CA2269656A1 - Receptor specific brain natriuretic peptide (bnp) - Google Patents
Receptor specific brain natriuretic peptide (bnp) Download PDFInfo
- Publication number
- CA2269656A1 CA2269656A1 CA002269656A CA2269656A CA2269656A1 CA 2269656 A1 CA2269656 A1 CA 2269656A1 CA 002269656 A CA002269656 A CA 002269656A CA 2269656 A CA2269656 A CA 2269656A CA 2269656 A1 CA2269656 A1 CA 2269656A1
- Authority
- CA
- Canada
- Prior art keywords
- arg
- ser
- gly
- seq
- lys
- 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.)
- Abandoned
Links
- 101800000407 Brain natriuretic peptide 32 Proteins 0.000 title description 3
- 102400000667 Brain natriuretic peptide 32 Human genes 0.000 title 1
- 101800002247 Brain natriuretic peptide 45 Proteins 0.000 title 1
- HPNRHPKXQZSDFX-OAQDCNSJSA-N nesiritide Chemical compound C([C@H]1C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CO)C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 HPNRHPKXQZSDFX-OAQDCNSJSA-N 0.000 title 1
- 230000027455 binding Effects 0.000 claims abstract description 32
- 150000001413 amino acids Chemical group 0.000 claims description 170
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 84
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 claims description 54
- 238000000034 method Methods 0.000 claims description 46
- 238000006467 substitution reaction Methods 0.000 claims description 45
- 239000000203 mixture Substances 0.000 claims description 24
- 239000013598 vector Substances 0.000 claims description 15
- 108020004414 DNA Proteins 0.000 claims description 14
- 239000013604 expression vector Substances 0.000 claims description 13
- 230000003247 decreasing effect Effects 0.000 claims description 9
- 102000053602 DNA Human genes 0.000 claims description 7
- 241000124008 Mammalia Species 0.000 claims description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 208000004880 Polyuria Diseases 0.000 claims description 4
- 230000035619 diuresis Effects 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- 230000012495 positive regulation of renal sodium excretion Effects 0.000 claims description 4
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000003792 electrolyte Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
- 230000024883 vasodilation Effects 0.000 claims description 3
- 125000001589 carboacyl group Chemical group 0.000 claims description 2
- 239000008221 sterile excipient Substances 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 75
- 102100039339 Atrial natriuretic peptide receptor 1 Human genes 0.000 abstract description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 16
- 108010038640 atrial natriuretic factor receptor A Proteins 0.000 abstract description 10
- 230000002829 reductive effect Effects 0.000 abstract description 6
- 238000011282 treatment Methods 0.000 abstract description 5
- 230000001976 improved effect Effects 0.000 abstract description 4
- 206010014418 Electrolyte imbalance Diseases 0.000 abstract description 3
- 230000001575 pathological effect Effects 0.000 abstract description 3
- 102100034605 Atrial natriuretic peptide receptor 3 Human genes 0.000 abstract description 2
- 101000924488 Homo sapiens Atrial natriuretic peptide receptor 3 Proteins 0.000 abstract description 2
- 238000011321 prophylaxis Methods 0.000 abstract description 2
- 235000001014 amino acid Nutrition 0.000 description 182
- 229940024606 amino acid Drugs 0.000 description 173
- 108020003175 receptors Proteins 0.000 description 53
- 102000005962 receptors Human genes 0.000 description 53
- 108090000623 proteins and genes Proteins 0.000 description 49
- -1 Ala Chemical compound 0.000 description 45
- 102000004196 processed proteins & peptides Human genes 0.000 description 40
- HEPLXMBVMCXTBP-QWRGUYRKSA-N Cys-Phe-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O HEPLXMBVMCXTBP-QWRGUYRKSA-N 0.000 description 36
- NMZXJDSKEGFDLJ-DCAQKATOSA-N Ser-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CCCCN)C(=O)O NMZXJDSKEGFDLJ-DCAQKATOSA-N 0.000 description 36
- 210000004027 cell Anatomy 0.000 description 34
- CNFMPVYIVQUJOO-NHCYSSNCSA-N Met-Val-Gln Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(N)=O CNFMPVYIVQUJOO-NHCYSSNCSA-N 0.000 description 33
- 108010078144 glutaminyl-glycine Proteins 0.000 description 33
- 101710102163 Atrial natriuretic peptide receptor 1 Proteins 0.000 description 31
- VTTSANCGJWLPNC-ZPFDUUQYSA-N Glu-Arg-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VTTSANCGJWLPNC-ZPFDUUQYSA-N 0.000 description 30
- 235000018102 proteins Nutrition 0.000 description 30
- 102000004169 proteins and genes Human genes 0.000 description 30
- 108010056582 methionylglutamic acid Proteins 0.000 description 29
- OTJMMKPMLUNTQT-AVGNSLFASA-N Val-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N OTJMMKPMLUNTQT-AVGNSLFASA-N 0.000 description 27
- 125000003275 alpha amino acid group Chemical group 0.000 description 26
- 108010017391 lysylvaline Proteins 0.000 description 25
- 229920001184 polypeptide Polymers 0.000 description 22
- LGQZOQRDEUIZJY-YUMQZZPRSA-N Gly-Cys-Lys Chemical compound NCCCC[C@H](NC(=O)[C@H](CS)NC(=O)CN)C(O)=O LGQZOQRDEUIZJY-YUMQZZPRSA-N 0.000 description 20
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 20
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 19
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 19
- 150000007523 nucleic acids Chemical class 0.000 description 19
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 17
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 17
- 125000000539 amino acid group Chemical group 0.000 description 16
- 239000002253 acid Substances 0.000 description 15
- 102000037865 fusion proteins Human genes 0.000 description 15
- 108020001507 fusion proteins Proteins 0.000 description 15
- 239000011347 resin Substances 0.000 description 15
- 229920005989 resin Polymers 0.000 description 15
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 14
- 239000002934 diuretic Substances 0.000 description 14
- MTYLORHAQXVQOW-AVGNSLFASA-N Arg-Lys-Met Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(O)=O MTYLORHAQXVQOW-AVGNSLFASA-N 0.000 description 13
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 12
- 230000004071 biological effect Effects 0.000 description 12
- 230000004927 fusion Effects 0.000 description 12
- 150000003839 salts Chemical class 0.000 description 12
- DPNHSNLIULPOBH-GUBZILKMSA-N Arg-Asn-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N DPNHSNLIULPOBH-GUBZILKMSA-N 0.000 description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 description 11
- FRBAHXABMQXSJQ-FXQIFTODSA-N Arg-Ser-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O FRBAHXABMQXSJQ-FXQIFTODSA-N 0.000 description 10
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 10
- 108020001621 Natriuretic Peptide Proteins 0.000 description 10
- 102000004571 Natriuretic peptide Human genes 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 239000000692 natriuretic peptide Substances 0.000 description 10
- 125000006239 protecting group Chemical group 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 238000003556 assay Methods 0.000 description 9
- 239000002131 composite material Substances 0.000 description 9
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 9
- ZXCAQANTQWBICD-DCAQKATOSA-N Cys-Lys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N ZXCAQANTQWBICD-DCAQKATOSA-N 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 230000000638 stimulation Effects 0.000 description 8
- 101500026735 Homo sapiens Brain natriuretic peptide 32 Proteins 0.000 description 7
- 125000001176 L-lysyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 description 7
- NTRAGDHVSGKUSF-AVGNSLFASA-N Leu-Arg-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NTRAGDHVSGKUSF-AVGNSLFASA-N 0.000 description 7
- WOUIMBGNEUWXQG-VKHMYHEASA-N Ser-Gly Chemical compound OC[C@H](N)C(=O)NCC(O)=O WOUIMBGNEUWXQG-VKHMYHEASA-N 0.000 description 7
- JURQXQBJKUHGJS-UHFFFAOYSA-N Ser-Ser-Ser-Ser Chemical compound OCC(N)C(=O)NC(CO)C(=O)NC(CO)C(=O)NC(CO)C(O)=O JURQXQBJKUHGJS-UHFFFAOYSA-N 0.000 description 7
- 230000001882 diuretic effect Effects 0.000 description 7
- 230000001452 natriuretic effect Effects 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 6
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 6
- 125000000010 L-asparaginyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C(=O)N([H])[H] 0.000 description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 6
- 230000009471 action Effects 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 6
- 210000004899 c-terminal region Anatomy 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 239000000543 intermediate Substances 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 229910052708 sodium Inorganic materials 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 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 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 5
- 239000004475 Arginine Substances 0.000 description 5
- HMQDRBKQMLRCCG-GMOBBJLQSA-N Asp-Arg-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HMQDRBKQMLRCCG-GMOBBJLQSA-N 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- 206010007559 Cardiac failure congestive Diseases 0.000 description 5
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 5
- 206010019280 Heart failures Diseases 0.000 description 5
- 206010020772 Hypertension Diseases 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 5
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 5
- 241000700159 Rattus Species 0.000 description 5
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 5
- 125000003118 aryl group Chemical group 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 238000002703 mutagenesis Methods 0.000 description 5
- 231100000350 mutagenesis Toxicity 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- 238000010647 peptide synthesis reaction Methods 0.000 description 5
- 239000008194 pharmaceutical composition Substances 0.000 description 5
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 5
- 230000002883 vasorelaxation effect Effects 0.000 description 5
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- SVHRPCMZTWZROG-DCAQKATOSA-N Arg-Cys-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCN=C(N)N)N SVHRPCMZTWZROG-DCAQKATOSA-N 0.000 description 4
- 241000282465 Canis Species 0.000 description 4
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- SSHIXEILTLPAQT-WHFBIAKZSA-N Gln-Asp Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(O)=O SSHIXEILTLPAQT-WHFBIAKZSA-N 0.000 description 4
- 108010078321 Guanylate Cyclase Proteins 0.000 description 4
- 102000014469 Guanylate cyclase Human genes 0.000 description 4
- MDCTVRUPVLZSPG-BQBZGAKWSA-N His-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CNC=N1 MDCTVRUPVLZSPG-BQBZGAKWSA-N 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 4
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 4
- QJUWBDPGGYVRHY-YUMQZZPRSA-N Leu-Gly-Cys Chemical compound CC(C)C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N QJUWBDPGGYVRHY-YUMQZZPRSA-N 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- ZYTPOUNUXRBYGW-YUMQZZPRSA-N Met-Met Chemical compound CSCC[C@H]([NH3+])C(=O)N[C@H](C([O-])=O)CCSC ZYTPOUNUXRBYGW-YUMQZZPRSA-N 0.000 description 4
- YPIGGYHFMKJNKV-UHFFFAOYSA-N N-ethylglycine Chemical compound CC[NH2+]CC([O-])=O YPIGGYHFMKJNKV-UHFFFAOYSA-N 0.000 description 4
- 108010065338 N-ethylglycine Proteins 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- IBIDRSSEHFLGSD-YUMQZZPRSA-N Val-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-YUMQZZPRSA-N 0.000 description 4
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 4
- 230000036772 blood pressure Effects 0.000 description 4
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 4
- 125000004432 carbon atom Chemical group C* 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 108010050848 glycylleucine Proteins 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 229910000040 hydrogen fluoride Inorganic materials 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 108010000761 leucylarginine Proteins 0.000 description 4
- 125000005647 linker group Chemical group 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 229930182817 methionine Natural products 0.000 description 4
- 108010085203 methionylmethionine Proteins 0.000 description 4
- 231100000252 nontoxic Toxicity 0.000 description 4
- 230000003000 nontoxic effect Effects 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 102200067655 rs80358961 Human genes 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 4
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 3
- PECYZEOJVXMISF-UHFFFAOYSA-N 3-aminoalanine Chemical compound [NH3+]CC(N)C([O-])=O PECYZEOJVXMISF-UHFFFAOYSA-N 0.000 description 3
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 description 3
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 241000024188 Andala Species 0.000 description 3
- NVCIXQYNWYTLDO-IHRRRGAJSA-N Arg-His-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCCN=C(N)N)N NVCIXQYNWYTLDO-IHRRRGAJSA-N 0.000 description 3
- CRCCTGPNZUCAHE-DCAQKATOSA-N Arg-His-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CN=CN1 CRCCTGPNZUCAHE-DCAQKATOSA-N 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- OCQUNKSFDYDXBG-QXEWZRGKSA-N Gly-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OCQUNKSFDYDXBG-QXEWZRGKSA-N 0.000 description 3
- MXXXVOYFNVJHMA-IUCAKERBSA-N Gly-Arg-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)CN MXXXVOYFNVJHMA-IUCAKERBSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- LBRCLQMZAHRTLV-ZKWXMUAHSA-N Ile-Gly-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LBRCLQMZAHRTLV-ZKWXMUAHSA-N 0.000 description 3
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- SCIFESDRCALIIM-VIFPVBQESA-N N-methyl-L-phenylalanine Chemical compound C[NH2+][C@H](C([O-])=O)CC1=CC=CC=C1 SCIFESDRCALIIM-VIFPVBQESA-N 0.000 description 3
- 206010029164 Nephrotic syndrome Diseases 0.000 description 3
- 102000003729 Neprilysin Human genes 0.000 description 3
- 108090000028 Neprilysin Proteins 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 241000282320 Panthera leo Species 0.000 description 3
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 3
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 description 3
- ZPHBZEQOLSRPAK-UHFFFAOYSA-N Phosphoramidon Natural products C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O ZPHBZEQOLSRPAK-UHFFFAOYSA-N 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 3
- JJUNLJTUIKFPRF-BPUTZDHNSA-N Ser-Met-Trp Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CO)N JJUNLJTUIKFPRF-BPUTZDHNSA-N 0.000 description 3
- JAWGSPUJAXYXJA-IHRRRGAJSA-N Ser-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=CC=C1 JAWGSPUJAXYXJA-IHRRRGAJSA-N 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- 229960002478 aldosterone Drugs 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 239000000908 ammonium hydroxide Substances 0.000 description 3
- 239000012736 aqueous medium Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000012148 binding buffer Substances 0.000 description 3
- 230000000975 bioactive effect Effects 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 208000019425 cirrhosis of liver Diseases 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000009833 condensation Methods 0.000 description 3
- 230000005494 condensation Effects 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000024924 glomerular filtration Effects 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 3
- 230000010807 negative regulation of binding Effects 0.000 description 3
- BWSDNRQVTFZQQD-AYVHNPTNSA-N phosphoramidon Chemical compound O([P@@](O)(=O)N[C@H](CC(C)C)C(=O)N[C@H](CC=1[C]2C=CC=CC2=NC=1)C(O)=O)[C@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@@H]1O BWSDNRQVTFZQQD-AYVHNPTNSA-N 0.000 description 3
- 108010072906 phosphoramidon Proteins 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 102220082839 rs149700042 Human genes 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 108010071207 serylmethionine Proteins 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 230000000087 stabilizing effect Effects 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 2
- OYIFNHCXNCRBQI-UHFFFAOYSA-N 2-aminoadipic acid Chemical compound OC(=O)C(N)CCCC(O)=O OYIFNHCXNCRBQI-UHFFFAOYSA-N 0.000 description 2
- RDFMDVXONNIGBC-UHFFFAOYSA-N 2-aminoheptanoic acid Chemical compound CCCCCC(N)C(O)=O RDFMDVXONNIGBC-UHFFFAOYSA-N 0.000 description 2
- 125000006181 4-methyl benzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])C([H])([H])* 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- JKRPBTQDPJSQIT-RCWTZXSCSA-N Arg-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O JKRPBTQDPJSQIT-RCWTZXSCSA-N 0.000 description 2
- CBHVAFXKOYAHOY-NHCYSSNCSA-N Asn-Val-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O CBHVAFXKOYAHOY-NHCYSSNCSA-N 0.000 description 2
- 102100039341 Atrial natriuretic peptide receptor 2 Human genes 0.000 description 2
- 101710102159 Atrial natriuretic peptide receptor 2 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 150000008574 D-amino acids Chemical class 0.000 description 2
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical compound C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 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 2
- GZUKEVBTYNNUQF-WDSKDSINSA-N Gly-Ala-Gln Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GZUKEVBTYNNUQF-WDSKDSINSA-N 0.000 description 2
- YDWZGVCXMVLDQH-WHFBIAKZSA-N Gly-Cys-Asn Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CC(N)=O YDWZGVCXMVLDQH-WHFBIAKZSA-N 0.000 description 2
- XPJBQTCXPJNIFE-ZETCQYMHSA-N Gly-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)CN XPJBQTCXPJNIFE-ZETCQYMHSA-N 0.000 description 2
- CCBIBMKQNXHNIN-ZETCQYMHSA-N Gly-Leu-Gly Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CCBIBMKQNXHNIN-ZETCQYMHSA-N 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 229910004373 HOAc Inorganic materials 0.000 description 2
- UMYZBHKAVTXWIW-GMOBBJLQSA-N Ile-Asp-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N UMYZBHKAVTXWIW-GMOBBJLQSA-N 0.000 description 2
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 2
- JUQLUIFNNFIIKC-YFKPBYRVSA-N L-2-aminopimelic acid Chemical compound OC(=O)[C@@H](N)CCCCC(O)=O JUQLUIFNNFIIKC-YFKPBYRVSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- SNDPXSYFESPGGJ-UHFFFAOYSA-N L-norVal-OH Natural products CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 2
- YKNBJXOJTURHCU-DCAQKATOSA-N Leu-Asp-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YKNBJXOJTURHCU-DCAQKATOSA-N 0.000 description 2
- RGUXWMDNCPMQFB-YUMQZZPRSA-N Leu-Ser-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RGUXWMDNCPMQFB-YUMQZZPRSA-N 0.000 description 2
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 2
- 208000019693 Lung disease Diseases 0.000 description 2
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 2
- SCIFESDRCALIIM-UHFFFAOYSA-N N-Me-Phenylalanine Natural products CNC(C(O)=O)CC1=CC=CC=C1 SCIFESDRCALIIM-UHFFFAOYSA-N 0.000 description 2
- PYUSHNKNPOHWEZ-YFKPBYRVSA-N N-formyl-L-methionine Chemical group CSCC[C@@H](C(O)=O)NC=O PYUSHNKNPOHWEZ-YFKPBYRVSA-N 0.000 description 2
- KSPIYJQBLVDRRI-UHFFFAOYSA-N N-methylisoleucine Chemical compound CCC(C)C(NC)C(O)=O KSPIYJQBLVDRRI-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 208000001647 Renal Insufficiency Diseases 0.000 description 2
- 108090000783 Renin Proteins 0.000 description 2
- 102100028255 Renin Human genes 0.000 description 2
- 108010077895 Sarcosine Proteins 0.000 description 2
- XSYJDGIDKRNWFX-SRVKXCTJSA-N Ser-Cys-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XSYJDGIDKRNWFX-SRVKXCTJSA-N 0.000 description 2
- MHVXPTAMDHLTHB-IHPCNDPISA-N Ser-Phe-Trp Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CC=CC=C1 MHVXPTAMDHLTHB-IHPCNDPISA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- AJNUKMZFHXUBMK-GUBZILKMSA-N Val-Ser-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N AJNUKMZFHXUBMK-GUBZILKMSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 230000029936 alkylation Effects 0.000 description 2
- 238000005804 alkylation reaction Methods 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 108010082685 antiarrhythmic peptide Proteins 0.000 description 2
- 108010043240 arginyl-leucyl-glycine Proteins 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 235000010633 broth Nutrition 0.000 description 2
- 229960001948 caffeine Drugs 0.000 description 2
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 2
- 210000002837 heart atrium Anatomy 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 235000011167 hydrochloric acid Nutrition 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000001077 hypotensive effect Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 150000007529 inorganic bases Chemical class 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 201000006370 kidney failure Diseases 0.000 description 2
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 2
- 108010052968 leupeptin Proteins 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 239000002833 natriuretic agent Substances 0.000 description 2
- 102000027424 natriuretic peptide receptors Human genes 0.000 description 2
- 108091008599 natriuretic peptide receptors Proteins 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 108010091212 pepstatin Proteins 0.000 description 2
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 235000011007 phosphoric acid Nutrition 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 201000011461 pre-eclampsia Diseases 0.000 description 2
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 2
- 229960004919 procaine Drugs 0.000 description 2
- 238000001525 receptor binding assay Methods 0.000 description 2
- 102220088072 rs771342044 Human genes 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 210000002460 smooth muscle Anatomy 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- YAPQBXQYLJRXSA-UHFFFAOYSA-N theobromine Chemical compound CN1C(=O)NC(=O)C2=C1N=CN2C YAPQBXQYLJRXSA-UHFFFAOYSA-N 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 2
- 230000000304 vasodilatating effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 description 1
- GIDNRVZDUMNLRK-GFCCVEGCSA-N (2S)-2-amino-2,3-dimethyl-3-phenylbutanoic acid Chemical compound CC([C@](N)(C(=O)O)C)(C1=CC=CC=C1)C GIDNRVZDUMNLRK-GFCCVEGCSA-N 0.000 description 1
- WOVRTBFSWOVRST-ROUUACIJSA-N (2s)-2-[[(2s)-1-(2-carboxyethylamino)-1-oxo-3-phenylpropan-2-yl]amino]-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)NCCC(=O)O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 WOVRTBFSWOVRST-ROUUACIJSA-N 0.000 description 1
- QAQRHURCJWHRJU-FHWLQOOXSA-N (2s)-2-[[(2s)-1-[[(2s)-2-carboxy-2-hydroxyethyl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-4-phenylbutanoic acid Chemical compound C([C@@H](C(=O)NC[C@H](O)C(O)=O)N[C@@H](CCC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 QAQRHURCJWHRJU-FHWLQOOXSA-N 0.000 description 1
- XPRCPVGCTGELMN-QMMMGPOBSA-N (2s)-2-amino-3-(4-carbamimidoylphenyl)propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=C(C(N)=N)C=C1 XPRCPVGCTGELMN-QMMMGPOBSA-N 0.000 description 1
- CNBUSIJNWNXLQQ-NSHDSACASA-N (2s)-3-(4-hydroxyphenyl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CNBUSIJNWNXLQQ-NSHDSACASA-N 0.000 description 1
- ONOURAAVVKGJNM-SCZZXKLOSA-N (2s,3r)-2-azaniumyl-3-phenylmethoxybutanoate Chemical compound [O-]C(=O)[C@@H]([NH3+])[C@@H](C)OCC1=CC=CC=C1 ONOURAAVVKGJNM-SCZZXKLOSA-N 0.000 description 1
- CUKWUWBLQQDQAC-VEQWQPCFSA-N (3s)-3-amino-4-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s,3s)-1-[[(2s)-1-[(2s)-2-[[(1s)-1-carboxyethyl]carbamoyl]pyrrolidin-1-yl]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-ox Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 CUKWUWBLQQDQAC-VEQWQPCFSA-N 0.000 description 1
- YPGMOWHXEQDBBV-QWWZWVQMSA-N (4S,5S)-1,2-dithiane-4,5-diol Chemical compound O[C@@H]1CSSC[C@H]1O YPGMOWHXEQDBBV-QWWZWVQMSA-N 0.000 description 1
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- LKQWZAKMZIAUCH-DUFHHEBNSA-N 121051-68-1 Chemical compound C([C@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)CNC(=O)[C@H](CS)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CO)[C@@H](C)CC)[C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 LKQWZAKMZIAUCH-DUFHHEBNSA-N 0.000 description 1
- YQTCQNIPQMJNTI-UHFFFAOYSA-N 2,2-dimethylpropan-1-one Chemical group CC(C)(C)[C]=O YQTCQNIPQMJNTI-UHFFFAOYSA-N 0.000 description 1
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-UHFFFAOYSA-N 0.000 description 1
- LXUNZSDDXMPKLP-UHFFFAOYSA-N 2-Methylbenzenethiol Chemical compound CC1=CC=CC=C1S LXUNZSDDXMPKLP-UHFFFAOYSA-N 0.000 description 1
- YQAXFVHNHSPUPO-RNJOBUHISA-N 2-[[(2s)-2-[[2-[[(2s,4r)-1-[(2s)-1-(2-aminoacetyl)pyrrolidine-2-carbonyl]-4-hydroxypyrrolidine-2-carbonyl]amino]acetyl]amino]propanoyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1C[C@@H](O)CN1C(=O)[C@H]1N(C(=O)CN)CCC1 YQAXFVHNHSPUPO-RNJOBUHISA-N 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 229940013085 2-diethylaminoethanol Drugs 0.000 description 1
- 125000004493 2-methylbut-1-yl group Chemical group CC(C*)CC 0.000 description 1
- 125000005916 2-methylpentyl group Chemical group 0.000 description 1
- QTWJRLJHJPIABL-UHFFFAOYSA-N 2-methylphenol;3-methylphenol;4-methylphenol Chemical compound CC1=CC=C(O)C=C1.CC1=CC=CC(O)=C1.CC1=CC=CC=C1O QTWJRLJHJPIABL-UHFFFAOYSA-N 0.000 description 1
- 125000000094 2-phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 1
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 description 1
- WCPTXJJVVDAEMW-XVFCMESISA-N 3',5'-cyclic CMP Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H]2OP(O)(=O)OC[C@H]2O1 WCPTXJJVVDAEMW-XVFCMESISA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N 5-hydroxylysine Chemical group NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- 102220587867 Acidic mammalian chitinase_S22T_mutation Human genes 0.000 description 1
- 101100013118 Agrobacterium fabrum (strain C58 / ATCC 33970) fliN gene Proteins 0.000 description 1
- SVBXIUDNTRTKHE-CIUDSAMLSA-N Ala-Arg-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O SVBXIUDNTRTKHE-CIUDSAMLSA-N 0.000 description 1
- VHVVPYOJIIQCKS-QEJZJMRPSA-N Ala-Leu-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 VHVVPYOJIIQCKS-QEJZJMRPSA-N 0.000 description 1
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 101800000733 Angiotensin-2 Proteins 0.000 description 1
- 102400000345 Angiotensin-2 Human genes 0.000 description 1
- GXCSUJQOECMKPV-CIUDSAMLSA-N Arg-Ala-Gln Chemical compound C[C@H](NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O GXCSUJQOECMKPV-CIUDSAMLSA-N 0.000 description 1
- OMLWNBVRVJYMBQ-YUMQZZPRSA-N Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OMLWNBVRVJYMBQ-YUMQZZPRSA-N 0.000 description 1
- JGDGLDNAQJJGJI-AVGNSLFASA-N Arg-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N JGDGLDNAQJJGJI-AVGNSLFASA-N 0.000 description 1
- YUIGJDNAGKJLDO-JYJNAYRXSA-N Arg-Arg-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YUIGJDNAGKJLDO-JYJNAYRXSA-N 0.000 description 1
- IIABBYGHLYWVOS-FXQIFTODSA-N Arg-Asn-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O IIABBYGHLYWVOS-FXQIFTODSA-N 0.000 description 1
- ZTKHZAXGTFXUDD-VEVYYDQMSA-N Arg-Asn-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZTKHZAXGTFXUDD-VEVYYDQMSA-N 0.000 description 1
- NAARDJBSSPUGCF-FXQIFTODSA-N Arg-Cys-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)CN=C(N)N NAARDJBSSPUGCF-FXQIFTODSA-N 0.000 description 1
- PTVGLOCPAVYPFG-CIUDSAMLSA-N Arg-Gln-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PTVGLOCPAVYPFG-CIUDSAMLSA-N 0.000 description 1
- BJNUAWGXPSHQMJ-DCAQKATOSA-N Arg-Gln-Met Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O BJNUAWGXPSHQMJ-DCAQKATOSA-N 0.000 description 1
- SYAUZLVLXCDRSH-IUCAKERBSA-N Arg-Gly-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N SYAUZLVLXCDRSH-IUCAKERBSA-N 0.000 description 1
- WVNFNPGXYADPPO-BQBZGAKWSA-N Arg-Gly-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O WVNFNPGXYADPPO-BQBZGAKWSA-N 0.000 description 1
- BNODVYXZAAXSHW-IUCAKERBSA-N Arg-His Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 BNODVYXZAAXSHW-IUCAKERBSA-N 0.000 description 1
- MSILNNHVVMMTHZ-UWVGGRQHSA-N Arg-His-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CN=CN1 MSILNNHVVMMTHZ-UWVGGRQHSA-N 0.000 description 1
- YQGZIRIYGHNSQO-ZPFDUUQYSA-N Arg-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N YQGZIRIYGHNSQO-ZPFDUUQYSA-N 0.000 description 1
- RIQBRKVTFBWEDY-RHYQMDGZSA-N Arg-Lys-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RIQBRKVTFBWEDY-RHYQMDGZSA-N 0.000 description 1
- QBQVKUNBCAFXSV-ULQDDVLXSA-N Arg-Lys-Tyr Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QBQVKUNBCAFXSV-ULQDDVLXSA-N 0.000 description 1
- VRTWYUYCJGNFES-CIUDSAMLSA-N Arg-Ser-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O VRTWYUYCJGNFES-CIUDSAMLSA-N 0.000 description 1
- DDBMKOCQWNFDBH-RHYQMDGZSA-N Arg-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O DDBMKOCQWNFDBH-RHYQMDGZSA-N 0.000 description 1
- XMGVWQWEWWULNS-BPUTZDHNSA-N Arg-Trp-Ser Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N XMGVWQWEWWULNS-BPUTZDHNSA-N 0.000 description 1
- LLQIAIUAKGNOSE-NHCYSSNCSA-N Arg-Val-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N LLQIAIUAKGNOSE-NHCYSSNCSA-N 0.000 description 1
- XYOVHPDDWCEUDY-CIUDSAMLSA-N Asn-Ala-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O XYOVHPDDWCEUDY-CIUDSAMLSA-N 0.000 description 1
- PTNFNTOBUDWHNZ-GUBZILKMSA-N Asn-Arg-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O PTNFNTOBUDWHNZ-GUBZILKMSA-N 0.000 description 1
- HCAUEJAQCXVQQM-ACZMJKKPSA-N Asn-Glu-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HCAUEJAQCXVQQM-ACZMJKKPSA-N 0.000 description 1
- OWUCNXMFJRFOFI-BQBZGAKWSA-N Asn-Gly-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O OWUCNXMFJRFOFI-BQBZGAKWSA-N 0.000 description 1
- KHCNTVRVAYCPQE-CIUDSAMLSA-N Asn-Lys-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O KHCNTVRVAYCPQE-CIUDSAMLSA-N 0.000 description 1
- QCVXMEHGFUMKCO-YUMQZZPRSA-N Asp-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O QCVXMEHGFUMKCO-YUMQZZPRSA-N 0.000 description 1
- KGHLGJAXYSVNJP-WHFBIAKZSA-N Asp-Ser-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O KGHLGJAXYSVNJP-WHFBIAKZSA-N 0.000 description 1
- 244000186140 Asperula odorata Species 0.000 description 1
- 206010003658 Atrial Fibrillation Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 208000006017 Cardiac Tamponade Diseases 0.000 description 1
- 208000031229 Cardiomyopathies Diseases 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 208000002330 Congenital Heart Defects Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 235000017274 Diospyros sandwicensis Nutrition 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 101100234002 Drosophila melanogaster Shal gene Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101710194146 Ecotin Proteins 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 102000002045 Endothelin Human genes 0.000 description 1
- 108050009340 Endothelin Proteins 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001522878 Escherichia coli B Species 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 208000007530 Essential hypertension Diseases 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 240000002989 Euphorbia neriifolia Species 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 102000008857 Ferritin Human genes 0.000 description 1
- 108050000784 Ferritin Proteins 0.000 description 1
- 238000008416 Ferritin Methods 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 108010058643 Fungal Proteins Proteins 0.000 description 1
- 235000008526 Galium odoratum Nutrition 0.000 description 1
- XOKGKOQWADCLFQ-GARJFASQSA-N Gln-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XOKGKOQWADCLFQ-GARJFASQSA-N 0.000 description 1
- RMOCFPBLHAOTDU-ACZMJKKPSA-N Gln-Asn-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RMOCFPBLHAOTDU-ACZMJKKPSA-N 0.000 description 1
- KKCUFHUTMKQQCF-SRVKXCTJSA-N Glu-Arg-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O KKCUFHUTMKQQCF-SRVKXCTJSA-N 0.000 description 1
- NUSWUSKZRCGFEX-FXQIFTODSA-N Glu-Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O NUSWUSKZRCGFEX-FXQIFTODSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- JXYMPBCYRKWJEE-BQBZGAKWSA-N Gly-Arg-Ala Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O JXYMPBCYRKWJEE-BQBZGAKWSA-N 0.000 description 1
- AQLHORCVPGXDJW-IUCAKERBSA-N Gly-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)CN AQLHORCVPGXDJW-IUCAKERBSA-N 0.000 description 1
- FSPVILZGHUJOHS-QWRGUYRKSA-N Gly-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CNC=N1 FSPVILZGHUJOHS-QWRGUYRKSA-N 0.000 description 1
- MVORZMQFXBLMHM-QWRGUYRKSA-N Gly-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 MVORZMQFXBLMHM-QWRGUYRKSA-N 0.000 description 1
- LBDXVCBAJJNJNN-WHFBIAKZSA-N Gly-Ser-Cys Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(O)=O LBDXVCBAJJNJNN-WHFBIAKZSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 101100082540 Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) pcp gene Proteins 0.000 description 1
- ORERHHPZDDEMSC-VGDYDELISA-N His-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N ORERHHPZDDEMSC-VGDYDELISA-N 0.000 description 1
- VIJMRAIWYWRXSR-CIUDSAMLSA-N His-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 VIJMRAIWYWRXSR-CIUDSAMLSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000961044 Homo sapiens Atrial natriuretic peptide receptor 1 Proteins 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 108700039609 IRW peptide Proteins 0.000 description 1
- TZCGZYWNIDZZMR-UHFFFAOYSA-N Ile-Arg-Ala Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(C)C(O)=O)CCCN=C(N)N TZCGZYWNIDZZMR-UHFFFAOYSA-N 0.000 description 1
- NZOCIWKZUVUNDW-ZKWXMUAHSA-N Ile-Gly-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O NZOCIWKZUVUNDW-ZKWXMUAHSA-N 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- AGPKZVBTJJNPAG-UHNVWZDZSA-N L-allo-Isoleucine Chemical compound CC[C@@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-UHNVWZDZSA-N 0.000 description 1
- 125000000570 L-alpha-aspartyl group Chemical group [H]OC(=O)C([H])([H])[C@]([H])(N([H])[H])C(*)=O 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 125000002059 L-arginyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(=N[H])N([H])[H] 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 1
- QEFRNWWLZKMPFJ-ZXPFJRLXSA-N L-methionine (R)-S-oxide Chemical compound C[S@@](=O)CC[C@H]([NH3+])C([O-])=O QEFRNWWLZKMPFJ-ZXPFJRLXSA-N 0.000 description 1
- QEFRNWWLZKMPFJ-UHFFFAOYSA-N L-methionine sulphoxide Natural products CS(=O)CCC(N)C(O)=O QEFRNWWLZKMPFJ-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- 125000000769 L-threonyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])[C@](O[H])(C([H])([H])[H])[H] 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241000282838 Lama Species 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- DSFYPIUSAMSERP-IHRRRGAJSA-N Leu-Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DSFYPIUSAMSERP-IHRRRGAJSA-N 0.000 description 1
- IDGRADDMTTWOQC-WDSOQIARSA-N Leu-Trp-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IDGRADDMTTWOQC-WDSOQIARSA-N 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- AIRZWUMAHCDDHR-KKUMJFAQSA-N Lys-Leu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O AIRZWUMAHCDDHR-KKUMJFAQSA-N 0.000 description 1
- URGPVYGVWLIRGT-DCAQKATOSA-N Lys-Met-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O URGPVYGVWLIRGT-DCAQKATOSA-N 0.000 description 1
- SBQDRNOLGSYHQA-YUMQZZPRSA-N Lys-Ser-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SBQDRNOLGSYHQA-YUMQZZPRSA-N 0.000 description 1
- ZOKVLMBYDSIDKG-CSMHCCOUSA-N Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN ZOKVLMBYDSIDKG-CSMHCCOUSA-N 0.000 description 1
- PSVAVKGDUAKZKU-BZSNNMDCSA-N Lys-Tyr-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CCCCN)N)O PSVAVKGDUAKZKU-BZSNNMDCSA-N 0.000 description 1
- YQAIUOWPSUOINN-IUCAKERBSA-N Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN YQAIUOWPSUOINN-IUCAKERBSA-N 0.000 description 1
- DRRXXZBXDMLGFC-IHRRRGAJSA-N Lys-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN DRRXXZBXDMLGFC-IHRRRGAJSA-N 0.000 description 1
- RIPJMCFGQHGHNP-RHYQMDGZSA-N Lys-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCCCN)N)O RIPJMCFGQHGHNP-RHYQMDGZSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- UASDAHIAHBRZQV-YUMQZZPRSA-N Met-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N UASDAHIAHBRZQV-YUMQZZPRSA-N 0.000 description 1
- YHBHDYYHOUAKLR-AVGNSLFASA-N Met-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N YHBHDYYHOUAKLR-AVGNSLFASA-N 0.000 description 1
- LNXGEYIEEUZGGH-JYJNAYRXSA-N Met-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CCSC)CC1=CC=CC=C1 LNXGEYIEEUZGGH-JYJNAYRXSA-N 0.000 description 1
- UZBQXELAFPCGRV-SZMVWBNQSA-N Met-Trp-Arg Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UZBQXELAFPCGRV-SZMVWBNQSA-N 0.000 description 1
- VOAKKHOIAFKOQZ-JYJNAYRXSA-N Met-Tyr-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CCSC)CC1=CC=C(O)C=C1 VOAKKHOIAFKOQZ-JYJNAYRXSA-N 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 1
- HTLZVHNRZJPSMI-UHFFFAOYSA-N N-ethylpiperidine Chemical compound CCN1CCCCC1 HTLZVHNRZJPSMI-UHFFFAOYSA-N 0.000 description 1
- NSTPXGARCQOSAU-VIFPVBQESA-N N-formyl-L-phenylalanine Chemical compound O=CN[C@H](C(=O)O)CC1=CC=CC=C1 NSTPXGARCQOSAU-VIFPVBQESA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- RFEXGCASCQGGHZ-STQMWFEESA-N Phe-Gly-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O RFEXGCASCQGGHZ-STQMWFEESA-N 0.000 description 1
- KXUZHWXENMYOHC-QEJZJMRPSA-N Phe-Leu-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O KXUZHWXENMYOHC-QEJZJMRPSA-N 0.000 description 1
- DMEYUTSDVRCWRS-ULQDDVLXSA-N Phe-Lys-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 DMEYUTSDVRCWRS-ULQDDVLXSA-N 0.000 description 1
- KCIKTPHTEYBXMG-BVSLBCMMSA-N Phe-Trp-Arg Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCIKTPHTEYBXMG-BVSLBCMMSA-N 0.000 description 1
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 1
- 208000010067 Pituitary ACTH Hypersecretion Diseases 0.000 description 1
- 208000020627 Pituitary-dependent Cushing syndrome Diseases 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010036618 Premenstrual syndrome Diseases 0.000 description 1
- RVQDZELMXZRSSI-IUCAKERBSA-N Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 RVQDZELMXZRSSI-IUCAKERBSA-N 0.000 description 1
- WFIVLLFYUZZWOD-RHYQMDGZSA-N Pro-Lys-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WFIVLLFYUZZWOD-RHYQMDGZSA-N 0.000 description 1
- SVXXJYJCRNKDDE-AVGNSLFASA-N Pro-Pro-His Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H]1NCCC1)C1=CN=CN1 SVXXJYJCRNKDDE-AVGNSLFASA-N 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 1
- 201000003099 Renovascular Hypertension Diseases 0.000 description 1
- 108010035990 SC 46542 Proteins 0.000 description 1
- 108010085867 SCH 32615 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 1
- 108010065922 Sch 39370 Proteins 0.000 description 1
- 201000004239 Secondary hypertension Diseases 0.000 description 1
- IDQFQFVEWMWRQQ-DLOVCJGASA-N Ser-Ala-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IDQFQFVEWMWRQQ-DLOVCJGASA-N 0.000 description 1
- CDVFZMOFNJPUDD-ACZMJKKPSA-N Ser-Gln-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CDVFZMOFNJPUDD-ACZMJKKPSA-N 0.000 description 1
- ULVMNZOKDBHKKI-ACZMJKKPSA-N Ser-Gln-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ULVMNZOKDBHKKI-ACZMJKKPSA-N 0.000 description 1
- XWCYBVBLJRWOFR-WDSKDSINSA-N Ser-Gln-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O XWCYBVBLJRWOFR-WDSKDSINSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- OVQZAFXWIWNYKA-GUBZILKMSA-N Ser-Pro-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CO)N OVQZAFXWIWNYKA-GUBZILKMSA-N 0.000 description 1
- NADLKBTYNKUJEP-KATARQTJSA-N Ser-Thr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O NADLKBTYNKUJEP-KATARQTJSA-N 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 244000166071 Shorea robusta Species 0.000 description 1
- 235000015076 Shorea robusta Nutrition 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108090000787 Subtilisin Proteins 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 108010036928 Thiorphan Proteins 0.000 description 1
- LVHHEVGYAZGXDE-KDXUFGMBSA-N Thr-Ala-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(=O)O)N)O LVHHEVGYAZGXDE-KDXUFGMBSA-N 0.000 description 1
- QQWNRERCGGZOKG-WEDXCCLWSA-N Thr-Gly-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O QQWNRERCGGZOKG-WEDXCCLWSA-N 0.000 description 1
- OHDXOXIZXSFCDN-RCWTZXSCSA-N Thr-Met-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O OHDXOXIZXSFCDN-RCWTZXSCSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- GDPDVIBHJDFRFD-RNXOBYDBSA-N Trp-Tyr-Tyr Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GDPDVIBHJDFRFD-RNXOBYDBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- ZQOOYCZQENFIMC-STQMWFEESA-N Tyr-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)C1=CC=C(O)C=C1 ZQOOYCZQENFIMC-STQMWFEESA-N 0.000 description 1
- 108010001957 Ularitide Proteins 0.000 description 1
- PAPWZOJOLKZEFR-AVGNSLFASA-N Val-Arg-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N PAPWZOJOLKZEFR-AVGNSLFASA-N 0.000 description 1
- XEYUMGGWQCIWAR-XVKPBYJWSA-N Val-Gln-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)NCC(=O)O)N XEYUMGGWQCIWAR-XVKPBYJWSA-N 0.000 description 1
- PYXQBKJPHNCTNW-CYDGBPFRSA-N Val-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](C(C)C)N PYXQBKJPHNCTNW-CYDGBPFRSA-N 0.000 description 1
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 1
- 108010004977 Vasopressins Proteins 0.000 description 1
- 102000002852 Vasopressins Human genes 0.000 description 1
- IXKSXJFAGXLQOQ-XISFHERQSA-N WHWLQLKPGQPMY Chemical compound C([C@@H](C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CNC=N1 IXKSXJFAGXLQOQ-XISFHERQSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000009102 absorption Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 125000005076 adamantyloxycarbonyl group Chemical group C12(CC3CC(CC(C1)C3)C2)OC(=O)* 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000003470 adrenal cortex hormone Substances 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 238000012867 alanine scanning Methods 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 238000011166 aliquoting Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 229950006323 angiotensin ii Drugs 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 229960003121 arginine Drugs 0.000 description 1
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 125000003710 aryl alkyl group Chemical group 0.000 description 1
- 108010038633 aspartylglutamate Proteins 0.000 description 1
- 206010003668 atrial tachycardia Diseases 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- RROBIDXNTUAHFW-UHFFFAOYSA-N benzotriazol-1-yloxy-tris(dimethylamino)phosphanium Chemical compound C1=CC=C2N(O[P+](N(C)C)(N(C)C)N(C)C)N=NC2=C1 RROBIDXNTUAHFW-UHFFFAOYSA-N 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 125000004063 butyryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000012219 cassette mutagenesis Methods 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 208000028831 congenital heart disease Diseases 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 229930003836 cresol Natural products 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- WZHCOOQXZCIUNC-UHFFFAOYSA-N cyclandelate Chemical compound C1C(C)(C)CC(C)CC1OC(=O)C(O)C1=CC=CC=C1 WZHCOOQXZCIUNC-UHFFFAOYSA-N 0.000 description 1
- GCFAUZGWPDYAJN-UHFFFAOYSA-N cyclohexyl 3-phenylprop-2-enoate Chemical compound C=1C=CC=CC=1C=CC(=O)OC1CCCCC1 GCFAUZGWPDYAJN-UHFFFAOYSA-N 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 125000005982 diphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- ZUBDGKVDJUIMQQ-UBFCDGJISA-N endothelin-1 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)NC(=O)[C@H]1NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@@H](CC=2C=CC(O)=CC=2)NC(=O)[C@H](C(C)C)NC(=O)[C@H]2CSSC[C@@H](C(N[C@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N2)=O)NC(=O)[C@@H](CO)NC(=O)[C@H](N)CSSC1)C1=CNC=N1 ZUBDGKVDJUIMQQ-UBFCDGJISA-N 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 229940012017 ethylenediamine Drugs 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 235000011087 fumaric acid Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 1
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010038983 glycyl-histidyl-lysine Proteins 0.000 description 1
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 238000005534 hematocrit Methods 0.000 description 1
- 230000000004 hemodynamic effect Effects 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 239000003688 hormone derivative Substances 0.000 description 1
- XGIHQYAWBCFNPY-AZOCGYLKSA-N hydrabamine Chemical compound C([C@@H]12)CC3=CC(C(C)C)=CC=C3[C@@]2(C)CCC[C@@]1(C)CNCCNC[C@@]1(C)[C@@H]2CCC3=CC(C(C)C)=CC=C3[C@@]2(C)CCC1 XGIHQYAWBCFNPY-AZOCGYLKSA-N 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- QJHBJHUKURJDLG-UHFFFAOYSA-N hydroxy-L-lysine Natural products NCCCCC(NO)C(O)=O QJHBJHUKURJDLG-UHFFFAOYSA-N 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 101150074251 lpp gene Proteins 0.000 description 1
- 229960003646 lysine Drugs 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 229960002510 mandelic acid Drugs 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229940098779 methanesulfonic acid Drugs 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- IOPLHGOSNCJOOO-UHFFFAOYSA-N methyl 3,4-diaminobenzoate Chemical compound COC(=O)C1=CC=C(N)C(N)=C1 IOPLHGOSNCJOOO-UHFFFAOYSA-N 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 208000009928 nephrosis Diseases 0.000 description 1
- 231100001027 nephrosis Toxicity 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 208000008494 pericarditis Diseases 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 125000001792 phenanthrenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3C=CC12)* 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 108010018625 phenylalanylarginine Proteins 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 125000001557 phthalyl group Chemical group C(=O)(O)C1=C(C(=O)*)C=CC=C1 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 208000007232 portal hypertension Diseases 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 125000001501 propionyl group Chemical group O=C([*])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 208000002815 pulmonary hypertension Diseases 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 239000006176 redox buffer Substances 0.000 description 1
- 230000004648 relaxation of smooth muscle Effects 0.000 description 1
- 230000033904 relaxation of vascular smooth muscle Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 208000004124 rheumatic heart disease Diseases 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 102220040657 rs199773433 Human genes 0.000 description 1
- 102220019738 rs397507909 Human genes 0.000 description 1
- 102220064253 rs779277447 Human genes 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- ZERULLAPCVRMCO-UHFFFAOYSA-N sulfure de di n-propyle Natural products CCCSCCC ZERULLAPCVRMCO-UHFFFAOYSA-N 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000001935 tetracenyl group Chemical group C1(=CC=CC2=CC3=CC4=CC=CC=C4C=C3C=C12)* 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 229960004559 theobromine Drugs 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- LJJKNPQAGWVLDQ-SNVBAGLBSA-N thiorphan Chemical compound OC(=O)CNC(=O)[C@@H](CS)CC1=CC=CC=C1 LJJKNPQAGWVLDQ-SNVBAGLBSA-N 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 208000012175 toxemia of pregnancy Diseases 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 229960004319 trichloroacetic acid Drugs 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- YFTHZRPMJXBUME-UHFFFAOYSA-N tripropylamine Chemical compound CCCN(CCC)CCC YFTHZRPMJXBUME-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 125000003774 valeryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229960003726 vasopressin Drugs 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/10—Antioedematous agents; Diuretics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/08—Vasodilators for multiple indications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- Cardiology (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Obesity (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Heart & Thoracic Surgery (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Compounds related to BNP have reduced binding affinity for the natriuretic peptide receptor-C but possess improved or equivalent affinity to the wild type BNP for natriuretic peptide receptor-A. The BNP related compounds are suitable for use in the treatment or prophylaxis of various pathological conditions associated with water or electrolyte imbalance.
Description
RECEPTOR SPECIFIC BRAIN NATRI1;JRETIC PEPTIDE (BNP) Field of the Invention The present invention relates to compounds having natriuretic, diuretic, and/or vasodilatory activity in mammals. The compounds of this invention are related to B-type natriuretic peptide (BNP). In preferred embodiments the compounds exhibit decreased binding affinity to the natriuretic peptide clearance receptor (NPR-C) compared to wild-type BNP or the wild-type type A natriuretic peptide (ANP) and advantageously have enhanced potency. The invention also relates to pharmaceutical compositions comprising the novel compounds as well as their use in diagnostic, therapeutic, and prophylactic methods.
Description of Related Disclosures l0 Three natriuretic peptides, type A, type B and type C, have been identified which play a role in hemodynamics (Rosenzweig, A., and Seidman, C.E., (1992) Annu. Rev. Biochem.
60:229-255 (1991);
Ruskoaho, H. ( 1992) Pharmacol. Rev. 44:479-602). The bioactive peptides are derived from prohormones each of which arises from a distinct gene (Steinhelper, M., (1993) Circ. Res.
72:984-992). The genes are expressed in a tissue specific manner (Seilhamer et al., ( 1989) Biochem.
Biophys. Res. Commun. 165, 650-658) and are present in the systemic circulation. The type A (ANP) and type B
(BNP) peptides are derived from the myocardium, whereas the type C (CNP) peptide is thought to be derived from endothelial cells. Each of the bioactive peptides, derived from the C-terminal portion of the respective prohormone, exhibit a high degree of sequence homology within their 17 amino acid ring structures.
Both ANP and BNP are primarily cardiac hormones that are released by the heart in its role as an endocrine organ regulating fluid and electrolyte hemostasis. Human BNP is stored in the atrium and ventricle (Hosodo et al., (1991) Hypertension 17:1152-1156) as the mature hormone whereas ANP is stored in cardiocytes as the precursor. BNP was first isolated from porcine brain (Sudoh et al., ( 1988) Nature 332:78-81 ) although it is synthesized in and secreted into the circulation from the porcine heart (Aburaya et al., ( 1989}
Biochem. Biophys. Res. Commun. 158:360-368). Porcine BNP circulates as a 26 or 32 amino acid poiypeptide and exhibits structural homology with ANP (Sudoh et al., (1988) Nature 332:78-81 ). The cDNA sequence encoding human BNP precursor, hBNP[1-108] has been determined (Sudoh, et al., (1989) Biochem. Biophys.
Res. Commun. 159:1427-1434). Human BNP has also been isolated as a 32 amino acid poiypeptide from human atrium the circulating form being identical to sequence residues [77-108] of the human BNP precursor molecule.
The action of the natriuretic peptides is mediated through the binding of the peptides to cellular receptors. There are at least three structurally distinct cellular receptors (Chang et al., ( 1989) Nature 341:68-72). NPR-A (Chinkers et al., ( 1990) Nature 338:78-83) and NPR-B (Lowe et al., (1990) Nucleic Acids Res.
18:3412) receptors have homologous extracellular domains of approximately 440 amino acid residues with a single transmembrane domain. Both possess intracellular guanyl cyclase and tyrosine kinase-like domains (Chang et al. ( 1989), supra). The extracellular domain of the third receptor NPR-C is homologous to the NPR-A and NPR-B extracellular domains but contains only a 37 amino acid intracellular domain of unknown function (Fuller et al. (1988) J. Blot. Chem. 263:9395-9401 ). The role of these receptors in regulating blood pressure is unclear but NPR-A is thought to be responsible for most of the biological effects of ANP and BNP
(Maack T., ( 1992) Annu. Rev. Physiol.. 54, I 1-27).
8NP has peripheral and central nervous system action similar to those of ANP
including natriuretic, diuretic and hypotensive effect and smooth muscle relaxation (Yoshimura et al., (1991) Circulation 84:1581-1588; Mukoyama et al., ( 1991 ) J. Clin. Invest. 87:1402-1412). Binding of the bioactive peptide to the NPR-A
cellular receptor results in a number of actions including a decrease in blood pressure due in part to relaxation of vascular smooth muscle, an increase in sail and water excretion, transduction of plasma water to the interstitium and an inhibition of the release or action of aldosterone, angiotensin II, endothelin, renin and vasopressin.
Maintenance of normal extracellular fluid volume depends primarily on the excretion of sodium (natriuresis) and water (diuresis) by the kidneys. These are, in turn, primarily determined by (1) the rate at which plasma is filtered at the glomeruius (glomerular filtration rate, or GFR) and (2) the degree to which sodium is actively reabsorbed along the renal tubule (with water presumably following passively). Sodium reabsorption is regulated, in part, by the adrenal steroid hormone aldosterone, in part by blood pressure, hematocrit and plasma viscosity and in part by the various natriuretic factors or hormones (del3old, A.J. et al., (1981) Life: Sciences 28:89-94; Garcia R., (1982) Experientia 38:1071-73;
Currie, M.S. et al., (1983) Science 221:71-73; Flym~. T.G. et al., (1983) Biochem. Biophys. Res. Commun. 117:859-865; Currie, M.G. et al., ( 1984) Science 223:67-69; and Kangawa, K. et al., (1984) Biochem. Biophys.
Res. Commun. 118:131-139;
Mukoyama et al., (1991) J. Clin. Invest. 87:1402-1412).
It is known that the serum half life of natriuretic peptides that act as hypotensive regulators is relatively short (Crozier, LG. et al., (1986) The Lancet II 1242-1245) and that these peptides are removed from the blood stream by several mechanisms including proteolysis and receptor mediated endocytosis by NPR-C
(Maack ( 1992) supra). Since both ANP and BNP have a short half life with poor or non-existent peroral absorption, synthetic analogues of BNP with longer circulatory duration and favorable in vivo characteristics are desirable.
Summary of the Invention The present invention provides compounds which have natriuretic, diuretic, and/or vasodilatory activity in mammals. The compounds of this invention are related to the B-type natriuretic peptide (BNP) and exhibit decreased binding affinity to the natriuretic peptide clearance receptor (NPR-C) compared to BNP.
Advantageously, the compounds have enhanced potency defined in terms of NPR-A
receptor affinity and cGMP production. In preferred embodiments, the compounds and related compositions allow for a potent NPR-A receptor stimulation providing for low dose pharmaceutical formulations.
The compositions of the present invention are useful in therapeutic and prophylactic methods for inducing natruresis, diuresis or vasodilation or associated processes.
In one embodiment, the compounds of the present invention have a decreased binding affinity for the human clearance receptor (hNPR-C) compared to wild-type BNP. Preferably, according to this aspect of the invention, the compounds exhibit the same or an increased affinity for the type-A natriuretic peptide receptor.
More preferably, according to this aspect of the invention, at least one of amino acid residues Xaal9, Xaa23, Xaa24 or Xaa25 of wild-type 8NP is selected according to the following scheme in order to produce a BNP
variant or related compound of the invention:
_2_ WO 9811?690 PCT/US97/18384 Xaal9 is selected from the group consisting of 5er, Arg, Ala, Asn, Gly and conservative substitutions thereof;
Xaa23 is selected from the group Gly, Met, Phe, Leu and conservative substitutions thereof Xaa24 is selected from the group Trp, Tyr, Phe, and conservative substitutions thereof; and Xaa25 is selected from the group Gly, Arg, and conservative substitutions thereof.
In a further preferred embodiment, the compounds of the present invention are represented by Formula A-R 1-Cys-Phe-G ly-Arg-Lys-Met-Asp-Arg-I le S-S
B-R2-Cys-Xaa~s-Xaa24-Xaa~3-Ser-Ser-Ser-Xaal9 wherein:
A is selected from the group consisting of H, C 1-C6 alkanoyl and Thr-Ala-Pro-Arg (SEQ ID NO: 30);
RI is absent or selected from the group consisting of a peptide from between 1 and 10 amino acids, Gly-Ser-Gly-, Val-Gln-Gly-Ser-Giy (SEQ ID NO: 3 I ) and Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly (SEQ ID
NO: 32);
Xaal9 is selected from the group consisting of Arg, Ala, Asn, Gly and Ser;
Xaa23 is selected from the group Gly, Met, Phe, Leu;
Xaa24 is selected from the group Trp, Tyr, Phe;
Xaa25 is selected from the group Gly, and Arg;
R2 is selected from the group consisting of a 1 to 6 amino acid peptide and Lys-Val-Leu-Arg-Arg-His-(SEQ ID NO: 33); and B is selected from OR3 and NR3R4 where R3 and R4 are independently selected from H, C I-C6alkyl, C6-Cl2aryl and C6-Cl2aryl-CI-C6alkyl.
In one embodiment, the composition of the present invention is a polypeptide and the invention encompasses a composition of matter comprising a nucleic acid, preferably DNA, encoding the polypeptide of the invention. According to this aspect, the invention further comprises an expression control sequence operably linked to the DNA molecule, an expression vector, preferably a plasmid, comprising the DNA
molecule, where the control sequence is recognized by a host cell transformed with the vector, and a host cell transformed with the vector.
In one embodiment, the polypeptide compositions of the present invention may be made by a process which includes the steps of synthesizing nucleic acid sequences encoding any of the amino acid sequences of the invention, ligating the nucleic acid sequence into a suitable expression vector capable of expressing the nucleic acid sequence in a suitable host, transforming the host with the expression vector into which the nucleic acid sequence has been ligated, and culturing the host under conditions suitable for expression of the nucleic i acid sequence, whereby the protein encoded by the selected nucleic acid sequence is expressed by the host.
Preferably, the polypeptide is then recovered from the host cell culture. In this process, the ligating step may further contemplate ligating the nucleic acid into a suitable expression vector such that the nucleic acid is operably linked to a suitable secretory signal, whereby the amino acid sequence is secreted by the host.
The present invention further extends to therapeutic applications for the compounds described herein.
Thus the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound of the invention. Pharmaceutical compositions comprising these compounds can be used in the treatment or prophylaxis of various pathological conditions associated with water or electrolyte imbalance and hypertension, especially renovascuiar hypertension. Such conditions include, for example, congestive heart failure (CHF), nephrotic syndrome and hepatic cirrhosis, pulmonary disease, and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
Brief Description of the Drawings Figure 1: Concentration responses for cGMP second messenger production on human natriuretic peptide receptor-A expressed in 293 cells. Values for the concentration of natriuretic peptide giving half maximal stimulation of c-GMP production are given in the Figure. Values are plotted as average t standard deviation (n=3). In the Figure ANP refers to the 28 amino acid residue peptide reported by Kangawa et al., Biochem. Biophys. Res. Comm. ( 1984) 118(/ ):13 i-139; BNP refers to SEQ ID NO: ( ; BNP02 refers to SEQ ID NO: 2; BNP04 refers to SEQ ID NO: 7; BNPOS refers to SEQ ID NO: 7 and ANF 131 refers to SEQ ID NO: 50. The results show that the peptides are foil agonists on this receptor, with potency similar to BNP.
Figure 2: Stimulation of cGMP second messenger production on human natriuretic peptide receptor-A
expressed in 293 cells. cGMP values for natriuretic peptide stimulation of natriuretic peptide receptor-A at 4 x 10-8 M are given in the Figure. cGMP values are plotted as average t standard deviation (n=3). In the Figure ANP refers to the 28 amino acid residue peptide reported by Kangawa et al., Biochem. Biophys. Res.
Comm. ( 1984) 118(1 ):131-139; BNP refers to SEQ ID NO: 1; BNP02 refers to SEQ
ID NO: 2; BNP04 refers to SEQ ID NO: 7; BNPOS refers to SEQ ID NO: 7; ANF 131 refers to SEQ ID NO:
S0; BNP06 refers to SEQ
ID NO: 48; and BNP07 refers to SEQ ID NO: 49. The results show that the peptides are full agonists on this receptor, with potency similar to BNP.
Figure 3: Alignment of the amino acid sequence of BNP from mammalian species;
human (Sudoh, et al., (1989) Biochem Biophy. Res. Common. 159:1427-1434) (SEQ ID NO 1), canine (Seilhamer et al., (1989) Biochem. Biophys. Res. Common 165:650-658) (SEQ ID NO 44), porcine (Sudoh, T., et al., (1988) Nature 332:78-81 ) (SEQ ID NO 45), rat (Kojima et al., ( 1989) Biophys.
Biochem. Res. Common. 159:1420-1426) (SEQ ID NO 46) and mouse (Ogawa et al., ( 1994) J. Clin. Invest 93:19/ I-1921 ) (SEQ ID NO 47). Gaps (-) have been introduced to maximize alignment.
Detailed Description of the Preferred Embodiments Definitions Terms used in the claims and specifcation are defined as set forth below unless otherwise specified.
The term amino acid or amino acid residue, as used herein, refers to naturally occurring L amino acids or to D amino acids as described further below with respect to variants. The commonly used one- and three-KL1 . 1u\.I~.I'\-vll I.\l:lll..\ t):p : ~;S- t m>1 - ~:-,. ~.t ~ ,-.l_.L1 1 ~:~ _t,,,i L 1- r t:~ ts;~ =:C3;1-l~l~~.;,:1~ .;
. , _ .a:,~~_ _:... CA 02269656 1999-04-22 .i., _ _. . ..
.., .~.. .___ ._ letter abbreviations for amino acids are used herein (H~ce Alberts et aL, :vtofecular Biology of the CeII, Garland Publishing. Inc., New York (3d ed. 1994)).
As used herein the turns "BNP", "rype-B nattiuretic peptide", "wild-type H"Tp"
and the like are used interchangeably and refer to a naturahy occurring :mammalian BNP or recombinant BNP. The sequence ef 13NP from mammalian species is known, for example, human (Sudoh, et al..
(19891 Biochem Biophy. Res.
Common. 159:1421-1434) (SEQ ID NO 1), canine (Seilhamer et al., (1989) Biochem. t3iophys. Res. Comrnun 165:650-658) (SEQ ID NO 44), porcine (Sudoh, T., et al_, ( 1988) Nature 332:16-81) (SEQ ID NO 45) and rat (Kojima et ai., (1989) Hiophys. l3iochem. RPs_ Common. 159:1420-1426 (SEQ 1D
NO 4~. 'Ihe terms ~~hu~ g~Tp," "hBNP" and the like, mean ttte 32 amino acid residue pep::de reported by Sudoh, T., et at., (lava) s~p~a.
1~ ~~~.3~.~, having the following primary amino acid sequence:
I 2 3 4 5 6 7 8 9 10 11 I2 13 ld 15 ~6 Ser-Pro-1.ys-filet-val-Gin-Gty-Set-Giy-Cys-Pbe-Gly-Arg->rys-MCt-G
t7 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 erg-lie-Ser-5er-Ser-Ser-Gly-Leo-uly-Cys-Lys-'~ ai-Lea-Arg-Arg-His (5EQ ID NO: 1) The integers above specific residues of this structure define the residue position number. ?his residue position number is used in conjunction wire t:ze threo letter amino acid nomenclature to designate d;e residue at which a substitution is :made in the BNP related compounds of this invention. Thus for exarnple,.in a BNP :elated compound in which arginine (.4rg) replace serine (Scr) at residue position number i 9 of wild-type BNP, tide ~G nomenclature 'BNP Seri 9Arg" or the like is used. utuldple substitutions are designated in the same manner with a comma separating each substitution. Thus far cxanpte in a HNP :'elated compound in which phenylalanine (Phe), tryptophan (Trp) and arginine (A:g) replace amino acids 23, Z4 a.-td 25 respectively of auman BNP t.'~e nomenclature "hBNP GlyZ3Phe, ;.euZ4Trp, GIy25Arg" is sued.
The terms "BNP variant" and "BNP related cempaund" are dot-teed as a compound having at least qualitative biological activity in cotrunon witft tire polypeptide of SEQ iD
N0: 1 and having at least one amino acid substitution as dafined he: tin for conferring receptor specificity.
Preferred arnino acid substitutions arc outlined in 'Cable I of Example L The qualitating biological activity refeaed to a the capacity to stimulate cyclic CMP production :n ells expressing the guaaylate cyclsse-linked naa:uretic peptide recaptor NPR-A.
'fhe NPR-A receptor amino acid sequence deduc.~.d from eDNAs encoding the receptor is Jmown (Chinkers et al., (1989) Nature 338:78-83; Lowe et aJ_ (1989) EMBO 1. 8:1377-1384), Functional expression of the NPR-A receptor cDNA in cultured r~arttrnalian ec!!s which confess both ANP and HT,IP birtdir<g and guanylate cyclase activity is also known (Chinkets, es ai., ( 1989) Nature 338:TB-83).
Assay systems for tneastuernent of birtdiag affinities for NPR receptors as wall as eGMP accumulation are known (see, International , Application No. PCTlUS94I12591).
Additionally, irt prei'erred embo~dimenrs, the $NP variant or $NP related campound of the present 1 invention is believed to tampon with axy wild-type.~..:'~1P (Kaagawa etal..
(1984) Biochera- Biophys. Ras.
Corsmu~. 118(1):13'-I39) of BNP for binding to the NPR-A receptor and thereby inducing cGMP
accumt:latic;n. Therefore, without limitation to any one theory, ~qualitating biological activity may be defined as the ability to compete with a.~y wild-t-~e BNP or ANP for binding to NPR-A
r:ceptor thereby inducing AMENDED SHEET
cGMP accumulation. As will be appreciated from the foregoing, the term "compete" and "ability to compete"
are relative terms. Thus the terms, when used to describe the activity of the BNP variant, mean a BNP variant that when added in a 10-fold molar excess to wild-type BNP or wild-type ANP in a standard receptor binding assay produces at least a 50% inhibition of binding of the wild-type BNP or wild-type ANP. Preferably the BNP variant will produce at least a 50% inhibition of binding in a 5-fold molar excess and most preferably at least a 2-fold molar excess. A most preferred BNP variant or BNP related compound of the present invention will produce at least a 50% inhibition of binding when present in a 1:1 stoichiometric ratio with wild-type BNP
or wild-type ANP. For assay systems to measure binding of hBNP, hANP and the compounds of this invention to hNPR-C and hNPR-A see: Bennett et al., ( 1991 ) J. Biol. Chem. 266:23060-23067; Cunningham et al., (1994) J. EMBO 13:2508-2515; Chinkers et al., (1989) Nature 338:78-83 and Fuller et al., (1988) J. Biol.
Chem. 263:9395-9401; Schenk, D. B., et al., (1985) Biochem. Biophys. Res.
Comm. 127:433-442;
Scarborough, R. M., (1986) J. Biol. Chem. 261:12960-12964 and WO 90/01940.
A characteristic of the BNP related compound or BNP variant of the present invention is a decreased affcnity for the natriuretic clearance receptor NPR-C. Therefore variants or compounds of the present invention I S have at least qualitative biological activity in common with wild-type BNP
but have a decreased affinity for the clearance receptor NPR-C when compared to BNP in a standard receptor binding assay as described herein (see Bennett et al., ( 1991 ) J. Biol. Chem. 266:23060-23067).
The BNP variants and compounds of the present invention are, in general, homologous amino acid sequences of the rat, porcine, canine or other mammalian BNP's or homologous amino acid sequences of the sequence of SEQ ID NO: 1 including homologous in vitro generated variants having the qualitative biological activity defined above. Homology with respect to the BNP variants of the present invention is defined as the percentage of amino acid residues in a candidate sequence that are identical with either the amino acid residues in SEQ ID NO: I, the amino acid sequence of a mammalian BNP or a composite sequence as defined herein after aligning the sequences and introducing gaps if necessary to achieve the maximum identity (Figure 3}, No N- or C- terminal extension or deletion in the candidate sequence shal) be construed as reducing identity.
"Composite amino acid" within the present invention refers to an alternate amino acid having the same position in the 32 amino acid residue structure as human BNP from other mammalian vertebrate species. Therefore, an amino acid substitution referred to as a composite amino acid substitution replaces the identified amino acid with the equivalent or composite amino acid from another mammalian species. A
composite BNP sequence is defined as having at least one amino acid from the wild-type sequence replaced with a composite amino acid from another mammalian species.
Therefore, the invention contemplates a BNP variant having at least the qualitative biological activity as defined above and having, for example, at least about 75% amino acid homology with the polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: I lacking the 5 carboxyl terminal amino acid residues and/or the 9 amino terminal residues. The BNP variant amino acid sequence preferably will share at least 80%, more preferably, greater than 85% sequence homology with the sequence of SEQ ID NO:
I. However, a BNP
variant or related compound may exhibit less than SO% sequence homology with the sequence of SEQ ID NO:
1 and still retain the characteristics of the BNP variant or BNP related compound as defined above.
Included in the definition of BNP variant or BNP related compound of the present invention are amino acid sequence variants of the SEQ ID NO: 1 wherein an amino acid in addition to those described herein which confer receptor specificity has been substituted by another residue, including predetermined mutations (e.g.
site directed PCR mutagenesis); other composite amino acid substitutions from other mammalian species of BNP such as those listed above and other naturally occurring variants of the foregoing and human sequences.
According to this aspect of the present invention reference is made to Figure 3 and the amino acid sequence data presented therein. Also included is a BNP variant as described above wherein the BNP variant has been modified by substitution, chemically, enzymatically, or by other appropriate means with a moiety other than a naturally occurring amino acid, it being understood that the variant will have the qualitative biological activity described above. Exemplary non-naturally occurring amino acid substitution include those described herein below.
Additionally, amino acid sequence variants can be generated by substitutions which utilize the D rather than L form of an amino acid. This is especially useful in stabilizing the BNP
variant of the present invention against enrymatic degradation. Such stabilizing amino acid substitutions have been described for ANP (Mutt and Veber, ( 1987) Endocrin. and Metab. Clin. N. Amer. 16(1):19-41 ). Such structure/activity studies of ANP
analogues including the introduction of D-amino acids where appropriate can be conducted for BNP and are included in the definition of BNP variant or BNP related compound. Thus the term variant is meant to include, in addition to those substitutions described herein for receptor specificity, substitutions that confer increased stability of BNP molecules such as chiral amino acid replacements which may offer resistance to metabolic degradation. The present invention is meant to include BNP variants and related compounds that have decreased susceptibility to hydrolysis by neutral endopeptidase 24. I 1 (EC3.4.24.1 1 ) (NEP) and angiotensin converting enzyme (ACE) as well as improved potency or duration of action. For example, the replacement of amide bonds optionally with an amide isostere has been demonstrated for ANP. Replacements which decrease susceptibility to hydrolysis by the neutral endopeptidase 24.11 or ACE produce a BNP variant that is resistant to enzymatic degradation. Strategies include the substitution of amino acids with N-alkylated amino acids such as N-Me-Phe and addition of the urodilatin peptide tail Thr-Ala-Pro-Arg (SEQ ID NO: 30).
Also included in the scope of the BNP variant and related compounds is a BNP
variant wherein an amino acid has been added to or deleted from the N-terminal or C-terminal 17 member ring structure of wiid type BNP. Amino acid sequence insertions include carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues including pre and pro amino acid sequences as described (see for example Ogawa et al., (1994) J. Clin. Invest. 93:1911-1921). Other examples of terminal insertions include BNP variants with a composite N-terminal or C-terminal sequence from another mammalian species or from another natriuretic peptide (see Figure 3). Such amino terminal substitutions have been described (Shimekake et al., (1992) FEBS, 309(2):185-189). Suitable N-terminal sequences are described in International Publication No WO 89112060 and include Gly; Ser Gly;
Asp/Lys/Gly-Ser-Gly; Arg/His/Gln-Asp/Lys/Gly-Ser-Gly {SEQ ID NO 34); Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly (SEQ ID NO 35);
Thr/Met-Met/Val-ArglHisIGln-Asp/Lys/Gly-Ser-Gly (SEQ ID NO 36); Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly (SEQ ID NO 37); Pro-Lys-Thr/Met-Met/Val-Arg/HislGln-Asp/Lys/Gly-Ser-Gly (SEQ
_7_ ~ I
WO 98!17690 PCTIUS97/18384 ID NO 38); and Ser-Pro-Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly (SEQ ID NO 39), and are based upon the native upstream sequence for porcine, canine or human BNP or a composite thereof.
Exemplary C-terminal insertions are described in International Publication No.
89/12069 and included (OH), NH, or NR'R" where R' and R" are independently H, lower alkyl, or Asn/Lys; Asn/Lys-Val; Asn/Lys-VaI-Leu; Asn/Lys-Val-Leu-Arg (SEQ ID NO 40); Asn/Lys-Val-Leu-Arg-Arg/Lys (SEQ
ID NO 41 ); Asn/Lys-Val-Leu-Arg-Arg/Lys-Tyr-His (SEQ ID NO 42); or the amides (NH or NR'R") thereof.
As noted, in one embodiment, amino acid substitution variants have at least one amino acid residue in addition to those described herein for conferring receptor specificity in the BNP variant molecule removed and a different residue inserted in its place. The sites for substitutional mutagenesis include sites where amino acids found in the BNP variant from various species are substantially different in terms of side chain bulk, charge and or hydrophobicity. These amino acids are substituted with the exemplary conservative substitutions as described herein below including the exemplary non-naturally occurring amino acids.
Other sites of interest are those in which particular residues of wild-type BNP and the variants obtained from various species are identical. These positions may be important for the biological activity of the BNP variant or related compound. These sites, especially those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitution are shown below under the heading of preferred conservative substitutions. If such substitutions are shown to preserve qualitative biological activity as defined herein then more substantial changes denominated below as exemplary conservative substitutions may be generated and tested for biological activity.
In this regard, it is understood that amino acids are substituted on the basis of side chain bulk, charge and/or hydrophobicity. Amino acid residues are classifed into four major groups as described in, for example, International Publication No. 90/01940 the disclosure of which is hereby specifically incorporated by reference and which consists of the following groups:
Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous solution.
Basic: The residue has a positive charge due to association with H ion at physiological pH and the residue is amacted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
Neutral/non-polar: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. These residues are also designated "hydrophobic residues."
Neutral/polar: The residues are not charged at physiological pH, but the residue is amacted by aqueous solution so as to seek the outer positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
Amino acid residues can be further classified as cyclic or noncyclic, aromatic or non aromatic with respect to their side chain groups these designations being commonplace to the skilled artisan.
-g_ Original Exemplary Conservative Preferred Conservative Residue Substitution Substitution Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys 5er Ser Gln Asn Asn Glu Asp Asp 10Gly Pro Pro His Asn, Gln, Lys, Arg Arg lle Leu, Val, Met, Ala Leu Phe Leu Ile, Val Ile Met, Ala, Phe Lys Arg, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly 20Ser T"hr ~r Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe Leu Ala Peptides synthesized by the standard solid phase synthesis techniques described here, for example, are not limited to amino acids encoded by genes for substitutions involving the amino acids. Commonly encountered amino acids which are not encoded by the genetic code, include, for example, those described in International Publication No. WO 90/01940, as well as 2-amino adipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric (Abu) acid for Met, Leu, and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminoisobutyric acid (Aib) for Gly; cyclohexylaianine (Cha) for Val, and Leu and Ile; homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dpr) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylasparigine (EtAsn) for Asn, and Gln; Hydroxyllysine (Hyl) for Lys; aliohydroxyllysine (AHyI) for Lys; 3-(and 4)hydoxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr; allo-isoleucine (Alley for lle. Leu, and Val; p-amidinophenylalanine for Ala; N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala; N-methylisoleucine (Melle) for lle; Norvaline (Nva) for Met and other aliphatic amino acids; Norleucine (Nle) for Met and other aliphatic amino acids; Ornithine (Orn) for Lys, Arg and His;
Citrulline (City and methionine sulfoxide (MSO} for Thr, Asn and Gln; N-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F, Cl, Br, and I)phenylalanine, triflourylphenylalanine, for Phe.
A useful method for identification of certain residues or regions of the BNP
variant for amino acid substitution other than those described herein for receptor specificity is called alanine scanning mutagenesis as described by Cunningham and Wells ( 1989) Science, 244:1081-1085. Here a residue or group of target residues are identified (e.g. charged residues such as Arg, Asp, His, Lys, and Glu} and replaced by a neutral or negatively charged amino acid to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitution then are refined by introducing further or other variations at or for the sites of substitution. Thus while the site for ~ I
introducing an amino acid sequence variation is predetermined the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, Ala scanning or random mutagenesis may be conducted at the target codon or region and the expressed BNP variants screened for the optimal combination of desired activity.
Phage display of protein or peptide libraries offers another methodology for the selection of BNP
variants or BNP related compounds with improved affinity, altered specificity, or improved stability (Smith, G.P., (1991} Curr. Opin. Biotechnol. 2:668-673). High affinity proteins, displayed in a monovalent fashion as fusions with the M13 gene III coat protein {Clackson, T., (1994) et al.,Trends Biotechnol. 12:173-183), can be identified by cloning and sequencing the corresponding DNA packaged in the phagemid particles after a number of rounds of binding selection.
Other insertional BNP variants or BNP related compounds include the fusion to the N- or C- terminus of the BNP molecule of immunogenic polypeptides, e.g., bacterial poiypeptides such as beta lactamase or an enzyme encoded by E coli Trp locus or yeast protein, and C-terminal fusion with proteins having a long half life such w~: immunoglobulin constant region or other immunoglobulin regions, albumin, or ferritin as described in WO 89/02922 published 6 April 1989.
The term "C 1-C6alkyl" when used to describe a BNP variant means a branched, unbranched or cyclic, saturated aliphatic hydrocarbon radical, having the number of carbon atoms specified. Representative examples of these alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl, cycolhexyl and the like.
The terms "lower alkyl" and"C1-C6alkyl" are synonymous and used interchangeably. A preferred "C1-C6alkyl" group is methyl.
The term "C1-C6alkanoyl" when used to describe a BNP variant encompasses groups such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, caproyl, and the like.
The term "C6-Cl2aryl" when used to describe a BNP variant means a homocyclic hydrocarbon aromatic radical, whether or not fused, having the number of carbon atoms designated. Preferred aryl groups include phenyl, napthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g. Lang's Handbook of Chemistry (Dean, J. A., ed) 13th ed. Table 7-2 ( 1985]).
The term "C6-Cl2aryl-C1-C6alkyl" when used to describe a BNP variant means one, two, or three aryl groups having the number of carbon atoms designated, appended to an alkyl radical having the number of carbon atoms designated including but not limited to; benzyl, napthylmethyl, phenethyl, benzyhydryl (diphenylmethyl), florenyl, trityl, and the like. A preferred arylalkyl group is the benzyl group.
"Pharmaceutically acceptable salts" include both acid and base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malefic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toiuenesulfonic acid, salicyciic acid and the like.
"Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.
Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethyiamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.
Receptor Specific BNP Variants and Related Compounds The BNP variants and related compounds of this invention have at least one amino acid of wild-type BNP substituted as described herein for conferring NPR-A receptor specificity.
Therefore, preferred I S compounds of this invention are synthetic hormone agonists of the human natriuretic peptide receptor-A
(hNPR-A). This receptor is found in kidney, smooth muscle, adrenal and other tissues (Napier M.A. et al, (1984) Proc. Nat. Acad. Sci. USA, 81:5946-5950; Hori et al., (1985) Biochem.
Biophys. Res. Commun.
129:773-779; Quirion et al., (1986) Proc. Natl. Acad. Sci. 83:174-175) and contains an intracellular guanyl cyclase domain. The action of hNPR-A is mediated by hydrolysis of GTP to produce the second messenger cGMP. Accordingly, preferred compounds of this invention stimulate hNPR-A to produce cGMP to at least the same extent as wild-type BNP.
The preferred compounds of this invention do not however bind to the human natriuretic peptide clearance receptor-C (hNPR-C) to the same extent as wild-type BNP. Both in vitro and i~r vivo systems can be used to identify the compounds of the present invention. Since binding of BNP to the NPR-A cellular receptor is presumptively a prerequisite to qualitative biological activity the BNP related compounds of the present invention are identified by screening for binding of the candidate compound to the NPR-A and NPR-C
receptors. Primary screens for the compounds of the present invention therefore include vasorelaxant assays in rat and rabbit aorta tissues, relaxation of chick rectum intestinal smooth muscle tissue, natriuresis in rats, as well as the receptor specific binding assays and cGMP formation in cell culture assays described herein above and in the following Example sections.
For example, assays have been developed for the screening of BNP which evaluate the ability of the BNP to compete with a labeled native BNP for binding to intact cultured cells or membrane preparations derived from cultured cells expressing the cellular receptors. By way of example, NPR-A expressing 293 cells produced as described by Lowe and Fendly (1992) J. Biol. Chem. 267:21691-21697 may be utilized. Likewise the cDNA for NPR-C (Lowe et al., ( 1990) Nucleic acids Res. 18:3412) may be transfected into suitable host cells for continuous expression. These competitive displacement assays are considered commonplace for examining receptor ligand interactions.
According to a particular cellular assay, radiolabeled or florescent labeled BNP is incubated with immobilized NPR-A or NPR-C receptor-immunoglobulin chimeras in varying concentration of unlabeled candidate compound. Increasing concentrations of successful candidate compound effectively prevent binding of labeled BNP to immobilized receptor chimeras. The concentration of unlabeled peptide at which 50%
maximal BNP is displaced is referred to as the EC50 and reflects the receptor binding affinity. Therefore a candidate compound with an EC50 of 100nM displays a substantially weaker interaction with a receptor than peptide with an ECSO of 10 nM.
As noted above, BNP related compounds or variants within the present invention have a decreased binding affinity for NPR-C and corresponding NPR-A receptor specificity.
Therefore, according to the present invention a BNP variant with an EC50 greater than wild type BNP for the NPR-C
is said to display a weaker affinity for the NPR-C receptor and therefore a decreased affinity for the clearance receptor.
In a preferred embodiment, receptor specificity is reflected in the ratio of binding affinity of the candidate compound for NPR-A and NPR-C. A change in the ratio of binding affinity for the candidate compound to NPR-A compared to NPR-C is referred to as a change in receptor specificity. According to this aspect of the present invention, it is a characteristic of the BNP variant or BNP related compound to exhibit a decrease in the ratio of binding affinity for NPR-A compared to NPR-C as compared to wild-type BNP in the same assay. Preferred compounds and variants according to this aspect of the invention exhibit at least a 3 0 fold decrease in the ratio of the binding affinities compared to wild-type BNP and more preferably greater that a 100 fold decrease in the ratio.
This is preferably achieved by substituting an amino acid from wild-type BNP
according to the scheme provided in Table I of Example I. Accordingly, substituting residue 19 with a basic amino acid, more preferably Arg, Har, Lys, Orn, and p-amidinophenyl-Ala and most preferably Arg; substituting residue 23 with a neutral, nonpolar, large, aromatic or nonaromatic amino acid, more preferably Val, lle, Leu, Met, Phe, Trp, t-BuA, T-BuG, N-MeIle, Nle and Cha, and more preferably, Met, Phe or Leu;
substituting residue 24 with an aromatic amino acid, more preferably Phe, Trp, and Tyr; and substituting residue 25 with a basic amino acid, more preferably Arg, Har, Lys, Orn, and p-amidinophenyl-Ala and most preferably Arg results in a BNP
variant or related compound.
The above noted substitutions can occur individually or in combination.
Preferred among the individual substitutions conferring receptor specificity is a BNP variant or related compound which has a substitution for amino acid residue 19 of wild-type BNP as provided in the preceding paragraph. Preferred among combination substitutions conferring receptor specificity is a BNP
variant or related compound where each of amino acids 23, 24, and 25 of wild type BNP are substituted according to the scheme presented in the preceding paragraph. Such preferred combination substitutions may be accompanied by a substitution of residue l9 according to the scheme provided in the preceding paragraph.
Exemplary peptides are given below:
Ser-Pro-Lys-Met-V al-G In-G ly-Ser-G ly-Cys-Phe-G ly-Arg-Lys-Met-G lu-Arg-I le-Ser-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 2) or hBNP G1y23Phe, Leu24Trp, G1y25Arg;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Ser-Ser-Ser-Ser-Met-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 3) or hBNP G1y23Met, Leu24Trp, Gly25Arg;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-G ly-Cys-Phe-G ly-Arg-Lys-Met-Glu-Arg-I le-Arg-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 4) or hBNP Serl9Arg;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Arg-His-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 5) or hBNP Serl9Arg, Ser20His;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-lle-Arg-His-Leu-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 6) or hBNP Serl9Arg, Ser20His, Ser2iLeu;
Ser-Pro-Lys-Met-Val-Gln-G ly-Ser-Gly-Cys-Phe-G ly-Arg-Lys-Met-Glu-Arg-l le-Arg-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 7) hBNP Serl9Arg GIy23Phe, Leu24Trp, Gly25Arg;
Ser-Pro-Lys-Met-Val-Gln-G ly-Ser-Gly-Cys-Phe-G ly-Arg-Lys-Met-G lu-Arg-Ile-Arg-Ser-Ser-Ser-Met-Trp-Arg-Cys-Lys-Vat-Leu-Arg-Arg-His (SEQ ID NO: 8) or hBNP Serl9Arg, GIy23Met, Leu24Trp, GIy25Arg;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Arg-His-Ser-Ser-Met-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 9) or hBNP Serl9Arg, Ser20His, G1y23Met, Leu24Trp, GIy25Arg; and Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-G ly-Cys-Phe-G ly-Arg-Lys-Met-Glu-Arg-I le-Arg-His-Leu-Ser-Met-Trp Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 10) or hBNP Serl9Arg, Ser20His, Ser21 Leu, G1y23Met, Leu24Trp, Gly25Arg.
Further compounds within the scope of the present invention are:
hBNP Arg l3Ser, Lys l4Pro, Serl9Arg; (SEQ ID NO: 1 I ) hBNP Lysl4Thr, Serl9Arg, Ser2lGln; (SEQ ID NO: 12) hBNP Lys l4Asn, Serl9Arg, Ser20His, Ser21 Leu; (SEQ ID NO: 13) hBNP Lysl4Asn, Serl9Ala, Ser20Arg, Ser2lGlu; (SEQ 1D NO: 14) hBNP Lysl4Asn, Serl9Arg, Ser20Lys, Ser2lThr; (SEQ ID NO: 15) hBNP Lysl4Asn, Serl9Arg, Ser20Gly; (SEQ ID NO: 16) hBNP Lysl4Asn, Serl9Gly, Ser20Arg, Ser2lAla; (SEQ ID NO: 17) hBNP Lysl4Asn, Serl9Arg, Ser2lTrp; (SEQ ID NO: 18) hBNP Lys l4Asn, Serl9Arg, Ser20Asn, Ser21 Thr; (SEQ ID NO: 19) hBNP Lys l4GIn, Serl9Arg, Ser20His, Ser22Thr; {SEQ ID NO: 20) hBNP Lysl4Asn, Serl9Gly, Ser20His, Ser2lLeu; (SEQ ID NO: 21) hBNP Lysl4Asn, Serl9Arg, Ser20Thr, Ser2lLys; (SEQ 1D NO: 22) hBNP Lysl4Asn, Serl9Gly, Ser20Gly, Ser2lLeu; (SEQ ID NO: 23) hBNP Lysl4Asn, Serl9Arg; {SEQ ID NO: 24}
hBNP Lysl4Thr, Serl9Arg, Ser20His, Ser2lGly, Ser22Gly;(SEQ ID NO: 25) hBNP Lysl4Asn, SerI9Arg, Ser20Gly, Ser2lMet; (SEQ ID NO: 26) hBNP GIy23Met, Leu24Tyr, GIy25Arg; (SEQ ID NO: 27) hBNP GIy23Leu, Leu24Trp, Gly25Arg; (SEQ ID NO: 28) and hBNP GIy23Met, Leu24Phe, GIy25Arg (SEQ ID NO: 29).
I
Chemical Synthesis One method of producing compounds of the present invention involves chemical synthesis of the polypeptide, followed by treatment under oxidizing conditions appropriate to obtain the native conformation, that is, the correct disulfide bond linkages. This can be accomplished using methodologies well known to those skilled in the art (see Atherton, E., and Sheppard R.C., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford England (1989); Kelley, R.F. & Winkler, M.E. in Genetic Engineering Principles and Methods, Setlow, J. K., ed., Plenum Press, N.Y., vol. 12, pp 1-19 (1990);
Stewart, J. M. & Young, J. D. Solid Phase Peptide Synthesis, Pierce Chemical Co. Rockford, IL(1984)).
Polypeptides of the invention may be conveniently prepared using solid phase peptide synthesis (Merrifield, (1964) J. Am. Chem. Soc., 85:2149; Houghten, (1985) Proc. Natl.
Acad. Sci. USA 82:5132).
Solid phase synthesis begins at the carboxy-terminus of the putative peptide by coupling a protected amino acid to a suitable resin (e.g. chloromethylated polystyrene resin) as shown in Figures 1-1 and 1-2, on pages 2 and 4 of Stewart and Young supra. After removal of the a-amino protecting group with, for example, trifluoroar~tic acid (TFA) in methyiene chloride and neutralizing in, for example TEA, the next a-amino- and side-chain protected amino acid in the synthesis is added. The remaining a-amino- and, if necessary, side-chain-protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the resin. Alternatively, some amino acids may be coupled to one another forming a peptide prior to addition of the peptide to the growing solid phase polypeptide chain.
The condensation between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as the azide method, mixed acid anhydride method, DCC (N,N'-dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide)methods, active ester method (p-nitrophenyl ester method, BOP [benzotriazole-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate] method, N-hydroxysuccinic acid imido ester method, etc, and Woodward reagent K
method.
Common to chemical synthesis of peptides is the protection of any reactive side-chain groups of the amino acids with suitable protecting groups. Ultimately these protecting groups are removed after the desired polypeptide chain has been sequentially assembled. Also common is the protection of the a-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group followed by the selective removal of the a-amino-protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common in polypeptide synthesis that an intermediate compound is produced which contains each of the amino acid residues located in the desired sequence in the peptide chain with various of these residues having side-chain protecting groups attached. These protecting groups are then commonly removed substantially at the same time so as to produce the desired resultant product following removal from the resin.
Suitable protective groups for protecting the a-and e- amino side chain groups are exemplified by benzyioxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), O-chlorobenzyloxycarbonyl [Z(2Cl)], p-nitrobenzyloxycarbonyl [Z(N02)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl, (Boc), t amyloxycarbonyl (Aoc), isoboronyloxycarbonyl, adamatyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonylethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt), dimethylophosphinothioyi (Mpt) and the like.
Protective groups for the carboxy functional group are exemplified by; benzyl ester (OBzI), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that specific amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxyl groups are protected by a suitable protective group. For example, the guanidino group of arginine may be protected with vitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Mds}, 1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group of cysteine may be protected with p-methoxybenzyi, triphenylmethyl, acetylaminomethyl ethylcarbamoyle, 4-methylbenryl, 2, 4, 6-trimethy-benzyi (Tmb) etc, and the hydroxyl group of serine can be protected with benzyl, t-butyl, acetyl, tetrahydropyranyl and the like.
Stewart and Young supra provides detailed information regarding procedures for preparing peptides.
Protection of a-amino groups is described on pages 14-18, and side-chain blockage is described on pages 18-28. A table of protecting groups for amine, hydroxyl and sulfhydryl functions is provided on pages 149-151.
After the desired amino acid sequence has been completed, the intermediate peptide is removed from the resin support by treatment with a reagent, such as liquid HF and one or more thio-containing scavengers, which not only cleaves the peptide from the resin, but also cleaves all the remaining side-chain protecting groups. Following HF cleavage, the protein sequence is washed with ether, transferred to a large volume of dilute acetic acid, and stirred at pH adjusted to about 8.0 with ammonium hydroxide.
Preferably in order to avoid alkylation of residues in the polypeptide, (for example, alkylation of methionine, cysteine, and tyrosine residues) a thio-cresol and cresol scavenger mixture is used. The resin is washed with ether, and immediately transferred to a large volume of dilute acetic acid to solubilize and minimize intermolecular cross-linking. A 250 pM polypeptide concentration is diluted in about 2 liters of 0.1 M acetic acid solution. The solution is then stirred and its pH adjusted to about 8.0 using ammonium hydroxide. Upon pH adjustment, the polypeptide takes its desired conformational arrangement.
Non-peptide (amide isostere) Linkages In one embodiment of the invention, the amide linkages of BNP {-C(=O)-NH-) may be replaced with amide isostere linkages such as; -CH2-NH-, -CH2-S-, -CH2-O-, -CH2-CH2-, -CH=CH-(cis and trans), -C(=O)-CH2-, -CH(OH)-CH2-, -CH(CN)-NH-, -O-C(=O)-NH- and -CH2-SO-, by methods known in the art.
The following references describe preparation of peptide analogs which include these alternative-linking moieties: Spatola, A.F., Vega Data 1(3): "Peptide Backbone Modifications"
(General Review) (Mar 1983), Spatola, A.F., in "Chemistry and Biochemistry of Amino Acids Peptides and Proteins", B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983); Hudson, D. et al. Int. J. Pept. Prot.
Res. 14:177-185 {1979) (-CH2NH-, -CH2CH2-); Tourwe, D., et al., Structural Biology 331-333 (1989) (E, -CH=CH-); Spatola, A.F., et al., Life Sci. 38:1243-1249 (1986) (-CH2-S-); Almquist, R.G., et al., J.
Med. Chem. 23:1392-1398 ( 1980) (-C(=O)-CH2-); Jennings-White C., et al., Tetrahedron Left 23:( 1982) (-C(=O)-CH2-); Szelke, M., et al., EP
Application No.45655 {-CH(OH)-CHI); Holladay, M.W., et al., Tetrahedron Lett 24:4401-4404 (1983) (-C(OH)-CH2-); Hruby, V.J. Life Sci 31:189-199 (1982) (-CH2S-); and Cho, C.Y. et a1, Science 261:1303-1305 ( 1993 )(-O-C(=O)-NH-).
Recombinant Synthesis In a further embodiment, the present invention encompasses a nucleic acid, preferably DNA, encoding the protein component of a BNP variant that contain the amino acid substitutions described herein. The invention further comprises an expression control sequence operably linked to the DNA molecule, an expression vector, preferably a plasmid, comprising the DNA molecule, where the control sequence is recognized by a host cell transformed with the vector.
The compounds of the present invention may be made by a process which includes the steps of synthesizing (by art standard techniques) nucleic acid sequences encoding any of the amino acid sequences described herein, ligating the nucleic acid sequence into a suitable expression vector capable of expressing the nucleic acid sequence in a suitable host, transforming the host with the expression vector into which the nucleic acid sequence has been ligated, culturing the host under conditions suitable for expression of the nucleic acid sequence, whereby the protein encoded by the selected nucleic acid sequence is expressed by the host. In this process, the ligating step may further contemplate ligating the nucleic acid into a suitable expression vector such that the nucleic acid is operably linked to a suitable secretory signal, whereby the amino acid sequence is secreted by the host. The secretory signal may be selected, for example, from the group consisting of the leader sequence of stlI, ecotin, lama, herpes gD, lpp, alkaline phosphatase, invertase, and alpha factor and is preferably stlI.
By way of illustration, with expression vectors encoding wild-type BNP in hand, site specific mutagenesis (Kunkel et al., Methods Enzymol. 204:125-139 [1991]; Carter, P., et al.. Nucl. Acids. Res.
13:4331 [1986]; Zoller, M. J. et al.,Nucl. Acids Res. 10:6487 [1982]), cassette mutagenesis (Wells, J. A., et al., Gene 34:315 [1985]), restriction selection mutagenesis (Wells, J. A., et al., Philos. Trans, R. Soc. London SerA 317, 415 [ 1986]) or other known techniques may be performed on the BNP
DNA. The mutant DNA can then be used in place of the parent DNA by insertion into the aforementioned expression vectors. Growth of host bacteria containing the expression vectors with the mutant DNA allows the production of mutant BNP, which can be isolated as described herein.
The host cell may be prokaryotic or eukaryotic. Prokaryotes are preferred for cloning and expressing DNA sequences to produce parent polypeptides, segment substituted polypeptides, residue-substituted polypeptides and polypeptide variants. For example, E. coli K12 strain 294 (ATCC No. 31446) may be used as E. coli B, E. toll- X 1776 (ATCC No. 31537), and E. toll c600 and c600hf1, E. toll W3110 (F-, gamma-, prototrophic /ATCG No. 27325), bacilli such as Bacillus subrilis, and other enterobacteriaceae such as Salmonella -typhimurium or Serratia marcesans, and various pseudomonas species. The preferred prokaryote is E. toll W3110 (ATCC 27325). When expressed by prokaryotes the polypeptides typically contain an N-3S terminal methionine or a formyl methionine and are not glycosylated. In the case of fusion proteins, the N-terminal methionine or formyl methionine resides on the amino terminus of the fusion protein or the signal sequence of the fusion protein. These examples are, of course, intended to be illustrative rather than limiting.
In addition to prokaryotes, eukaryotic organisms, such as yeast cultures, or cells derived from multicellular organisms may be used. In principle, any such cell culture is workable. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a reproducible procedure (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of such useful host cell lines are VERO and HeLa cells, Chinese Hamster Ovary (CHO) cell lines, W138, 293, BHK, COS-7 and MDCK cell lines.
Preferred expression vectors of the present invention may be selected from, for example, pBR322, phGHl, pB0475, pB1537, pRITS, pRlT2T, pKK233-2, pDR540, pPL-lambda and pB1537, with the most preferred vector being pB1537.
A preferred vector for direct expression of the BNP variants of the present invention is pB1537 (Cunningham et al. ( 1994) EMBO J. 13:2508-2515) and contains origins of replication for E. toll, the alkaline phosphatase promoter, the stlI signal sequence, the BNP variant gene, and the ampicillin resistance gene. Other preferred vectors are pRlTS and pRlT2T (Pharmacia Biotechnology). These vectors contain appropriate promoters followed by the Z domain of protein A, allowing genes inserted into the vectors to be expressed as fusion proteins.
Other preferred vectors can be constructed using standard techniques by combining the relevant traits of the vectors described herein. In this instance a vector containing the origins of replication for phage and E.
toll, which allow it to be shuttled between such hosts, is used thereby facilitating both mutagenesis and expression (Cunningham, B., et al., Science 243:1330-1336 [1989]; Wells, J. &
Cunningham, B., WO
90/04788 published 3 May 1990). Relevant traits of the vector include the promoter, the ribosome binding site, the variant gene or gene fusion (the Z domain of protein A and a BNP variant and its linker), the signal sequence, the antibiotic resistance markers, the copy number, and the appropriate origins of replication.
Gene Fusions A variation on the above procedures contemplates the use of gene fusions, wherein the gene encoding a BNP variant is associated, in the vector, with a gene encoding another protein or a fragment of another protein. This results in a BNP variants being produced by the host cell as a fusion with another protein. The "other" protein is often a protein or peptide which can be secreted by the cell, making it possible to isolate and purify the desired protein from the culture medium and eliminating the necessity of destroying the host cells which arises when the desired protein remains inside the cell. Alternatively, the fusion protein can be expressed intracellularly. It is useful to use fusion proteins that are highly expressed.
The use of gene fusions, though not essential, can facilitate the expression of heterologous proteins in E. toll as well as the subsequent purification of those gene products (Harris, T. J. R. in Genetic Engineering, Williamson, R., Ed., Academic, London, Vol. 4, p. 127[1983]; Uhlen, M. & Moks, T., Methods Enzymol.
185:129-143 [1990]). Protein A fusions are often used because the binding of protein A, or more specifically the Z domain of protein A, to IgG provides an "affinity handle" for the purification of the fused protein (Nilsson, B. & Abrahmsen, L. Methods Enrymol. 185:144-161 [1990]). It has also been shown that many heterologous proteins are degraded when expressed directly in E. toll, but are stable when expressed as fusion proteins (Marston, F. A. O., Biochem J. 240: 1 [1986]).
BNP variants expressed as fusion proteins may be properly folded or may require folding to obtain the native structure. The properly folded fusion protein may be active and useful as pharmaceutical drug.
_ 17-When denaturing and refolding are needed, typically the protein is treated with a chaotrope, such a guanidine HCI, and is then treated with a redox buffer, containing, for example, reduced and oxidized dithiothreitol or glutathione at the appropriate ratios, pH, and temperature, such that the protein of interest is refolded to its native structure.
More preferred would be the correctly folded native protein that is obtained from the fusion protein by methods known in the art. Fusion proteins can be cleaved using chemicals, such as cyanogen bromide, which cleaves at a methionine, or hydroxylamine, which cleaves between an Asn and Gly. Using standard recombinant DNA methodology, the nucleotide base pairs encoding these amino acids may be inserted just prior to the 5' end of the BNP variant gene.
Alternatively, one can employ proteolytic cleavage of fusion proteins, which has been recently reviewed (Carter, P. (1990) in Protein Purification: From Molecular Mechanisms to Large-Scale Processes, Ladisch, M. R., Willson, R. C., Painton, C. C., and Builder, S. E., eds., American Chemical Society Symposium Series No. 427, Ch 13, 181-193).
I'roteases such as Factor Xa, thrombin, subtilisin and mutants thereof, have been successfully used to cleave fusion ~~roteins. Typically, a peptide linker that is amenable to cleavage by the protease used is inserted between the "other" protein (e.g. the Z domain of protein A) and the protein of interest, such as a BNP
variant. Using recombinant DNA methodology, the nucleotide base pairs encoding the linker are inserted between the genes or gene fragments coding for the other proteins. Proteotytic cleavage of the partially purified fusion protein containing the correct linker can then be carried out on either the native fusion protein, or the reduced or denatured fusion protein.
Purification and characterization Purification and characterization of BNP variants may be carried out by any art standard technique.
For example, BNP variants may be purified from the broths of E. toll cultures grown in 10 liter fermentors by batch reverse phase chromatography, cation exchange chromatography and C 18 reverse phase HPLC.
Utility and Administration Compounds of the present invention have natriuretic, diuretic and vasorelaxant activity and may inhibit the release of aldosterone and renin. Thus_ these cnmnnunrlc fir, mcP
ae thor~..o..+:.. .,..,....,. ._ .L_ treatment of various pathological conditions associated with water or electrolyte imbalance and hypertension, especially renovascular hypertension. The compounds of the present invention can be used analogous to ANP
peptides in therapeutic and prophylactic measures. BNP has potent diuretic and systemic vasorelaxant properties (Kambayashi et al., (1990) Biochem. Biophys. Res. Commun. 173:599-605). Increased levels of BNP have been reported in congestive heart failure and during acute exercise (Kohno, M., et al., ( l 992) Metabolism 41:1273-1275; Mukoyama M., et al., ( 1991 ) J. Clin. Invest.
87:1402-1412). Both BNP and ANP
are unregulated proportionally during chronic hypoxia (Am. J. Physiol. (1994) 266:L308-L315). Therefore any condition resulting in blood volume or blood pressure overload is a candidate. Such conditions include, for example, cardiac diseases such as congestive heart failure (CHF), cardiomyopathies, myocarditis, valve diseases such as rheumatic heart disease, coronary heart disease such as myocardial ischemia and infarction, arrhythmias such as paroxysmal atrial tachycardia and atrial fibrillation and flutter, congenital heart defects, restrictive disorders such as pericarditis and cardiac tamponade, drug induced cardiac diseases, hypertension, including essential hypertension, secondary, pulmonary and portal hypertension, kidney diseases such as chronic renal failure and nephrosis, cirrhosis, primary atdosteronism, Cushing's disease, preeclampsia, toxemia of pregnancy, premenstrual syndrome, nephrotic syndrome and hepatic cirrhosis, pulmonary disease, and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
The compounds and compositions can be administered to humans in a manner similar to ANP and other similar therapeutic agents. The dosage to be administered will depend on the usual factors including; age, weight, sex, condition of the patient, specific disorder being treated, and route of administration. In general, the dosage required for therapeutic efficacy will range from about 0.01 to 1000 mg/kg, more usually 0. I to 25 mg/kg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time until the desired therapeutic benefits have been obtained.
The present invention also provides compositions containing an effective amount of compounds of the present invention, including the nontoxic addition salts, amides and esters thereof, which may, alone, serve to provide the above-recited therapeutic benefits. The peptide compounds may be formulated into the compositions as neutral or salt forms. Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, 2-ethylamino ethanol, histidine, procaine, and the like. Such compositions can be provided together with physiologically tolerable Liquid, gel or solid diluents, adjuvants and excipients.
Typically, such compositions are prepared as sterile, injectable liquid solutions or suspensions.
Compositions may also be emulsified. The active ingredient is often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient.
Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like. For a more detailed description of the foregoing see a standard pharmaceutical text such as Remingron's Pharmaceutical Sciences. Mack Publishing Co. Easton, PA. (1970).
In addition to the compounds of the present invention which display natriuretic, diuretic or vasorelaxant activity, compounds of the present invention can also be employed as intermediates in the synthesis of such useful compounds. Alternatively, by appropriate selection, compounds of the present invention whose activity levels are reduced or eliminated entirely can serve to modulate the activity of other diuretic, natriuretic or vasorelaxant compounds, including compounds outside the scope of the present invention, by, for example, binding to alternate receptors, stimulating receptor turnover, or providing alternate substrates for degradative enzyme of receptor activity and thus inhibiting these enzymes or receptors. When employed in this manner, such compounds can be delivered as admixtures with other active compounds or can be delivered separately, for example, in their own carriers.
WO 98/17b90 PCT/US97I18384 Pharmaceutical compositions may be prepared alone or in combination with a C
receptor ligand such as, for example SC-46542, or an inhibitor of the enzyme primarily responsible for the degradation of BNP such as SCH 32615, SCH 344826, SCH 39370, SQ 2900072, thiorfan and UK 69578.
Sythetic compounds which occupy the C-receptor increase the concentrations of circulating BNP and may be used in combination with the peptides of the present invention. U.S. Patent No 4,740,499 describes a method of prolonging or enhancing the bioactivity of an atrial peptide using two specific inhibitor of endopeptidase 24. I 1, thiorphan or kelotorphan administered simultaneous with the atrial peptides.
The following examples are offered by way of illustration and not by way of limitation. The disclosures of all citations in the specification are expressly incorporated herein by reference.
EXAMPLES
EXAMPLE I
Monovaient phage display (Bass et al., (1990) Protein Struct. Funct. Genent., 8:309-314; Lowman et al., (1991) Biochemistry, 30:10832-10838) was used to screen variants of BNP for ones that bind tightly to human NPR-A and not to NPR-C. After multiple enrichment screen BNP variants that bound preferentially to NPR-A were isolated.
Construction of Libraries: Monovalent phagemid libraries (Lowman, H. B. et al., { 1991 ) Methods:
Companion Methods Enzymol. 3:205-216) of BNP were generated by site directed mutagenesis (Kunkel, T.
A., et al., {1991) Methods Enzymol. 204:125-139). Each library contained three, four or five codons fully randomized for all 20 amino acids (as shown in Table 1 below) using oligonucleotides that mutated target codons to NNS sequences (where N represents a mixture of all four bases and S
is a mixture of both C and G).
Stocks of approximately 1014 phagemid per mL were prepared from PEG
precipitates of culture broths from XL-1 Blue cells containing the plasmid and superinfected with K07 helper phage.
Selection of receptor specific BNP variants: Before negatively selecting for NPR-A specific mutants the libraries were pre-enriched for NPR-A binders using NPR-A IgG fusion (A-IgG) (Bennett et al. ( 1991 } J.
Biol. Chem. 266:23060-23067). Wells of Maxisorb microtiter plates (Nunc cat.
#439454) were coated with 100 pl of 2 pg/ml Rabbit anti-human Fc Antibody (Jackson Immuno Research cat.
#309-006-008) in 50mM
sodium bicarbonate (pH 9.6) at 4° C overnight. The coat solution was discarded and the wells were blocked for one hour at room temperature with 5% skim milk in 25mM sodium carbonate (pH9.6). After washing with 0.01% Tween~ in PBS, NPR-A IgG was added at 1 pg/ml in binding buffer (2% skim milk in PBS) for 1 hour at room temperature. The wells were washed again. 10 pl of phage stock (250 fold concentrated from growth media, stored in PBS) was brought up to 100 ~ l with binding buffer and incubated in the wells for 2 hours at room temperature. After washing 20-30 times, the phage were eluted with 100 p l 0.2M glycine (pH 2.0), then neutralized with 13 pl 1M Tris Base and titered by infecting E. toll (XL1-blue Stratagene, Inc., San Diego, CA). The remaining selected phagemid particles were propagated for the use in the next selection cycle.
Receptor specific selection was done by adding competing NPRC IgG (Bennett et al. (1991) supra) during the phage binding step in the NPR-A IgG coated wells. This was done in seven rounds of selection with the amount of competing CIgG increasing from 20nM to 200nM. For each library, there were control wells that lacked either the competing CIgG or both the AIgG coat and the CIgG.
ELISA measurement of phagemic amities for NPR-A and NPR-C: Phage ELISA were determined according to the method of Cunningham, B. C. et al., ( 1994) EMBO J., 13:2508-25 I 5. Microtiter plates (Nunc, Maxisorb, 96 wells) were coated with purified A-IgG (at lltg/ml) in 50 mM
sodium bicarbonate (pH 9.6) at 4 C overnight. The plates were blocked with 5% skimmed milk and washed with PBS, 0.01% TWEEN 20.
Serial dilutions of competing receptor {A-IgG or C-IgG) and a subsaturating concentration of BNP-phagemid were added to wells in 100 pi of binding buffer (2% skim milk in PBS). After 2 h the plates were washed, and the bound phagemid stained with sheep anti-M 13 HRP conjugate antibody (Pharmacia #279402-Ol ), and assayed. Affinities (EC50) were calculated as the concentration of competing receptor that resulted in half maximal phagemid binding.
Results The amino acid residues found at each randomized position after 7 rounds of selection are shown in Table I. 8 segments of 3 to 5 amino acids randomized by primer mutagenisis are indicated at the top of the figure, immediately above the numbering for 32 amino acid human BNP, the sequence of which is shown in single letter amino acid code. Cysteine residues 10 and 26 were not mutagenized since they form a disulfide bond essential for binding to NPR-A. The substitution of K3 for D (Display BNP) is analogous to the acidic substitution R3D in ANP used to facilitate secretion expression in E. coli (Cunningham et al. (1994)).
Individual clones were picked and the DNA sequence was determined. The number of times a particular sequence was observed is shown on the left. The ratio of mutant BNP phagemid binding affinity to ANP
phagemid binding to A-IgG is shown on the right. Mutations that occur outside of the mutagenesis window result from polymerase misincorporation mutations that render a selection advantage to the mutant BNP
phagemid (Cunningham et al. ( 1994)).
Description of Related Disclosures l0 Three natriuretic peptides, type A, type B and type C, have been identified which play a role in hemodynamics (Rosenzweig, A., and Seidman, C.E., (1992) Annu. Rev. Biochem.
60:229-255 (1991);
Ruskoaho, H. ( 1992) Pharmacol. Rev. 44:479-602). The bioactive peptides are derived from prohormones each of which arises from a distinct gene (Steinhelper, M., (1993) Circ. Res.
72:984-992). The genes are expressed in a tissue specific manner (Seilhamer et al., ( 1989) Biochem.
Biophys. Res. Commun. 165, 650-658) and are present in the systemic circulation. The type A (ANP) and type B
(BNP) peptides are derived from the myocardium, whereas the type C (CNP) peptide is thought to be derived from endothelial cells. Each of the bioactive peptides, derived from the C-terminal portion of the respective prohormone, exhibit a high degree of sequence homology within their 17 amino acid ring structures.
Both ANP and BNP are primarily cardiac hormones that are released by the heart in its role as an endocrine organ regulating fluid and electrolyte hemostasis. Human BNP is stored in the atrium and ventricle (Hosodo et al., (1991) Hypertension 17:1152-1156) as the mature hormone whereas ANP is stored in cardiocytes as the precursor. BNP was first isolated from porcine brain (Sudoh et al., ( 1988) Nature 332:78-81 ) although it is synthesized in and secreted into the circulation from the porcine heart (Aburaya et al., ( 1989}
Biochem. Biophys. Res. Commun. 158:360-368). Porcine BNP circulates as a 26 or 32 amino acid poiypeptide and exhibits structural homology with ANP (Sudoh et al., (1988) Nature 332:78-81 ). The cDNA sequence encoding human BNP precursor, hBNP[1-108] has been determined (Sudoh, et al., (1989) Biochem. Biophys.
Res. Commun. 159:1427-1434). Human BNP has also been isolated as a 32 amino acid poiypeptide from human atrium the circulating form being identical to sequence residues [77-108] of the human BNP precursor molecule.
The action of the natriuretic peptides is mediated through the binding of the peptides to cellular receptors. There are at least three structurally distinct cellular receptors (Chang et al., ( 1989) Nature 341:68-72). NPR-A (Chinkers et al., ( 1990) Nature 338:78-83) and NPR-B (Lowe et al., (1990) Nucleic Acids Res.
18:3412) receptors have homologous extracellular domains of approximately 440 amino acid residues with a single transmembrane domain. Both possess intracellular guanyl cyclase and tyrosine kinase-like domains (Chang et al. ( 1989), supra). The extracellular domain of the third receptor NPR-C is homologous to the NPR-A and NPR-B extracellular domains but contains only a 37 amino acid intracellular domain of unknown function (Fuller et al. (1988) J. Blot. Chem. 263:9395-9401 ). The role of these receptors in regulating blood pressure is unclear but NPR-A is thought to be responsible for most of the biological effects of ANP and BNP
(Maack T., ( 1992) Annu. Rev. Physiol.. 54, I 1-27).
8NP has peripheral and central nervous system action similar to those of ANP
including natriuretic, diuretic and hypotensive effect and smooth muscle relaxation (Yoshimura et al., (1991) Circulation 84:1581-1588; Mukoyama et al., ( 1991 ) J. Clin. Invest. 87:1402-1412). Binding of the bioactive peptide to the NPR-A
cellular receptor results in a number of actions including a decrease in blood pressure due in part to relaxation of vascular smooth muscle, an increase in sail and water excretion, transduction of plasma water to the interstitium and an inhibition of the release or action of aldosterone, angiotensin II, endothelin, renin and vasopressin.
Maintenance of normal extracellular fluid volume depends primarily on the excretion of sodium (natriuresis) and water (diuresis) by the kidneys. These are, in turn, primarily determined by (1) the rate at which plasma is filtered at the glomeruius (glomerular filtration rate, or GFR) and (2) the degree to which sodium is actively reabsorbed along the renal tubule (with water presumably following passively). Sodium reabsorption is regulated, in part, by the adrenal steroid hormone aldosterone, in part by blood pressure, hematocrit and plasma viscosity and in part by the various natriuretic factors or hormones (del3old, A.J. et al., (1981) Life: Sciences 28:89-94; Garcia R., (1982) Experientia 38:1071-73;
Currie, M.S. et al., (1983) Science 221:71-73; Flym~. T.G. et al., (1983) Biochem. Biophys. Res. Commun. 117:859-865; Currie, M.G. et al., ( 1984) Science 223:67-69; and Kangawa, K. et al., (1984) Biochem. Biophys.
Res. Commun. 118:131-139;
Mukoyama et al., (1991) J. Clin. Invest. 87:1402-1412).
It is known that the serum half life of natriuretic peptides that act as hypotensive regulators is relatively short (Crozier, LG. et al., (1986) The Lancet II 1242-1245) and that these peptides are removed from the blood stream by several mechanisms including proteolysis and receptor mediated endocytosis by NPR-C
(Maack ( 1992) supra). Since both ANP and BNP have a short half life with poor or non-existent peroral absorption, synthetic analogues of BNP with longer circulatory duration and favorable in vivo characteristics are desirable.
Summary of the Invention The present invention provides compounds which have natriuretic, diuretic, and/or vasodilatory activity in mammals. The compounds of this invention are related to the B-type natriuretic peptide (BNP) and exhibit decreased binding affinity to the natriuretic peptide clearance receptor (NPR-C) compared to BNP.
Advantageously, the compounds have enhanced potency defined in terms of NPR-A
receptor affinity and cGMP production. In preferred embodiments, the compounds and related compositions allow for a potent NPR-A receptor stimulation providing for low dose pharmaceutical formulations.
The compositions of the present invention are useful in therapeutic and prophylactic methods for inducing natruresis, diuresis or vasodilation or associated processes.
In one embodiment, the compounds of the present invention have a decreased binding affinity for the human clearance receptor (hNPR-C) compared to wild-type BNP. Preferably, according to this aspect of the invention, the compounds exhibit the same or an increased affinity for the type-A natriuretic peptide receptor.
More preferably, according to this aspect of the invention, at least one of amino acid residues Xaal9, Xaa23, Xaa24 or Xaa25 of wild-type 8NP is selected according to the following scheme in order to produce a BNP
variant or related compound of the invention:
_2_ WO 9811?690 PCT/US97/18384 Xaal9 is selected from the group consisting of 5er, Arg, Ala, Asn, Gly and conservative substitutions thereof;
Xaa23 is selected from the group Gly, Met, Phe, Leu and conservative substitutions thereof Xaa24 is selected from the group Trp, Tyr, Phe, and conservative substitutions thereof; and Xaa25 is selected from the group Gly, Arg, and conservative substitutions thereof.
In a further preferred embodiment, the compounds of the present invention are represented by Formula A-R 1-Cys-Phe-G ly-Arg-Lys-Met-Asp-Arg-I le S-S
B-R2-Cys-Xaa~s-Xaa24-Xaa~3-Ser-Ser-Ser-Xaal9 wherein:
A is selected from the group consisting of H, C 1-C6 alkanoyl and Thr-Ala-Pro-Arg (SEQ ID NO: 30);
RI is absent or selected from the group consisting of a peptide from between 1 and 10 amino acids, Gly-Ser-Gly-, Val-Gln-Gly-Ser-Giy (SEQ ID NO: 3 I ) and Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly (SEQ ID
NO: 32);
Xaal9 is selected from the group consisting of Arg, Ala, Asn, Gly and Ser;
Xaa23 is selected from the group Gly, Met, Phe, Leu;
Xaa24 is selected from the group Trp, Tyr, Phe;
Xaa25 is selected from the group Gly, and Arg;
R2 is selected from the group consisting of a 1 to 6 amino acid peptide and Lys-Val-Leu-Arg-Arg-His-(SEQ ID NO: 33); and B is selected from OR3 and NR3R4 where R3 and R4 are independently selected from H, C I-C6alkyl, C6-Cl2aryl and C6-Cl2aryl-CI-C6alkyl.
In one embodiment, the composition of the present invention is a polypeptide and the invention encompasses a composition of matter comprising a nucleic acid, preferably DNA, encoding the polypeptide of the invention. According to this aspect, the invention further comprises an expression control sequence operably linked to the DNA molecule, an expression vector, preferably a plasmid, comprising the DNA
molecule, where the control sequence is recognized by a host cell transformed with the vector, and a host cell transformed with the vector.
In one embodiment, the polypeptide compositions of the present invention may be made by a process which includes the steps of synthesizing nucleic acid sequences encoding any of the amino acid sequences of the invention, ligating the nucleic acid sequence into a suitable expression vector capable of expressing the nucleic acid sequence in a suitable host, transforming the host with the expression vector into which the nucleic acid sequence has been ligated, and culturing the host under conditions suitable for expression of the nucleic i acid sequence, whereby the protein encoded by the selected nucleic acid sequence is expressed by the host.
Preferably, the polypeptide is then recovered from the host cell culture. In this process, the ligating step may further contemplate ligating the nucleic acid into a suitable expression vector such that the nucleic acid is operably linked to a suitable secretory signal, whereby the amino acid sequence is secreted by the host.
The present invention further extends to therapeutic applications for the compounds described herein.
Thus the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the compound of the invention. Pharmaceutical compositions comprising these compounds can be used in the treatment or prophylaxis of various pathological conditions associated with water or electrolyte imbalance and hypertension, especially renovascuiar hypertension. Such conditions include, for example, congestive heart failure (CHF), nephrotic syndrome and hepatic cirrhosis, pulmonary disease, and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
Brief Description of the Drawings Figure 1: Concentration responses for cGMP second messenger production on human natriuretic peptide receptor-A expressed in 293 cells. Values for the concentration of natriuretic peptide giving half maximal stimulation of c-GMP production are given in the Figure. Values are plotted as average t standard deviation (n=3). In the Figure ANP refers to the 28 amino acid residue peptide reported by Kangawa et al., Biochem. Biophys. Res. Comm. ( 1984) 118(/ ):13 i-139; BNP refers to SEQ ID NO: ( ; BNP02 refers to SEQ ID NO: 2; BNP04 refers to SEQ ID NO: 7; BNPOS refers to SEQ ID NO: 7 and ANF 131 refers to SEQ ID NO: 50. The results show that the peptides are foil agonists on this receptor, with potency similar to BNP.
Figure 2: Stimulation of cGMP second messenger production on human natriuretic peptide receptor-A
expressed in 293 cells. cGMP values for natriuretic peptide stimulation of natriuretic peptide receptor-A at 4 x 10-8 M are given in the Figure. cGMP values are plotted as average t standard deviation (n=3). In the Figure ANP refers to the 28 amino acid residue peptide reported by Kangawa et al., Biochem. Biophys. Res.
Comm. ( 1984) 118(1 ):131-139; BNP refers to SEQ ID NO: 1; BNP02 refers to SEQ
ID NO: 2; BNP04 refers to SEQ ID NO: 7; BNPOS refers to SEQ ID NO: 7; ANF 131 refers to SEQ ID NO:
S0; BNP06 refers to SEQ
ID NO: 48; and BNP07 refers to SEQ ID NO: 49. The results show that the peptides are full agonists on this receptor, with potency similar to BNP.
Figure 3: Alignment of the amino acid sequence of BNP from mammalian species;
human (Sudoh, et al., (1989) Biochem Biophy. Res. Common. 159:1427-1434) (SEQ ID NO 1), canine (Seilhamer et al., (1989) Biochem. Biophys. Res. Common 165:650-658) (SEQ ID NO 44), porcine (Sudoh, T., et al., (1988) Nature 332:78-81 ) (SEQ ID NO 45), rat (Kojima et al., ( 1989) Biophys.
Biochem. Res. Common. 159:1420-1426) (SEQ ID NO 46) and mouse (Ogawa et al., ( 1994) J. Clin. Invest 93:19/ I-1921 ) (SEQ ID NO 47). Gaps (-) have been introduced to maximize alignment.
Detailed Description of the Preferred Embodiments Definitions Terms used in the claims and specifcation are defined as set forth below unless otherwise specified.
The term amino acid or amino acid residue, as used herein, refers to naturally occurring L amino acids or to D amino acids as described further below with respect to variants. The commonly used one- and three-KL1 . 1u\.I~.I'\-vll I.\l:lll..\ t):p : ~;S- t m>1 - ~:-,. ~.t ~ ,-.l_.L1 1 ~:~ _t,,,i L 1- r t:~ ts;~ =:C3;1-l~l~~.;,:1~ .;
. , _ .a:,~~_ _:... CA 02269656 1999-04-22 .i., _ _. . ..
.., .~.. .___ ._ letter abbreviations for amino acids are used herein (H~ce Alberts et aL, :vtofecular Biology of the CeII, Garland Publishing. Inc., New York (3d ed. 1994)).
As used herein the turns "BNP", "rype-B nattiuretic peptide", "wild-type H"Tp"
and the like are used interchangeably and refer to a naturahy occurring :mammalian BNP or recombinant BNP. The sequence ef 13NP from mammalian species is known, for example, human (Sudoh, et al..
(19891 Biochem Biophy. Res.
Common. 159:1421-1434) (SEQ ID NO 1), canine (Seilhamer et al., (1989) Biochem. t3iophys. Res. Comrnun 165:650-658) (SEQ ID NO 44), porcine (Sudoh, T., et al_, ( 1988) Nature 332:16-81) (SEQ ID NO 45) and rat (Kojima et ai., (1989) Hiophys. l3iochem. RPs_ Common. 159:1420-1426 (SEQ 1D
NO 4~. 'Ihe terms ~~hu~ g~Tp," "hBNP" and the like, mean ttte 32 amino acid residue pep::de reported by Sudoh, T., et at., (lava) s~p~a.
1~ ~~~.3~.~, having the following primary amino acid sequence:
I 2 3 4 5 6 7 8 9 10 11 I2 13 ld 15 ~6 Ser-Pro-1.ys-filet-val-Gin-Gty-Set-Giy-Cys-Pbe-Gly-Arg->rys-MCt-G
t7 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 erg-lie-Ser-5er-Ser-Ser-Gly-Leo-uly-Cys-Lys-'~ ai-Lea-Arg-Arg-His (5EQ ID NO: 1) The integers above specific residues of this structure define the residue position number. ?his residue position number is used in conjunction wire t:ze threo letter amino acid nomenclature to designate d;e residue at which a substitution is :made in the BNP related compounds of this invention. Thus for exarnple,.in a BNP :elated compound in which arginine (.4rg) replace serine (Scr) at residue position number i 9 of wild-type BNP, tide ~G nomenclature 'BNP Seri 9Arg" or the like is used. utuldple substitutions are designated in the same manner with a comma separating each substitution. Thus far cxanpte in a HNP :'elated compound in which phenylalanine (Phe), tryptophan (Trp) and arginine (A:g) replace amino acids 23, Z4 a.-td 25 respectively of auman BNP t.'~e nomenclature "hBNP GlyZ3Phe, ;.euZ4Trp, GIy25Arg" is sued.
The terms "BNP variant" and "BNP related cempaund" are dot-teed as a compound having at least qualitative biological activity in cotrunon witft tire polypeptide of SEQ iD
N0: 1 and having at least one amino acid substitution as dafined he: tin for conferring receptor specificity.
Preferred arnino acid substitutions arc outlined in 'Cable I of Example L The qualitating biological activity refeaed to a the capacity to stimulate cyclic CMP production :n ells expressing the guaaylate cyclsse-linked naa:uretic peptide recaptor NPR-A.
'fhe NPR-A receptor amino acid sequence deduc.~.d from eDNAs encoding the receptor is Jmown (Chinkers et al., (1989) Nature 338:78-83; Lowe et aJ_ (1989) EMBO 1. 8:1377-1384), Functional expression of the NPR-A receptor cDNA in cultured r~arttrnalian ec!!s which confess both ANP and HT,IP birtdir<g and guanylate cyclase activity is also known (Chinkets, es ai., ( 1989) Nature 338:TB-83).
Assay systems for tneastuernent of birtdiag affinities for NPR receptors as wall as eGMP accumulation are known (see, International , Application No. PCTlUS94I12591).
Additionally, irt prei'erred embo~dimenrs, the $NP variant or $NP related campound of the present 1 invention is believed to tampon with axy wild-type.~..:'~1P (Kaagawa etal..
(1984) Biochera- Biophys. Ras.
Corsmu~. 118(1):13'-I39) of BNP for binding to the NPR-A receptor and thereby inducing cGMP
accumt:latic;n. Therefore, without limitation to any one theory, ~qualitating biological activity may be defined as the ability to compete with a.~y wild-t-~e BNP or ANP for binding to NPR-A
r:ceptor thereby inducing AMENDED SHEET
cGMP accumulation. As will be appreciated from the foregoing, the term "compete" and "ability to compete"
are relative terms. Thus the terms, when used to describe the activity of the BNP variant, mean a BNP variant that when added in a 10-fold molar excess to wild-type BNP or wild-type ANP in a standard receptor binding assay produces at least a 50% inhibition of binding of the wild-type BNP or wild-type ANP. Preferably the BNP variant will produce at least a 50% inhibition of binding in a 5-fold molar excess and most preferably at least a 2-fold molar excess. A most preferred BNP variant or BNP related compound of the present invention will produce at least a 50% inhibition of binding when present in a 1:1 stoichiometric ratio with wild-type BNP
or wild-type ANP. For assay systems to measure binding of hBNP, hANP and the compounds of this invention to hNPR-C and hNPR-A see: Bennett et al., ( 1991 ) J. Biol. Chem. 266:23060-23067; Cunningham et al., (1994) J. EMBO 13:2508-2515; Chinkers et al., (1989) Nature 338:78-83 and Fuller et al., (1988) J. Biol.
Chem. 263:9395-9401; Schenk, D. B., et al., (1985) Biochem. Biophys. Res.
Comm. 127:433-442;
Scarborough, R. M., (1986) J. Biol. Chem. 261:12960-12964 and WO 90/01940.
A characteristic of the BNP related compound or BNP variant of the present invention is a decreased affcnity for the natriuretic clearance receptor NPR-C. Therefore variants or compounds of the present invention I S have at least qualitative biological activity in common with wild-type BNP
but have a decreased affinity for the clearance receptor NPR-C when compared to BNP in a standard receptor binding assay as described herein (see Bennett et al., ( 1991 ) J. Biol. Chem. 266:23060-23067).
The BNP variants and compounds of the present invention are, in general, homologous amino acid sequences of the rat, porcine, canine or other mammalian BNP's or homologous amino acid sequences of the sequence of SEQ ID NO: 1 including homologous in vitro generated variants having the qualitative biological activity defined above. Homology with respect to the BNP variants of the present invention is defined as the percentage of amino acid residues in a candidate sequence that are identical with either the amino acid residues in SEQ ID NO: I, the amino acid sequence of a mammalian BNP or a composite sequence as defined herein after aligning the sequences and introducing gaps if necessary to achieve the maximum identity (Figure 3}, No N- or C- terminal extension or deletion in the candidate sequence shal) be construed as reducing identity.
"Composite amino acid" within the present invention refers to an alternate amino acid having the same position in the 32 amino acid residue structure as human BNP from other mammalian vertebrate species. Therefore, an amino acid substitution referred to as a composite amino acid substitution replaces the identified amino acid with the equivalent or composite amino acid from another mammalian species. A
composite BNP sequence is defined as having at least one amino acid from the wild-type sequence replaced with a composite amino acid from another mammalian species.
Therefore, the invention contemplates a BNP variant having at least the qualitative biological activity as defined above and having, for example, at least about 75% amino acid homology with the polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: I lacking the 5 carboxyl terminal amino acid residues and/or the 9 amino terminal residues. The BNP variant amino acid sequence preferably will share at least 80%, more preferably, greater than 85% sequence homology with the sequence of SEQ ID NO:
I. However, a BNP
variant or related compound may exhibit less than SO% sequence homology with the sequence of SEQ ID NO:
1 and still retain the characteristics of the BNP variant or BNP related compound as defined above.
Included in the definition of BNP variant or BNP related compound of the present invention are amino acid sequence variants of the SEQ ID NO: 1 wherein an amino acid in addition to those described herein which confer receptor specificity has been substituted by another residue, including predetermined mutations (e.g.
site directed PCR mutagenesis); other composite amino acid substitutions from other mammalian species of BNP such as those listed above and other naturally occurring variants of the foregoing and human sequences.
According to this aspect of the present invention reference is made to Figure 3 and the amino acid sequence data presented therein. Also included is a BNP variant as described above wherein the BNP variant has been modified by substitution, chemically, enzymatically, or by other appropriate means with a moiety other than a naturally occurring amino acid, it being understood that the variant will have the qualitative biological activity described above. Exemplary non-naturally occurring amino acid substitution include those described herein below.
Additionally, amino acid sequence variants can be generated by substitutions which utilize the D rather than L form of an amino acid. This is especially useful in stabilizing the BNP
variant of the present invention against enrymatic degradation. Such stabilizing amino acid substitutions have been described for ANP (Mutt and Veber, ( 1987) Endocrin. and Metab. Clin. N. Amer. 16(1):19-41 ). Such structure/activity studies of ANP
analogues including the introduction of D-amino acids where appropriate can be conducted for BNP and are included in the definition of BNP variant or BNP related compound. Thus the term variant is meant to include, in addition to those substitutions described herein for receptor specificity, substitutions that confer increased stability of BNP molecules such as chiral amino acid replacements which may offer resistance to metabolic degradation. The present invention is meant to include BNP variants and related compounds that have decreased susceptibility to hydrolysis by neutral endopeptidase 24. I 1 (EC3.4.24.1 1 ) (NEP) and angiotensin converting enzyme (ACE) as well as improved potency or duration of action. For example, the replacement of amide bonds optionally with an amide isostere has been demonstrated for ANP. Replacements which decrease susceptibility to hydrolysis by the neutral endopeptidase 24.11 or ACE produce a BNP variant that is resistant to enzymatic degradation. Strategies include the substitution of amino acids with N-alkylated amino acids such as N-Me-Phe and addition of the urodilatin peptide tail Thr-Ala-Pro-Arg (SEQ ID NO: 30).
Also included in the scope of the BNP variant and related compounds is a BNP
variant wherein an amino acid has been added to or deleted from the N-terminal or C-terminal 17 member ring structure of wiid type BNP. Amino acid sequence insertions include carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues including pre and pro amino acid sequences as described (see for example Ogawa et al., (1994) J. Clin. Invest. 93:1911-1921). Other examples of terminal insertions include BNP variants with a composite N-terminal or C-terminal sequence from another mammalian species or from another natriuretic peptide (see Figure 3). Such amino terminal substitutions have been described (Shimekake et al., (1992) FEBS, 309(2):185-189). Suitable N-terminal sequences are described in International Publication No WO 89112060 and include Gly; Ser Gly;
Asp/Lys/Gly-Ser-Gly; Arg/His/Gln-Asp/Lys/Gly-Ser-Gly {SEQ ID NO 34); Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly (SEQ ID NO 35);
Thr/Met-Met/Val-ArglHisIGln-Asp/Lys/Gly-Ser-Gly (SEQ ID NO 36); Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly (SEQ ID NO 37); Pro-Lys-Thr/Met-Met/Val-Arg/HislGln-Asp/Lys/Gly-Ser-Gly (SEQ
_7_ ~ I
WO 98!17690 PCTIUS97/18384 ID NO 38); and Ser-Pro-Lys-Thr/Met-Met/Val-Arg/His/Gln-Asp/Lys/Gly-Ser-Gly (SEQ ID NO 39), and are based upon the native upstream sequence for porcine, canine or human BNP or a composite thereof.
Exemplary C-terminal insertions are described in International Publication No.
89/12069 and included (OH), NH, or NR'R" where R' and R" are independently H, lower alkyl, or Asn/Lys; Asn/Lys-Val; Asn/Lys-VaI-Leu; Asn/Lys-Val-Leu-Arg (SEQ ID NO 40); Asn/Lys-Val-Leu-Arg-Arg/Lys (SEQ
ID NO 41 ); Asn/Lys-Val-Leu-Arg-Arg/Lys-Tyr-His (SEQ ID NO 42); or the amides (NH or NR'R") thereof.
As noted, in one embodiment, amino acid substitution variants have at least one amino acid residue in addition to those described herein for conferring receptor specificity in the BNP variant molecule removed and a different residue inserted in its place. The sites for substitutional mutagenesis include sites where amino acids found in the BNP variant from various species are substantially different in terms of side chain bulk, charge and or hydrophobicity. These amino acids are substituted with the exemplary conservative substitutions as described herein below including the exemplary non-naturally occurring amino acids.
Other sites of interest are those in which particular residues of wild-type BNP and the variants obtained from various species are identical. These positions may be important for the biological activity of the BNP variant or related compound. These sites, especially those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitution are shown below under the heading of preferred conservative substitutions. If such substitutions are shown to preserve qualitative biological activity as defined herein then more substantial changes denominated below as exemplary conservative substitutions may be generated and tested for biological activity.
In this regard, it is understood that amino acids are substituted on the basis of side chain bulk, charge and/or hydrophobicity. Amino acid residues are classifed into four major groups as described in, for example, International Publication No. 90/01940 the disclosure of which is hereby specifically incorporated by reference and which consists of the following groups:
Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous solution.
Basic: The residue has a positive charge due to association with H ion at physiological pH and the residue is amacted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
Neutral/non-polar: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. These residues are also designated "hydrophobic residues."
Neutral/polar: The residues are not charged at physiological pH, but the residue is amacted by aqueous solution so as to seek the outer positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
Amino acid residues can be further classified as cyclic or noncyclic, aromatic or non aromatic with respect to their side chain groups these designations being commonplace to the skilled artisan.
-g_ Original Exemplary Conservative Preferred Conservative Residue Substitution Substitution Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys 5er Ser Gln Asn Asn Glu Asp Asp 10Gly Pro Pro His Asn, Gln, Lys, Arg Arg lle Leu, Val, Met, Ala Leu Phe Leu Ile, Val Ile Met, Ala, Phe Lys Arg, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly 20Ser T"hr ~r Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe Leu Ala Peptides synthesized by the standard solid phase synthesis techniques described here, for example, are not limited to amino acids encoded by genes for substitutions involving the amino acids. Commonly encountered amino acids which are not encoded by the genetic code, include, for example, those described in International Publication No. WO 90/01940, as well as 2-amino adipic acid (Aad) for Glu and Asp; 2-aminopimelic acid (Apm) for Glu and Asp; 2-aminobutyric (Abu) acid for Met, Leu, and other aliphatic amino acids; 2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids; 2-aminoisobutyric acid (Aib) for Gly; cyclohexylaianine (Cha) for Val, and Leu and Ile; homoarginine (Har) for Arg and Lys; 2,3-diaminopropionic acid (Dpr) for Lys, Arg and His; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylglycine (EtGly) for Gly, Pro, and Ala; N-ethylasparigine (EtAsn) for Asn, and Gln; Hydroxyllysine (Hyl) for Lys; aliohydroxyllysine (AHyI) for Lys; 3-(and 4)hydoxyproline (3Hyp, 4Hyp) for Pro, Ser, and Thr; allo-isoleucine (Alley for lle. Leu, and Val; p-amidinophenylalanine for Ala; N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala; N-methylisoleucine (Melle) for lle; Norvaline (Nva) for Met and other aliphatic amino acids; Norleucine (Nle) for Met and other aliphatic amino acids; Ornithine (Orn) for Lys, Arg and His;
Citrulline (City and methionine sulfoxide (MSO} for Thr, Asn and Gln; N-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F, Cl, Br, and I)phenylalanine, triflourylphenylalanine, for Phe.
A useful method for identification of certain residues or regions of the BNP
variant for amino acid substitution other than those described herein for receptor specificity is called alanine scanning mutagenesis as described by Cunningham and Wells ( 1989) Science, 244:1081-1085. Here a residue or group of target residues are identified (e.g. charged residues such as Arg, Asp, His, Lys, and Glu} and replaced by a neutral or negatively charged amino acid to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitution then are refined by introducing further or other variations at or for the sites of substitution. Thus while the site for ~ I
introducing an amino acid sequence variation is predetermined the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, Ala scanning or random mutagenesis may be conducted at the target codon or region and the expressed BNP variants screened for the optimal combination of desired activity.
Phage display of protein or peptide libraries offers another methodology for the selection of BNP
variants or BNP related compounds with improved affinity, altered specificity, or improved stability (Smith, G.P., (1991} Curr. Opin. Biotechnol. 2:668-673). High affinity proteins, displayed in a monovalent fashion as fusions with the M13 gene III coat protein {Clackson, T., (1994) et al.,Trends Biotechnol. 12:173-183), can be identified by cloning and sequencing the corresponding DNA packaged in the phagemid particles after a number of rounds of binding selection.
Other insertional BNP variants or BNP related compounds include the fusion to the N- or C- terminus of the BNP molecule of immunogenic polypeptides, e.g., bacterial poiypeptides such as beta lactamase or an enzyme encoded by E coli Trp locus or yeast protein, and C-terminal fusion with proteins having a long half life such w~: immunoglobulin constant region or other immunoglobulin regions, albumin, or ferritin as described in WO 89/02922 published 6 April 1989.
The term "C 1-C6alkyl" when used to describe a BNP variant means a branched, unbranched or cyclic, saturated aliphatic hydrocarbon radical, having the number of carbon atoms specified. Representative examples of these alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 2,2-dimethylbutyl, cycolhexyl and the like.
The terms "lower alkyl" and"C1-C6alkyl" are synonymous and used interchangeably. A preferred "C1-C6alkyl" group is methyl.
The term "C1-C6alkanoyl" when used to describe a BNP variant encompasses groups such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, caproyl, and the like.
The term "C6-Cl2aryl" when used to describe a BNP variant means a homocyclic hydrocarbon aromatic radical, whether or not fused, having the number of carbon atoms designated. Preferred aryl groups include phenyl, napthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g. Lang's Handbook of Chemistry (Dean, J. A., ed) 13th ed. Table 7-2 ( 1985]).
The term "C6-Cl2aryl-C1-C6alkyl" when used to describe a BNP variant means one, two, or three aryl groups having the number of carbon atoms designated, appended to an alkyl radical having the number of carbon atoms designated including but not limited to; benzyl, napthylmethyl, phenethyl, benzyhydryl (diphenylmethyl), florenyl, trityl, and the like. A preferred arylalkyl group is the benzyl group.
"Pharmaceutically acceptable salts" include both acid and base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malefic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toiuenesulfonic acid, salicyciic acid and the like.
"Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts.
Salts derived from pharmaceutically acceptable organic nontoxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethyiamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline, and caffeine.
Receptor Specific BNP Variants and Related Compounds The BNP variants and related compounds of this invention have at least one amino acid of wild-type BNP substituted as described herein for conferring NPR-A receptor specificity.
Therefore, preferred I S compounds of this invention are synthetic hormone agonists of the human natriuretic peptide receptor-A
(hNPR-A). This receptor is found in kidney, smooth muscle, adrenal and other tissues (Napier M.A. et al, (1984) Proc. Nat. Acad. Sci. USA, 81:5946-5950; Hori et al., (1985) Biochem.
Biophys. Res. Commun.
129:773-779; Quirion et al., (1986) Proc. Natl. Acad. Sci. 83:174-175) and contains an intracellular guanyl cyclase domain. The action of hNPR-A is mediated by hydrolysis of GTP to produce the second messenger cGMP. Accordingly, preferred compounds of this invention stimulate hNPR-A to produce cGMP to at least the same extent as wild-type BNP.
The preferred compounds of this invention do not however bind to the human natriuretic peptide clearance receptor-C (hNPR-C) to the same extent as wild-type BNP. Both in vitro and i~r vivo systems can be used to identify the compounds of the present invention. Since binding of BNP to the NPR-A cellular receptor is presumptively a prerequisite to qualitative biological activity the BNP related compounds of the present invention are identified by screening for binding of the candidate compound to the NPR-A and NPR-C
receptors. Primary screens for the compounds of the present invention therefore include vasorelaxant assays in rat and rabbit aorta tissues, relaxation of chick rectum intestinal smooth muscle tissue, natriuresis in rats, as well as the receptor specific binding assays and cGMP formation in cell culture assays described herein above and in the following Example sections.
For example, assays have been developed for the screening of BNP which evaluate the ability of the BNP to compete with a labeled native BNP for binding to intact cultured cells or membrane preparations derived from cultured cells expressing the cellular receptors. By way of example, NPR-A expressing 293 cells produced as described by Lowe and Fendly (1992) J. Biol. Chem. 267:21691-21697 may be utilized. Likewise the cDNA for NPR-C (Lowe et al., ( 1990) Nucleic acids Res. 18:3412) may be transfected into suitable host cells for continuous expression. These competitive displacement assays are considered commonplace for examining receptor ligand interactions.
According to a particular cellular assay, radiolabeled or florescent labeled BNP is incubated with immobilized NPR-A or NPR-C receptor-immunoglobulin chimeras in varying concentration of unlabeled candidate compound. Increasing concentrations of successful candidate compound effectively prevent binding of labeled BNP to immobilized receptor chimeras. The concentration of unlabeled peptide at which 50%
maximal BNP is displaced is referred to as the EC50 and reflects the receptor binding affinity. Therefore a candidate compound with an EC50 of 100nM displays a substantially weaker interaction with a receptor than peptide with an ECSO of 10 nM.
As noted above, BNP related compounds or variants within the present invention have a decreased binding affinity for NPR-C and corresponding NPR-A receptor specificity.
Therefore, according to the present invention a BNP variant with an EC50 greater than wild type BNP for the NPR-C
is said to display a weaker affinity for the NPR-C receptor and therefore a decreased affinity for the clearance receptor.
In a preferred embodiment, receptor specificity is reflected in the ratio of binding affinity of the candidate compound for NPR-A and NPR-C. A change in the ratio of binding affinity for the candidate compound to NPR-A compared to NPR-C is referred to as a change in receptor specificity. According to this aspect of the present invention, it is a characteristic of the BNP variant or BNP related compound to exhibit a decrease in the ratio of binding affinity for NPR-A compared to NPR-C as compared to wild-type BNP in the same assay. Preferred compounds and variants according to this aspect of the invention exhibit at least a 3 0 fold decrease in the ratio of the binding affinities compared to wild-type BNP and more preferably greater that a 100 fold decrease in the ratio.
This is preferably achieved by substituting an amino acid from wild-type BNP
according to the scheme provided in Table I of Example I. Accordingly, substituting residue 19 with a basic amino acid, more preferably Arg, Har, Lys, Orn, and p-amidinophenyl-Ala and most preferably Arg; substituting residue 23 with a neutral, nonpolar, large, aromatic or nonaromatic amino acid, more preferably Val, lle, Leu, Met, Phe, Trp, t-BuA, T-BuG, N-MeIle, Nle and Cha, and more preferably, Met, Phe or Leu;
substituting residue 24 with an aromatic amino acid, more preferably Phe, Trp, and Tyr; and substituting residue 25 with a basic amino acid, more preferably Arg, Har, Lys, Orn, and p-amidinophenyl-Ala and most preferably Arg results in a BNP
variant or related compound.
The above noted substitutions can occur individually or in combination.
Preferred among the individual substitutions conferring receptor specificity is a BNP variant or related compound which has a substitution for amino acid residue 19 of wild-type BNP as provided in the preceding paragraph. Preferred among combination substitutions conferring receptor specificity is a BNP
variant or related compound where each of amino acids 23, 24, and 25 of wild type BNP are substituted according to the scheme presented in the preceding paragraph. Such preferred combination substitutions may be accompanied by a substitution of residue l9 according to the scheme provided in the preceding paragraph.
Exemplary peptides are given below:
Ser-Pro-Lys-Met-V al-G In-G ly-Ser-G ly-Cys-Phe-G ly-Arg-Lys-Met-G lu-Arg-I le-Ser-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 2) or hBNP G1y23Phe, Leu24Trp, G1y25Arg;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Ser-Ser-Ser-Ser-Met-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 3) or hBNP G1y23Met, Leu24Trp, Gly25Arg;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-G ly-Cys-Phe-G ly-Arg-Lys-Met-Glu-Arg-I le-Arg-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 4) or hBNP Serl9Arg;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Arg-His-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 5) or hBNP Serl9Arg, Ser20His;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-lle-Arg-His-Leu-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 6) or hBNP Serl9Arg, Ser20His, Ser2iLeu;
Ser-Pro-Lys-Met-Val-Gln-G ly-Ser-Gly-Cys-Phe-G ly-Arg-Lys-Met-Glu-Arg-l le-Arg-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 7) hBNP Serl9Arg GIy23Phe, Leu24Trp, Gly25Arg;
Ser-Pro-Lys-Met-Val-Gln-G ly-Ser-Gly-Cys-Phe-G ly-Arg-Lys-Met-G lu-Arg-Ile-Arg-Ser-Ser-Ser-Met-Trp-Arg-Cys-Lys-Vat-Leu-Arg-Arg-His (SEQ ID NO: 8) or hBNP Serl9Arg, GIy23Met, Leu24Trp, GIy25Arg;
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Arg-His-Ser-Ser-Met-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 9) or hBNP Serl9Arg, Ser20His, G1y23Met, Leu24Trp, GIy25Arg; and Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-G ly-Cys-Phe-G ly-Arg-Lys-Met-Glu-Arg-I le-Arg-His-Leu-Ser-Met-Trp Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 10) or hBNP Serl9Arg, Ser20His, Ser21 Leu, G1y23Met, Leu24Trp, Gly25Arg.
Further compounds within the scope of the present invention are:
hBNP Arg l3Ser, Lys l4Pro, Serl9Arg; (SEQ ID NO: 1 I ) hBNP Lysl4Thr, Serl9Arg, Ser2lGln; (SEQ ID NO: 12) hBNP Lys l4Asn, Serl9Arg, Ser20His, Ser21 Leu; (SEQ ID NO: 13) hBNP Lysl4Asn, Serl9Ala, Ser20Arg, Ser2lGlu; (SEQ 1D NO: 14) hBNP Lysl4Asn, Serl9Arg, Ser20Lys, Ser2lThr; (SEQ ID NO: 15) hBNP Lysl4Asn, Serl9Arg, Ser20Gly; (SEQ ID NO: 16) hBNP Lysl4Asn, Serl9Gly, Ser20Arg, Ser2lAla; (SEQ ID NO: 17) hBNP Lysl4Asn, Serl9Arg, Ser2lTrp; (SEQ ID NO: 18) hBNP Lys l4Asn, Serl9Arg, Ser20Asn, Ser21 Thr; (SEQ ID NO: 19) hBNP Lys l4GIn, Serl9Arg, Ser20His, Ser22Thr; {SEQ ID NO: 20) hBNP Lysl4Asn, Serl9Gly, Ser20His, Ser2lLeu; (SEQ ID NO: 21) hBNP Lysl4Asn, Serl9Arg, Ser20Thr, Ser2lLys; (SEQ 1D NO: 22) hBNP Lysl4Asn, Serl9Gly, Ser20Gly, Ser2lLeu; (SEQ ID NO: 23) hBNP Lysl4Asn, Serl9Arg; {SEQ ID NO: 24}
hBNP Lysl4Thr, Serl9Arg, Ser20His, Ser2lGly, Ser22Gly;(SEQ ID NO: 25) hBNP Lysl4Asn, SerI9Arg, Ser20Gly, Ser2lMet; (SEQ ID NO: 26) hBNP GIy23Met, Leu24Tyr, GIy25Arg; (SEQ ID NO: 27) hBNP GIy23Leu, Leu24Trp, Gly25Arg; (SEQ ID NO: 28) and hBNP GIy23Met, Leu24Phe, GIy25Arg (SEQ ID NO: 29).
I
Chemical Synthesis One method of producing compounds of the present invention involves chemical synthesis of the polypeptide, followed by treatment under oxidizing conditions appropriate to obtain the native conformation, that is, the correct disulfide bond linkages. This can be accomplished using methodologies well known to those skilled in the art (see Atherton, E., and Sheppard R.C., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford England (1989); Kelley, R.F. & Winkler, M.E. in Genetic Engineering Principles and Methods, Setlow, J. K., ed., Plenum Press, N.Y., vol. 12, pp 1-19 (1990);
Stewart, J. M. & Young, J. D. Solid Phase Peptide Synthesis, Pierce Chemical Co. Rockford, IL(1984)).
Polypeptides of the invention may be conveniently prepared using solid phase peptide synthesis (Merrifield, (1964) J. Am. Chem. Soc., 85:2149; Houghten, (1985) Proc. Natl.
Acad. Sci. USA 82:5132).
Solid phase synthesis begins at the carboxy-terminus of the putative peptide by coupling a protected amino acid to a suitable resin (e.g. chloromethylated polystyrene resin) as shown in Figures 1-1 and 1-2, on pages 2 and 4 of Stewart and Young supra. After removal of the a-amino protecting group with, for example, trifluoroar~tic acid (TFA) in methyiene chloride and neutralizing in, for example TEA, the next a-amino- and side-chain protected amino acid in the synthesis is added. The remaining a-amino- and, if necessary, side-chain-protected amino acids are then coupled sequentially in the desired order by condensation to obtain an intermediate compound connected to the resin. Alternatively, some amino acids may be coupled to one another forming a peptide prior to addition of the peptide to the growing solid phase polypeptide chain.
The condensation between two amino acids, or an amino acid and a peptide, or a peptide and a peptide can be carried out according to the usual condensation methods such as the azide method, mixed acid anhydride method, DCC (N,N'-dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide)methods, active ester method (p-nitrophenyl ester method, BOP [benzotriazole-1-yl-oxy-tris (dimethylamino) phosphonium hexafluorophosphate] method, N-hydroxysuccinic acid imido ester method, etc, and Woodward reagent K
method.
Common to chemical synthesis of peptides is the protection of any reactive side-chain groups of the amino acids with suitable protecting groups. Ultimately these protecting groups are removed after the desired polypeptide chain has been sequentially assembled. Also common is the protection of the a-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group followed by the selective removal of the a-amino-protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common in polypeptide synthesis that an intermediate compound is produced which contains each of the amino acid residues located in the desired sequence in the peptide chain with various of these residues having side-chain protecting groups attached. These protecting groups are then commonly removed substantially at the same time so as to produce the desired resultant product following removal from the resin.
Suitable protective groups for protecting the a-and e- amino side chain groups are exemplified by benzyioxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), O-chlorobenzyloxycarbonyl [Z(2Cl)], p-nitrobenzyloxycarbonyl [Z(N02)], p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl, (Boc), t amyloxycarbonyl (Aoc), isoboronyloxycarbonyl, adamatyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc), methylsulfonylethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt), dimethylophosphinothioyi (Mpt) and the like.
Protective groups for the carboxy functional group are exemplified by; benzyl ester (OBzI), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It is often desirable that specific amino acids such as arginine, cysteine, and serine possessing a functional group other than amino and carboxyl groups are protected by a suitable protective group. For example, the guanidino group of arginine may be protected with vitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzenesulfonyl, 4-methoxy-2, 6-dimethylbenzenesulfonyl (Mds}, 1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group of cysteine may be protected with p-methoxybenzyi, triphenylmethyl, acetylaminomethyl ethylcarbamoyle, 4-methylbenryl, 2, 4, 6-trimethy-benzyi (Tmb) etc, and the hydroxyl group of serine can be protected with benzyl, t-butyl, acetyl, tetrahydropyranyl and the like.
Stewart and Young supra provides detailed information regarding procedures for preparing peptides.
Protection of a-amino groups is described on pages 14-18, and side-chain blockage is described on pages 18-28. A table of protecting groups for amine, hydroxyl and sulfhydryl functions is provided on pages 149-151.
After the desired amino acid sequence has been completed, the intermediate peptide is removed from the resin support by treatment with a reagent, such as liquid HF and one or more thio-containing scavengers, which not only cleaves the peptide from the resin, but also cleaves all the remaining side-chain protecting groups. Following HF cleavage, the protein sequence is washed with ether, transferred to a large volume of dilute acetic acid, and stirred at pH adjusted to about 8.0 with ammonium hydroxide.
Preferably in order to avoid alkylation of residues in the polypeptide, (for example, alkylation of methionine, cysteine, and tyrosine residues) a thio-cresol and cresol scavenger mixture is used. The resin is washed with ether, and immediately transferred to a large volume of dilute acetic acid to solubilize and minimize intermolecular cross-linking. A 250 pM polypeptide concentration is diluted in about 2 liters of 0.1 M acetic acid solution. The solution is then stirred and its pH adjusted to about 8.0 using ammonium hydroxide. Upon pH adjustment, the polypeptide takes its desired conformational arrangement.
Non-peptide (amide isostere) Linkages In one embodiment of the invention, the amide linkages of BNP {-C(=O)-NH-) may be replaced with amide isostere linkages such as; -CH2-NH-, -CH2-S-, -CH2-O-, -CH2-CH2-, -CH=CH-(cis and trans), -C(=O)-CH2-, -CH(OH)-CH2-, -CH(CN)-NH-, -O-C(=O)-NH- and -CH2-SO-, by methods known in the art.
The following references describe preparation of peptide analogs which include these alternative-linking moieties: Spatola, A.F., Vega Data 1(3): "Peptide Backbone Modifications"
(General Review) (Mar 1983), Spatola, A.F., in "Chemistry and Biochemistry of Amino Acids Peptides and Proteins", B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983); Hudson, D. et al. Int. J. Pept. Prot.
Res. 14:177-185 {1979) (-CH2NH-, -CH2CH2-); Tourwe, D., et al., Structural Biology 331-333 (1989) (E, -CH=CH-); Spatola, A.F., et al., Life Sci. 38:1243-1249 (1986) (-CH2-S-); Almquist, R.G., et al., J.
Med. Chem. 23:1392-1398 ( 1980) (-C(=O)-CH2-); Jennings-White C., et al., Tetrahedron Left 23:( 1982) (-C(=O)-CH2-); Szelke, M., et al., EP
Application No.45655 {-CH(OH)-CHI); Holladay, M.W., et al., Tetrahedron Lett 24:4401-4404 (1983) (-C(OH)-CH2-); Hruby, V.J. Life Sci 31:189-199 (1982) (-CH2S-); and Cho, C.Y. et a1, Science 261:1303-1305 ( 1993 )(-O-C(=O)-NH-).
Recombinant Synthesis In a further embodiment, the present invention encompasses a nucleic acid, preferably DNA, encoding the protein component of a BNP variant that contain the amino acid substitutions described herein. The invention further comprises an expression control sequence operably linked to the DNA molecule, an expression vector, preferably a plasmid, comprising the DNA molecule, where the control sequence is recognized by a host cell transformed with the vector.
The compounds of the present invention may be made by a process which includes the steps of synthesizing (by art standard techniques) nucleic acid sequences encoding any of the amino acid sequences described herein, ligating the nucleic acid sequence into a suitable expression vector capable of expressing the nucleic acid sequence in a suitable host, transforming the host with the expression vector into which the nucleic acid sequence has been ligated, culturing the host under conditions suitable for expression of the nucleic acid sequence, whereby the protein encoded by the selected nucleic acid sequence is expressed by the host. In this process, the ligating step may further contemplate ligating the nucleic acid into a suitable expression vector such that the nucleic acid is operably linked to a suitable secretory signal, whereby the amino acid sequence is secreted by the host. The secretory signal may be selected, for example, from the group consisting of the leader sequence of stlI, ecotin, lama, herpes gD, lpp, alkaline phosphatase, invertase, and alpha factor and is preferably stlI.
By way of illustration, with expression vectors encoding wild-type BNP in hand, site specific mutagenesis (Kunkel et al., Methods Enzymol. 204:125-139 [1991]; Carter, P., et al.. Nucl. Acids. Res.
13:4331 [1986]; Zoller, M. J. et al.,Nucl. Acids Res. 10:6487 [1982]), cassette mutagenesis (Wells, J. A., et al., Gene 34:315 [1985]), restriction selection mutagenesis (Wells, J. A., et al., Philos. Trans, R. Soc. London SerA 317, 415 [ 1986]) or other known techniques may be performed on the BNP
DNA. The mutant DNA can then be used in place of the parent DNA by insertion into the aforementioned expression vectors. Growth of host bacteria containing the expression vectors with the mutant DNA allows the production of mutant BNP, which can be isolated as described herein.
The host cell may be prokaryotic or eukaryotic. Prokaryotes are preferred for cloning and expressing DNA sequences to produce parent polypeptides, segment substituted polypeptides, residue-substituted polypeptides and polypeptide variants. For example, E. coli K12 strain 294 (ATCC No. 31446) may be used as E. coli B, E. toll- X 1776 (ATCC No. 31537), and E. toll c600 and c600hf1, E. toll W3110 (F-, gamma-, prototrophic /ATCG No. 27325), bacilli such as Bacillus subrilis, and other enterobacteriaceae such as Salmonella -typhimurium or Serratia marcesans, and various pseudomonas species. The preferred prokaryote is E. toll W3110 (ATCC 27325). When expressed by prokaryotes the polypeptides typically contain an N-3S terminal methionine or a formyl methionine and are not glycosylated. In the case of fusion proteins, the N-terminal methionine or formyl methionine resides on the amino terminus of the fusion protein or the signal sequence of the fusion protein. These examples are, of course, intended to be illustrative rather than limiting.
In addition to prokaryotes, eukaryotic organisms, such as yeast cultures, or cells derived from multicellular organisms may be used. In principle, any such cell culture is workable. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a reproducible procedure (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)). Examples of such useful host cell lines are VERO and HeLa cells, Chinese Hamster Ovary (CHO) cell lines, W138, 293, BHK, COS-7 and MDCK cell lines.
Preferred expression vectors of the present invention may be selected from, for example, pBR322, phGHl, pB0475, pB1537, pRITS, pRlT2T, pKK233-2, pDR540, pPL-lambda and pB1537, with the most preferred vector being pB1537.
A preferred vector for direct expression of the BNP variants of the present invention is pB1537 (Cunningham et al. ( 1994) EMBO J. 13:2508-2515) and contains origins of replication for E. toll, the alkaline phosphatase promoter, the stlI signal sequence, the BNP variant gene, and the ampicillin resistance gene. Other preferred vectors are pRlTS and pRlT2T (Pharmacia Biotechnology). These vectors contain appropriate promoters followed by the Z domain of protein A, allowing genes inserted into the vectors to be expressed as fusion proteins.
Other preferred vectors can be constructed using standard techniques by combining the relevant traits of the vectors described herein. In this instance a vector containing the origins of replication for phage and E.
toll, which allow it to be shuttled between such hosts, is used thereby facilitating both mutagenesis and expression (Cunningham, B., et al., Science 243:1330-1336 [1989]; Wells, J. &
Cunningham, B., WO
90/04788 published 3 May 1990). Relevant traits of the vector include the promoter, the ribosome binding site, the variant gene or gene fusion (the Z domain of protein A and a BNP variant and its linker), the signal sequence, the antibiotic resistance markers, the copy number, and the appropriate origins of replication.
Gene Fusions A variation on the above procedures contemplates the use of gene fusions, wherein the gene encoding a BNP variant is associated, in the vector, with a gene encoding another protein or a fragment of another protein. This results in a BNP variants being produced by the host cell as a fusion with another protein. The "other" protein is often a protein or peptide which can be secreted by the cell, making it possible to isolate and purify the desired protein from the culture medium and eliminating the necessity of destroying the host cells which arises when the desired protein remains inside the cell. Alternatively, the fusion protein can be expressed intracellularly. It is useful to use fusion proteins that are highly expressed.
The use of gene fusions, though not essential, can facilitate the expression of heterologous proteins in E. toll as well as the subsequent purification of those gene products (Harris, T. J. R. in Genetic Engineering, Williamson, R., Ed., Academic, London, Vol. 4, p. 127[1983]; Uhlen, M. & Moks, T., Methods Enzymol.
185:129-143 [1990]). Protein A fusions are often used because the binding of protein A, or more specifically the Z domain of protein A, to IgG provides an "affinity handle" for the purification of the fused protein (Nilsson, B. & Abrahmsen, L. Methods Enrymol. 185:144-161 [1990]). It has also been shown that many heterologous proteins are degraded when expressed directly in E. toll, but are stable when expressed as fusion proteins (Marston, F. A. O., Biochem J. 240: 1 [1986]).
BNP variants expressed as fusion proteins may be properly folded or may require folding to obtain the native structure. The properly folded fusion protein may be active and useful as pharmaceutical drug.
_ 17-When denaturing and refolding are needed, typically the protein is treated with a chaotrope, such a guanidine HCI, and is then treated with a redox buffer, containing, for example, reduced and oxidized dithiothreitol or glutathione at the appropriate ratios, pH, and temperature, such that the protein of interest is refolded to its native structure.
More preferred would be the correctly folded native protein that is obtained from the fusion protein by methods known in the art. Fusion proteins can be cleaved using chemicals, such as cyanogen bromide, which cleaves at a methionine, or hydroxylamine, which cleaves between an Asn and Gly. Using standard recombinant DNA methodology, the nucleotide base pairs encoding these amino acids may be inserted just prior to the 5' end of the BNP variant gene.
Alternatively, one can employ proteolytic cleavage of fusion proteins, which has been recently reviewed (Carter, P. (1990) in Protein Purification: From Molecular Mechanisms to Large-Scale Processes, Ladisch, M. R., Willson, R. C., Painton, C. C., and Builder, S. E., eds., American Chemical Society Symposium Series No. 427, Ch 13, 181-193).
I'roteases such as Factor Xa, thrombin, subtilisin and mutants thereof, have been successfully used to cleave fusion ~~roteins. Typically, a peptide linker that is amenable to cleavage by the protease used is inserted between the "other" protein (e.g. the Z domain of protein A) and the protein of interest, such as a BNP
variant. Using recombinant DNA methodology, the nucleotide base pairs encoding the linker are inserted between the genes or gene fragments coding for the other proteins. Proteotytic cleavage of the partially purified fusion protein containing the correct linker can then be carried out on either the native fusion protein, or the reduced or denatured fusion protein.
Purification and characterization Purification and characterization of BNP variants may be carried out by any art standard technique.
For example, BNP variants may be purified from the broths of E. toll cultures grown in 10 liter fermentors by batch reverse phase chromatography, cation exchange chromatography and C 18 reverse phase HPLC.
Utility and Administration Compounds of the present invention have natriuretic, diuretic and vasorelaxant activity and may inhibit the release of aldosterone and renin. Thus_ these cnmnnunrlc fir, mcP
ae thor~..o..+:.. .,..,....,. ._ .L_ treatment of various pathological conditions associated with water or electrolyte imbalance and hypertension, especially renovascular hypertension. The compounds of the present invention can be used analogous to ANP
peptides in therapeutic and prophylactic measures. BNP has potent diuretic and systemic vasorelaxant properties (Kambayashi et al., (1990) Biochem. Biophys. Res. Commun. 173:599-605). Increased levels of BNP have been reported in congestive heart failure and during acute exercise (Kohno, M., et al., ( l 992) Metabolism 41:1273-1275; Mukoyama M., et al., ( 1991 ) J. Clin. Invest.
87:1402-1412). Both BNP and ANP
are unregulated proportionally during chronic hypoxia (Am. J. Physiol. (1994) 266:L308-L315). Therefore any condition resulting in blood volume or blood pressure overload is a candidate. Such conditions include, for example, cardiac diseases such as congestive heart failure (CHF), cardiomyopathies, myocarditis, valve diseases such as rheumatic heart disease, coronary heart disease such as myocardial ischemia and infarction, arrhythmias such as paroxysmal atrial tachycardia and atrial fibrillation and flutter, congenital heart defects, restrictive disorders such as pericarditis and cardiac tamponade, drug induced cardiac diseases, hypertension, including essential hypertension, secondary, pulmonary and portal hypertension, kidney diseases such as chronic renal failure and nephrosis, cirrhosis, primary atdosteronism, Cushing's disease, preeclampsia, toxemia of pregnancy, premenstrual syndrome, nephrotic syndrome and hepatic cirrhosis, pulmonary disease, and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.
The compounds and compositions can be administered to humans in a manner similar to ANP and other similar therapeutic agents. The dosage to be administered will depend on the usual factors including; age, weight, sex, condition of the patient, specific disorder being treated, and route of administration. In general, the dosage required for therapeutic efficacy will range from about 0.01 to 1000 mg/kg, more usually 0. I to 25 mg/kg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time until the desired therapeutic benefits have been obtained.
The present invention also provides compositions containing an effective amount of compounds of the present invention, including the nontoxic addition salts, amides and esters thereof, which may, alone, serve to provide the above-recited therapeutic benefits. The peptide compounds may be formulated into the compositions as neutral or salt forms. Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, 2-ethylamino ethanol, histidine, procaine, and the like. Such compositions can be provided together with physiologically tolerable Liquid, gel or solid diluents, adjuvants and excipients.
Typically, such compositions are prepared as sterile, injectable liquid solutions or suspensions.
Compositions may also be emulsified. The active ingredient is often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient.
Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH-buffering agents, and the like. For a more detailed description of the foregoing see a standard pharmaceutical text such as Remingron's Pharmaceutical Sciences. Mack Publishing Co. Easton, PA. (1970).
In addition to the compounds of the present invention which display natriuretic, diuretic or vasorelaxant activity, compounds of the present invention can also be employed as intermediates in the synthesis of such useful compounds. Alternatively, by appropriate selection, compounds of the present invention whose activity levels are reduced or eliminated entirely can serve to modulate the activity of other diuretic, natriuretic or vasorelaxant compounds, including compounds outside the scope of the present invention, by, for example, binding to alternate receptors, stimulating receptor turnover, or providing alternate substrates for degradative enzyme of receptor activity and thus inhibiting these enzymes or receptors. When employed in this manner, such compounds can be delivered as admixtures with other active compounds or can be delivered separately, for example, in their own carriers.
WO 98/17b90 PCT/US97I18384 Pharmaceutical compositions may be prepared alone or in combination with a C
receptor ligand such as, for example SC-46542, or an inhibitor of the enzyme primarily responsible for the degradation of BNP such as SCH 32615, SCH 344826, SCH 39370, SQ 2900072, thiorfan and UK 69578.
Sythetic compounds which occupy the C-receptor increase the concentrations of circulating BNP and may be used in combination with the peptides of the present invention. U.S. Patent No 4,740,499 describes a method of prolonging or enhancing the bioactivity of an atrial peptide using two specific inhibitor of endopeptidase 24. I 1, thiorphan or kelotorphan administered simultaneous with the atrial peptides.
The following examples are offered by way of illustration and not by way of limitation. The disclosures of all citations in the specification are expressly incorporated herein by reference.
EXAMPLES
EXAMPLE I
Monovaient phage display (Bass et al., (1990) Protein Struct. Funct. Genent., 8:309-314; Lowman et al., (1991) Biochemistry, 30:10832-10838) was used to screen variants of BNP for ones that bind tightly to human NPR-A and not to NPR-C. After multiple enrichment screen BNP variants that bound preferentially to NPR-A were isolated.
Construction of Libraries: Monovalent phagemid libraries (Lowman, H. B. et al., { 1991 ) Methods:
Companion Methods Enzymol. 3:205-216) of BNP were generated by site directed mutagenesis (Kunkel, T.
A., et al., {1991) Methods Enzymol. 204:125-139). Each library contained three, four or five codons fully randomized for all 20 amino acids (as shown in Table 1 below) using oligonucleotides that mutated target codons to NNS sequences (where N represents a mixture of all four bases and S
is a mixture of both C and G).
Stocks of approximately 1014 phagemid per mL were prepared from PEG
precipitates of culture broths from XL-1 Blue cells containing the plasmid and superinfected with K07 helper phage.
Selection of receptor specific BNP variants: Before negatively selecting for NPR-A specific mutants the libraries were pre-enriched for NPR-A binders using NPR-A IgG fusion (A-IgG) (Bennett et al. ( 1991 } J.
Biol. Chem. 266:23060-23067). Wells of Maxisorb microtiter plates (Nunc cat.
#439454) were coated with 100 pl of 2 pg/ml Rabbit anti-human Fc Antibody (Jackson Immuno Research cat.
#309-006-008) in 50mM
sodium bicarbonate (pH 9.6) at 4° C overnight. The coat solution was discarded and the wells were blocked for one hour at room temperature with 5% skim milk in 25mM sodium carbonate (pH9.6). After washing with 0.01% Tween~ in PBS, NPR-A IgG was added at 1 pg/ml in binding buffer (2% skim milk in PBS) for 1 hour at room temperature. The wells were washed again. 10 pl of phage stock (250 fold concentrated from growth media, stored in PBS) was brought up to 100 ~ l with binding buffer and incubated in the wells for 2 hours at room temperature. After washing 20-30 times, the phage were eluted with 100 p l 0.2M glycine (pH 2.0), then neutralized with 13 pl 1M Tris Base and titered by infecting E. toll (XL1-blue Stratagene, Inc., San Diego, CA). The remaining selected phagemid particles were propagated for the use in the next selection cycle.
Receptor specific selection was done by adding competing NPRC IgG (Bennett et al. (1991) supra) during the phage binding step in the NPR-A IgG coated wells. This was done in seven rounds of selection with the amount of competing CIgG increasing from 20nM to 200nM. For each library, there were control wells that lacked either the competing CIgG or both the AIgG coat and the CIgG.
ELISA measurement of phagemic amities for NPR-A and NPR-C: Phage ELISA were determined according to the method of Cunningham, B. C. et al., ( 1994) EMBO J., 13:2508-25 I 5. Microtiter plates (Nunc, Maxisorb, 96 wells) were coated with purified A-IgG (at lltg/ml) in 50 mM
sodium bicarbonate (pH 9.6) at 4 C overnight. The plates were blocked with 5% skimmed milk and washed with PBS, 0.01% TWEEN 20.
Serial dilutions of competing receptor {A-IgG or C-IgG) and a subsaturating concentration of BNP-phagemid were added to wells in 100 pi of binding buffer (2% skim milk in PBS). After 2 h the plates were washed, and the bound phagemid stained with sheep anti-M 13 HRP conjugate antibody (Pharmacia #279402-Ol ), and assayed. Affinities (EC50) were calculated as the concentration of competing receptor that resulted in half maximal phagemid binding.
Results The amino acid residues found at each randomized position after 7 rounds of selection are shown in Table I. 8 segments of 3 to 5 amino acids randomized by primer mutagenisis are indicated at the top of the figure, immediately above the numbering for 32 amino acid human BNP, the sequence of which is shown in single letter amino acid code. Cysteine residues 10 and 26 were not mutagenized since they form a disulfide bond essential for binding to NPR-A. The substitution of K3 for D (Display BNP) is analogous to the acidic substitution R3D in ANP used to facilitate secretion expression in E. coli (Cunningham et al. (1994)).
Individual clones were picked and the DNA sequence was determined. The number of times a particular sequence was observed is shown on the left. The ratio of mutant BNP phagemid binding affinity to ANP
phagemid binding to A-IgG is shown on the right. Mutations that occur outside of the mutagenesis window result from polymerase misincorporation mutations that render a selection advantage to the mutant BNP
phagemid (Cunningham et al. ( 1994)).
-2 I -WO 98!17690 PCT/US97/18384 Q z Q O ~ '~ 4lN N N1O M N
d ~~
000 ~ filN ~ Y ?
E
d ~~
000 ~ filN ~ Y ?
E
3 c M x _ M
O
M
N
N
r N :G
N U
N J VI
Z
N
V1 :1.1~ W W [--' ~ a ~ s ~
~ a z 00 _ a ~ -~~ z ~--z z z '-~ ~ ~ V ~ z N
- '- J
U
O~ V N W W
N ap ~ W C C
r C7 W CL
'o d z W W
L
W -> >~ D
M :L W O' C
D
N a. > W ", 3 .. _ ~ ~o W o ~ z a n. w.
z m m G ~ o A _ N O
3t ~
A a Q z Q w x ~, U ooc n O . ef Os~n o O~ w x atO O
O
M
N
N
r N :G
N U
N J VI
Z
N
V1 :1.1~ W W [--' ~ a ~ s ~
~ a z 00 _ a ~ -~~ z ~--z z z '-~ ~ ~ V ~ z N
- '- J
U
O~ V N W W
N ap ~ W C C
r C7 W CL
'o d z W W
L
W -> >~ D
M :L W O' C
D
N a. > W ", 3 .. _ ~ ~o W o ~ z a n. w.
z m m G ~ o A _ N O
3t ~
A a Q z Q w x ~, U ooc n O . ef Os~n o O~ w x atO O
4 ~ o 0o p, z ~ M ~T~' ~ ~ ~ ~ f'1M V'1 N C N N M Vj O
en E
c c m N
t1 _ M
O
P
N
N
r N :G
W
N
N CJ G:p(,C:~
- 3 3 >'3 V ~ u.
N
N
v~ < 3 t-yr ..~~ a V
N V c~
~ N z z x H V x _' w x V ~ ~ ~ V ~ V c: x v -D
- z :e z z z z o- z z z z ~-z M M
=V V
- u.
U
o V
N o0 V
d v M ~ A
N a w~ _ c ' E - w ~' o L7 a " ~ a ~ I
WO 98!17690 PCT/US97/18384 y..
a a a S~ ~ ~ ~ ~ a ~ ~ m oa m E
C
.Q
G_ W
N y Q a ~I
r x , ~
w w w a N G ~ 0.
~ t-~-'w a. ~ v~
N ;t 4 2 Z 6.L1.d G.
N U
N U
vo v ~
N 4L4. IL
N
N
N
N
O
-r b N
- L
M
V ou c W v U a a v v T
C
N x N v c II
M k CJ
N n.
- (n C
a n. 4.
~ c r~ A o E
'" o ----- r, o U a ~. ~.
In the following Examples, amino acids are described by the standard three letter amino acid code when referring to intermediates and final products. The linear peptides and intermediates described herein were prepared by the solid phase method of peptide synthesis (R. Merrifield, ( I
964) J. Am. Chem. Soc. 85:2149 and . 5 M. Bodansky, "Principles of Peptide Synthesis." Springer-Verlag, (1984).
Abbreviations used are as follows:
tert-butyloxycarbonyl (Boc); p-toiuenesulfonyl (Tos); 4-methylbenzyl (MeBzl);
benzyl (Bzl); 2-bromobenzyloxycarbonyl (Br-Z); cyclohexyi ester (Ochex); 4-methoxylbenzyl (MeOBzI), 2-chlorobenzyloxycarbonyl (C1-Z); hydrogen fluoride (HF);
benzotriazolyloxytris(dimethylamino)phosphoniumhexaftuorophosphate (BOP);
methyiene chloride (DCM);
trifluoroacetic acid (TFA); and dimethyfacetamide (DMA).
Peptides were assembled using an Applied Biosystems 430A automated peptide synthesizer.
Protected amino acids were obtained from various vendors. Side chain protection was Asp (Ochex), Cys (MeOBzI), Arg (Tos), Ser(Bzl), Thr (Bzl), Lys (Cl-Z), and Tyr (Br-Z).
The peptides were assembled on 0.7 g ( 1.0 mmol) of Boc-Tyr(Br-Z) PAM resin (Applied Biosystems).
The coupling protocols were those recommended by Applied Biosystems and DMA
was used instead of DMF
as solvent. After chain assembly the N-terminal Boc group was removed using TFA. The peptide resin was then washed with dichloromethane followed by methanol and then dried under vacuum. The peptide was then deprotected and removed from the resin by stirring the peptide-resin in 20 ml of a mixture of HF (92.5%), anisole (5%) and ethylmethylsulfide (2.5%) for I hr at 0 degrees. The HF was then removed in vacuo and the resin washed with ether. The resin was washed with 10% acetic acid in water, acetonitrile, water, and the fltrate was recovered and lyophilized. The crude linear peptide (300 mg) was dissolved in 10 L of distilled water with the pH adjusted to 7.4-7.6 with concentrated ammonium hydroxide.
The cyclization was begun immediately by adding 0.003 M K3[Fe(CN)6] (I g dissolved in 100 mL
of distilled water) dropwise (ca. 1 drop/ 10 sec.) with vigorous stirring.
Addition of the iron solution was halted when a bright yellow color persisted, and the reaction mixture was stirred for an additional 4 to 5 h. The pH
of the cyclized solution was adjusted to 4.5 with HOAc.
Bio Rex 70 cation exchange resin , 200-400 mesh in the Na+ form was washed with I N HCl and then with distilled water. Approximately 110 g of this resin was added to the cyclized peptide solution and stirred overnight. The resin was filtered, washed exhaustively with distilled water, and slurry packed into a column.
The peptide was eluted using 70% aqueous HOAc, identified by TLC (ninhydrin visualization) and lyophilized.
The peptide was dissolved in 0.1 % TFA in water, loaded onto a 2.5 cm x 50 cm C- I 8 reversed phase column {15 micron, 300A} and eluted with a shallow gradient of increasing acetonitrile. The elution conditions consisted of a 10% to 50% acetonitrile (containing 0.1 % TFA) gradient at 0.5%
min.' 1. The aqueous phase was 0.1% TFA in water. Product containing fractions were then lyophilized to afford the pure title peptide.
Following the procedure in Example I , the compounds of Examples 3 - 6 were analogously prepared.
Ser-Pro-Lys-Met-Val-G ln-G ly-Ser-G ly-Cys-Phe-G ly-Arg-Lys-Met-Glu-Arg-I le-Ser-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 2) ~ I
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Arg-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 4) Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Arg-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 7) Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Gly-Arg-Met-Glu-Arg-Ile-Arg-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 43) Measurement of binding affinities for NPR-A and NPR-C.
Methods Membrane Preparation Membranes from stable 293 cell lines expressing NPR-A
(Lowe and Fendly ( 1992) J. i? iol. Chem.) or NPR-C (Cunningham et al. ( 1994) supra) were prepared by removing the cells from tissue culture platcs with O.SmM EDTA in PBS, centrifuging at 225xg, 4°
C, and resuspending in 50mM Hepes Ph7.4, 1mM EDTA, ImM DTT, 250mM Sucrose, 0.7ug/ml Pepstatin A, 0.5ug/ml Leupeptin, 1mM PMSF, 1 uM Phosphoramidon. The resuspended cells were homogenized for 30 seconds with a Brinkman polytron on setting 5. The homogenate was centrifuged in 30m1 corex tubes in Sorval HB-4 rotor at 400xg ( 1500rpm) for 10 minutes at 4°C. The supernatant was centrifuged again in screw cap polycarbonate tubes (Beckman 355618) at 100,000xg (31,500 rpm) for 30 minutes, 4° C in a Beckman T160 rotor. The pellet was resuspended in 50mM Hepes, O.ImM EDTA, S.OmM MgCl2, 100mM NaCI, 0.7~cglml Pepstatin A, 0.5ug/ml Leupeptin, 1 mM PMSF, 1 uM Phosphoramidon and homogenized again with 15 strokes in a Dounce homogenizer (Wheaton 7m1, #357542). The membranes were passed through a 22 gauge needle several times before aliquoting into eppendorf tubes for storage at -80°C.
Measurement binding affrnities for NPR-A and NPR-C Membranes were diluted in 50mM Hepes, 5mM
MgCl2, 1 OOmM NaCI, 0.1 mM EDTA, 0.2% BSA, 1 uM Phosphoramidon and added to an equal volume of diluted pure synthetic peptide plus 30pM I 125 labeled ratANP (Amersham). The binding was allowed to go for 2 hours at room temperature in 96 well U-bottomed microtiter polypropylene plates (Sigma M-4029) with gentle agitation. The bound hormone was separated from the free using a Packard Filtermate 196 cell harvester and 1 % polyethyleneimine-saturated Packard unifilter-96 GF/B plates by vacuum filtration. The plates were dried under vacuum chamber for 10 minutes prior to adding 401 Microscint-20 scintillant and counting in a Packard Topcount scintillation counter. Each affinity represents points measured in duplicate within three separate assays.
Results:
Assay # 1 N PR-A NPR-C
hANP (SEQ ID NO: 51 43.55 f 12.9 198 f 90.4 ) hBNP (SEQ ID NO: 1 645 t 298 1791 t 145 }
hBNP G1y23Phe, Leu24Trp,378 t 136 9363 t 2067 G1y25Arg (SEQ ID NO:
2) hBNP Serl9Arg (SEQ 155 t 55.3 95,498 f 14,603 1D NO: 4) hBNP Argl3Gly, Lysl4Arg43,723 t 5915 13,918 t 12,901 (SEQ
ID N0:48) hBNP Argl3Gly, Lysl4Arg,518 t 72.2 28,370 f 9,654 Serl9Arg (SEQ ID N0:49) hANP Metl2Ile, GIy20Phe,507 t 118 295 ~ 56.8 Leu2lTrp, GIy22Arg (SEQ ID
N0:50) hBNP Serl9Arg, Gly23Phe,287 t 27 172,400 t 120,096 Leu24Trp, G1y25Arg (SEQ ID NO:
7) hANP GIyl6Arg (SEQ 17.2 ~ 7.6 10,601 t 1271 ID NO: 52) Example 7 The peptides were assayed for stimulation CGMP production.
Whole Cell Stimulation of cGMP Production, and cGMP RIA
Cell Culture: Cells were maintained in F12 (w/o GHT)/Low Glucose DMEM (50/50), 25 mM Hepes pH 7.2, 2mM Glutamine, 400 microgramslml 6418 and 10% dialyzed calf serum. Log phase cells were removed from plates with 0.5 mM EDTA in PBS, taken up in media and cell concentration measured with a coulter counter. Cells were plated to 12 well dishes at a density of 300,000 cells/well, 18 to 24 hours prior to stimulation.
Whole Cell Stimulation: Plates were placed on a 37°C warm plate, media aspirated and 37°C
incubation media added for IO min. (50/50, 25 mM Hepes pH 7.2, 0. I mM
isobutyl methyl Xanthine, 0.1%
(w/v) BSA). The reaction was terminated for control value - see below, or media was aspirated and incubation media with test sample was added. The reaction was continued for 10 min., then 0.5 ml of ice cold 12%
Trichloro Acetic Acid was added to stop the reaction and extract the cyclic nucleotides. Plates were quick frozen on a bed of dry ice, thawed and the TCA with cell debris taken into a 12 x 75 mm disposable glass tube and spun at 2000 x G for 10 min. The supernatant was transferred to a fresh glass tube and the TCA removed by extracting 3 times with water saturated Ether. The residual Ether was removed by boiling off at 50° for 20 minutes. The sample is now ready for RIA determination of cGMP content.
_27-I
RIA: i00 microliters of sample (neat or dilutions thereof) was measured in duplicate. The standard curve was set up with highest concentration of cGMP first. The protocol for Acetylation is followed as described (Biomedical Techniques Inc., Stoughton, MA). After overnight incubation, and centrifugation, pellets were counted in an isodata counter. Data was analyzed using a cGMP RIA
analysis program on RS/1.
Results Figures 1 and 2 demonstrate the measurement of cGMP production in 293 cells expressing hNPR-A.
Wild-type and variants stimulate guanyl cyclase activity in the membrane preparations as measured by cGMP
production.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Genentech, Inc.
(ii) TITLE OF INVENTION: Receptor Selective BNP
(iii) NUMBER OF SEQUENCES: 52 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Dennison Associates (B) STREET: 133 Richmond Street West, Suite 301 (C) CITY: Toronto (D) PROVINCE: Ontario (E) COUNTRY: Canada (F) POSSTAL CODE: M5H 2L7 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WinPatin (Genentech) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,296,656 (B) FILING DATE: 09-OCT-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Dennison Associates (B) REFERENCE/DOCKET NUMBER: JJ-10 474CA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 368-8313 (B) TELEFAX: (416) 368-1645 (2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Phe Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Met Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met 1 5 '10 15 Glu Arg Ile Arg His Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg His Leu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg Ser Ser Ser Phe Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg Ser Ser Ser Met Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg His Ser Ser Met Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg His Leu Ser Met Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Ser Pro Met Glu Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Thr Met Glu Arg Ile Arg Ser Gln Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg His Leu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Ala Arg Glu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:15:
en E
c c m N
t1 _ M
O
P
N
N
r N :G
W
N
N CJ G:p(,C:~
- 3 3 >'3 V ~ u.
N
N
v~ < 3 t-yr ..~~ a V
N V c~
~ N z z x H V x _' w x V ~ ~ ~ V ~ V c: x v -D
- z :e z z z z o- z z z z ~-z M M
=V V
- u.
U
o V
N o0 V
d v M ~ A
N a w~ _ c ' E - w ~' o L7 a " ~ a ~ I
WO 98!17690 PCT/US97/18384 y..
a a a S~ ~ ~ ~ ~ a ~ ~ m oa m E
C
.Q
G_ W
N y Q a ~I
r x , ~
w w w a N G ~ 0.
~ t-~-'w a. ~ v~
N ;t 4 2 Z 6.L1.d G.
N U
N U
vo v ~
N 4L4. IL
N
N
N
N
O
-r b N
- L
M
V ou c W v U a a v v T
C
N x N v c II
M k CJ
N n.
- (n C
a n. 4.
~ c r~ A o E
'" o ----- r, o U a ~. ~.
In the following Examples, amino acids are described by the standard three letter amino acid code when referring to intermediates and final products. The linear peptides and intermediates described herein were prepared by the solid phase method of peptide synthesis (R. Merrifield, ( I
964) J. Am. Chem. Soc. 85:2149 and . 5 M. Bodansky, "Principles of Peptide Synthesis." Springer-Verlag, (1984).
Abbreviations used are as follows:
tert-butyloxycarbonyl (Boc); p-toiuenesulfonyl (Tos); 4-methylbenzyl (MeBzl);
benzyl (Bzl); 2-bromobenzyloxycarbonyl (Br-Z); cyclohexyi ester (Ochex); 4-methoxylbenzyl (MeOBzI), 2-chlorobenzyloxycarbonyl (C1-Z); hydrogen fluoride (HF);
benzotriazolyloxytris(dimethylamino)phosphoniumhexaftuorophosphate (BOP);
methyiene chloride (DCM);
trifluoroacetic acid (TFA); and dimethyfacetamide (DMA).
Peptides were assembled using an Applied Biosystems 430A automated peptide synthesizer.
Protected amino acids were obtained from various vendors. Side chain protection was Asp (Ochex), Cys (MeOBzI), Arg (Tos), Ser(Bzl), Thr (Bzl), Lys (Cl-Z), and Tyr (Br-Z).
The peptides were assembled on 0.7 g ( 1.0 mmol) of Boc-Tyr(Br-Z) PAM resin (Applied Biosystems).
The coupling protocols were those recommended by Applied Biosystems and DMA
was used instead of DMF
as solvent. After chain assembly the N-terminal Boc group was removed using TFA. The peptide resin was then washed with dichloromethane followed by methanol and then dried under vacuum. The peptide was then deprotected and removed from the resin by stirring the peptide-resin in 20 ml of a mixture of HF (92.5%), anisole (5%) and ethylmethylsulfide (2.5%) for I hr at 0 degrees. The HF was then removed in vacuo and the resin washed with ether. The resin was washed with 10% acetic acid in water, acetonitrile, water, and the fltrate was recovered and lyophilized. The crude linear peptide (300 mg) was dissolved in 10 L of distilled water with the pH adjusted to 7.4-7.6 with concentrated ammonium hydroxide.
The cyclization was begun immediately by adding 0.003 M K3[Fe(CN)6] (I g dissolved in 100 mL
of distilled water) dropwise (ca. 1 drop/ 10 sec.) with vigorous stirring.
Addition of the iron solution was halted when a bright yellow color persisted, and the reaction mixture was stirred for an additional 4 to 5 h. The pH
of the cyclized solution was adjusted to 4.5 with HOAc.
Bio Rex 70 cation exchange resin , 200-400 mesh in the Na+ form was washed with I N HCl and then with distilled water. Approximately 110 g of this resin was added to the cyclized peptide solution and stirred overnight. The resin was filtered, washed exhaustively with distilled water, and slurry packed into a column.
The peptide was eluted using 70% aqueous HOAc, identified by TLC (ninhydrin visualization) and lyophilized.
The peptide was dissolved in 0.1 % TFA in water, loaded onto a 2.5 cm x 50 cm C- I 8 reversed phase column {15 micron, 300A} and eluted with a shallow gradient of increasing acetonitrile. The elution conditions consisted of a 10% to 50% acetonitrile (containing 0.1 % TFA) gradient at 0.5%
min.' 1. The aqueous phase was 0.1% TFA in water. Product containing fractions were then lyophilized to afford the pure title peptide.
Following the procedure in Example I , the compounds of Examples 3 - 6 were analogously prepared.
Ser-Pro-Lys-Met-Val-G ln-G ly-Ser-G ly-Cys-Phe-G ly-Arg-Lys-Met-Glu-Arg-I le-Ser-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 2) ~ I
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Arg-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 4) Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Glu-Arg-Ile-Arg-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 7) Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Gly-Arg-Met-Glu-Arg-Ile-Arg-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO: 43) Measurement of binding affinities for NPR-A and NPR-C.
Methods Membrane Preparation Membranes from stable 293 cell lines expressing NPR-A
(Lowe and Fendly ( 1992) J. i? iol. Chem.) or NPR-C (Cunningham et al. ( 1994) supra) were prepared by removing the cells from tissue culture platcs with O.SmM EDTA in PBS, centrifuging at 225xg, 4°
C, and resuspending in 50mM Hepes Ph7.4, 1mM EDTA, ImM DTT, 250mM Sucrose, 0.7ug/ml Pepstatin A, 0.5ug/ml Leupeptin, 1mM PMSF, 1 uM Phosphoramidon. The resuspended cells were homogenized for 30 seconds with a Brinkman polytron on setting 5. The homogenate was centrifuged in 30m1 corex tubes in Sorval HB-4 rotor at 400xg ( 1500rpm) for 10 minutes at 4°C. The supernatant was centrifuged again in screw cap polycarbonate tubes (Beckman 355618) at 100,000xg (31,500 rpm) for 30 minutes, 4° C in a Beckman T160 rotor. The pellet was resuspended in 50mM Hepes, O.ImM EDTA, S.OmM MgCl2, 100mM NaCI, 0.7~cglml Pepstatin A, 0.5ug/ml Leupeptin, 1 mM PMSF, 1 uM Phosphoramidon and homogenized again with 15 strokes in a Dounce homogenizer (Wheaton 7m1, #357542). The membranes were passed through a 22 gauge needle several times before aliquoting into eppendorf tubes for storage at -80°C.
Measurement binding affrnities for NPR-A and NPR-C Membranes were diluted in 50mM Hepes, 5mM
MgCl2, 1 OOmM NaCI, 0.1 mM EDTA, 0.2% BSA, 1 uM Phosphoramidon and added to an equal volume of diluted pure synthetic peptide plus 30pM I 125 labeled ratANP (Amersham). The binding was allowed to go for 2 hours at room temperature in 96 well U-bottomed microtiter polypropylene plates (Sigma M-4029) with gentle agitation. The bound hormone was separated from the free using a Packard Filtermate 196 cell harvester and 1 % polyethyleneimine-saturated Packard unifilter-96 GF/B plates by vacuum filtration. The plates were dried under vacuum chamber for 10 minutes prior to adding 401 Microscint-20 scintillant and counting in a Packard Topcount scintillation counter. Each affinity represents points measured in duplicate within three separate assays.
Results:
Assay # 1 N PR-A NPR-C
hANP (SEQ ID NO: 51 43.55 f 12.9 198 f 90.4 ) hBNP (SEQ ID NO: 1 645 t 298 1791 t 145 }
hBNP G1y23Phe, Leu24Trp,378 t 136 9363 t 2067 G1y25Arg (SEQ ID NO:
2) hBNP Serl9Arg (SEQ 155 t 55.3 95,498 f 14,603 1D NO: 4) hBNP Argl3Gly, Lysl4Arg43,723 t 5915 13,918 t 12,901 (SEQ
ID N0:48) hBNP Argl3Gly, Lysl4Arg,518 t 72.2 28,370 f 9,654 Serl9Arg (SEQ ID N0:49) hANP Metl2Ile, GIy20Phe,507 t 118 295 ~ 56.8 Leu2lTrp, GIy22Arg (SEQ ID
N0:50) hBNP Serl9Arg, Gly23Phe,287 t 27 172,400 t 120,096 Leu24Trp, G1y25Arg (SEQ ID NO:
7) hANP GIyl6Arg (SEQ 17.2 ~ 7.6 10,601 t 1271 ID NO: 52) Example 7 The peptides were assayed for stimulation CGMP production.
Whole Cell Stimulation of cGMP Production, and cGMP RIA
Cell Culture: Cells were maintained in F12 (w/o GHT)/Low Glucose DMEM (50/50), 25 mM Hepes pH 7.2, 2mM Glutamine, 400 microgramslml 6418 and 10% dialyzed calf serum. Log phase cells were removed from plates with 0.5 mM EDTA in PBS, taken up in media and cell concentration measured with a coulter counter. Cells were plated to 12 well dishes at a density of 300,000 cells/well, 18 to 24 hours prior to stimulation.
Whole Cell Stimulation: Plates were placed on a 37°C warm plate, media aspirated and 37°C
incubation media added for IO min. (50/50, 25 mM Hepes pH 7.2, 0. I mM
isobutyl methyl Xanthine, 0.1%
(w/v) BSA). The reaction was terminated for control value - see below, or media was aspirated and incubation media with test sample was added. The reaction was continued for 10 min., then 0.5 ml of ice cold 12%
Trichloro Acetic Acid was added to stop the reaction and extract the cyclic nucleotides. Plates were quick frozen on a bed of dry ice, thawed and the TCA with cell debris taken into a 12 x 75 mm disposable glass tube and spun at 2000 x G for 10 min. The supernatant was transferred to a fresh glass tube and the TCA removed by extracting 3 times with water saturated Ether. The residual Ether was removed by boiling off at 50° for 20 minutes. The sample is now ready for RIA determination of cGMP content.
_27-I
RIA: i00 microliters of sample (neat or dilutions thereof) was measured in duplicate. The standard curve was set up with highest concentration of cGMP first. The protocol for Acetylation is followed as described (Biomedical Techniques Inc., Stoughton, MA). After overnight incubation, and centrifugation, pellets were counted in an isodata counter. Data was analyzed using a cGMP RIA
analysis program on RS/1.
Results Figures 1 and 2 demonstrate the measurement of cGMP production in 293 cells expressing hNPR-A.
Wild-type and variants stimulate guanyl cyclase activity in the membrane preparations as measured by cGMP
production.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Genentech, Inc.
(ii) TITLE OF INVENTION: Receptor Selective BNP
(iii) NUMBER OF SEQUENCES: 52 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Dennison Associates (B) STREET: 133 Richmond Street West, Suite 301 (C) CITY: Toronto (D) PROVINCE: Ontario (E) COUNTRY: Canada (F) POSSTAL CODE: M5H 2L7 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WinPatin (Genentech) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,296,656 (B) FILING DATE: 09-OCT-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Dennison Associates (B) REFERENCE/DOCKET NUMBER: JJ-10 474CA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 368-8313 (B) TELEFAX: (416) 368-1645 (2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Phe Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Met Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met 1 5 '10 15 Glu Arg Ile Arg His Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg His Leu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg Ser Ser Ser Phe Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg Ser Ser Ser Met Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg His Ser Ser Met Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Arg His Leu Ser Met Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Ser Pro Met Glu Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Thr Met Glu Arg Ile Arg Ser Gln Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg His Leu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Ala Arg Glu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:15:
- 5 -(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Ser Pro Lys Met Val Gln Gly Ser Cys Phe Gly Arg Asn Gly Met Glu Arg Ile Arg Lys Thr Ser Gly Gly Cys Lys Val Leu Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE SEQ ID N0:16:
DESCRIPTION:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Gly Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID
N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 acids amino (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear (xi) SEQUENCE DESCRIPTION:SEQ ID N0:17:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Gly Arg Ala Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID
N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 acids amino (B) TYPE: Amino Acid
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Ser Pro Lys Met Val Gln Gly Ser Cys Phe Gly Arg Asn Gly Met Glu Arg Ile Arg Lys Thr Ser Gly Gly Cys Lys Val Leu Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE SEQ ID N0:16:
DESCRIPTION:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Gly Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID
N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 acids amino (B) TYPE: Amino Acid (D) TOPOLOGY:
Linear (xi) SEQUENCE DESCRIPTION:SEQ ID N0:17:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Gly Arg Ala Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID
N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 acids amino (B) TYPE: Amino Acid
- 6 -(D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Trp Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Asn Thr Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Gln Met Glu Arg Ile Arg His Ser Thr Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Gly His Leu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Thr Lys Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Gly Gly Leu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg _ g _ Arg His (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Thr Met Glu Arg Ile Arg His Gly Gly Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Gly Met Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Met Tyr Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Leu Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Met Phe Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Thr Ala Pro Arg (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Val Gln Gly Ser Gly (2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Ser Pro Lys Met Val Gln Gly Ser Gly (2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1..2 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1..3 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1..4 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Xaa Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 2..5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
Lys Xaa Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 3..6 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Pro Lys Xaa Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 4..7 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Ser Pro Lys Xaa Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Xaa Val Leu Arg (2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1, 5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Xaa Val Leu Arg Xaa (2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1, 5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Xaa Val Leu Arg Xaa Tyr His (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Gly Arg Met Glu Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
Ser Pro Lys Met Met His Lys Ser Gly Cys Phe Gly Arg Arg Leu Asp Arg Ile Gly Ser Leu Ser Gly Leu Gly Cys Asn Val Leu Arg Lys Tyr (2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Ser Pro Lys Thr Met Arg Asp Ser Gly Cys Phe Gly Arg Arg Leu Asp Arg Ile Gly Ser Leu Ser Gly Leu Gly Cys Asn Val Leu Arg Arg Tyr (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
Ser Gln Asp Ser Ala Phe Arg Ile Gln Glu Arg Leu Arg Asn Ser Lys Met Ala His Ser Ser Ser Cys Phe Gly Gln Lys Ile Asp Arg Ile Gly Ala Val Ser Arg Leu Gly Cys Asp Gly Leu Arg Leu Phe (2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Ser Gln Gly Ser Thr Leu Arg Val Gln Gln Arg Pro Gln Asn Ser Lys Val Thr His Ile Ser Ser Cys Phe Gly His Lys Ile Asp Arg Ile Gly Ser Val Ser Arg Leu Gly Cys Asn Ala Leu Lys Leu Leu (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Gly Arg Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Gly Arg Met Asp Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Phe Trp Arg Cys Asn Ser Phe Arg Tyr (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr (2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Arg Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Trp Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Asn Thr Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Gln Met Glu Arg Ile Arg His Ser Thr Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Gly His Leu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Thr Lys Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Gly Gly Leu Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg _ g _ Arg His (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Thr Met Glu Arg Ile Arg His Gly Gly Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Asn Met Glu Arg Ile Arg Gly Met Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Met Tyr Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Leu Trp Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Glu Arg Ile Ser Ser Ser Ser Met Phe Arg Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Thr Ala Pro Arg (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Val Gln Gly Ser Gly (2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Ser Pro Lys Met Val Gln Gly Ser Gly (2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1..2 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1..3 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1..4 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Xaa Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 2..5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
Lys Xaa Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 3..6 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
Pro Lys Xaa Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 4..7 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Ser Pro Lys Xaa Xaa Xaa Xaa Ser Gly (2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
Xaa Val Leu Arg (2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1, 5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Xaa Val Leu Arg Xaa (2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (ix) FEATURE:
(A) NAME/KEY: Unknown amino acid (B) LOCATION: 1, 5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Xaa Val Leu Arg Xaa Tyr His (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Gly Arg Met Glu Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
Ser Pro Lys Met Met His Lys Ser Gly Cys Phe Gly Arg Arg Leu Asp Arg Ile Gly Ser Leu Ser Gly Leu Gly Cys Asn Val Leu Arg Lys Tyr (2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Ser Pro Lys Thr Met Arg Asp Ser Gly Cys Phe Gly Arg Arg Leu Asp Arg Ile Gly Ser Leu Ser Gly Leu Gly Cys Asn Val Leu Arg Arg Tyr (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
Ser Gln Asp Ser Ala Phe Arg Ile Gln Glu Arg Leu Arg Asn Ser Lys Met Ala His Ser Ser Ser Cys Phe Gly Gln Lys Ile Asp Arg Ile Gly Ala Val Ser Arg Leu Gly Cys Asp Gly Leu Arg Leu Phe (2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Ser Gln Gly Ser Thr Leu Arg Val Gln Gln Arg Pro Gln Asn Ser Lys Val Thr His Ile Ser Ser Cys Phe Gly His Lys Ile Asp Arg Ile Gly Ser Val Ser Arg Leu Gly Cys Asn Ala Leu Lys Leu Leu (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Gly Arg Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Gly Arg Met Asp Arg Ile Arg Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His (2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Phe Trp Arg Cys Asn Ser Phe Arg Tyr (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr (2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Arg Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr
Claims (24)
1. A BNP variant having a decreased binding affinity for one human clearance receptor (hNPR-C) compared to human wild-type BNP wherein at least one of amino acids residues Xaa19, Xaa23, Xaa24 or Xaa25 of wild-type BNP is selected according to the following scheme:
Xaa19 is selected from the group consisting of Ser, Ala, Ash, Gly and Arg, and conservative substitutions thereof;
Xaa23 is selected from the group Gly, bet, Leu and Phe, and conservative substitutions thereof;
Xaa24 is selected from the group Leu, Trp, Tyr, and Phe, and conservative substitutions therefore; and Xaa25 is selected from the group Gly and Arg and conservative substitutions thereof.
Xaa19 is selected from the group consisting of Ser, Ala, Ash, Gly and Arg, and conservative substitutions thereof;
Xaa23 is selected from the group Gly, bet, Leu and Phe, and conservative substitutions thereof;
Xaa24 is selected from the group Leu, Trp, Tyr, and Phe, and conservative substitutions therefore; and Xaa25 is selected from the group Gly and Arg and conservative substitutions thereof.
2. The BNP variant of claim 1 wherein Xaa19 is Arg.
3. The BNP variant of Claim 1 wherein Xaa23 is Phe.
4. The BNP variant of claim 1 wherein Xaa24 is Trp.
5. The BNP variant of claim 1 wherein Xaa25 is Arg.
6. The BNP variant of claim 2 wherein Xaa23 is selected from the group Gly, Met, Phe and Leu;
Xaa24 is selected from the group Trp, Tyr and Phe; and Xaa25 is selected from the group Gly and Arg.
Xaa24 is selected from the group Trp, Tyr and Phe; and Xaa25 is selected from the group Gly and Arg.
7. The BNP variant of claim 1 represented by Formula I:
wherein:
A is selected from the group consisting of H, C1-C6 alkanoyl and Thr-Ala-Pro-Arg (SEQ ID NO: 30);
R1 is absent ar selected from the group consisting of a peptide from between 1 and 10 amino acids, Gly-Ser -Gly, Val-Gln-Gly-Ser-Gly (SEQ ID NO: 31) and Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly (SEQ ID NO: 32);
Xaa19 is selected from the group consisting of Arg, Ala, Asn, Gly and Ser;
Xaa23 is selected from the group Gly, Met, Phe, Leu, Nle;
Xaa24 is selected from the group Leu, Trp, Tyr, Phe;
Xaa25 is selected from the group Gly and Arg;
R2 is selected from the group consisting of a 1 to 6 amino acid peptide and Lys-Val-Leu-Arg-Awg-His (SEQ ID NO:
33); and B is selected from OR3 and NR3R4, where R6 and R4 are independently selected from H, C1-C6alkyl, C6-C12aryl and C6-12aryl-C1-6alkyl.
wherein:
A is selected from the group consisting of H, C1-C6 alkanoyl and Thr-Ala-Pro-Arg (SEQ ID NO: 30);
R1 is absent ar selected from the group consisting of a peptide from between 1 and 10 amino acids, Gly-Ser -Gly, Val-Gln-Gly-Ser-Gly (SEQ ID NO: 31) and Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly (SEQ ID NO: 32);
Xaa19 is selected from the group consisting of Arg, Ala, Asn, Gly and Ser;
Xaa23 is selected from the group Gly, Met, Phe, Leu, Nle;
Xaa24 is selected from the group Leu, Trp, Tyr, Phe;
Xaa25 is selected from the group Gly and Arg;
R2 is selected from the group consisting of a 1 to 6 amino acid peptide and Lys-Val-Leu-Arg-Awg-His (SEQ ID NO:
33); and B is selected from OR3 and NR3R4, where R6 and R4 are independently selected from H, C1-C6alkyl, C6-C12aryl and C6-12aryl-C1-6alkyl.
The BNP variant of claim 7 having the following composition:
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His(SEQ ID NO:2)
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His(SEQ ID NO:2)
9. The BNP variant of claim 7 having the following composition:
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Met-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His(SEQ ID NO:3)
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Met-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His(SEQ ID NO:3)
10. The BNP variant of claim 7 having the following composition:
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Arg-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His(SEQ ID NO:4)
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Arg-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-His(SEQ ID NO:4)
11. The BNP variant of claim 7 having the following composition:
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Arg-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His(SEQ ID NO:7)
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Arg-Ser-Ser-Ser-Phe-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His(SEQ ID NO:7)
12. The BNP variant of claim 7 having the following composition:
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Arg-Ser-Ser-Ser-Met-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO:8)
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Arg-Ser-Ser-Ser-Met-Trp-Arg-Cys-Lys-Val-Leu-Arg-Arg-His (SEQ ID NO:8)
13. A composition comprising a sterile excipient and the BNP variant of claim 1.
14. A method of treating a disorder of electrolyte balance in a mammal comprising administering a pharmaceutically effective amount of the composition of claim 13 to the mammal.
15. A method for inducing natriuresis, diuresis or vasodilation comprising administering to a mammal in need thereof a pharmaceutically effective amount of the composition of claim 13.
16. A BNP variant according to any one of claims 1 to 12 for medical use.
17. Use of a BNP variant according to any one of claims 1 to 12 in the preparation or a medicament to treat a disorder of electrolyte balance in a mammal.
18. Use of a BNP variant according to any one of claims 1 to 12 in the prepartition of a medicament to induce natriuresis, diuresis or vasodilation in a mammal.
19. An isolated DNA molecule which encodes a BNP variant according to any one of claims 1 to 12.
20. An isolated DNA molecule according to claim 19 which is operably linked to an expression control sequence.
21. An expression vector which comprises an isolated DNA
molecule according to claim 20 wherein the expression control sequence may be recognised by a host cell transformed with the vector.
molecule according to claim 20 wherein the expression control sequence may be recognised by a host cell transformed with the vector.
22. A host cell which is transformed with a vector according to claim 21.
23. A method which comprises culturing a host cell according to claim 22 under conditions suitable for expression of said BNP variant.
24. A method which following culture according to claim 23 comprises recovering said BNP variant from culture medium.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US73188096A | 1996-10-22 | 1996-10-22 | |
US08/731,880 | 1996-10-22 | ||
PCT/US1997/018384 WO1998017690A1 (en) | 1996-10-22 | 1997-10-09 | Receptor specific brain natriuretic peptide (bnp) |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2269656A1 true CA2269656A1 (en) | 1998-04-30 |
Family
ID=24941306
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002269656A Abandoned CA2269656A1 (en) | 1996-10-22 | 1997-10-09 | Receptor specific brain natriuretic peptide (bnp) |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0939770A1 (en) |
JP (1) | JP2001503256A (en) |
AU (1) | AU4816497A (en) |
CA (1) | CA2269656A1 (en) |
WO (1) | WO1998017690A1 (en) |
ZA (1) | ZA979060B (en) |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004047871A2 (en) | 2002-11-26 | 2004-06-10 | Nobex Corporation | Modified naturetic compounds, conjugates, and uses thereof |
ES2887039T3 (en) | 2004-04-21 | 2021-12-21 | Alexion Pharma Inc | Conjugates for bone delivery and method of using them to target proteins to bone |
CA2823066A1 (en) | 2010-12-27 | 2012-07-05 | Alexion Pharma International Sarl | Compositions comprising natriuretic peptides and methods of use thereof |
US10052366B2 (en) | 2012-05-21 | 2018-08-21 | Alexion Pharmaceuticsl, Inc. | Compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof |
US10822596B2 (en) | 2014-07-11 | 2020-11-03 | Alexion Pharmaceuticals, Inc. | Compositions and methods for treating craniosynostosis |
WO2016090251A1 (en) | 2014-12-05 | 2016-06-09 | Alexion Pharmaceuticals, Inc. | Treating seizure with recombinant alkaline phosphatase |
EP3250227A2 (en) | 2015-01-28 | 2017-12-06 | Alexion Pharmaceuticals, Inc. | Methods of treating a subject with an alkaline phosphatase deficiency |
MX2018002121A (en) | 2015-08-17 | 2018-06-18 | Alexion Pharma Inc | Manufacturing of alkaline phosphatases. |
US11229686B2 (en) | 2015-09-28 | 2022-01-25 | Alexion Pharmaceuticals, Inc. | Reduced frequency dosage regimens for tissue non-specific alkaline phosphatase (TNSALP)-enzyme replacement therapy of hypophosphatasia |
JP2018533571A (en) | 2015-10-30 | 2018-11-15 | アレクシオン ファーマシューティカルズ, インコーポレイテッド | Methods for treating cranial suture early healing disease in a patient |
EP3426286A4 (en) | 2016-03-08 | 2019-12-04 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia in children |
EP3436020A4 (en) | 2016-04-01 | 2019-12-25 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia in adolescents and adults |
JP2019513711A (en) | 2016-04-01 | 2019-05-30 | アレクシオン ファーマシューティカルズ, インコーポレイテッド | Treating muscle weakness with alkaline phosphatase |
US10988744B2 (en) | 2016-06-06 | 2021-04-27 | Alexion Pharmaceuticals, Inc. | Method of producing alkaline phosphatase |
US11116821B2 (en) | 2016-08-18 | 2021-09-14 | Alexion Pharmaceuticals, Inc. | Methods for treating tracheobronchomalacia |
EP3600383A4 (en) | 2017-03-31 | 2020-10-28 | Alexion Pharmaceuticals, Inc. | Methods for treating hypophosphatasia (hpp) in adults and adolescents |
WO2019190752A1 (en) | 2018-03-30 | 2019-10-03 | Alexion Pharmaceuticals, Inc. | Manufacturing of glycoproteins |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2194895T3 (en) * | 1993-11-12 | 2003-12-01 | Genentech Inc | SPECIAL ATRIAL NATURAL PEPTIDES OF A RECEIVER. |
-
1997
- 1997-10-09 JP JP51943698A patent/JP2001503256A/en not_active Ceased
- 1997-10-09 AU AU48164/97A patent/AU4816497A/en not_active Abandoned
- 1997-10-09 WO PCT/US1997/018384 patent/WO1998017690A1/en not_active Application Discontinuation
- 1997-10-09 ZA ZA979060A patent/ZA979060B/en unknown
- 1997-10-09 CA CA002269656A patent/CA2269656A1/en not_active Abandoned
- 1997-10-09 EP EP97910899A patent/EP0939770A1/en not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
ZA979060B (en) | 1999-04-09 |
WO1998017690A1 (en) | 1998-04-30 |
AU4816497A (en) | 1998-05-15 |
EP0939770A1 (en) | 1999-09-08 |
JP2001503256A (en) | 2001-03-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6028055A (en) | Receptor selective BNP | |
US5846932A (en) | Receptor specific atrial natriuretic peptides | |
US5665704A (en) | Receptor specific atrial natriuretic peptides | |
EP0728147B1 (en) | Receptor specific atrial natriuretic peptides | |
US6525022B1 (en) | Receptor specific atrial natriuretic peptides | |
US5583108A (en) | Vasonatrin peptide and analogs thereof | |
CA2269656A1 (en) | Receptor specific brain natriuretic peptide (bnp) | |
US6025467A (en) | Parathyroid hormone derivatives and their use | |
AU632043B2 (en) | Cyclic analogs of atrial natriuretic peptides | |
EP0543425B1 (en) | Endothelin DNA and use thereof | |
JPH05255392A (en) | A-c-b proinsulin, its production, method for using it and intermediate in insulin production | |
EP1709065A2 (en) | A counterion exchange process for peptides | |
JPH0690780A (en) | Human relaxin gene | |
WO1988006598A1 (en) | Vasoactive intestinal peptide analogs | |
JP4166289B2 (en) | Cyclic CRF agonist | |
EP0323740A2 (en) | Linear analogs of atrial natriuretic peptides | |
Sohma et al. | Comparative properties of insulin-like growth factor 1 (IGF-1) and [Gly7D-Ala] IGF-1 prepared by total chemical synthesis | |
AU8237187A (en) | Derivatives of atrial natriuretic peptides | |
JPH0725799B2 (en) | High calcium humoral factor antagonist | |
EP0315118A2 (en) | DNA coding for endothelin and use thereof | |
Morris et al. | Synthesis of mouse renin as a 2‐5‐33‐5 kilodalton pre‐pro‐two‐chain molecule and use of its cDNA to identify the human gene | |
DK2209798T3 (en) | modified væksthormonpolypeptider | |
JPH09157294A (en) | Parathyroid hormone derivative | |
CA1340505C (en) | Bovine placental lactogen | |
Raingeaud et al. | Production, analysis and bioactivity of recombinant vasoactive intestinal peptide analogs |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Dead |