Lys-Cys-Ala-Leu-Gly-Tyr-Asn-Lys-Arg-Pro-Leu-Pro-Lys(NH2)-ε-(-AAn)。其中,AAn为Leu-Gly-Ala-Ser-Trp-His-Arg-Pro-Asp,或Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg-Cys-Pro,或以上序列通过缺失、加入、插入和/或替换一个或多个氨基酸残基所得到的多肽序列。上述氨基酸为D型氨基酸或L型氨基酸。
可在所述多肽的N端或残基侧链上被聚乙二醇化,所述的聚乙二醇分子的分子量在2,000到60,000之间。
可在所述的分支多肽的氨基酸侧链基团上、氨基端或羧基端进行一个或多个羟基化、羧基化、羰基化、甲基化、乙基化、磷酸化、酯化或糖基化,得到所述分支多肽衍生物。
所述的分支多肽可以与酸或碱反应形成药学上可接受的盐类化合物。
本发明的另一个目的是提供所述分支多肽及衍生物的应用。
所述分支多肽可以用于治疗艾滋病及相关免疫疾病的药物分子。
所述分支多肽可以用于进行干细胞动员的药物分子。
所述分支多肽可以用于治疗急性髓系白血病的药物分子。
与现有发明相比,本发明具有以下优点:
本发明的多肽为分枝状多肽,能显著提高多肽与蛋白的结合效率,具有很强的抗趋化因子受体CXCR4的作用,表现出较强的抗肿瘤细胞转移和抗HIV-1病毒入侵的作用。且为D型多肽,生物稳定性好。
附图说明
图1:多肽与蛋白结合模式图。
图2:多肽与蛋白结合位点。
图3:DD2多肽HPLC色谱图。
图4:DS1多肽的HPLC色谱图。
图5:DD2多肽MALDI质谱图。
图6:DS1多肽MALDI质谱图。
图7:DD2多肽的亲合活性。
图8:DS1多肽的亲合活性。
图9:DD2多肽的趋化活性。
图10:DS1多肽的趋化活性。
图11:DD2多肽的抗HIV-1病毒活性。
图12:DS1多肽的抗HIV-1病毒活性
图13:DD2多肽抑制细胞内钙流的活性
图14:DS1多肽抑制细胞内钙流的活性
具体实施方式
实施例1多肽的设计。
多肽设计使用分子动力学模拟的方法来进行。具体是使用SYBYL-x软件包中Biopolymer模块进行模拟计算,用以预测设计多肽与CXCR4蛋白的结合构象。CXCR4蛋白模型由已发表的晶体结构所构建(PDB:3ODU)。用软件对CXCR4晶体结构进行整理,去掉多余的配体分子,水分子和其他小分子,对蛋白文件加氢和电荷,并得到mol2文件。多肽分子的结构经过模拟退火和能量最小化得到能量相对较低的构象。通过手动调整,将多肽分子移至蛋白分子的表面,并调整二面角避免分子碰撞。在分子动力学模拟的过程中,对多肽和蛋白的结构进行溶剂化,选取水分子作为溶剂,水分子模型使用TIP3P模型。在Tripos力场下,分子动力学模拟预先进行10psec,使系统温度参数梯度升高至300K。然后,分子动力学模拟在300K的温度参数下保持1nsec。在分子动力学模拟过程中,只有多肽配体分子和CXCR4蛋白中ECL的氨基酸残基可以运动,而其他的残基均保持坐标不变。运算结束之后,使蛋白-多肽配体的系统重新能量最低化,并写入输出文件,并分析模拟过程中能量的变化以及蛋白-多肽的结合方式。
基于已知CXCR4的抑制剂多肽DV1。DV1是由CXCR4的另一个天然配体vMIP-II的衍生化所得到的。vMIP-II是由人类疱疹病毒8所编码的一个类趋化因子蛋白,是CXCR4的天然抑制性配体。同时,vMIP-II也可以和CCR5/CCR3等其他的趋化因子受体结合。根据对vMIP-II的构效关系研究发现,vMIP-II的N端21个氨基酸残基的多肽转变为D型氨基酸时,D型多肽的活性比V1更强,且具有很好的受体选择性。我们选取该多肽作为设计新型多肽的一个模板。
实施例2多肽的合成和纯化。
采用固相合成方法来合成多肽。固相树脂采用TentaGel Amide树脂,通过分离得到的多肽为C端氨基化的多肽。所有的氨基酸均使用9-芴甲氧羰基(9-Fmoc)保护氨基。在合成过程中,第一个氨基酸的羧基与固相树脂的活化氨基形成酰胺键,连接在固相上。通过20%的哌啶80%二甲基甲酰胺(DMF)的脱保护作用,将Fmoc基团脱去,露出氨基,并在缩合剂DIC(5当量)和HOBt(5当量)的作用下与下一个氨基酸(5当量)的羧基反应。多余的反应物和缩合剂在反应后通过用DMF清洗固相树脂而除去。这样,多肽的序列就从C端到N端一个个连接在固相树脂上面。当最后一个氨基酸连接完毕后,使用多肽分离试剂使多肽从固相树脂上分离下来。多肽分离试剂使用90%三氟乙酸,5%硫代苯酚和5%的水经混合后得到。多肽分离试剂同时也能脱掉氨基酸残基上面多余的保护基团。通常,反应时间为2小时,期间用玻璃棒轻轻搅拌数次,如多肽序列里面有精氨酸等则能观察到树脂的颜色变成深红色。分离结束后,用空气吹去多余的溶剂至残留1~2mL,使用预冷的乙醚(10mL)沉淀多肽30min。沉淀完毕后,使用离心机将多肽收集在试管底部,倒掉上清。沉淀后的多肽用双蒸水溶解,放置在冻干机中干燥,得到粗品多肽。
赖氨酸的4个碳的侧链作为连接桥连接两个多肽模板。赖氨酸的侧链由Dde(N-[1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene ethyl]))基团保护,脱保护时使用温和的脱保护剂肼的DMF溶液(2%)。脱保护反应重复3次,每次2min,之后用DMF清洗树脂并用Kaiser测试检验脱保护反应是否完成。反应完毕后,赖氨酸的侧链氨基即与新的氨基酸的羧基反应,得到分枝状的多肽。
多肽合成完毕后得到的粗品肽,使用制备型高效液相色谱仪进行纯化。在检测好的纯化条件进行纯化,收集色谱中出现的色谱峰,并置于冻干机中干燥。干燥后的多肽用分析型高效液相色谱进行纯度鉴定,并用基质辅助激光解吸/电离质谱检测其分子量来确认。所有的多肽的纯度均达95%以上。
实施例3多肽的亲合活性测试。
HEK293细胞外源性的插入了CXCR4的质粒,能够稳定表达CXCR4蛋白。该细胞在RPMI1640(10%小牛血清,100IU青霉素,0.1mg/mL链霉素,2mM谷氨酰胺)中培养,并加入0.4mg/mL的G418加以筛选。实验之前,HEK293细胞用胰酶消化并收集,用4℃的FACS缓冲液(0.5%BSA,0.05%叠氮化钠的PBS溶液)洗涤两遍,并最终用缓冲液稀释成1×107细胞/mL。细胞被混合均匀后置于锥形孔的96孔板,每孔5×105细胞。一抗(12G5,鼠抗人CXCR4抗体,1∶3000)和不同浓度的多肽与细胞在冰上孵育40min后,用FACS缓冲液洗涤细胞2次。完毕后,加二抗(抗鼠抗体-FITC,1∶250)冰上避光孵育30min。反应完毕后,用FACS缓冲液洗涤细胞两次。细胞的荧光强度(485EX/528EM)由荧光检测仪(Synergy 2,BioTek)所记录。实验数据是由至少三次独立实验所得到,每次实验均有两次重复。CXCR4的结合曲线用Sigmoidaldose-response模型所得到,IC50值则由GraphPad Prism 4计算得出。
实施例4多肽的趋化活性测试。
本实验所用的细胞是天然表达CXCR4蛋白的SUP-T1细胞系。SUP-T1细胞在RPMI1640(10%小牛血清,100IU青霉素,0.1mg/mL链霉素,2mM谷氨酰胺)中培养,实验开始前收集细胞,确定细胞密度。SUP-T1细胞用缓冲液洗涤两次,并稀释成终密度为1.25×107/mL的细胞混悬液。将1×106的细胞置于Transwell(Corning)的上层孔中。下层孔加入200uL的缓冲液和SDF-1a作为促使细胞迁移的趋化物。在抑制剂实验模式中,上层孔中的细胞在进行细胞迁移之前,与不同浓度的抑制剂预先37℃孵育2hr。孵育后,将上层孔板置于下层孔中,置于细胞孵育箱中,使细胞迁移3hr。在激活剂实验模式中,下层孔的缓冲液除了加入SDF-1a之外,也在平行孔中加入不同浓度的多肽。然后将上层孔板置于下层孔中,置于细胞孵育箱中,使细胞迁移3hr。孵育结束后,移走上层孔板,在下层孔中加入40uL/孔的CellTiter Blue(Promoga),用来定量迁移至下层孔的细胞。实验数据由至少3次独立实验所得到,每次实验均有2次重复。
实施例5多肽的抗HIV-1病毒活性测试。
本实验所需要的细胞是Cf2TH-CD4-CXCR4或Cf2Th-CXCR4细胞系。在感染实验之前,细胞以6×103/孔的密度置于96孔板中,并孵育24hr。感染实验当天,将不同浓度的抑制剂(1~100uM)加入病毒中(10000个逆转录酶单位),37℃孵育30min。将细胞中的培养液移走,加入病毒-抑制剂混合物,在37℃中孵育48hr。孵育结束后,将细胞的培养液移走并加入细胞裂解液,冻融3次后完全裂解细胞。在细胞裂解物中加入100uL荧光素缓冲液(15mMMgSO4,15mM KPO4,1mM ATP,1mM DTT)和50uL的D-荧光素钾(1mN,BD Pharmingen),并用生物发光酶标仪(EG&G Berthold MicroplateLuminometer LB 96V)记录荧光信号。
实施例6多肽对细胞钙流的测试。
本实验所用的细胞是SUP-T1细胞系。实验前收集细胞,给细胞计数以确定细胞密度,用实验缓冲液(20mM HEPES的HBSS溶液)洗涤细胞,并最终将细胞液稀释成1×106/mL。本实验使用Fura-2AM(Molecular Probes)用于追踪钙离子的信号,同时使用丙磺舒作为离子通道抑制剂阻止细胞将Fura-2AM泵出。当SDF-1a或CXCR4激活剂与CXCR4结合并触发信号转导通路时,钙离子从内质网流出,与细胞质内的Fura-2AM结合,形成高强度的荧光螯合物,被荧光检测仪所检测,以此确定受体是否被激活。CXCR4钙离子内流实验也分为抑制剂模式和激活剂模式。
CXCR4激动剂模式:稀释好的细胞悬浊液用Fura-2AM Dye(2uM)与丙磺舒(5uM)染色,放于37℃的细胞孵育箱中45min,每10min混匀一次。染色结束后,用实验缓冲液洗涤细胞2次。将细胞液加入石英分光液槽中,置入荧光检测仪中,调整波长为340EX/510EM,记录荧光信号。手动加入不同浓度的SDF-1a(50nM)或CXCR4激动剂(10nM~10uM)后,观察是否能够增加荧光强度。
CXCR4抑制剂模式:细胞处理方法同激动剂模式。在细胞液加入石英分光液槽后,先加入一定浓度的抑制剂(10nM~10uM),等待120sec,再加入SDF-1a(50nM)。观察荧光信号与没有加抑制剂组的差别。
表1本发明中包括的多种分支多肽的序列
序列表
<110> 徐岩,黄子为
<120> 一种具有拮抗趋化因子受体CXCR4的活性多肽的及其设计制备和生物医学应用
<130> MP1707735Z
<141> 2019-10-18
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (1)..(22)
<223> Leu是D-氨基酸; Ala是D-氨基酸; Ser是D-氨基酸;Trp是D-氨基酸; His是D-氨基酸; Arg是D-氨基酸; Pro是D-氨基酸; Asp是D-氨基酸; Lys是D-氨基酸; Cys是D-氨基酸; Gly是D-氨基酸; Tyr是D-氨基酸; Asn是D-氨基酸;
<220>
<221> MOD_RES
<222> (22)..(22)
<223> Lys的侧链氨基上连接一个多肽支链,多肽支链的氨基酸序列为:Lys ProVal Ser Leu Ser Tyr Arg; Lys的侧链氨基与Arg的羧基形成酰胺键
<400> 1
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly Tyr Asn
1 5 10 15
Lys Arg Pro Leu Pro Lys
20
<210> 2
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (1)..(22)
<223> Leu是D-氨基酸; Ala是D-氨基酸; Ser是D-氨基酸; Trp是D-氨基酸; His是D-氨基酸; Arg是D-氨基酸; Pro是D-氨基酸; Asp是D-氨基酸; Lys是D-氨基酸;Cys是D-氨基酸; Tyr是D-氨基酸; Asn是D-氨基酸;
<220>
<221> MOD_RES
<222> (22)..(22)
<223> Lys的侧链氨基上连接一个多肽支链,多肽支链的氨基酸序列为:Lys ProVal Ser Leu Ser Tyr Arg Cys Pro; Lys的侧链氨基与Pro的羧基形成酰胺键
<400> 2
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly Tyr Asn
1 5 10 15
Lys Arg Pro Leu Pro Lys
20
<210> 3
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (1)..(22)
<223> Leu是D-氨基酸; Ala是D-氨基酸; Ser是D-氨基酸; Trp是D-氨基酸; His是D-氨基酸; Arg是D-氨基酸; Pro是D-氨基酸; Asp是D-氨基酸; Lys是D-氨基酸; Cys是D-氨基酸; Tyr是D-氨基酸; Asn是D-氨基酸; Val是D-氨基酸;
<220>
<221> MOD_RES
<222> (22)..(22)
<223> Lys的侧链氨基上连接一个多肽支链,多肽支链的氨基酸序列为:Lys ProVal Ser Leu Ser Tyr Arg,Lys是D-氨基酸;Pro是D-氨基酸; Val是D-氨基酸; Ser是D-氨基酸; Leu是D-氨基酸; Ser是D-氨基酸; Tyr是D-氨基酸; Arg是D-氨基酸; Lys的侧链氨基与Arg的羧基形成酰胺键
<400> 3
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly Tyr Asn
1 5 10 15
Lys Arg Pro Leu Pro Lys
20
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (1)..(10)
<223> Leu是D-氨基酸;Ala是D-氨基酸;Ser是D-氨基酸;Trp是D-氨基酸;His是D-氨基酸;Arg是D-氨基酸;Pro是D-氨基酸;Asp是D-氨基酸;Lys是D-氨基酸;
<400> 4
Leu Gly Ala Ser Trp His Arg Pro Asp Lys
1 5 10
<210> 5
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (1)..(10)
<223> Leu是D-氨基酸;Ala是D-氨基酸;Ser是D-氨基酸;Trp是D-氨基酸;His是D-氨基酸;Arg是D-氨基酸;Pro是D-氨基酸;Asp是D-氨基酸;Lys是D-氨基酸;
<220>
<221> MOD_RES
<222> (10)..(10)
<223> Lys侧链氨基上连接一个多肽支链,多肽支链的序列为:Leu Gly Ala SerTrp His Arg Pro Asp Lys,Leu是D-氨基酸; Gly是D-氨基酸; Ala是D-氨基酸; Ser是D-氨基酸; Trp是D-氨基酸; His是D-氨基酸; Arg是D-氨基酸; Pro是D-氨基酸; Asp是D-氨基酸; Lys是D-氨基酸;Lys侧链氨基与多肽链末端Lys的羧基形成酰胺键;
<400> 5
Leu Gly Ala Ser Trp His Arg Pro Asp Lys
1 5 10
<210> 6
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (1)..(22)
<223> Leu是D-氨基酸; Ala是D-氨基酸; Ser 是D-氨基酸;Trp是D-氨基酸; His是D-氨基酸; Arg是D-氨基酸; Pro是D-氨基酸; Asp是D-氨基酸; Lys是D-氨基酸; Cys是D-氨基酸; Tyr 是D-氨基酸;Asn是D-氨基酸;
<220>
<221> MOD_RES
<222> (22)..(22)
<223> Lys侧链氨基上连接一个多肽支链,多肽支链的序列为:Leu Gly Ala SerTrp His Arg Pro Asp Lys,Leu是D-氨基酸; Gly是D-氨基酸; Ala是D-氨基酸; Ser是D-氨基酸; Trp是D-氨基酸; His是D-氨基酸; Arg是D-氨基酸; Pro是D-氨基酸; Asp是D-氨基酸; Lys是D-氨基酸;Lys侧链氨基与多肽链末端Lys的羧基形成酰胺键;
<400> 6
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly Tyr Asn
1 5 10 15
Lys Arg Pro Leu Pro Lys
20
<210> 7
<211> 22
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (1)..(22)
<223> Leu是D-氨基酸; Ala是D-氨基酸; Ser是D-氨基酸; Trp是D-氨基酸; His是D-氨基酸; Arg是D-氨基酸; Pro是D-氨基酸; Asp是D-氨基酸; Lys是D-氨基酸; Cys是D-氨基酸; Tyr是D-氨基酸; Asn是D-氨基酸; Val是D-氨基酸;
<220>
<221> MOD_RES
<222> (22)..(22)
<223> Lys的侧链氨基上连接一个多肽支链,多肽支链的氨基酸序列为:Lys ProVal Ser Leu Ser Tyr Arg Ser Ala; Lys的侧链氨基与Ala的羧基形成酰胺键
<400> 7
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly Tyr Asn
1 5 10 15
Lys Arg Pro Leu Pro Lys
20
<210> 8
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (1)..(11)
<223> Leu是D-氨基酸;Ala是D-氨基酸;Ser是D-氨基酸;Trp是D-氨基酸;His是D-氨基酸;Arg是D-氨基酸;Pro是D-氨基酸;Asp是D-氨基酸;Lys是D-氨基酸;
<220>
<221> MOD_RES
<222> (11)..(11)
<223> Lys的侧链氨基上连接一个多肽支链,多肽支链的氨基酸序列为:Lys ProVal Ser Leu Ser Tyr Arg Ser Ala; Lys的侧链氨基与Ala的羧基形成酰胺键
<400> 8
Leu Gly Ala Ser Trp His Arg Pro Asp Lys Lys
1 5 10
Claims (11)
1.具有CXCR4拮抗活性的分支多肽,其特征在于:所述分支多肽的序列从氨基端到羧基端的序列如下:
Leu-Gly-Ala-Ser-Trp-His-Arg-Pro-Asp-Lys-Cys-Ala-Leu-Gly-Tyr-Asn-Lys-Arg- Pro-Leu-Pro-Lys(NH 2 )-ε-(-AAn),
其中,AAn从N端到C端的序列如下:
Lys-Pro-Val-Ser-Leu-Ser-Tyr-Arg,或Leu-Gly-Ala-Ser-Trp-His-Arg-Pro-Asp- Lys;
其中,Lys(NH 2 )-ε与AAn的羧基端的Arg或Lys的羧基形成酰胺键;
斜体表示D型氨基酸。
2.一种分支多肽衍生物,是权利要求1所述的分支多肽的药学上可接受的盐。
3.根据权利要求1所述分支多肽的制备方法,其特征在于:
(1)采用分子动力学模拟的方法,预测多肽的空间构象,并确定多肽的序列;
(2)固相合成方法合成目标多肽,并使用HPLC进行纯化;
(3)检验多肽的生物活性,筛选目标多肽。
4.根据权利要求2所述分支多肽衍生物的制备方法,其特征在于:
(1)采用分子动力学模拟的方法,预测多肽的空间构象,并确定多肽的序列;
(2)固相合成方法合成目标多肽,并使用HPLC进行纯化;
(3)检验多肽的生物活性,筛选目标多肽,将所述目标多肽与酸或碱反应形成药学上可接受的盐类化合物。
5.根据权利要求3所述分支多肽的制备方法,其特征在于步骤(3)中所述生物活性检测,包括受体亲和性实验,趋化抑制实验,细胞内钙流实验和抗HIV-1侵入实验。
6.根据权利要求1所述分支多肽作为CXCR4受体拮抗剂在制备作为抗艾滋病的先导药物的应用。
7.根据权利要求2所述分支多肽衍生物作为CXCR4受体拮抗剂在制备作为抗艾滋病的先导药物的应用。
8.根据权利要求1所述分支多肽作为CXCR4受体拮抗剂在制备作为造血干细胞动员中的先导药物的应用。
9.根据权利要求2所述分支多肽衍生物作为CXCR4受体拮抗剂在制备作为造血干细胞动员中的先导药物的应用。
10.根据权利要求1所述分支多肽作为CXCR4受体拮抗剂在制备作为急性髓系白血病中的先导药物的应用,所述急性髓系白血病为阳性表达CXCR4的急性髓系白血病。
11.根据权利要求2所述分支多肽衍生物作为CXCR4受体拮抗剂在制备作为急性髓系白血病中的先导药物的应用,所述急性髓系白血病为阳性表达CXCR4的急性髓系白血病。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510037065.8A CN104892744B (zh) | 2015-01-26 | 2015-01-26 | 一种具有拮抗趋化因子受体cxcr4的活性多肽的及其设计制备和生物医学应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510037065.8A CN104892744B (zh) | 2015-01-26 | 2015-01-26 | 一种具有拮抗趋化因子受体cxcr4的活性多肽的及其设计制备和生物医学应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104892744A CN104892744A (zh) | 2015-09-09 |
CN104892744B true CN104892744B (zh) | 2020-01-31 |
Family
ID=54025751
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510037065.8A Expired - Fee Related CN104892744B (zh) | 2015-01-26 | 2015-01-26 | 一种具有拮抗趋化因子受体cxcr4的活性多肽的及其设计制备和生物医学应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN104892744B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105534896B (zh) * | 2015-12-11 | 2019-01-04 | 国家纳米科学中心 | 一种多肽和化疗药物联合的载药胶束及其制备方法和应用 |
CN107325187B (zh) * | 2017-07-19 | 2021-11-09 | 黄子为 | 一种具有cxcr4蛋白激动活性的多肽及其应用和药物组合物 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002022657A2 (en) * | 2000-09-12 | 2002-03-21 | University Of British Columbia | Peptide antagonist of multiple chemokine receptors and uses thereof |
CN102869675A (zh) * | 2010-01-26 | 2013-01-09 | 国家研究委员会 | 结合cxcr4受体的环状肽和相对医疗和诊断用途 |
-
2015
- 2015-01-26 CN CN201510037065.8A patent/CN104892744B/zh not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002022657A2 (en) * | 2000-09-12 | 2002-03-21 | University Of British Columbia | Peptide antagonist of multiple chemokine receptors and uses thereof |
CN102869675A (zh) * | 2010-01-26 | 2013-01-09 | 国家研究委员会 | 结合cxcr4受体的环状肽和相对医疗和诊断用途 |
Non-Patent Citations (5)
Title |
---|
"A novel synthetic bivalent ligand to probe chemokine receptor CXCR4 dimerization and inhibit HIV-1 entry";Choi WT 等;《Biochemistry》;20120911;第51卷(第36期);第7078-7086页 * |
"A synthetic bivalent ligand of CXCR4 inhibits HIV infection";Xu Y 等;《Biochem Biophys Res Commun》;20130614;第435卷(第4期);第646-650页 * |
"Drug discovery research targeting the CXC chemokine receptor 4 (CXCR4)";Choi WT 等;《J Med Chem》;20120209;第55卷(第3期);第977-994页 * |
"Exploring the Stereochemistry of CXCR4-Peptide Recognition and Inhibiting HIV-1 Entry with D-Peptides Derived from Chemokines";Naiming Zhou 等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20020517;第277卷(第20期);第17476-17485页 * |
"vMIP-Ⅱ通过CXCR4拮抗乳腺癌转移作用的初步研究";刘富金 等;《癌症》;20041105;第23卷(第11期);第1283-1287页 * |
Also Published As
Publication number | Publication date |
---|---|
CN104892744A (zh) | 2015-09-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
ES2770361T3 (es) | Un polipéptido antagonista del receptor 4 de quimiocina CXC (CXCR4) | |
JP7427287B2 (ja) | 二重il-2rおよびil-7r結合化合物 | |
JP7427286B2 (ja) | IL-2RβγC結合化合物 | |
JP2023514128A (ja) | IL-7Rαγc結合化合物 | |
Xu et al. | A synthetic bivalent ligand of CXCR4 inhibits HIV infection | |
CN104892744B (zh) | 一种具有拮抗趋化因子受体cxcr4的活性多肽的及其设计制备和生物医学应用 | |
CN114728039A (zh) | Kv1.3阻断剂 | |
CN116964072A (zh) | IL-2Rβγc结合化合物和其用途 | |
Boulais et al. | Photolabeling identifies transmembrane domain 4 of CXCR4 as a T140 binding site | |
WO2002057313A2 (en) | Receptor-binding compounds and methods for identifying them | |
US20030167129A1 (en) | Binding compounds and methods for identifying binding compounds | |
CN114524870A (zh) | 源自于ssx2抗原的短肽 | |
Decalf et al. | A novel method to produce synthetic murine CXCL10 for efficient screening of functional variants | |
Strong et al. | Synthetic chemokines directly labeled with a fluorescent dye as tools for studying chemokine and chemokine receptor interactions | |
CN116199744B (zh) | 一种结合fgfr2受体的多肽及其用途 | |
CN110862449B (zh) | 衍生自ssx的肿瘤抗原短肽 | |
CN107325187A (zh) | 一种具有cxcr4蛋白激动活性的多肽及其应用和药物组合物 | |
CN111138522B (zh) | 衍生自afp的肿瘤抗原短肽 | |
CN112300261B (zh) | 一种衍生自afp的肿瘤抗原短肽 | |
US20030018438A1 (en) | Binding compounds and methods for identifying binding compounds | |
WO2001070768A2 (en) | Receptor-binding compounds and method for identifying them | |
KR20230145939A (ko) | 케모카인 수용체 cxcr4에 대한 고 친화성 항체 | |
WO2024146707A1 (en) | Variant ligand conjugates for payload delivery | |
CN116829579A (zh) | Il-2r和il-7r双重结合化合物 | |
CN116997562A (zh) | 异串联双环肽复合物 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | 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 | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200131 |
|
CF01 | Termination of patent right due to non-payment of annual fee |