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WO1998037910A1 - Inhibiteurs peptidiques de propeptide/prohormone convertases - Google Patents

Inhibiteurs peptidiques de propeptide/prohormone convertases Download PDF

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Publication number
WO1998037910A1
WO1998037910A1 PCT/US1998/003642 US9803642W WO9837910A1 WO 1998037910 A1 WO1998037910 A1 WO 1998037910A1 US 9803642 W US9803642 W US 9803642W WO 9837910 A1 WO9837910 A1 WO 9837910A1
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seq
peptide
ala
val
thr
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PCT/US1998/003642
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English (en)
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Bryan D. Noe
Mark E. Rothenberg
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Emory University
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Priority to AU63378/98A priority Critical patent/AU6337898A/en
Publication of WO1998037910A1 publication Critical patent/WO1998037910A1/fr

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    • 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
    • C07K14/62—Insulins
    • 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
    • C07K14/655—Somatostatins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention pertains to peptide inhibitors of propeptide/prohormone convertases, to a method of inhibiting propeptide/prohormone convertases, to a method of treating cancer, endocrine disorders, and viral infections including AIDS, and, more specifically, to a method of treating cancer, endocrine disorders, and viral infections by means of peptide inhibitors of propeptide/prohormone convertases.
  • PCs Prohormone/Propeptide Convertases
  • PCI/3 and PC2 are expressed predominantly within endocrine and neuroendocrine cells and tissues, while furin, PACE4 and PC6 isoforms are expressed ubiquitously.
  • PC6A has been localized only within a subset of endocrine and non-endocrine cells (for example, pancreatic islets and gut endocrine cells), while PC4 is expressed primarily within testicular germ cells. See P. A.
  • PCs are involved in exacerbating the pathophysiology of many different types of cancer.
  • Prime candidates are PCI/3, PC2, and PC6A. See j. Lusson et al. (1993) Proc. Natl. Acad. Sci. USA 90:6691-6695 and T. Nakagawa et al. (1993) J. Biochem. 113: 132-135.
  • PCs are involved.
  • tumors of neuroendocrine cell origin often hypersecrete peptide hormones. See IF Rehfield et al. (1995) Front.
  • Examples include insulinomas, glucagonomas, somatostatinomas, gastrinomas (Zollinger-Ellison Syndrome), tumors of the anterior pituitary (ACTH or growth hormone hypersecretion), among others.
  • glucagonomas glucagonomas
  • somatostatinomas gastrinomas (Zollinger-Ellison Syndrome)
  • tumors of the anterior pituitary afflicted individuals suffer severe physiologic consequences as a result of peptide hormone hypersecretion.
  • the PCs play an essential role in mediating the metabolic consequences of peptide hormone hypersecretion in that their activity is responsible for cleaving the biologically inactive precursors of the bioactive peptides.
  • the substrates are precursors to growth factors such as epidermal growth factor (EGF), the family of insulin-like growth factors (IGFs), nerve growth factors such as NGF- ⁇ , neurotrophin, platelet derived growth factors (PDGFs), or transforming growth factor beta (TGF- ⁇ ).
  • EGF epidermal growth factor
  • IGFs insulin-like growth factors
  • nerve growth factors such as NGF- ⁇ , neurotrophin, platelet derived growth factors (PDGFs), or transforming growth factor beta (TGF- ⁇ ).
  • TGF- ⁇ transforming growth factor beta
  • gpl20 and the non-covalently associated gp41, are cleavage products of gpl60, a precursor protein initially synthesized in the host cell and sorted into the constitutive secretory pathway. See R. Wukket et al. (1988) Proc. Natl. Acad. Sci USA 85:9580-9584 and M. Kowalski et al. (1987) Science 237: 1351-1355.
  • the gpl60 cleavage site, REKR l AN fits the furin cleavage site consensus sequence REKR. F. Veronese et al. (1985) Science 229: 1402-1405 and Hatsuzawa et al. (1992) J.
  • furin can mediate cleavage of gpl60. See S. Hallenberger et al. (1992) Nature 360: 358-361 and E. Decroly et al.(1997) FEBS Lett. 405: 68-72. However, the observation that g l60 is also cleaved in furin- deficient cells suggests that proteases other than fiirin may be responsible for gpl60 processing in some cell types. See M. Inocencio (1997) J. Biol. Chem. 272: 1344- 1348. Experimental evidence from CD4+ cells suggests that furin, PC6 and/or PC7 are primarily responsible for host cell- mediated cleavage of gpl60 in HIV target cells. See S. Zarkik et al. (1997) FEBS Lett. 406: 205-210 and E. Decroly et al. (1996) J. Biol. Chem. 271: 30442-30450.
  • inhibitors are expressed in intact cells and targeted to the regulated secretory pathway. In this way, a constantly renewable supply of inhibitor is supplied in the trans Golgi network (to inhibit furin) and in secretory granules (to inhibit PCs) by constitutive or regulated expression.
  • inhibitors is expressed and targeted into the regulated secretory pathway of mammalian neuroendocrine cells where they are suitable as reagents in reducing malignant transformation and tumorigenesis in cancer cells.
  • Inhibition of peptide prohormone conversion also reduces the serious physiologic consequences of tumors that produce and release bioactive peptides such as those derived from insulinomas, gastrinomas, or lung cancer cells which often hypersecrete a variety of hormonally active peptides. Inhibition of prohormone conversion would reduce or block the release of the bioactive product peptides from such tumors.
  • inhibition of peptide prohormone conversion could inhibit neoplasia by blocking PC- mediated processing of growth factors which are produced in many types of tumor cells. These endogenously produced growth factors operate in an autocrine fashion to stimulate malignant transformation and tumorigenesis. If maturation of these growth factors are reduced or prevented, growth and proliferation of tumor cells would be inhibited. However, such results had not been obtained in the prior art.
  • Peptide inhibitors are suitable in the treatment of viral infections, including HIV infections.
  • PC inhibitors are expressed and targeted into the regulated secretory pathway of HIV target cells, where they inhibit processing of gpl60, block formation of gpl20, thereby diminishing the infectivity of newly synthesized virions. Formation of syncytia also decreases, contributing to a stabilization of the infected patient's immune system. Thus, if processing of gpl60 are reduced or prevented, HIV infection is inhibited. Such results have not been obtained in the prior art.
  • an object of the present invention is to provide a novel peptide inhibitor of propeptide/prohormone convertase.
  • a peptide comprising at least one peptide selected from the group consisting of the peptides RGDVACTKQFDPVWT (SEQ ID NO: 1 ),
  • GEDVMCPMVYDPVLLT (SEQ ID NO: 2 ), DTGRVCTREYRPVTNS (SEQ ID NO: 3), RDGNICNKLYDPNWT (SEQ ID NO: 4), ADGVMCTREYAPWVT (SEQ ID NO: 5), SEGVMCPMIYDPVLLT (SEQ ID NO: 6),
  • CALEGSLQKRGIVEQCC SEQ ID NO: 7
  • CALEGSLQKHGIVEQCC SEQ ID NO: 8
  • CRDGVICNKLYDPVWTC SEQ ID NO: 9
  • CADGNMCTREYAPWVTC SEQ ID NO: 10
  • CDTGRNCTREYRPNTNSC SEQ ID NO: 11
  • CRGDVACTKQFDPVWTC SEQ ID NO: 13
  • CGEDVMCPMVYDPVLLTC SEQ ID NO: 14
  • CSEGVMCPMI YDP VLLTC SEQ ID NO : 15
  • RDGVICNKNYDP VWT SEQ ID NO : 16
  • DDAVMCTREYAPVWT (SEQ ED NO: 17), DQDRACIKIYDPLNVT (SEQ ID NO: 18), DAGVMCTREYAPWVT (SEQ ID NO: 19), RDGVMCTKQYDP VT (SEQ ID NO: 20), EPGRMCTKEWRPITNT (SEQ ID NO: 21),
  • RDDVWCNKLYDPWVT SEQ ID NO: 22
  • EDSVMCTREYAPVVVT SEQ ID NO: 23
  • TGDVMCTKQYDVWT SEQ ID NO: 24
  • longer peptides containing said peptides comprising the steps of treating a propeptide/prohormone convertase with a peptide selected from the above-described group and monitoring the degree of inhibition of propeptide/prohormone convertase.
  • a method of treating a tumor cell comprising the steps of treating one or more tumor cells with a peptide selected from the above-described group and monitoring the degree of inhibition of propeptide/prohormone convertase.
  • the above objects, among others, have also been achieved by means of a method of treating a viral infection comprising the steps of treating one or more cells infected with a viral pathogen with at least one peptide selected from the above- described group and monitoring the degree of inhibition of propeptide/prohormone convertase. Further, the above objects, among others, have been achieved by means of a kit for inhibiting propeptide/prohormone convertase comprising at least one peptide selected from the above-described group.
  • Figure 1 depicts several representative members of the furin/PC family along with their tissue distribution
  • Figures 2 A and 2B represent the effect of the inhibitor Z-Thr-(4-Amph- Gly)P(oph) 2 on the processing of glucagon;
  • Figure 3 illustrates the effects of natural and Streptomyces inhibitor peptides on the ability of PC 1/3 to cleave a fluorogenic substrate
  • FIGS. 4 A, 4B, 4C, and 4D depict the effects of Streptomyces inhibitors on PC2 at two concentrations
  • Figures 4E, 4F, and 4G represent the comparison of different concentrations of Streptomyces inhibitors
  • Figure 5 illustrates the comparison of the effects of full-length STI-2 and 16-amino-acid-long peptides (based on sequences of PSN and SSI) on recombinant PC 1/3 cleavage of a fluorogenic substrate;
  • Figures 6 A, 6B, 6C, and 6D depict the effect of proinsulin C-A (KR) and proinsulin C-A (KH) on the ability of PC 1/3 and PC2 to cleave a fluorogenic substrate;
  • Figures 7 A, 7B, 7C, 7D and 7E illustrate the effects of the STI-2 peptide and the modified STI-2(KN) peptide on PC 1/3 and PC2 activity;
  • Figures 8 A, 8B, 8C, 8D, 8E, and 8F represent the effects of the STI-2 peptide on PC 1/3- or PC2-mediated proglucagon conversion;
  • Figures 9 A, 9B, 9C, 9D, 9E, and 9F illustrate the effects of STI-2(KN) on PC 1/3 -mediated proglucagon conversion;
  • Figures 10 A, 10B, IOC, 10D, 10E, and 10F illustrate the effects of mutated forms (N72K and N72R) of full-length STI-2 SSI-like inhibitors on PCl/3 and PC2 activity.
  • Figures 11 A, 1 IB, 11C, and 1 ID illustrate the effects on full length STI-2 [N72R] on furin or PC6A mediated cleavage.
  • Figure 12 depicts a schematic of the "carrier" preprosomatostatin and the peptide products of the chimeric cDNA constructs coding for amino acid sequences representing PC inhibitors.
  • the present invention relates to a peptide inhibitor of propeptide/prohormone convertase selected from the group consisting of
  • CTRXYXPWVT (SEQ ID NO: 43), CTKXYXPWVT (SEQ ID NO: 44), CNRXYXPVWT (SEQ ID NO: 45), CNKXYXPWVT (SEQ ID NO: 46), CTRXWXPVWT (SEQ ID NO: 47),
  • CTKXWXPWVT (SEQ ID NO: 48), wherein X is any of the 20 common amino acids, and longer peptides containing said peptides.
  • the peptide inhibitor of propeptide/prohormone convertase is selected from the group consisting of:
  • RGDVACTKQFDPWVT (SEQ ID NO: 1 ), GEDVMCPMVYDPVLLT (SEQ ID NO: 2 ), DTGRVCTREYRPVTVS (SEQ ID NO: 3), RDGVICNKLYDPVWT (SEQ ID NO: 4), ADGVMCTREYAPWVT (SEQ ID NO: 5), SEGVMCPMIYDPNLLT (SEQ ID NO: 6), CALEGSLQKRGIVEQCC (SEQ ID NO: 7), CALEGSLQKHGIVEQCC (SEQ ID NO: 8), CRDGVICNKLYDPWVTC (SEQ ID NO: 9),
  • CADGVMCTREYAPVWTC (SEQ ID NO: 10), CDTGRVCTREYRPVTVSC (SEQ ID NO: 11), CRGDVACTKQFDPWVTC (SEQ ID NO: 13), CGEDVMCPMVYDPVLLTC (SEQ ID NO: 14), CSEGVMCPMIYDPVLLTC (SEQ ID NO: 15),
  • RDGVICNKNYDPVWT (SEQ ID NO: 16), DDAVMCTREYAPWVT (SEQ ID NO: 17), DQDRACKIYDPLWT (SEQ ID NO: 18), DAGVMCTREYAPWVT (SEQ ID NO: 19), RDGVMCTKQYDPWVT (SEQ ID NO: 20),
  • EPGRMCTKEWRPITVT (SEQ ID NO: 21), RDDVWCNKLYDPWVT (SEQ ID NO: 22), EDSVMCTREYAPVVVT (SEQ ID NO: 23), TGDVMCTKQYDWVT (SEQ ID NO: 24), and longer peptides containing said peptides, such as SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ ID NO: 41; or SEQ ID NO: 42.
  • the peptide inhibitor has the following sequence:
  • CADLRGVGGDEDALKARDGVICNKL YDPVWTVDGVWQGKRVSYERTFGNE CVKNSYGTSLFAF, (SEQ ID NO:25) and derivatives and mutants thereof.
  • the propeptide/prohormone convertase is selected from the group consisting of PCl/3, PC2, PC6A, PC7 and furin.
  • the peptide inhibitor is useful in the treatment of a tumor cell.
  • the tumor cell may be a hormone-secreting tumor cell, a tumor cell of a neuroendocrine neoplastic disease, or a tumor cell of a neoplastic disease involving overproduction of a growth factor.
  • the peptide inhibitor is useful in the treatment of a cell infected with a viral pathogen, or in the treatment of a cell infected with HIV.
  • a method of suppressing propeptide/prohormone convertase activity comprises the steps of treating a propeptide/prohormone convertase with a peptide selected from the group consisting of
  • CTKXYXPVWT (SEQ ID NO: 44), CNRXYXPVWT (SEQ ID NO: 45),
  • CNKXYXPWVT (SEQ ID NO: 46), CTRXWXPVVNT (SEQ ID NO: 47), CTKXWXPVWT (SEQ ID NO: 48), wherein X is any of the 20 common amino acids, and longer peptides containing said peptides.
  • a method of suppressing propeptide/prohormone convertase acitivity comprises the steps of treating a propeptide/prohormone convertase with a peptide selected from the group consisting of RGDNACTKQFDPNVNT (SEQ ID NO: 1 ), GEDVMCPMVYDPVLLT (SEQ ID NO: 2 ), DTGRVCTREYRPVTVS (SEQ ID NO: 3), RDGVICNKLYDPVWT (SEQ ID NO: 4), ADGVMCTREYAPNVNT (SEQ ID NO: 5),
  • SEGVMCPMIYDPVLLT (SEQ ID NO: 6), CALEGSLQKRGIVEQCC (SEQ ID NO: 7), CALEGSLQKHGIVEQCC (SEQ ID NO: 8), CRDGVICNKLYDPWVTC (SEQ ID NO: 9), CADGNMCTREYAPWVTC (SEQ ID NO: 10),
  • CDTGRVCTREYRPVTVSC SEQ ID NO: 11
  • CRGDVACTKQFDPWVTC SEQ ID NO: 13
  • CGEDVMCPMVYDPVLLTC SEQ ID NO: 14
  • CSEGVMCPMIYDPVLLTC SEQ ID NO: 15
  • RDGVICNKNYDPVWT SEQ ID NO: 16
  • DDAVMCTREYAPNWT SEQ ID NO: 17
  • DQDRACIKIYDPLNVT SEQ ID NO: 18
  • DAGVMCTREYAPWVT SEQ ID NO: 19
  • RDGVMCTKQYDPWVT SEQ ID NO: 20
  • EPGRMCTKEWRPITVT SEQ ID NO: 21
  • RDDVWCNKLYDPVVVT (SEQ ID NO: 22), EDSVMCTREYAPWVT (SEQ ID NO: 23), TGDVMCTKQYDWVT (SEQ ID NO: 24), and longer peptides containing said peptides, such as SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ DD NO: 30; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ DD NO: 30; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ DD NO: 30; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ DD NO: 30; SEQ ID NO: 25; SEQ ID NO: 26;
  • the propeptide/prohormone convertase is selected from the group consisting of PCl/3, PC2, PC6A, PC7, and furin.
  • a method of treating a tumor cell comprises the steps of treating a tumor cell with at least one peptide selected from the group consisting of
  • a method of treating a tumor cell comprises the steps of treating a tumor cell with at least one peptide selected from the group consisting of
  • RGDVACTKQFDPNVVT SEQ ID NO: 1
  • GEDVMCPMVYDPVLLT SEQ ID NO: 2
  • DTGRVCTREYRPVTVS SEQ ID NO: 3
  • RDGVICNKLYDPWVT (SEQ ID NO: 4), ADGVMCTREYAPWVT (SEQ ID NO: 5), SEGVMCPMIYDP VLLT (SEQ ID NO: 6), CALEGSLQKRGIVEQCC (SEQ ID NO: 7), CALEGSLQKHGIVEQCC (SEQ ID NO: 8),
  • CRDGVICNKLYDPVWTC (SEQ ID NO: 9), CADGNMCTREYAPNVVTC (SEQ ID NO: 10), CDTGRVCTREYRPNTVSC (SEQ ID NO: 11), CRGDVACTKQFDPNWTC (SEQ ID NO: 13), CGEDVMCPMVYDPVLLTC (SEQ ID NO: 14), CSEGVMCPMIYDPVLLTC (SEQ ID NO : 15), RDGVICNKNYDPVWT (SEQ ID NO: 16), DDAVMCTREYAPVWT (SEQ ID NO: 17), DQDRACIKIYDPLWT (SEQ ID NO: 18),
  • DAGNMCTREYAPVNVT (SEQ ID NO: 19), RDGVMCTKQYDPVWT (SEQ ID NO: 20), EPGRMCTKEWRPITVT (SEQ ID NO: 21), RDDVWCNKLYDPWVT (SEQ ID NO: 22), EDSVMCTREYAPVWT (SEQ ID NO: 23),
  • TGDVMCTKQYDVWT (SEQ ID NO: 24), and longer peptides containing said peptides; such as SEQ ID NO: 25; SEQ D NO: 26; SEQ ED NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ED NO: 32; SEQ ED NO: 33; SEQ ED NO: 34; SEQ ED NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ED NO: 40; SEQ ED NO: 41; or SEQ ED NO: 42; and monitoring the degree of inhibition of propeptide/prohormone convertase.
  • SEQ ID NO: 25 SEQ D NO: 26; SEQ ED NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ED NO: 32; SEQ ED NO: 33
  • the propeptide/prohormone convertase is PCl/3 or PC2.
  • the tumor cell is a hormone-secreting tumor cell, a tumor cell of a neuroendocrine neoplastic disease, or a tumor cell of a neoplastic disease involving overproduction of a growth factor.
  • CTRXYXPVWT (SEQ ID NO: 43), CTKXYXPVWT (SEQ ID NO: 44), CNRXYXPVWT (SEQ ID NO: 45), CNKXYXPVWT (SEQ ID NO: 46), CTRXWXPVWT (SEQ ID NO: 47), CTKXWXPVWT (SEQ ED NO: 48), wherein X is any of the 20 common amino acids, and longer peptides containing said peptides.
  • RGDVACTKQFDPVWT (SEQ ID NO: 1 )
  • GEDVMCPMVYDPVLLT (SEQ ID NO: 2 )
  • DTGRVCTREYRPVTVS SEQ ID NO: 3
  • RDGVICNKLYDPVWT SEQ ID NO: 4
  • ADGVMCTREYAPWVT SEQ ID NO: 5
  • SEGVMCPMIYDPVLLT SEQ ED NO: 6
  • CALEGSLQKRGIVEQCC SEQ ID NO: 7
  • CALEGSLQKHGIVEQCC SEQ ID NO: 8
  • CRDGVICNKLYDPVVVTC SEQ ID NO: 9
  • CADGVMCTREYAPVVVTC SEQ ID NO: 10
  • CDTGRVCTREYRPVTVSC SEQ ID NO: 11
  • CRGDVACTKQFDPWVTC SEQ ID NO: 13
  • CGEDVMCPMVYDPVLLTC (SEQ ID NO: 14), CSEGVMCPMIYDPVLLTC (SEQ ID NO: 15), RDGVICNKNYDPVWT (SEQ ID NO: 16), DDAVMCTREYAPVWT (SEQ ID NO: 17), DQDRACIKIYDPLWT (SEQ ID NO : 18),
  • DAGVMCTREYAPVWT (SEQ ID NO: 19), RDGVMCTKQYDPVWT (SEQ ID NO: 20), EPGRMCTKEWRPITVT (SEQ ID NO: 21), RDDVWCNKLYDPWVT (SEQ ID NO: 22), EDSVMCTREYAPVWT (SEQ ED NO: 23), TGDVMCTKQYDVWT (SEQ ED NO: 24), and longer peptides containing said peptides; such as SEQ ID NO: 25; SEQ ID NO: 26; SEQ ED NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ED NO: 38; SEQ ID NO: 39; SEQ ID NO: 40; SEQ m NO: 41; or SEQ ID NO: 42
  • the propeptide/prohormone is PC6A, PC7, or furin.
  • the cell is selected from the group consisting of lymphoid and monocyte/macrophage cells.
  • Another embodiment of the present invention relates to the nucleic acid encoding the peptide inhibitors of the present invention.
  • an expression vector for a peptide inhibitor comprises at least one nucleic acid encoding at least one peptide inhibitor of the present invention, operatively linked to a nucleic acid encoding a signal peptide.
  • the expression vector of the present invention comprises a nucleic acid encoding a Golgi retention signal.
  • the expression vector of the present invention further comprises a nucleic acid encoding an inducible element.
  • the inducible element of the expression vector of the present invention is a 5' LTR component of HJV-1.
  • Another embodiment of the present invention relates to a chimeric protein comprising at least one peptide inhibitor of the present invention, fused at its amino terminus to a signal peptide.
  • Another embodiment of the present invention relates to an expression vector for a peptide inhibitor comprising at least one nucleic acid encoding at least one peptide of the present invention, operatively linked to a nucleic acid encoding a carrier peptide.
  • the carrier peptide is a preproregion of a prohormone.
  • the preprohormone is pro somato statin.
  • the expression vector further comprises a nucleic acid encoding a Golgi retention signal.
  • the expression vector further comprises a nucleic acid encoding an inducible element, which may be a 5' LTR of HIN.
  • Another embodiment of the present invention relates to a chimeric protein comprising at least one peptide inhibitor of the present invention, fused at its amino terminus to the preproregion of a prohormone.
  • PCs propeptide/prohormone convertases
  • PCs are generally specific for cleaving at pairs of basic amino acid residues (for example Lys-Arg), although PCs can also cleave at sites containing only one basic amino acid (for example Arg) or at sites with more than two basic amino acids. Identification and characterization of the PCs was difficult, because their low abundance and the lack of specific assays made it difficult to separate the PCs from more abundant and nonspecific proteases which contaminated sample preparations.
  • Kex2 a cleavage enzyme with a catalytic region which is 28% identical with the bacterial subtilisin serine proteases which cleave at the C-terminus of both Lys-Arg and Arg-Arg pairs with approximately equal efficiency, but cleave poorly after single arginine residues or at pairs of lysines.
  • the discovery and characterization of Kex2 represents the first identification of an authentic propeptide/prohormone convertase. Subsequently, a mammalian homolog of Kex2, furin, was identified.
  • PCI Kex2-like endoprotease
  • PC mRNAs encode proteins which, while highly homologous to furin, exhibit some distinct differences.
  • the predicted size of PCI 13 is 87 kDa while PC2 is expressed as either a 66 kDa or a 64-66 kDa form.
  • the PCs do not contain the cysteine-rich region, the transmembrane domain, or the cytoplasmic tail observed in furin.
  • the catalytic domain contains the subtilisin-like active site Asp, His, Asn (Asp for PC2), and Ser residues.
  • Both PCl/3 and PC2, like Kex2 and furin, contain a hydrophobic N-terminal signal peptide which is cleaved in the endoplasmic reticulum.
  • Figure 1 provides a diagrammatic representation of some of the known members of the furin/PC family along with their tissue distribution. Filled boxes represent signal peptide; arrows, pro-enzyme cleavage site; diagonal shading, domain with subtilisin homology showing the location of residues corresponding to active site amino acids in subtilisin BPN'; STR, serine-threonine rich domain; dotted boxes, cysteine rich region; cross-hatched area, transmembrane domain (TM); AH, amphipathic helix.
  • the available cleavage specificity data demonstrate that PCl/3 and PC2 cleave precursors both at specific single basic residues and at pairs of basic residues, and that the four combinations Lys-Arg, Arg- Arg, Lys-Lys, and Arg-Lys are cleavage sites for these convertases.
  • the present inventors investigated naturally occurring inhibitors of the subtilisin BPN' proteases produced by the gram positive bacteria Streptomyces as possible PC inhibitors.
  • Streptomyces Subtilisin Inhibitor which was originally purified from the culture medium of the bacterial strain Streptomyces albogriseolus S-3253, exhibits a strong inhibitory preference toward the subtilisin BPN' proteinase (the putative ancestor of the mammalian PCs).
  • subtilisin BPN' proteinase the putative ancestor of the mammalian PCs.
  • All of the SSI inhibitors characterized to date exist as dimeric proteins consisting of two identical subunits connected by disulfide bridges. The specificity and kinetics of the SSI inhibitors reside in the amino acid sequence surrounding the reactive site. SSI-like inhibitors function by forming complexes with the target protease but cleavage of the peptide bond at the reactive site of the inhibitor is prevented.
  • the reactive site of the SSI inhibitor is comprised of the amino acid pair Met(73)-Nal(74), whereas in the similar STI-1 and STI-2 inhibitors the reactive sites are comprised of Arg-Glu and Lys-Leu, respectively. See IE. Strickler et al. (1992) J. Biol. Chem. 267:3236-3241. All SSI-like inhibitors exhibit a strong inhibitory preference for the subtilisin-like over the trypsin-like proteinases.
  • the PCs are specific for cleavage at either monobasic or dibasic amino acid cleavage sites.
  • the first criterion was to select a mammalian endocrine cell line which synthesizes one or more precursor derived peptides and which co-expresses one or more of the known PCs. Such a culture system permits examination of the function of the PCs in relation to their substrates in intact cells. Second, the cell line should express a prohormone substrate which undergoes differential processing involving the activities of multiple PCs. The ⁇ TCl-6 cell line fulfills these criteria.
  • the ⁇ TCl cell line evolved from a glucagonoma developed in transgenic mice.
  • the mice express a hybrid gene consisting of a glucagon-promoter sequence fused to the SN40 T-antigen oncoprotein.
  • the ⁇ TCl -6 line was shown to maintain many of its differentiated pancreatic ⁇ -cell characteristics for greater than 40 passages and expresses high levels of preproglucagon mR ⁇ A. Radioimmunoassays were used to demonstrate that these cells synthesize large quantities of glucagon, some glucagon-like peptide-I (GLP-I), and small amounts of unprocessed proglucagon.
  • This cell line also expresses PCl/3, PC2, and PC6A.
  • Proglucagon is an excellent substrate to use in the proposed studies because of the potential for differential processing.
  • multiple PCs are involved in proglucagon processing in the ⁇ TCl -6 cell.
  • performance of the proposed experiments using a tumor cell line affords the opportunity to test inhibitors in an environment that mimics cellular conditions in neoplastic tissue.
  • Naturally occurring glucagonomas secrete hyperphysiologic amounts of glucagon and GLP-I which leads to an imbalance in the homeostatic regulation of blood glucose levels
  • the ⁇ TCl -6 cell line provides an ideal model system in which to test the efficacy of broad spectrum PC inhibitors.
  • inhibitors were tested in the in vitro assay.
  • the inhibitors, peptidyl derivatives of ( ⁇ -aminoalkyl) phosphonate diphenyl esters were designed to specifically inhibit subtilisin-like serine proteinases.
  • Recombinant PCl/3 or immunopurified PC2 were incubated ⁇ 50 ⁇ M inhibitor with HPLC purified 3 H- tryptophan labeled proglucagon derived from metabolically labeled ⁇ TCl -6 cells. After incubation, the reaction mixtures were subjected to RP-HPLC to separate proglucagon cleavage products.
  • Figures 2A and 2B depict reversed phase HPLC chromatograms from in vitro inhibitor studies utilizing recombinant PCl/3 (A) or PC2 (B), respectively, ⁇ 50 ⁇ M of Z-Thr-(4-AmphGly) p (oph) 2 as the inhibitor, to determine the effects of the inhibitor on processing of proglucagon.
  • Products are: 1 - glicentin, 2 - oxyntomodulin, 3 - glucagon, 4 - GLP-IQ-36) amide/(7-36) amide, 5 - proglucagon 54/55, 6 - proglucagon 56, 7 - proglucagon 58 (the three forms of proglucagon are differentially methionine sulfoxidized), and 8 - MPGF.
  • Solid lines with open circles depict PC incubations with the inhibitor, dashed lines with solid circles depict PC incubations without the inhibitor.
  • API-2C H 2 N-SEGVMCPMIYDPVLLT-COOH (SSI-like) (SEQ ID NO: 6)
  • the cleavage inhibitor reactive sites are indicated in bold type and underlined.
  • the full length inhibitors were obtained by means of expression and purification of full length SSI-like inhibitors in a prokaryotic expression system.
  • DH5 ⁇ cells were transformed with the inducible plasmid vectors containing the wild type STI-2 coding sequence (pOSTI-2) or a mutated sequence which encodes Lys (pOSTI- 2[N72K]) or Arg(pOSTI-2[N72R]) in place of Asn immediately upstream of the reactive site.
  • the host bacteria are then cultured in the presence of 100 ⁇ g/ml ampicillin and 1 mM IPTG for 16 hrs. Cells are collected by centrifugation and lysed by repeated freeze-thaw in phosphate buffered saline.
  • Soluble components are then dialyzed against water followed by partial purification using a concentrator with a molecular weight cutoff of 10,000.
  • the inibitor proteins were further purified by reversed phase HPLC. At a flow rate of 4 ml/min a 40 min linear gradient is run from 25% A (A is 80% acetonitrile/0.1% TFA; B is 0.1% TFA) to 44% A, then holding a 44% A for 10 min.
  • Fractions were analyzed for STI-2 immunoreactivity using SDS PAGE/ western analysis with a polyclonal SSI-antibody. Immunoreactive fractions were pooled and lyophilized, followed by resuspension in sterile water for inhibitor studies.
  • Procedures were also developed for expression, purification, and testing the activity of full-length PCl/3, PC2, and PC6A.
  • the Baculogold DNA reagent system (PharMingen) was utilized.
  • the cDNA for mouse PCl/3 was cloned into the plasmid pVL1392.
  • This plasmid was then co-transfected into Sf9 insect cells along with Baculovirus DNA (Baculogold DNA) from which an essential part of the Baculovirus genome had been removed.
  • Co-transfection of the plasmid and the altered genomic DNA was facilitated by lipofection (Lipofectin, Gibco/BRL).
  • the mPCl/3/pVL1392 plasmid construct contained the missing sequence of the Baculovirus genome.
  • media were collected and samples subjected to buffer exchange by passing them through a Sephadex G-25 column.
  • media from High Five cells infected with the wild-type virus, and from cells not exposed to viral infection were also collected and treated in parallel.
  • proteins from culture samples were precipitated and subjected to SDS PAGE.
  • the separated proteins were then electroblotted onto polyvinylidene difluoride (PVDF) membranes and PCl/3 proteins were detected by antibody staining.
  • PVDF polyvinylidene difluoride
  • two PC 1/3 -related proteins with approximate molecular weights of 80 and 88 kDa were detected using this procedure. It is probable that the larger molecular weight form represents the precursor to the active 80 kDa enzyme.
  • Samples were also tested for proteolytic activity using the substrate Pyr-Arg-Thr-Lys-Arg-AMC. Only the media from the PCl/3-virus infected cells contained proteolytic activity capable of recognizing and cleaving this substrate.
  • Reaction mixtures were diluted in concentrated Tris buffer, pH 8.7, containing 0.5 mM EDTA, 8 M urea (reducing and alkylating buffer) and DTT at a final concentration of 57.5 mM. Samples were sparged with argon gas and incubated at 50°C for 30 min. After cooling to ambient temperature, reducing and alkylating buffer containing sufficient iodoacetate to achieve a final concentration of 95 mM was added and the samples incubated for 7 min protected from light at ambient temperature. Reactions were terminated by adding glacial acetic acid to achieve a final concentration of 3.5 M.
  • the recombinant enzymes are further purified to eliminate any contaminating activity.
  • a purification protocol was developed, and purification of PCl/3 was performed as an example. Specifically, a PCl/3 concentrate was prepared using an Amicon Centriprep concentrator having a 30,000 molecular weight cut-off. The concentrate was then subjected to FPLC at 4°C on a Phamacia Hi Trap SP column with an elution buffer of 20mM sodium acetate/acetic acid, lmM 2-mercaptoethanol at pH 5. Prior to loading, the sample was preincubated in 5 volumes of elution buffer for 1 hour at 37°, to permit autocatalysis to remove the propeptide and C-terminal extension peptide.
  • the column was washed for 5 minutes and then eluted by applying a linear gradient of 0-200 mM sodium sulfate over 30 minutes at a flow rate of lml/min.
  • One minute fractions were collected in tubes containing sufficient glycerol to achieve a final concentration of 20%.
  • Aliquots of 25 ⁇ l from each tube are tested in the fiuorometric assay to determine enzyme activity. Samples were examined by SDS PAGE on duplicate gels, followed by Coomaassie staining and western analysis using a PCl/3 antibody at a concentration of 1 : 1000. The preparation was devoid of any activity other than that contributed by the PC.
  • the ability of furin, PC6A and PC7 to cleave either the fluorogenic substrate pGlu-Arg-Thr-Lys-Arg-AMC or the fluorogenic substrate pArg-Thr-Lys-Arg-AMC is compared.
  • the fluorometric inhibitor assays are performed as previously described, except that the incubations for furin and PC6A are performed at pH 7.5, while those for PC7 are performed at pH 6.5. Varying concentrations of each inhibitor peptide are analyzed to determine effective dose ranges. The substrate most efficiently cleaved is then used to screen the ability of inhibitors to block PC activity.
  • Figure 3 depicts the effects of natural and Streptomyces inhibitor peptides on the ability of PCl/3 to cleave a fluorogenic substrate. Control incubations were performed with substrate plus enzyme only. Relative fluorescence indicated on the Y-axis is in arbitrary units read directly from the fluorometer display at 15 minute intervals. The results demonstrate that PCl/3 was found to be much less susceptible than PC2 to the action of each of these inhibitor peptides. The effects on PCl/3 activity are shown in Figure 3. For the experiment shown, the inhibitor concentration was 200 ⁇ M. Clearly, PCl/3 is partially inhibited by any of these peptides at this peptide concentration.
  • ⁇ TCl -6 cells are incubated with 250 ⁇ Ci of 3 H-tryptophan for one hour, the cells are extracted, and radiolabeled proglucagon is isolated by reversed phase HPLC. After overnight incubation of the radiolabeled substrate in the presence of recombinant PC, reaction products are separated by HPLC, and radioactivity attributable to glucagon related cleavage products is quantitated by liquid scintillation spectrometry. When an inhibitor is being tested, it is incubated with the PC for 30 min prior to addition of the labeled proglucagon. The results from these assays demonstrate whether the inhibitor in question successfully competes with a natural substrate to inhibit PC activity.
  • both the [N72K] and [N72R] mutant forms of the STI-2 full length protein proved to be effective inhibitors of PC2 mediated cleavage than either the STI-2 peptide or the full length STI-2 ( Figure 1 OA-F).
  • the STI-1 [N72R] protein is by far the most potent inhibitor of both furin and PC6A. See Figures 11 A-D.
  • This modified inhibitor protein exhibits dose dependent inhibition of both recmbinant furin and PC6A in the fluorometric assay, with substantially complete inhibition of furin at concentrations as low as 50 ⁇ M, with an IC 50 of about 12.9 ⁇ M, also indicating its suitability for inhibition of gpl60 processing enzyme.
  • Figure 5 depicts the comparison of the effects of full-length STI-2 and 16-amino-acid-long peptides based on the sequences of PSN and SSI on recombinant PCl/3 cleavage of a fluorogenic substrate. Conditions were the same as described for Figure 3, except that the concentration of STI-2 was 100 ⁇ M and the concentrations of the PSN and SSI peptides were 200 ⁇ M. The 30 minute lag prior to initiation of substrate cleavage occurred because the PCl/3 preparation was not preincubated to activate the enzyme. The two 16-amino-acid-long Streptomyces inhibitor peptides were relatively ineffective in blocking PCl/3 activity. However, the full length STI-2 protein proved to be a very potent inhibitor of both PCl/3 and PC/2. These results demonstrate that STI-2, either as a peptide or full length protein, is effective in blocking both PCl/3 and PC2 activity.
  • STI-2 The full sequence of STI-2 is: ASLYAPSALVLTNGHGTSAAAATPLRAVTLNCAPTASGTHPAPALACADLRG VGGDroALKARDGVICNKLYDPVVVTVDGVWQGKRVSYERTFGNECVKNSY GTSLFAF. (SEQ ID NO: 25 )
  • SIL1 H 2 N-DTGRVCTREYRPVTVS-COOH SEQ ID NO: 3
  • derivatives thereof as well as the peptides
  • CADGVMCTREYAPVVVTC (SEQ ID NO: 10) CDTGRVCTREYRPVTVSC (SEQ ID NO: 11) CRDGVICNKLTDPWVTC (SEQ ID NO: 12) CRGDVACTKQFDPWVTC (SEQ ID NO: 13) CGEDVMCPMVYDPVLLTC (SEQ ro NO: 14)
  • CSEGVMCPMIYDPVLLTC SEQ ID NO: 15
  • derivatives shall encompass longer peptides which incorporate the peptide sequences identified. It will be apparent that some additional amino acid substitutions may increase inhibitor efficacy.
  • Figures 6 A - 6D illustrate the effects of proinsulin C-A (KR) and proinsulin C-A(KH) peptides on the ability of PCl/3 and PC2 to cleave the fluorogenic substrate. Control incubations were performed with substrate plus enzyme only. Relative fluorescence indicated on the Y-axis is in arbitrary units derived directly from the fluorometer readings.
  • proinsulin C- A(KR) peptide exhibited no inhibition of proglucagon conversion mediated by either PCl/3 or PC2 suggesting that both enzymes preferentially cleave the natural substrate, proglucagon.
  • the second proinsulin derived peptide, proinsulin C-A(KH) proved to be ineffective as an inhibitor of either PCl/3 or PC2 at concentrations up to 200 ⁇ M in both the fluorometric ( Figures 6C and 6D) and proglucagon conversion assays.
  • FIGS 7 A - 7E depict the effects of the STI-2 peptide and the modified STI-2(KN) peptide on PCl/3 and PC2 activity in the fluorometric assay.
  • the STI-2 peptide was demonstrated to be a very effective inhibitor of both PCl/3 and PC2 activities in these reactions. As shown in Figures 7A and 7B, this peptide exhibits strong dose dependent inhibition of both of these PCs.
  • Several experiments to determine the half-maximal inhibitory concentrations for the STI-2 peptide in the fluorometric assay were performed.
  • the resulting IC50 values were 30.1 ⁇ 2.9 ⁇ M and 10.0 ⁇ 1.2 ⁇ M (mean ⁇ S.D.) for PCl/3 and PC2, respectively, as illustrated in Figures 7D and 7E.
  • the substrate concentration for the IC50 experiments was 200 ⁇ M, indicating that the STI-2 peptide is a potent inhibitor of these PCs.
  • substitution of Asn for Leu in the C-terminal position of the reactive site completely abolished the ability of the STI-2 peptide to inhibit either PCl/3 or PC2 in the fluorometric assay, as illustrated in Figure 7C.
  • FIGS 8 A - 8F depict the effects of the STI-2 peptide on PCl/3 or PC2 mediated proglucagon conversion in vitro.
  • Reversed phase HPLC was employed to separate proglucagon cleavage products.
  • MPGF denotes Major Proglucagon Fragment.
  • Neither the PCl/3 nor the PC2 enzyme preparations generated random degradation of proglucagon. Incubations consistently produced identifiable proglucagon cleavage products.
  • STI-2 exhibited essentially complete inhibition of both PCl/3 ( Figures 8 A - 8C) and PC2 ( Figures 8D - 8F) mediated proglucagon conversion in vitro.
  • Figures 9A -9C depict the effects of STI-2(KN) on PCl/3 mediated proglucagon conversion in vitro.
  • Figures 9D - 9E depict the effects of the SIL-2 peptide on PCl/3 and PC2 activity in the fluorometric assay.
  • the modified STI-2(KN) peptide exhibited no capacity to inhibit either PC 1/3 -( Figures 9 A - 9C) or PC2- mediated proglucagon conversion.
  • Figures 9D - 9F demonstrate that a related molecule, SIL-2, is also a partial inhibitor of PC2, but that the inhibition becomes complete if the preparation is first subjected to size fractionation. This result demonstrates that the contaminating activities are smaller than about 30,000 daltons and that it should be possible to easily purify the PC2 isozyme.
  • Oligonucleotides that code for all or part of the sequences determined to be effective in PC inhibition in the in vitro studies are linked downstream of the coding region of a preprohormone.
  • the prepro- region of preprosomatostatin is chosen.
  • the primary structure of this precursor is depicted below with the peptide hormone cleavage sites shown in bold type and underlined.
  • This sequence is particularly suitable as the "carrier” to deliver inhibitor sequences to the regulated secretory pathway for several reasons.
  • the "pro- region” of this precursor is not normally processed in mammalian cells, and it does not have any demonstrated biologic activity.
  • the "pro- region” of prosomatostatin is known to target the precursor into the regulated secretory pathway, ultimately to be stored and processed in secretory granules.
  • the portion of the precursor that contains somatostatin-28 (SS-28) and somatostatin- 14 (SS-14), the bioactive products of this precursor can be easily eliminated from our proposed "carrier” by restriction digestion of the cDNA.
  • Dual digestion of the rat preprosomatostatin cDNA with Xbal and PvuII will cut the plasmid at base positions 53 and 329, respectively.
  • base 53 is located 58 bases upstream from the ATG start codon, and base 329 occurs 32 bases upstream from the codon that designates the single Arg residue that serves as the cleavage site for releasing SS-28 from prosomatostatin.
  • Use of the endonuclease cleavage site at base 329 is particularly important because it will eliminate the coding region for both of the bioactive products (SS-14 and SS-28) and their respective cleavage sites from the prepro- region that is intended to serve as the carrier for vectorial transport into the endoplasmic reticulum.
  • oligonucleotides that have been prepared to code for the specific inhibitors are ligated to the 3' end of the prepro- region of the "carrier".
  • the 3' end of these oligos will contain a Clal site to accommodate directional insertion into the vector.
  • This chimeric coding region is then ligated into the modified pOPRSV eukaryotic expression vector which has been modified to allow directional insertion of inserts with a 5' Xbal and a 3' Clal site.
  • the insert then codes for a chimeric peptide in the transfected cells.
  • Figure 12 depicts representative examples of several chimeric peptides of this type.
  • Figure 12 provides a schematic of the "carrier" preprosomatostatin
  • the inhibitors shown are: (A) STI-2 peptide, (B) the SIL-2 peptide, (C) API-2C peptide, (D) the SIL-4 peptide.
  • the reactive site residues of the subtilisin inhibitors are underlined.
  • the LacSwitch eukaryotic expression system (Stratagene) consists of a eukaryotic Lac-repressor expressing vector (p3'SS) and two eukaryotic lac-operator containing vectors into one of which the cDNA of interest is inserted by cloning. Normally both LacSwitch vectors are transfected into a cultured cell line where expression of the coding region inserted into pOPRSVCAT is repressed by the product of the second vector until an inducer (for example IPTG) is added to the media. Upon induction, expression of the sequence inserted into pOPRSVCAT resumes.
  • the pOPRSV plasmid can also be transfected alone, allowing it to constitutively express the product(s) coded for by the insert it carries.
  • a modified form of the pOPRSV vector is used for introduction of inserts.
  • the modified vector was constructed by ligating the multiple cloning site (MCS) of pBluescript KS+ with that of the MCS of pREP9 at the Kpnl site.
  • MCS multiple cloning site
  • the new construct is then digested with Notl and ligated into the Notl sites of the pOPRSV vector, replacing the chloramphenicol acetyl transferase (CAT) reporter gene.
  • the modified (pOPRSVmod) vector is then transformed into DH5 ⁇ F cells (Gibco/BRL).
  • the modified vector allows for directional incorporation of inserts in either the antisense or sense orientation using the Clal and Xbal restriction enzyme sites.
  • Cells to be transfected with the pOPRSV vector + chimeric inhibitor inserts are plated in triplicate into 60 mm culture dishes and cultured in normal growth media overnight prior to transfection. On the day of the transfections, cells in the dishes are washed twice with prewarmed Hanks Balanced Salt Solution (HBSS) prior to the addition of transfection media (the approximate cell density in each dish will not exceed 2-4 x 10 3 ).
  • the transfection media consists of 20 ⁇ g of plasmid and 60 ⁇ g of Lipofectamine (Gibco/BRL) in 1.5 ml DMEM with no antibiotics or serum. The protocol for these transfections is adapted from the Gibco/BRL transfection protocol.
  • the DNA-Lipofectamine solution incubates at room temperature for 45 min prior to addition to cells.
  • the cells are then incubated in the transfection media for 6 hours at 37°C under 5% CO 2 , after which normal growth medium is added and the cells incubated overnight.
  • the transfection efficiency of this protocol was determined to be approximately 84% using the pCHl 10 ⁇ -galactose expression vector (Pharmacia Biotech).
  • Important controls include "transfection" incubations of cells with transfection media without plasmid, plasmids without inserts, and cells which were not treated. One day after performing the transfections, incubation in media containing G418 is used to achieve selection of transfected cells to identify stable transfectants.
  • glucagon RIA of media samples, metabolic labeling, extraction, HPLC separations and peptide mapping analyses to determine proglucagon product/precursor ratios.
  • Processing in transfected cells can be initially assessed by glucagon RIA ofaliquots from media samples.
  • the glucagon RIA can be based on the 04A antiserum.
  • the RIA based on this antiserum detects only the free C-terminus of glucagon and is thus not reactive to proglucagon or any cleavage intermediate of the precursor.
  • media samples are taken for RIA starting at day 1 after transfection to determine the optimal culture time after transfection for assessing the effects of initiator expression.
  • Glucagon levels secreted from cells transfected with vector only and cells transfected with insert containing vector are compared. When reductions of glucagon release are observed, cells are taken for metabolic labeling and product analyses.
  • preprosomatostatin that does not exhibit its normal cleavage sites for processing somatostatin-28 and somatostatin-14 from the precursor could also serve as an effective inhibitor of the PCs in intact cells.
  • Both PCl/3 and PC2 can cleave at the RK site in prosomatostatin to release SS-14.
  • a non-PC aspartyl protease and PCl/3 can release SS-28 from prosomatostatin. Accordingly, if both of the susceptible cleavage sites in prosomatostatin were altered, and the resulting mutant precursor were introduced into the regulated secretory pathway of cells making bioactive peptides, it could compete with the natural substrate for the PCs and thereby inhibit PC activity.
  • the complete coding sequence will be recovered from its parent vector (pRTB 143) using dual digestion with Xbal and Clal. After gel purification of the resulting fragment, both the 5' and 3' ends will be blunt ended using the Klenow fragment of DNA polymerase I. This DNA fragment will be ligated into the pOPRSVmod expression vector predigested with Xbal and Clal and blunt ended. The vector carrying this insert will then be transformed into competent cells for amplification to verify insertion and determine correct orientation by either restriction digestion or sequencing. Clones containing the insert in the correct orientation are used for transfection into ⁇ TCl -6 cells as discussed previously.
  • immunocytochemistry may be employed using the N- terminally directed prosomatostatin antiserum as the primary antiserum. Transfections of vectors carrying either non-mutated preprosomatostatin cDNA inserts or no insert will be employed as controls. If sufficiently high levels of the modified prosomatostatin are expressed in these cells and targeted into the regulated secretory pathway, proglucagon processing would likely be impaired. This impairment could be exhibited initially by reduction of glucagon secreted into culture media of transfected cells as determined by glucagon RIA. If glucagon levels are reduced, then inhibition of PC mediated processing at specific cleavage sites will be assayed using the established metabolic labeling and HPLC product analysis protocol. Experiments are undertaken to identify peptide PC inhibitors of HIV.
  • RGDVACTKQFDPVWT (SEQ ID NO: 1 ), GEDVMCPMVYDPVLLT (SEQ ID NO: 2 ), DTGRVCTREYRPNTVS (SEQ ro NO: 3),
  • RDGNICNKLYDPWVT (SEQ ro NO: 4), ADGVMCTREYAPWVT (SEQ ID NO: 5), SEGVMCPMIYDPVLLT (SEQ ID NO: 6), CALEGSLQKRGIVEQCC (SEQ ID NO: 7), CALEGSLQKHGIVEQCC (SEQ ID NO: 8),
  • CRDGVICNKLYDPVWTC (SEQ ro NO: 9), CADGVMCTREYAPWVTC (SEQ ID NO: 10), CDTGRVCTREYRPNTVSC (SEQ ID NO: 11), CRGDVACTKQFDPVWTC (SEQ ID NO: 13), CGEDVMCPMVYDPVLLTC (SEQ ED NO: 14), CSEGVMCPMIYDPVLLTC (SEQ ID NO: 15), RDGVICNKNYDPWVT (SEQ ID NO: 16), DDAVMCTREYAPVWT (SEQ ID NO: 17), DQDRACIKIYDPLWT (SEQ ID NO : 18),
  • DAGVMCTREYAPWVT (SEQ ro NO: 19), RDGVMCTKQYDPVWT (SEQ ID NO: 20), EPGRMCTKEWRPITVT (SEQ ED NO: 21), RDDVWCNKLYDPWVT (SEQ ED NO: 22), EDSVMCTREYAPWVT (SEQ ID NO: 23), or
  • TGDVMCTKQYDVWT (SEQ ID NO: 24).
  • This fluorometric assay may also be adapted to testing the efficacy of inhibitors of HIV gp 160 surface envelope protein processing. Peptide and full length inhibitors shown to be effective in the fluorometric assay are then tested for their ability to inhibit cleavage of the target substrate, gpl60.
  • the gpl60 cleavage assay is based on similar techniques known in the art. See E. Decroley et al. (1994) J. Biol. Chem. 269: 12240-12247 and L. Miranda et al. (1996) Proc. Natl. Acad. Sci. USA 93: 7695-7700. Commercial gpl60 may be used, although immunoreactive epitopes could be differentially mapped on western blots, leading to inconsistent quantitation.
  • gpl60 is preferably prepared by infecting CV-1 cells with a W:gpl60 construct at a multiplicity of infection of 1-5 pfu/cell. After an 18-20 hours incubation, cells are placed in Met and Cys-depleted DMEM for 1 hour, and then cultured for 6 hours in the same medium supplemented with 150 ⁇ Ci 35 S-Met/ S-Cys. Cells are then lysed in 30mM Tris-HCL, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate.
  • the 35 S-labeled gpl60 is separated from the soluble components using a lentil lectin agarose column, and then eluted in lysis buffer containing 0.5M ⁇ -methylmannoside.
  • the 35 S-labeled gpl60 (10,000 cpm) is then incubated overnight with either furin, PC6A or PC7 in 50mM Tris/acetate supplemented with 1% Triton Z-100 and 2mM calcium acetate. The pH is adjusted as indicated for the previous fluorometric assay. Incubates are then subjected to SDS PAGE on 8% gels and quantification of gpl ⁇ O and its cleavage products is analyzed by a phosphoimager.
  • Enhanced sensitivity can be achieved by preparing fluorograms, scanning to generate digital images, and using the NIH IMAGE program to quantitate band intensity. Each inhibitor is incubated with the PC for 30 minutes prior to the addition of labeled gpl ⁇ O. The results determine whether an inhibitor can successfully compete with gpl ⁇ O to inhibit PC activity.
  • any peptide shown to be an inhibitor in the fluorometric assay or gpl ⁇ O conversion assays is then tested to determine the likely mechanisms of enzyme action.
  • the question of whether inhibitor proteins are cleaved by PCs is determined by HPLC. Incubates of the recombinant PC plus full length inhibitors are subjected to reverse-phase HPLC, which permits differentiation of cleavage products from the intact protein. For example, the STI-2 full length protein has a calculated value of 339, whereas the N-terminal fragment after cleavage is 290 and the C terminal fragment is 46. Therefore, cleavage products would be expected to elute earlier than the intact protein on HPLC. Peptide incubates are similarly analyzed by HPLC.
  • inhibitors may be iodinated to determine whether the inhibitor binds the enzyme tightly and is only cleaved very slowly.
  • STI-2 has 4 tyrosines, which would be distributed so that each of the potential cleavage products indicated above would be labeled.
  • the HPLC is then repeated with iodinated inhibitors to determine whether small amounts of cleavage products are released slowly. If the results suggest that the inhibitors are not cleaved by the PCs, it is assumed that they are bound to the enzymes but not cleaved. In either case, IC50 and V/Km values will be determined for the peptides and full length inhibitors.
  • the question of whether inhibition is highly sequence dependent is addressed using sequence modified peptides and full length inhibitors. The results of these two tests provide information useful in identifying the inhibitors that have potential to function most effectively in vivo.
  • An in vivo assay is performed to identify peptides and full length proteins that inhibit PC processing in HIN target cells. Any peptide or full length sequence identified as an inhibitor in both the fluorometric and gpl ⁇ O conversion assay is tested in vivo.
  • a mammalian expression vector is prepared for a peptide according to the previously presented protocol.
  • a chimeric cD ⁇ A construct is created comprising a nucleic acid coding sequence of the peptide(s) of interest linked downstream of the nucleic acid coding region of a carrier peptide.
  • the construct may comprise one or more nucleic acid coding sequences, encoding one or more peptides of interest.
  • the carrier peptide is preferably the prepro-region of a preprohormone, such as prosomatostatin. After insertion into the eukaryotic expression vector, the chimeric insert will then code for a chimeric peptide in transfected cells.
  • transfection vectors for full length inhibitors are prepared by ligating coding inserts into the multiple cloning site of the pSecTag2 mammalian expression vector (Invitrogen).
  • This vector incorporates a coding region for a signal peptide shown to be effective in targeting nascent polypeptide chains into the secretory pathway of mammalian cells.
  • This vector is provided in three forms, with two being extended in the multiple cloning site by one or two nucleotides, respectively, to facilitate incorporation of the insert for in frame reading. Oligonucleotide priming of coding sequences is used for D ⁇ A sequence analysis to verify that the sequences are correctly oriented in the vector.
  • the pSecTag 2 vector is useful for other reasons as well.
  • the vector contains six histidine residues which facilitate purification using a nickel resin column. This procedure can be used to verify synthesis of the inhibitor protein in transfected cells. Moreover, an antibody to C-myc can be used to monitor transfection efficiencies and expression levels when an antibody to the inhibitor is unavailable.
  • An alternative approach is to transfect the preprosomatostatin or inhibitor chimeras into HIN taget cells.
  • Complete, or nearly complete, inhibition of PC cleave of gp 160 may depend upon the level of expression of the inhibitor protein. It is desirable to achieve as high expression of inhibitor protein as is feasible. For example, inhibitor to PC ratios of 5 : 1 are desirable.
  • Inhibitor expression may be enhanced by using an inducible vector. Where vectors are used therapeutically, the TAT responsive element of the 5'LTR of HIN may be useful as an inducible element. The 5'LTR of HIN-1 is placed upstream of the coding sequences for either full-length SSI-like proteins or prepro-peptide/chimeric constructs yielding SSI reactive site sequences. There it can serve as a promoter, but expression of inhibitor remains latent until the host cell is infected with HIN. Levels of inhibitor may also be enhanced by using a vector comprising a
  • Golgi retention signal See, e.g., Pfeiffer, T. et al.(1997) J. Gen. Virol. 78: 1745-1753. This signal prevents a foreign protein like the inhibitor from being directed to the lysosomes for degradation. Incorporation of a Golgi retention signal into the inhibitor vector would ensure that inhibitor protein is retained in the Golgi.
  • the efficacy of the inhibitors is examined in both CD4+ lymphoid and monocyte/macrophage cells.
  • CD4+ cell lines may include Jurkat, SupTi, Ti, or T lymphoblasts.
  • Monocyte/macrophage cell lines may include U937 and THP-1.
  • W:gpl60 will be replaced by PCRCMN-gpl60 (Invitrogen).
  • Transfected cells are then washed and preincubated for 1 hour in Cys and Met-depleted media. Cells are then incubated for 4 hours in media supplemented with 35 S-Cys and 35 S-Met. Media is recovered and centrifuged for 10 minutes at 300g to remove cells and debris.
  • An equal volume of an inhibitor cocktail containing lmM EDTA, 2 ⁇ g/ml aprotinin, and lOO ⁇ g/ml PMSF is added to prevent proteolytic degradation of labeled products secreted into the media.
  • a lysis buffer 50mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl and 1% SDS.
  • Media and lysate are precleared by adding an equal volume of 50% (w/v) protein A-Sepharose beads and rocking for 4 hours at 4°C.
  • the beads are removed by centrifugation, and the supernatants placed in separate tubes to which an appropriate amount of gp 160/ 120/41 antisera is added. New protein A-Sepharose beads are added and rocked for an additional 4 hours at 4°C.
  • a cell fusion assay is performed to monitor the extent to which gpl20 is displayed on the surface of cells expressing inhibitors. See C. Yang et al. (1996) Virology 221 : 87- 97. Cells are seeded into six-well plates one day prior to infection with W:gpl60. One day after W:gpl60 infection, the infected cells are overlaid with CD4+ cells. Cell fusion is monitored and photographed 4-8 hours later by phase contrast microscopy. A reduction in syncytia formation in cells expressing inhibitors relative to those cells transfected with a control vector indicates diminished gpl20 expression. Alternatively, fusion can be monitored in cells transfected with the plasmid GlNT7 ⁇ - gal or infected with vTF7-3 by a colorimetric assay.
  • a biotinylation assay is conducted to determine cell surface gpl20 expression. After transfection and radiolabeling, cells are biotinylated in PBS with 0.5 mg/ml of sulfo-succinimidyl-2(biotinamido)ethyl-l,3-dithiopropionate (NHS- SS-biotin, Pierce) for 30 minutes at 4°C, followed by lysis in lysis buffer. Lysates are then immunoprecipated with the appropriate antisera/ Protein-A Agarose overnight at 4°C. Samples are washed twice in lysis buffer plus 0.4% SDS and heated at 95°C for 15 minutes.
  • Dissociated proteins are dissolved in lysis buffer and incubated with Streptavidin-agarose (Pierce) for 5 hours at 4°C. Biotinylated samples are then washed three times with lysis buffer, combined with reducing sample buffer,. heated and loaded onto SDS gels. The gpl ⁇ O protein and its cleavage products in media, cell lysates, and at the cell surface will be quantitated as previously described in C. Spies et al. J. Virol. 68:585-591, incorporated herein by reference. A reduction in surface expression of gpl20 indicates a finding of inhibitor efficacy.
  • CD4+ and/or CD8+ lymphocyte precursors or monocyte/macrophage precursors are isolated from mice, rats or non-human primates. These cells are transfected in vitro with expression vectors comprising the inhibitor of interest, according to Apperly, IF., et al. (1991) Blood 78: 310-317; Bunnell, B.A. et al. (1995) Proc. Natl. Acad. Sci. USA 92: 7739-7743; Morgan, R.A. (1996) Gene Therapy. In Immunology of HIV Infection. S. Gupta, editor.

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Abstract

Cette invention se rapporte à des peptides inhibiteurs de propeptides/prohormone convertases (PC) choisis dans le groupe constitué par (I) ainsi qu'à des peptides plus longs contenant ces peptides inhibiteurs. En plus de leur pouvoir d'inhibition de la PC, ces peptides sont utiles dans le traitement des cellules tumorales et dans le traitement des troubles endocriniens.
PCT/US1998/003642 1997-03-02 1998-02-25 Inhibiteurs peptidiques de propeptide/prohormone convertases WO1998037910A1 (fr)

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Cited By (1)

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WO2001036643A1 (fr) * 1999-11-19 2001-05-25 Transkaryotic Therapies, Inc. Produit d'assemblage d'acides nucleiques optimisant la production de produits

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WO1993017086A1 (fr) * 1992-02-25 1993-09-02 Novo Nordisk A/S Detergent contenant une protease, un inhibiteur de protease, et nouveaux inhibiteurs destines a etre utilises dans le detergent

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Title
CLARK D. A., ET AL.: "PROTEASE INHIBITORS SUPPRESS IN VITRO GROWTH OF HUMAN SMALL CELL LUNG CANCER.", PEPTIDES, ELSEVIER, AMSTERDAM, NL, vol. 14., no. 05., 1 January 1993 (1993-01-01), AMSTERDAM, NL, pages 1021 - 1028., XP002914836, ISSN: 0196-9781, DOI: 10.1016/0196-9781(93)90081-Q *
DATABASE MPSRCH GENBANK 1 January 1900 (1900-01-01), "PROTEASE INHIBITOR WITH VARIABLE POSITION 29 RESIDUE", XP002914838, Database accession no. R12814 *
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KOJIMA S., ET AL.: "PRIMARY STRUCTURE AND INHIBITORY PROPERTIES OF A PROTEINASE INHIBITOR PRODUCED BY STREPTOMYCES CACAOI.", BIOCHIMICA ET BIOPHYSICA ACTA. PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, ELSEVIER, AMSTERDAM; NL, vol. 1207., 1 January 1994 (1994-01-01), AMSTERDAM; NL, pages 120 - 125., XP002914834, ISSN: 0167-4838, DOI: 10.1016/0167-4838(94)90060-4 *
TAGUCHI S., ET AL.: "COMPARATIVE STUDIES ON THE PRIMARY STRUCTURES AND INHIBITORY PROPERTIES OF SUBTILISIN-TRYPSIN INHIBITORS FROM STREPTOMYCES.", EUROPEAN JOURNAL OF BIOCHEMISTRY, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 220., 1 January 1994 (1994-01-01), GB, pages 911 - 918., XP002914833, ISSN: 0014-2956, DOI: 10.1111/j.1432-1033.1994.tb18694.x *
TERABE M., ET AL.: "NEW SUBTILISIN-TRYPSIN INHIBITORS PRODUCED BY STREPTOMYCES: PRIMARY STRUCTURES AND THEIR RELATIONSHIP TO OTHER PROTEINASE INHIBITORS FROM STREPTOMYCES.", BIOCHIMICA ET BIOPHYSICA ACTA. PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, ELSEVIER, AMSTERDAM; NL, vol. 1292., 1 January 1996 (1996-01-01), AMSTERDAM; NL, pages 233 - 240., XP002914835, ISSN: 0167-4838, DOI: 10.1016/0167-4838(95)00207-3 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001036643A1 (fr) * 1999-11-19 2001-05-25 Transkaryotic Therapies, Inc. Produit d'assemblage d'acides nucleiques optimisant la production de produits
JP2003514532A (ja) * 1999-11-19 2003-04-22 トランスカーヨティック セラピーズ インコーポレイテッド 生成物の産生量を最適化するための核酸構築体
AU784477B2 (en) * 1999-11-19 2006-04-13 Shire Human Genetic Therapies, Inc. Nucleic acid construct for optimized production of products
US7229793B1 (en) 1999-11-19 2007-06-12 Shire Human Genetic Therapies, Inc. Constructs and cells for production of small peptides

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