EP0465495A1 - Ribozome inactivating proteins and derivatives thereof - Google Patents

Ribozome inactivating proteins and derivatives thereof

Info

Publication number
EP0465495A1
EP0465495A1 EP90904825A EP90904825A EP0465495A1 EP 0465495 A1 EP0465495 A1 EP 0465495A1 EP 90904825 A EP90904825 A EP 90904825A EP 90904825 A EP90904825 A EP 90904825A EP 0465495 A1 EP0465495 A1 EP 0465495A1
Authority
EP
European Patent Office
Prior art keywords
mol
molecular mass
abs
protein
rips
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.)
Pending
Application number
EP90904825A
Other languages
German (de)
French (fr)
Inventor
Fiorenzo Stirpe
Luigi Barbieri
Gianni Gromo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Italfarmaco SpA
Original Assignee
Italfarmaco SpA
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Italfarmaco SpA filed Critical Italfarmaco SpA
Publication of EP0465495A1 publication Critical patent/EP0465495A1/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention refers to ribosome inactivating proteins and their sulphydrylated derivatives useful for the preparation of immunotoxins.
  • Ribosome-inactivating proteins from plants (RIPs) (Cancer Surveys 1 , 489, 1982; J. Natural Products. 8, 446, 1985, FEBS Letters 1.95, 1, 1986) catalytically inactivate the 60 S ribosomal subunits of eukary ⁇ tic ribosomes. They can be either single-chain proteins (RIPs type 1) or two-chain proteins (RIPs type 2), in which one chain has the enzymic activity and the other one has the properties of a galactose-specific lectin. RIPs type 1 are more frequent, being present in several parts of many and possibly all plants, including seeds, roots, leaves and latices, sometimes in more than one form, possibly isoforms.
  • RIPs have an unusual N-glycosidase activity, and cleave the N-glycosidic bond of adenine 4324 of 28S rRNA introducing a lesion that renders RNA cleavable by aniline at a site adjacent to the cleavage induced by
  • immunotoxins hybrid molecules consisting of a toxic moiety linked to an antibody. Hence immunotoxins will be useful to eliminate harmful cells, neoplastic, immun ⁇ competent and parasitic cells being considered as possible targets (Cancer Immunol. Iramunother. 2J7, 95, 1988).
  • RIPs namely the pokeweed antiviral protein (PAP), was initially purified as an anti-viral protein. This property of RIPs was attributed to an easier penetration into virally infected cells with consequent xnactivation of their ribosomes and arrest of viral multiplication. Recently, however, a RIP type 1, trichosa thin, was found to inhibit replication of human immunodeficiency virus through a mechanism apparently independent of the effect on ribosomes (..) .
  • PAP pokeweed antiviral protein
  • proteins of the invention consists in a potent inhibition of protein synthesis, mainly in cell-free systems: although poorly toxic to most mammalian cells, they may be converted into highly ⁇ ytotoxic agents by conjugation - with suitable chemical agents - to suitable carriers ("haptomers") , e.g. monoclonal antibodies. If said carriers are antigen-specific for tumoral cells, said new RIPs may be used for the selective destruction of the latter.
  • haptomers e.g. monoclonal antibodies.
  • the proteins of the invention can inhibit the virus replication in cultured cells acutely infected with different viruses including the human immunodeficiency virus (HIV 1).
  • the invention concerns also the derivatives of said RIPs wherein sulphydryl groups were introduced, represented by the formula I. Said derivatives have the same or slightly lower inhibitory activity than the starting RIPs and therefore, since they are both the synthesis intermediates of immunotoxins and the inhibiting agents that the latter release into the cells, they are useful for the preparation of antibody-toxins conjugates.
  • Tox Y s R (wherein Tox - represents the considered RIP; Y, R and x have the meanings hereinbelow reported)
  • the derivatives of formula I are obtained according to conventional methods using bifunctional reagents such as, for instance, dimethyl 3, 3 '-dithiobispropionimidate or the reagents hereinbelow reported: N-succinimidyl-3-(2- -pyridyldithio)propionate (SPDP)
  • Y -NH- most pre ⁇ ferably 0 , 7-1 , 5 )
  • SAMSA S-acetyl-mercapto-succinic anhydride
  • the RIPs of the invention will be designed with the following names: momorcochin (from Momordica cochinchinensis seeds); bryodine-L (from Bryonia dioica leaves); asparin 1 and asparin 2 (from Asparagus of icinalis seeds).
  • Momorcochin and bryodin-L are glycoproteins, whereas asparin 1 and 2 are proteins.
  • the proteins of the invention whose characteristics are hereinafter reported, are obtained by conventional methods for the extraction of proteins from plants.
  • the proteins of the invention are therefore obtainable by a process comprising: a) grinding and extraction of seeds or leaves; b) centrifugation or filtration of the extract; c) chromatographies on cross-linked dextrane or dialysis of the surnatant of the step b) d) ion-exchange chromatography and/or gel filtrations in suitable sequences; e) final purification by means of dialysis, lyophilization, chromatography, precipitation or other usual methods.
  • the so obtained proteins may be modified by means of the previously cited reagents, according to known methods.
  • the obtained derivative may then be used for the preparation of immunotoxins which may be used in therapy, for instance for the treatment of tumoral forms in which case they will be administered by parenteral route in doses ranging from 1 to 100 mg, using suitable pharmaceutical formulations.
  • the seeds are wet-ground, in suited grinders and extracted; leaves and roots, sliced in small pieces are subjected to centrifugation with filtration before extraction, recovering the juice and extracting the residue.
  • the extraction is carried out with a 0.14 M sodium chloride/5mM sodium phosphate buffer, pH 7.4-7.5, under stirring for 12 hours at the temperature of 4-10 ⁇ C.
  • Clarification is carried out by centrifugation; for leaves and roots the corresponding juices, obtained as above described, are added to the clarified mixture and then clarified again by high g centrifugation.
  • the final clarified solutions (“crude extracts”) are subjected to the following phase.
  • the extracts are adjusted to pH 4.2-4.8, e.g. to pH 4.5, with acetic acid; after standing, a precipitate is formed which is centrifugated and discarded.
  • the clear supernatants (“acidic extracts”) p are charged on a S-Sepharose column, equilibrated with lOmM sodium acetate buffer, pH 4.5. After washing with the same buffer and then with 5mM sodium phosphate, pH 7.5, till neutrality of the effluent, the column is eluted with 1M sodium chloride/phosphate buffer.
  • the clear eluates are saturated with ammonium sulfate; after standing, the precipitates are collected by centrifugation.
  • the solids are dissolved in 5mM phosphate buffer, pH 7.0; the solutions are clarified by centrifugation, and subjected to the isolation step of the single RIPs ("S-£
  • said final extract may be prepared by subjecting the crude extracts to dialysis for at least 24 hours, at 4°C, against 50-500 volumes of 5mM phosphate buffer, pH 6.5, with at least two total changes. Each precipitate formed during the dialysis is removed by centrifugation and the clear extract is then subjected to the isolation procedure.
  • “crude extract”, “acidic extract”, “S-Sepharose percolate” are carried out determinations of total proteins content, specific and total activity, hereinafter described for each RIP.
  • the column is eluted with NaCl, in linear gradient from 0 to 300mM, in the same buffer, with percolation rate of 1.2 1/h, at 20 ⁇ C, for a total volume of 20 1, collecting fractions of 450 ml. Each fraction is assayed for both the absorption at 260 nm and conductimetry (mS:cra). Each fraction is also assayed for the activity inhibiting specific and total protein synthesis, as hereinafter reported in the biological characteristics. The comparison between the elution pattern and that known for momordin (from Momordica charantia: J. Chromatogr.
  • cytochro e C (12300), myoglobin (17200), carbonic anhydrase (30000), ovoalbumin (45000), bovine serum albumin (66250), ovotransferrin (76000-78000);
  • momorcochin 0.74 i.e. a yield of about 1% by weight in momorcochin relative to the total proteins first extracted and 0.07% by weight relative to the extracted seeds.
  • Electrophoresis shows also that a single chemical species is present.
  • the method is based on the "in vitro" induction of the synthesis with said sub-cellular preparation, in the presence of suitable cofactors and of the mixture of natural amino acids one of which is labelled with 3H or 14C.
  • suitable cofactors one of which is labelled with 3H or 14C.
  • the amount of incorporated radioactivity in the protein obtained, in function of time, allows to evaluate the protein synthesis rate of the system and the effects of different agents, such as RIPs, thereon.
  • Tris/HCl buffer solutions pH 7.4, containing lOOmM ammonium acetate, 2mM magnesium acetate, ImM AT, 0.2mM GTP, 15mM phosphocreatine, 0.5mM natural amino acids (excluding leucine), are added with: 3 ⁇ g creatine kinase, 89nCi of L-(14C)-leucine and 25 ⁇ l of rabbit reticulocyte lysate (prepared according to J. Biol. Chem. 237 , 760, 1982), the final volumes being 62.5 ⁇ l. After incubation at 28 °C for 5' the reaction is stopped by addition of 1 ml of 0.1 M KOH.
  • the method is similar to the previous one but, instead of rabbit reticulocyte mRNAs - directing the synthesis of complex proteins - a simplified polynucleotide is used which translates the synthesis of an homopolymer, polyphenylalanine, in the presence of purified ribosomes.
  • the trichloroacetic acid-insoluble-radioactivity i.e. the amount of polymerized labelled phenylalanine is measured (Biochem.
  • HeLa TG cells human oviduct carcinoma
  • JAR cells human choriocarcinoma
  • human fibr ⁇ blasts human fibr ⁇ blasts
  • NBlOO cells human neuroblastoma cell line
  • the method consists in culturing different cells, in RPMI 1640 medium free from foetal calf serum, in the presence of different amounts of RIPs. After a period of 18 hours, the activity of protein synthesis is measured by incubating the cells for 1-5 hours in an RPMI 1640 medium, free from serum and leucine and added with 0.1-1 ⁇ ci of L-(14C)-leucine/ml. The radioactivity incorporated in the cells is measured according to J. Biol. Chem. 257, 7495, 1982. The ID (dose inhibiting 50% of protein synthesis) is calculated by linear-regression analysis.
  • momorcochin 211.9 units/mg x 10 is about 201 times higher than that of momordin (reported to be 1.0526 x
  • Toxicity in animals C.2: LD50: 24.5 mg/kg body weight. Momordin has a LD of 4.30 mg/kg body weight (FEBS Letter, above cited).
  • D.l - With SPDP the method described in Biochem J. 240, 659, 1986 is used. The determination of introduced 2-pyridylsulphydryl groups is carried out according to Biochem. J. 173, 727, 1978. Momorcochin to be sulphydrylated is dissolved in borate buffer, pH 9.0, at the concentration of 10 mg/ml. The molar ratio of SPDP to momorcochin, the number of introduced groups, and the IC are reported in the table D.4. D.2 - With 2-iminothiolane: the method described in Biochemistry 2A_, 1517, 1985 is used. The number of introduced sulphydryl groups is determined, according to Arch. Biochem. Biophys 82,, 70, 1959. Momorcochin is in 1.5 mM solution in 50 mM borate buffer, pH 9.0. The purification of the final product requires a gel-filtration through Sephadex G 25. The molar ratio of
  • IC50 values of the modified products are calculated with reticulocyte lysate (Method C.l.l) and are expressed in nM. Each value is the mean of three determinations.
  • Example 1 12.5 kg of fresh leaves of Bryonia dioica, a cucurbitacea species, are extracted as in Example 1 up to the obtaining of "S-Sepharose percolate"; said solution is worked up as in Example 2, using, instead of Sephadex G-50 R , Sephacryl-S 200 R ("Sephacryl-S200 eluate”); after chromatography on CM-Sepharose Fast Flow , under the conditions described for momorcochin, the active fractions ("CM-Sepharose eluate”), are pooled, dialyzed extensively against water and charged on a
  • the electrophoresis further shows that the lyophilized is a single substance. Isoelectric point: > 9.5
  • Aminoacid composition (mols/mol protein)
  • bryodine-L 362.3 units/mg 10
  • CM 0.097 M and CM 0.112 M fractions from leaves, and 2.56 times higher then bryodine (CM 0.100 M fraction, from roots) as reported in Biochem. J. 240, cited.
  • the IC50 are determined according to the method C.l.l of Example 2 and are expressed in nM. Each value is the mean of three determinations.
  • SPDP 1.5 1 1.03 0.11 SPDP 2.0 : 1 1.32 0.12
  • CM-Sepharose extract 1 kg of Asparagus officinalis seeds is extracted as in Example 3, the CM-Sepharose extract is separated in two fractions ("CM-peak 1: fractions between 1.8 and 2.4
  • CM-peak 2 fractions between 3.3 and 3.8 1). The two fractions are then purified separately on two ⁇ >
  • Asparin 1 by gel filtration : 29700 by electrophoresis : 30500 asparin 2: by gel filtration : 28100 by electrophoresis : 29800
  • the electrophoresis shows also that the two fractions are single substances.
  • Amino acid composition (mol/mol protein) asparin 1: ,
  • the biological activity is assayed by the methods of Example 2, items C.l.l, C.1.2, C.1.3 and the toxicity is determined according to C.2.
  • Toxicity in animals (C.2): LD50: 20 mg/kg body weight
  • IC50 are determined according to the method C.l.l of
  • Example 2 and are expressed in nM. Each value is the mean of three determinations.
  • Asparin 1 Reagent Reagent/RIP Number of IC50 molar ratio introduced groups (nM)
  • Anti-HIV activity Activity inhibiting the replication of HIVl in lymphoblastoid cultured cells.
  • VB cells cultured under standard conditions, were inoculated with HIVl virus at known concentration for a period of about 60• at 37 ⁇ C. The excess virus not bound to the cells is removed by washing and the cells are then incubated (about 1 X 10 cells/ml) in the presence of
  • the anti HIVl activity has been evaluated, as usual, in the period of the maximum virus production.
  • the inhibition of replication was evaluated as percent of the content of p 24 with respect to the controls.
  • the cells were cultured after the HIVl infection as reported above. Once the amount of infection was measured according to Crowe S., Mills J. e Mc Grath M. 1987 AIDS Res. Hum.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Botany (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)
  • Manufacture Of Tobacco Products (AREA)
  • Diaphragms For Electromechanical Transducers (AREA)

Abstract

L'invention concerne de nouvelles protéines inactivant les ribozomes, tirées d'espèces de Momordica, Bryonia et Asparagus, ainsi que leurs dérivés sulfhydrylés, utiles pour la préparation d'immunotoxines.The invention relates to novel ribozome-inactivating proteins from Momordica, Bryonia and Asparagus species, as well as their sulfhydryl derivatives, useful for the preparation of immunotoxins.

Description

- I - RIBOSOME INACTIVATING PROTEINS AND DERIVATIVES THEREOF
The present invention refers to ribosome inactivating proteins and their sulphydrylated derivatives useful for the preparation of immunotoxins.
Ribosome-inactivating proteins from plants (RIPs) (Cancer Surveys 1 , 489, 1982; J. Natural Products. 8, 446, 1985, FEBS Letters 1.95, 1, 1986) catalytically inactivate the 60 S ribosomal subunits of eukaryσtic ribosomes. They can be either single-chain proteins (RIPs type 1) or two-chain proteins (RIPs type 2), in which one chain has the enzymic activity and the other one has the properties of a galactose-specific lectin. RIPs type 1 are more frequent, being present in several parts of many and possibly all plants, including seeds, roots, leaves and latices, sometimes in more than one form, possibly isoforms. RIPs have an unusual N-glycosidase activity, and cleave the N-glycosidic bond of adenine 4324 of 28S rRNA introducing a lesion that renders RNA cleavable by aniline at a site adjacent to the cleavage induced by
O^-sarcin. This makes ribosomes unable to bind the elongation factors 1 or 2, with consequent arrest of protein synthesis. In spite of the numerous similarities in their structure and of the identical enzymic activity, RIPs have different effects on ribosomes from different organisms (from plants, protozoa, insects and other metazoa).
Interest in RIPs is growing since they have been used as components of "immunotoxins", hybrid molecules consisting of a toxic moiety linked to an antibody. Hopefully immunotoxins will be useful to eliminate harmful cells, neoplastic, immunσcompetent and parasitic cells being considered as possible targets (Cancer Immunol. Iramunother. 2J7, 95, 1988).
Additional interest on RIPs comes from their antiviral activity. All RIPs tested inhibited the infectivity of plant and animal viruses. Actually the first identified RIP type 1, namely the pokeweed antiviral protein (PAP), was initially purified as an anti-viral protein. This property of RIPs was attributed to an easier penetration into virally infected cells with consequent xnactivation of their ribosomes and arrest of viral multiplication. Recently, however, a RIP type 1, trichosa thin, was found to inhibit replication of human immunodeficiency virus through a mechanism apparently independent of the effect on ribosomes (..) . it is useful to have several RIPs available for the preparation of immunotoxins, to have conjugates active against cells resistant to some RIPs, and to circumvent the problems arising from the neutralization due to the immunological reaction after administration and from the inactivation of some RIPs during the conjugation procedure.
It has now been found new ribosome inactivating proteins obtainable from the plants Momordica cochinchinensis, Bryonia dioica e Asparagus officinalis. Said plants were already previously studied without recognizing therein the presence of the proteins of the invention, which are characterized by a remarkably higher activity than that of known similar proteins, extracted from the same vegetal species. For instance, the protein extracted from Momordica cochinchinensis, a cucurbitacea of Indian origin, exhibit surprisingly different characteristics than the known momordine, extracted from the some Momordica genus (Momordica charantia) . Analogously, three glycoproteins are known from Asparagus officinalis L. (Biochem. J. 216, 617, 1983) whereas the substances of the invention extracted from the same species are proteins having different biological and chemico-physical characteristics. The activity of the proteins of the invention consists in a potent inhibition of protein synthesis, mainly in cell-free systems: although poorly toxic to most mammalian cells, they may be converted into highly σytotoxic agents by conjugation - with suitable chemical agents - to suitable carriers ("haptomers") , e.g. monoclonal antibodies. If said carriers are antigen-specific for tumoral cells, said new RIPs may be used for the selective destruction of the latter.
It has also been found that the proteins of the invention can inhibit the virus replication in cultured cells acutely infected with different viruses including the human immunodeficiency virus (HIV 1). The invention concerns also the derivatives of said RIPs wherein sulphydryl groups were introduced, represented by the formula I. Said derivatives have the same or slightly lower inhibitory activity than the starting RIPs and therefore, since they are both the synthesis intermediates of immunotoxins and the inhibiting agents that the latter release into the cells, they are useful for the preparation of antibody-toxins conjugates.
Tox Y s R (wherein Tox - represents the considered RIP; Y, R and x have the meanings hereinbelow reported) The derivatives of formula I are obtained according to conventional methods using bifunctional reagents such as, for instance, dimethyl 3, 3 '-dithiobispropionimidate or the reagents hereinbelow reported: N-succinimidyl-3-(2- -pyridyldithio)propionate (SPDP)
Y: -NH- most pre¬ ferably 0 , 7-1 , 5 )
2 ) 2-Iminothiolane (Traut ' s reagent ) :
Y : even more preferably
0 , 7 )
3) S-acetyl-mercapto-succinic anhydride (SAMSA):
Y: -NH-OC-CH— R: H; CH -CO- x: 0,2-3 (prefera-
1 "-■
CH2-COOH bly 0,4-1, most pre¬ ferably 0,7-1) The RIPs of the invention will be designed with the following names: momorcochin (from Momordica cochinchinensis seeds); bryodine-L (from Bryonia dioica leaves); asparin 1 and asparin 2 (from Asparagus of icinalis seeds). Momorcochin and bryodin-L are glycoproteins, whereas asparin 1 and 2 are proteins. The proteins of the invention, whose characteristics are hereinafter reported, are obtained by conventional methods for the extraction of proteins from plants. The proteins of the invention are therefore obtainable by a process comprising: a) grinding and extraction of seeds or leaves; b) centrifugation or filtration of the extract; c) chromatographies on cross-linked dextrane or dialysis of the surnatant of the step b) d) ion-exchange chromatography and/or gel filtrations in suitable sequences; e) final purification by means of dialysis, lyophilization, chromatography, precipitation or other usual methods.
The so obtained proteins may be modified by means of the previously cited reagents, according to known methods. The obtained derivative may then be used for the preparation of immunotoxins which may be used in therapy, for instance for the treatment of tumoral forms in which case they will be administered by parenteral route in doses ranging from 1 to 100 mg, using suitable pharmaceutical formulations. The following examples further illustrate the invention.
EXAMPLE 1 Preparation of crude extracts
The seeds are wet-ground, in suited grinders and extracted; leaves and roots, sliced in small pieces are subjected to centrifugation with filtration before extraction, recovering the juice and extracting the residue. The extraction is carried out with a 0.14 M sodium chloride/5mM sodium phosphate buffer, pH 7.4-7.5, under stirring for 12 hours at the temperature of 4-10βC.
Clarification is carried out by centrifugation; for leaves and roots the corresponding juices, obtained as above described, are added to the clarified mixture and then clarified again by high g centrifugation. The final clarified solutions ("crude extracts") are subjected to the following phase. The extracts are adjusted to pH 4.2-4.8, e.g. to pH 4.5, with acetic acid; after standing, a precipitate is formed which is centrifugated and discarded. The clear supernatants ("acidic extracts") p are charged on a S-Sepharose column, equilibrated with lOmM sodium acetate buffer, pH 4.5. After washing with the same buffer and then with 5mM sodium phosphate, pH 7.5, till neutrality of the effluent, the column is eluted with 1M sodium chloride/phosphate buffer.
The clear eluates are saturated with ammonium sulfate; after standing, the precipitates are collected by centrifugation.
The solids are dissolved in 5mM phosphate buffer, pH 7.0; the solutions are clarified by centrifugation, and subjected to the isolation step of the single RIPs ("S-£|epharose percolate"). Alternatively, said final extract may be prepared by subjecting the crude extracts to dialysis for at least 24 hours, at 4°C, against 50-500 volumes of 5mM phosphate buffer, pH 6.5, with at least two total changes. Each precipitate formed during the dialysis is removed by centrifugation and the clear extract is then subjected to the isolation procedure. During the steps of: "crude extract", "acidic extract", "S-Sepharose percolate", are carried out determinations of total proteins content, specific and total activity, hereinafter described for each RIP. EXAMPLE 2
Isolation, and characterization of momorcochin A) Extraction and isolation 1.2 kg of Momordica cochinchinensis seeds, a Cucurbitaceae species of indian origin, are extracted as disclosed in Example 1. The final solution "S-Sepharose percolate" is chromatographed on Sephadex G-50 ("Sephadex G-50 eluate") and then charged on a CM-Sepharose Fast Flow column, equilibrated with 5mM sodium phosphate buffer, pH 7.0, and provided with flow-cell and absorption detector at 280 nm. The column is washed with the equilibration buffer until the percolate does not absorb any longer at 280 nm. The column is eluted with NaCl, in linear gradient from 0 to 300mM, in the same buffer, with percolation rate of 1.2 1/h, at 20βC, for a total volume of 20 1, collecting fractions of 450 ml. Each fraction is assayed for both the absorption at 260 nm and conductimetry (mS:cra). Each fraction is also assayed for the activity inhibiting specific and total protein synthesis, as hereinafter reported in the biological characteristics. The comparison between the elution pattern and that known for momordin (from Momordica charantia: J. Chromatogr. 408, 235, 1987) confirms the non coincidence of the two substances (momordin: elution fractions from 12 to 17 1 about, momorcochin: fractions from 6 to 7.5 1). The mo orcochin-containing fractions were pooled and dialyzed against water until complete salting-out. The final solution is lyophilized and the residue ("momorcochin") is analyzed for chemico-physical and biological characteristics.
B) Chemico-physical characterization a) Protein content of the extraction solutions: results obtained according to the methods: - by Lowry (J. Biol. Chem. 193, 265, 1951) (standard: bovine serum albumin),
- by Kalb δ Bernlhor (Anal. Biochem. J3 , 362, 1977),
- spectrophotometric, using the absorption at 280nm of the purified protein, with molar extinction coefficient of 0,8. b) Relative molecular mass (rM) : determined by:
- electrophoresis on polyacrylamide gel (Nature 227 , 680, 1970) with the following markers (between brackets the relative rM) : cytochro e C (12300), myoglobin (17200), carbonic anhydrase (30000), ovoalbumin (45000), bovine serum albumin (66250), ovotransferrin (76000-78000);
- gel-filtration through Sephacryl S-200 (95 x 1.6 cm columns) equilibrated with 20mM sodium phosphate buffer, pH 7.5 containing 0.3M NaCl; elution-rate 8 ml/h at 20βC; calibration (between brackets the relative rM): dextran blue (2 x 10 ), bovine serum albumin (66250), ovoalbumin
(45000), chymotrypsinogen A (25000), ribonuclease A
(13700) . c) Isoelectric point, amino acid composition, amino sugar and neutral sugar compositions; determined according to
Biochem. J. 240, 659, 1986.
By applying said methods to the intermediate extracts (described in Example 1) to those of this Example and to the final lyophilized product
"momorcochin", the following results are obtained: Proteins at different purification steps (g/1 of kg starting seeds):
- "crude extract" : 52.26
- "acidic extract" 36.00 - "S-Sepharose percolate" 19.48
- "Sephadex G-50 eluate" 2.55
- "momorcochin" 0.74 i.e. a yield of about 1% by weight in momorcochin relative to the total proteins first extracted and 0.07% by weight relative to the extracted seeds.
Molecular mass of the final product: by gel filtration: 31000 by electrophoresis: 30700
Electrophoresis shows also that a single chemical species is present.
Isoelectric point > 9 (momordin: 8.60 - Biochem. J. 207,
505, 1982)
Extinction at 280 n (A280): 0.7
Amino acid compositions (mols/mole protein) (means values from hydrolysis at 24, 48 and 72 h; ratios calculated assuming rM=30700; error < 1%; IUPAC symbols, between brackets the corresponding values found for momordin - Biochem. J. 207, 505, 1982; aa: amino acid):
aa mol/ ol/pr. aa mol/mol/pr. aa mol/mol/pr.
Lys 15.1 (11.4) Glx : 21.7 (24.8) Met : 3.3 ( 7.0) His 2.7 ( 4.7) Pro : 8.8 ( 9.0) lie : 12.3 (13.8) Arg 8.1 (12.6) Gly : 11.3 (15.8) Leu : 26.6 (21.2) Asx 27.7 (27.6) Ala : 23.6 (24.6) Tyr • 11.4 (13.4) Thr 18.4 (16.2) Half-Cys:ass. (2.2) Phe • 10.8 ( 8.4) Ser 19.2 (16.8) Val : 18.7 (13.7) Trp : abs. ( 1.0)
The comparison with the composition of momordin shows the different compositions of the two RIPs, extracted from two species of the same genus Momordica. Total content in neutral sugars: 2.82%
(Momordin - Biochem. J. 207, above cited - has a content of 1.74%).
Sugar composition (mols/mol RIP):
(ratios calculated assuming rM: 30700, IUPAC symbols; between brackets the corresponding values for momordin Biochem. J. 207 , above cited): Sugars mols/mol RIP
Fuc 1.42 (0.90)
Glc : 0.98 (0.80)
Man 2.16 (1.30)
Xyl : 0.98 (0.50)
Glc-NH : abs. (2.00)
Also in this case the unexpected difference between momorcochin and momordin is evident. C) Biological characterization C.l - Inhibiting activity on protein synthesis: assayed by :
C.l.l - Rabbit reticulocyte lysate (Biochem. J. 240 , 659, 1986). By this method, the ability of RIPs to inhibit the protein synthesis in the presence of reticulocyte components (post-mitochondrial fraction) is determined.
The method is based on the "in vitro" induction of the synthesis with said sub-cellular preparation, in the presence of suitable cofactors and of the mixture of natural amino acids one of which is labelled with 3H or 14C. The amount of incorporated radioactivity in the protein obtained, in function of time, allows to evaluate the protein synthesis rate of the system and the effects of different agents, such as RIPs, thereon. lO M Tris/HCl buffer solutions, pH 7.4, containing lOOmM ammonium acetate, 2mM magnesium acetate, ImM AT, 0.2mM GTP, 15mM phosphocreatine, 0.5mM natural amino acids (excluding leucine), are added with: 3μg creatine kinase, 89nCi of L-(14C)-leucine and 25 μl of rabbit reticulocyte lysate (prepared according to J. Biol. Chem. 237 , 760, 1982), the final volumes being 62.5 μl. After incubation at 28 °C for 5' the reaction is stopped by addition of 1 ml of 0.1 M KOH. The measurement of the incorporated radioactivity is carried out as disclosed in Biochem. J. 174 , 491, 1978. Scalar amounts of RIP are added before the incubation under the same experimental conditions. The results are expressed as ιc cn (concentration inhibiting by 50% the synthesis) or in units and specific activities (units/mg) . An inhibition unit (or activity unit), the amount (in mg) of RIP, needed for inhibiting by 50% the protein synthesis, adjusting the reaction volume to 1 ml. C.1.2 - Polyuridylate (poly-U)-directed Polymerization with ribosomes from rabbit or Trypanosoma brucei reticulocytes - of phenylalanine (Proc. Natl. Acad. Sci.
U.S. 4 , 1588, 1961). This method allows the evaluation of the inhibiting activity of the RIPs on the polyphenylalanine synthesis, directed by the synthetic polynucleotide poly-U, consisting only of the uracil nucleotide, starting from t-RNA esterified phenylalanine.
The method is similar to the previous one but, instead of rabbit reticulocyte mRNAs - directing the synthesis of complex proteins - a simplified polynucleotide is used which translates the synthesis of an homopolymer, polyphenylalanine, in the presence of purified ribosomes.
An 80mM Tris/HCl buffer, pH 7.4, at a final volume of 250μl containing 120mM magnesium acetate, 2mM GTP, is added with: 200μg poly-U, 25 pmol of
14C-phenylalanyl-t-RNA, 20 pmol of rabbit reticulocyte ribosomes or Trypanosoma brucei ribosomes (J. Protozool.
35, 384, 1988), 250μg (as proteins) of "pH 5 supernatant", according to Biochem. J. 176, 265, 1978).
After incubation at 30βC for 30', the trichloroacetic acid-insoluble-radioactivity (i.e. the amount of polymerized labelled phenylalanine) is measured (Biochem.
J. 240, cited). The experiment is then carried out again in the presence of scalar amounts of RIPs. The IC_n is
50 calculated by linear-regression analysis.
C.1.3 - Human cell cultures: HeLa TG cells (human oviduct carcinoma), JAR cells (human choriocarcinoma) , human fibrόblasts, and NBlOO cells (human neuroblastoma cell line), were maintained as monolayer cultures in RPMI 1640 medium supplemented with non-essential amino acids, antibiotics and 10% foetal calf serum. This method allows the evaluation of the cytotoxic effect of RIPs in different integer cell systems, by comparing the protein synthesis in the presence and in the absence of toxins.
The method consists in culturing different cells, in RPMI 1640 medium free from foetal calf serum, in the presence of different amounts of RIPs. After a period of 18 hours, the activity of protein synthesis is measured by incubating the cells for 1-5 hours in an RPMI 1640 medium, free from serum and leucine and added with 0.1-1 μci of L-(14C)-leucine/ml. The radioactivity incorporated in the cells is measured according to J. Biol. Chem. 257, 7495, 1982. The ID (dose inhibiting 50% of protein synthesis) is calculated by linear-regression analysis. C.2 - Toxicity in animals The toxicity of pure RIPs was evaluated in Swiss female mice weighing 27-32 g, fed ad libitum, by i.p. route at 6 different doses ranging from 5.6 to 23.7 mg/kg body weight, 6 animals per dose. The LD was calculated 48 hours after by linear-regression analysis. The autoptic examination was carried out on the main organs. By applying said methods to the intermediate extracts of Example 1, to those of the present Example and to the final lyophilized product, ("momorcochin"), the following results are obtained: Inhibiting activity of protein synthesis with rabbit reticulocyte lysate (C.l.l.):
Fraction Specific Activity Extracted Yield*1
.-3
(unit/mg x 10 ) Total Activity*
(unit x 10"6)
-"crude extract" 16.8 877.2 (100 )
-"acidic extract" 29.1 1 , 049.6 119.6 _«S-Sepharose percolate" 27.5 536. 6 61.2
-"Sephadex G-50 eluate" 149.2 379.1 43.2
-"momorcochin" 285.7 211.9 24.0
* : referred to 1.2 kg of starting seeds
** : referred to the "crude extract",' assumed as 100.
It has to be pointed out that the specific activity
_3 of momorcochin (211.9 units/mg x 10 is about 201 times higher than that of momordin (reported to be 1.0526 x
_3 10 units/mg in J. Chromatogr. 408, 235, 1987).
IC50 for the protein synthesis in cell-free systems
Method Enzymatic systems IC50 (nM)
C.l.l Reticulocyte lysate 0.12 C.1.2 Purified ribosomes from reticulocytes 1.0 C.1.2 Purified ribosomes from T. Brucei 3,330.
Comparing the IC5_ for momordin (FEBS-Letters 195, 1, 1986) obtained with reticulocyte lysate (0.06nM) with that of momorcochin, a further differentiation between the two RIPs is evident. IC50 for the protein synthesis in integer cells (C.1.3): (The values are the mean of two different determinations; between brackets the ID for momordin; symbols disclosed in item C.1.3):
Cell ID50 (nM)
HeLa 2,870 (32,000) TG 2,040 Jar 3,330 NB 320
Human fibroblasts 109
Toxicity in animals (C.2): LD50: 24.5 mg/kg body weight. Momordin has a LD of 4.30 mg/kg body weight (FEBS Letter, above cited).
D) Chemical modifications
D.l - With SPDP: the method described in Biochem J. 240, 659, 1986 is used. The determination of introduced 2-pyridylsulphydryl groups is carried out according to Biochem. J. 173, 727, 1978. Momorcochin to be sulphydrylated is dissolved in borate buffer, pH 9.0, at the concentration of 10 mg/ml. The molar ratio of SPDP to momorcochin, the number of introduced groups, and the IC are reported in the table D.4. D.2 - With 2-iminothiolane: the method described in Biochemistry 2A_, 1517, 1985 is used. The number of introduced sulphydryl groups is determined, according to Arch. Biochem. Biophys 82,, 70, 1959. Momorcochin is in 1.5 mM solution in 50 mM borate buffer, pH 9.0. The purification of the final product requires a gel-filtration through Sephadex G 25. The molar ratio of
2-iminothiolane to momorcochin and the IC are shown in
50 Table D .4 .
D.3 - With SAMSA: according to J. Am. Chem. Soc. 81,
3802, 1959. The number of introduced sulphydrhyl groups is determined, after addition of hydroxylamine, according to Methods in Enzymology 2j , 457n 1972. Momorcochin is in solution of 125mM phosphate buffer pH 7.
D.4 - Determination of IC50 of modified RIPs
The IC50 values of the modified products are calculated with reticulocyte lysate (Method C.l.l) and are expressed in nM. Each value is the mean of three determinations.
Reagent Molar ratio Number of IC50 Reagent/RIP(*) introduced groups (nM)
None 0.12
SPDP 1.5 s 1 1.13 0.12
SPDP 2.0 : 1 1.44 0.20
2-Iminothiolane 1.25 mM 0.73 0.10 SAMSA 28 : 1 0.70 0.10
(*): tinder the above reported experimental conditions
EXAMPLE 3 Isolation and characterization of bryodine -L A) Extraction and isolation
12.5 kg of fresh leaves of Bryonia dioica, a cucurbitacea species, are extracted as in Example 1 up to the obtaining of "S-Sepharose percolate"; said solution is worked up as in Example 2, using, instead of Sephadex G-50R, Sephacryl-S 200R ("Sephacryl-S200 eluate"); after chromatography on CM-Sepharose Fast Flow , under the conditions described for momorcochin, the active fractions ("CM-Sepharose eluate"), are pooled, dialyzed extensively against water and charged on a
Blue-Sepharose column, equilibrated with lOmM Tris/HCl buffer, pH 8.0; the column is eluted with a 0-200mM NaCl gradient, in the same buffer, monitoring the absorption at 280 nm and the conductivity of the fractions (mS/cm) . The fractions from 6.8 to 7.6 1 are pooled and dialyzed extensively against water and then lyophilized
(bryodine-L). B) Chemico-physical characterization.
The used methods are those disclosed in B of Example
2, paragraphs a, b, c.
Proteins at different purification steps (g/12.5 kg of leaves). - "crude extract" 293.75
- "S-Sepharose percolate" 1.74
- "Sephacryl-S 200 eluate" 0.41
- "CM-Sepharose eluate" 0.035
- "Bryodine-L" 0.021 Molecular mass of the final product: by gel-filtration : 27300 by electrophoresis : 28800
The electrophoresis further shows that the lyophilized is a single substance. Isoelectric point: > 9.5
Extinction at 280 nm (A280): 0.8
Aminoacid composition (mols/mol protein)
(mean values of the hydrolysis at 24, 48, 72 hours; ratios calculated assuming rM = 28800, error < 1%; IUPAC symbols; between backets the corresponding values found for bryodine - Biochem. J. 240, 659, 1986; aa: amino acid) . aa η mol/mol/pr. aa mol/mol/pr. aa mol/mol/pr.
Lys 10.8 ( 8.6) Glx : 18.9 (17.7) Met 2.2 ( 1.6) His 1.0 ( 1.9) Pro : 7.2 ( 6.7) He 15.4 (15.1) Arg 11.0 (11.8) Gly : 11.4 (11.4) Leu 24.5 (28.3) Asx 25.5 (22.5) Ama : 24.1 (22.4) Tyr 11.7 (14.2) Thr 17.4 (15.1) Half-Cys:abs. (0.24) Phe 7.4 ( 8.3) Ser 24.4 (30.2) Val : 14.4 (15.6) Trp abs. ( 2.0)
The ^comparison between data shows the different compositions of the two RIPs. This difference is even more marked from the following data. Total content in neutral sugars: 2.72%
For bryodine (Biochem. J. 240, cited) the total content is 6.33%.
Sugar compositions (mols/mol RIP):
(ratios calculated assuming rM : 28800 ; IUPAC symbols; between brackets the corresponding values for bryodine Biochem. J. 240, cited) : Sugars mols/mol RIP
Fuc 1.52 (3.47) Glc 0.43 (1.55) Man 2.52 (6.27) Xyl 0.63 (0.93
C) Biological characterization
The biological characterization is assayed according to the methods described in Example 1, items C.l.l., C.1.2, C.1.3,. whereas the toxicity is evaluated according to C.2. Inhibiting activity of protein synthesis with rabbit reticulocyte lysate (C.l.l):
Fraction Specific Activity Extracted Yield**
_3 (unit/mg x 10 ) Total Activity*
(unit x 10~6)
-••crude extract" 0.6 170.0 (100)
-"S-Sepharose percolate" 81.4 141.0 83.2 -"Sephacryl S-200 eluate" 123.0 50.6 29.7
-"CM-Sepharose eluate" 251.9 8.7 5.1
-"Bryodine-L" 362.3 6.7 3.9
* : referred to 12.5 kg of fresh leaves ** : referred to "crude extract", assumed as 100.
It should be pointed out that the specific activity of bryodine-L (362.3 units/mg 10 ) is 2.8 and 1.6 times higher, respectively, than that of the CM 0.097 M and CM 0.112 M fractions, from leaves, and 2.56 times higher then bryodine (CM 0.100 M fraction, from roots) as reported in Biochem. J. 240, cited. IC50 for protein synthesis in cell-free systems
Method Enzymatic systems IC50 (nM)
C.l.l Rabbit reticulocyte lysate 0.09
C.1.2 Purified ribosomes from reticulocytes 1.3 C.1.2 Purified ribosomes from T. Brucei 3,330.
in this case the comparison with the data described in Biochem. J. 240 is not possible since either reported in incorporated d.p.m. or the ID (with the same meaning of IC5c0rι) was evaluated with different ribosome preparations (from wheat germs or from Bryonia dioica) . IC50 for protein synthesis in integer cell systems (C.1.3): (the values are the mean of two different determinations)
Cells ID50 (nM)
HeLa 3,330 TG 770
Jar 3,330
NB 50
Human fibroblasts 800
Toxicity in animals (C.2): LD50: 10 mg/kg body weight. Bryodine (Biochem. J. 240, cited): 14.5 mg/kg. D) Chemical modifications
The chemical modifications are carried out using the methods D.l, D.2, D.3 of Example 2. Determination of IC50 of modified bryodine-L:
The IC50 are determined according to the method C.l.l of Example 2 and are expressed in nM. Each value is the mean of three determinations.
Reagent Molar ratio Number of IC50 Reagent/RIP(*) introduced groups (nM)
None 0.10
SPDP 1.5 : 1 1.03 0.11 SPDP 2.0 : 1 1.32 0.12
2-Iminothiolane 1.25 mM 0.70 0.09
SAMSA 28 : 1 0.75 0.11 EXAMPLE 4
Isolation and characterization of asparin 1 and 2 A) Extraction and isolation
1 kg of Asparagus officinalis seeds is extracted as in Example 3, the CM-Sepharose extract is separated in two fractions ("CM-peak 1: fractions between 1.8 and 2.4
1; "CM-peak 2: fractions between 3.3 and 3.8 1). The two fractions are then purified separately on two τ>
Red-Sepharose columns, equilibrated with Tris/HCl buffer, pH 8.0, eluting with a NaCl gradient 0-300 mM in the same buffer.
The purified fraction from "CM-peak 1" ("asparin 1") is lyophilized, wheras that from "CM-peak 2" ("asparin
2") is first eluted through a Sephacryl S-200 column. B) Chemico-physical characterization'
The methods of item B of Example 2, paragraphs a, b, c, were used.
Proteins at different purification steps (g/lkg seeds):
-"crude extract" 23.23 -"acidic extract" 13.58
-"S-Sepharose percolate" 7.55
-"Sephacryl-S 200 eluate" 2.04
-"CM-peak 1" 0.159
-"CM-peak 2« 0.229 -"asparin 1" 0.054
-"asparin 2" 0.049
Molecular mass of the final products:
Asparin 1: by gel filtration : 29700 by electrophoresis : 30500 asparin 2: by gel filtration : 28100 by electrophoresis : 29800
The electrophoresis shows also that the two fractions are single substances.
Isoelectric point: -asparin 1 : 8.7 -asparin 2 : 9.2
Extinction, at 280 nm (A280): 1.0 (both)
Amino acid composition: (mol/mol protein) asparin 1: ,
(mean values of hydrolysis at 24, 48, 72 hours; ratios calculated assuming rM = 30500; error < 1%; IUPAC symbols; aa: amino acid):
aa mol/mol/pr. aa mol/mol/pr. aa mol/mol/pr. ———————-w, — — —— ___—_ Lys J 15.3 Glx : 24.3 Met : 4.1
His .: 3.4 Pro : 14.9 He : 11.4
Arg : 14.4 Gly : 15.0 Leu : 26.3
Asx : 26.5 Ala : 19.5 Tyr : 9.8
Thr : 13.9 Half-Cys: 3.3 Phe : 6.3 Ser : 11.0 Val : 16.7 Trp : abs.
Asparin 2:
(mean values of hydrolysis at 24, 48, 72 hours; ratios calculated assuming rM = 29800; error < 1%; IUPAC symbols; aa: amino acid): aa mol/mol/pr. aa mol/mol/pr. aa mol/mol/pr.
Lys - 14.9 Glx : 23.2 Met : 1.1
His : 2.9 Pro : 15.4 He : 11.5
Arg : 14.8 Gly : 15.2 Leu : 26.6
Asx • 27.2 Ala : 18.8 Tyr : 9.8
Thr • 14.2 Half-Cys: 1.3 Phe : 6.3
Ser : 11.3 Val : 16.9 Trp : abs.
Total content in neutral sugars: 0%
For the three glycoproteins described (Biochem. J. 216, cited) the contents are: 1.42%, 1.20%, 1.32% with a composition: Glycopro'tein
Sugar peak 1 peak 2 peak 3
(mol/mol protein)
Fucose traces 0 0 Galactose 0.3 traces 0.3 Glucose 2.1 2.1 2.1 Mannose 0.4 0.3 0.3
C) Biological characterization.
The biological activity is assayed by the methods of Example 2, items C.l.l, C.1.2, C.1.3 and the toxicity is determined according to C.2.
Inhibiting activity of protein synthesis with rabbit reticulocyte lysate (C.l.l): Fraction Specific Activity Extracted* Yield**
_3 (unit/mg x 10 ) Total Activity (unit x x 10 ")
-"crude extract" 7.9 183.5 (100)
-"acid extract" 16.3 221.0 120
-"S-Sepharose percolate" 35.3 266.5 145
-"Seρhacryl-S 200 eluate 69.4 141.8 77
-"CM-peak 1" 89.9 15.7 8.5
-"CM-peak 2" 218,0 49.9 27.2
-"asparin 1" 112.4 6.1 3.3
-"asparin 2" 224.2 10.9 5.9
* : referred to 1 kg of seeds
** : referred to the "crude extract", assumed as 100.
IC50 for the protein synthesis in cell-free systems
Asparin 1:
Method Enzymatic systems IC50 (nM)
C.l.l Rabbit reticulocyte lysate 0.27
C.1.2 Purified ribosomes from reticulocytes 8.8 C.1.2 Purified ribosomes from T. Brucei > 3,330.
Asparin 2:
Method Enzymatic systems IC50 (nM)
C.l.l Rabbit reticulocyte lysate 0.15
C.1.2 Purified ribosomes from reticulocytes 6.9 c.1.2 Purified ribosomes from T. Brucei > 3,330.
IC50 for the protein synthesis in integer cells (C.1.3): (the values are the mean of two different determinations) Asparin 1:
Cells ID50 (nM)
HeLa 3,330
TG 610
Jar 3,330
NB 180 Human fibroblasts 3,330
Asparin 2:
Cells ID50 (nM)
HeLa 3,330
TG 210
Jar 3,330
NB 180 Human fibroblasts 2,020
Toxicity in animals (C.2): LD50: 20 mg/kg body weight
(both).
D) Chemical modifications Chemical modifications are carried out as in Example
2, items D.l, D.2, D.3.
Determination of IC50 of modified asparin 1 and 2:
IC50 are determined according to the method C.l.l of
Example 2 and are expressed in nM. Each value is the mean of three determinations.
Asparin 1: Reagent Reagent/RIP Number of IC50 molar ratio introduced groups (nM)
None 0.27
SPDP 1.5 : 1 1.03 0.41
SPDP 2.0 : 1 1.32 0.67
2-Iminothiolane 1.25 mM 0.70 0.28
SAMSA 28 : 1 0.75 0.26
Asparin 2:
Reagent Reagent/RIP Number of IC50 molar ratio introduced groups (nM)
None 0.15
SPDP 1.5 : 1 1.03 0.19
SPDP 2.0 i 1 1.32 0.40
2-Iminothiolane 1.25 mM 0.70 0.26
SAMSA 28 : 1 0.75 0.21
EXAMPLE 5
Anti-HIV activity Activity inhibiting the replication of HIVl in lymphoblastoid cultured cells.
VB cells, cultured under standard conditions, were inoculated with HIVl virus at known concentration for a period of about 60• at 37βC. The excess virus not bound to the cells is removed by washing and the cells are then incubated (about 1 X 10 cells/ml) in the presence of
—8 —6 increasing concentrations (between 10 and 10~ M) of bryodine or asparin 1 or asparin 2 or momorcochin. The presence of HIVl in the culture is determined by the analysis of the expression of the viral antigen p 24 using a commercial immunoassay.
The anti HIVl activity has been evaluated, as usual, in the period of the maximum virus production.
The inhibition of replication was evaluated as percent of the content of p 24 with respect to the controls.
The obtained results show that bryodine, asparin 1, asparin 2 and momorcochin are able to inhibit the virus
—8 —6 replication at concentrations (10~ - 10 mols/1) not impairing the macromolecular synthesis of the cells, with inhibition values of 70-80%.
Inhibition of HIVl in infected monocyte machrophage cells Monocyte/macrophage cells were isolated from peripheral blood from healthy volunteers according to the standard method with Ficoll gradient.
The cells were cultured after the HIVl infection as reported above. Once the amount of infection was measured according to Crowe S., Mills J. e Mc Grath M. 1987 AIDS Res. Hum.
Retrov. 3_ 135-145, the cells were treated with the RIPs
—8 —6 of the invention at concentrations from 10 to 10 M and cultured for 4 days. The expression of the p 24 antigen was determined by cytofluorimetric analysis and the inhibition values expressed as percent versus controls. The results show that the RIPs of the invention can inhibit the virus replication at the tested concentrations, with inhibition values of 70-80%.

Claims

1. A ribosome inactivating protein, having the following characteristics:
- molecular mass by gel-filtration : 31.000
- molecular mass by electrophoresis : 30.700
- isoelectric point : > 9
- extinction at 280 nm : 0.7
- amino acid composition for rM 30.700
aa mol/mol/pr. aa mol/mol/pr. aa mol/mol/pr.
Lys : 15.1 Glx : 21.7 Met : 3.3
His : 2.7 Pro : 8.8 He : 12.3
Arg : 8.1 Gly : 11.3 Leu : 26.6
Asx : 27.7 Ala : 23.6 Tyr : 11.4
Thr : 18.4 Half-Cys:ass. Phe : 10.8
Ser : 19.2 Val : 18.7 Trp • abs.
- total content in neutral sugars : 2.82% - sugar composition (mols/mol RIP) for rM 30.700
Sugar mols/mol RIP
Fuc : 1.42
Glc : :. 0.98
Man : 2.16
Xyl : 0.98
Glc-NH : ass.
2. A ribosome inactivating protein, having the following characteristics: - molecular mass by gel-filtration : 27.300 molecular mass by electrophoresis : 28.800 isoelectric point : > 9.5 extinction at 280 nm : 0.8 amino acid composition for rM 28.800
aa mol/mol/pr. aa mol/mol/pr. aa mol/mol/pr.
Lys : 10.8 Glx : 18.9 Met : 2.2
His : 1.0 Pro : 7.2 He : 15.4
Arg : 11.0 Gly : 11.4 Leu : 24.5
Asx : 25.5 Ala : 24.1 Tyr : 11.7
Thr : 17.4 Half-Cys:abs. Phe : 7.4
Ser : 24.4 Val : 14.4 Trp : abs.
- total content in neutral sugars ': 2.72%
- sugar composition (mols/mol TIP) for rM 28.800
Sugar mols/mol RIP
Fuc 1.52 Glc 0.43 Man 2.52 Xyl 0.63
3. A ribosome inactivating protein, having the following characteristics:
- molecular mass by gel-filtration : 29.700
- molecular mass by electrophoresis : 30.500
- isoelectric point : > 8.7
- extinction at 280 nm : 1.0
- amino acid composition for rM 30.500 aa mol/mol/pr. aa mol/mol/pr. aa mol/mol/pr.
Lys : 15.3 Glx : 24.3 Met : 4.1
His : 3.4 Pro : 14.9 He : 11.4
Arg : 14.4 Gly : 15.0 Leu : 26.3
Asx : 26.5 Ala : 19.5 Tyr : 9.8
Thr : 13.9 Half-Cys: 3.3 Phe : 6.3
Ser : 11.0 Val : 16.7 Trp : abs.
4. A ribosome inactivating protein, having the following characteristics:
- molecular mass by gel-filtration : 28.100
- molecular mass by electrophoresis : 29.800
- isoelectric point :' > 9.2
- extinction at 280 nm : 1.0
- amino acid composition for rM 29.800
aa mol/mol/pr. aa mol/mol/pr. aa mol/mol/pr. —
Lys : 14.9 Glx : 23.2 Met : 1.1
His : 2.9 Pro : 15.4 He : 11.5
Arg : 14.8 Gly : 15.2 Leu : 26.6
Asx : 27.2 Ala : 18.8 Tyr : 9.8
Thr : 14.2 , Half-Cys: 1.3 Phe : 6.3
Ser : 11.3 Val : 16.9 Trp : abs.
5. Protein derivatives as claimed in claims 1 to 4, having formu
wherein Tox represents the protein, Y is selected from the group consisting of
-NHCOCH-.CH 2_.- ; -NH-OC-CH-CH COOH; R is selected from hydrogen, acetyl, 2-pyridyl and x is comprised from 0.2 to 3.
EP90904825A 1989-03-31 1990-03-26 Ribozome inactivating proteins and derivatives thereof Pending EP0465495A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT1996489 1989-03-31
IT8919964A IT1229216B (en) 1989-03-31 1989-03-31 PROTEINS INHIBIT RIBOSOMES AND THEIR DERIVATIVES.

Publications (1)

Publication Number Publication Date
EP0465495A1 true EP0465495A1 (en) 1992-01-15

Family

ID=11162688

Family Applications (2)

Application Number Title Priority Date Filing Date
EP90904825A Pending EP0465495A1 (en) 1989-03-31 1990-03-26 Ribozome inactivating proteins and derivatives thereof
EP90105725A Expired - Lifetime EP0390040B1 (en) 1989-03-31 1990-03-26 Ribosome inactivating proteins and derivatives thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP90105725A Expired - Lifetime EP0390040B1 (en) 1989-03-31 1990-03-26 Ribosome inactivating proteins and derivatives thereof

Country Status (17)

Country Link
EP (2) EP0465495A1 (en)
JP (1) JPH04504252A (en)
CN (1) CN1046335A (en)
AT (1) ATE89830T1 (en)
AU (1) AU5333490A (en)
CA (1) CA2050592A1 (en)
DE (1) DE69001701T2 (en)
DK (1) DK0390040T3 (en)
ES (1) ES2055203T3 (en)
IL (1) IL93924A0 (en)
IT (1) IT1229216B (en)
NZ (1) NZ233106A (en)
PH (1) PH27547A (en)
PL (2) PL286575A1 (en)
SG (1) SG52793G (en)
WO (1) WO1990012034A1 (en)
ZA (1) ZA902484B (en)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635384A (en) * 1990-06-11 1997-06-03 Dowelanco Ribosome-inactivating proteins, inactive precursor forms thereof, a process for making and a method of using
US5248606A (en) * 1990-06-11 1993-09-28 Dowelanco Dna encoding inactive precursor and active forms of maize ribosome inactivating protein
ES2074935B1 (en) * 1993-03-02 1996-05-16 Univ Valladolid EBULIN 1 PROTEIN FROM THE SAMBUCUS EBULUS L. PLANT, PROCEDURE FOR ITS OBTAINING AND USE
ES2074936B1 (en) * 1993-03-02 1996-05-16 Univ Valladolid PROTEIN NIGRINE D OF THE SAMBUCUS NIGRA L. PLANT, PROCEDURE FOR ITS OBTAINING AND USE.
DE69415026D1 (en) * 1993-09-06 1999-01-14 Menarini Richerche Sud S P A RIBOSOM INACTIVE VIRUS PROTEINS EXTRACTED FROM SAPONARIA OCYMOIDES AND VACCARIA PYRAMIDATA SEEDS AND THEIR PRODUCTION AND IMMUNOTOXINS THAT INCLUDE
US5597569A (en) * 1993-10-25 1997-01-28 Bristol-Myers Squibb Company Bryodin 2 a ribosome-inactivating protein isolated from the plant Bryonia dioica
US5541110A (en) 1994-05-17 1996-07-30 Bristol-Myers Squibb Cloning and expression of a gene encoding bryodin 1 from Bryonia dioica
CN1064370C (en) * 1994-10-22 2001-04-11 王崴 Extraction method and application of plant protein
EP1241267A3 (en) * 2001-03-14 2004-01-02 Viscum AG Enzyme activity test using fluorescently labeled oligonucleotide substrate
US8067045B2 (en) 2005-03-03 2011-11-29 Pharmanex, Llc Nutritional formulations and associated methods
CN108324924A (en) * 2018-04-23 2018-07-27 海南海泽惠生物科技有限公司 Application of the bitter melon protein in preparing anti-nipple tumor virus infection drug

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4869903A (en) * 1987-05-29 1989-09-26 Genelabs Incorporated Method of selectively inhibiting HIV

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9012034A1 *

Also Published As

Publication number Publication date
CA2050592A1 (en) 1990-10-01
IT1229216B (en) 1991-07-25
ES2055203T3 (en) 1994-08-16
AU5333490A (en) 1990-11-05
NZ233106A (en) 1992-05-26
EP0390040B1 (en) 1993-05-26
DK0390040T3 (en) 1993-08-16
CN1046335A (en) 1990-10-24
IT8919964A0 (en) 1989-03-31
DE69001701D1 (en) 1993-07-01
PL286575A1 (en) 1991-07-15
PL286574A1 (en) 1991-07-15
DE69001701T2 (en) 1993-09-23
PH27547A (en) 1993-08-18
SG52793G (en) 1993-06-25
JPH04504252A (en) 1992-07-30
WO1990012034A1 (en) 1990-10-18
IL93924A0 (en) 1990-12-23
ATE89830T1 (en) 1993-06-15
EP0390040A1 (en) 1990-10-03
ZA902484B (en) 1991-01-30

Similar Documents

Publication Publication Date Title
Irvin Pokeweed antiviral protein
Stirpe et al. Ribosome‐inactivating proteins up to date
Ramnath et al. Antitumour effect of abrin on transplanted tumours in mice
Bolognesi et al. New ribosome-inactivating proteins with polynucleotide: adenosine glycosidase and antiviral activities from Basella rubra L. and Bougainvillea spectabilis Willd.
Stirpe et al. Bryodin, a ribosome-inactivating protein from the roots of Bryonia dioica L.(white bryony)
Stirpe et al. Properties of volkensin, a toxic lectin from Adenia volkensii.
Lappi et al. Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies
JPH02500437A (en) Method for selectively inhibiting human immunodeficiency virus (HIV)
JPH07506842A (en) Double-stranded non-toxic ribosome inactivating protein (RIP), its production method and application
EP0390040B1 (en) Ribosome inactivating proteins and derivatives thereof
EP0647272B1 (en) Anti-hiv proteins gap 31, dap 30 and dap 32, dna coding therefor and therapeutic uses thereof
US4985541A (en) Novel cytotoxic protein
Wang et al. Trichosanthin-monoclonal antibody conjugate specifically cytotoxic to human hepatoma cells in vitro
Bolognesi et al. Purification and properties of a new ribosome-inactivation protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis
Battelli et al. Toxicity of, and histological lesions caused by, ribosome‐inactivating proteins, their IgG‐conjugates, and their homopolymers
Cenini et al. Ribosome‐Inactivating Proteins from Plants Inhibit Ribosome Activity of Trypanosoma and Leishmania 1
AU594482B2 (en) Inhibitor of protein synthesis, process for its isolation, its use and pharmaceutical compositions in which it is present
Vidal et al. Studies on components of immunotoxins: Purification of ricin and its subunits and influence of unreacted antibodies
CA1339111C (en) Antiviral agent
US5149528A (en) Cytotoxic protein from Trichosanthes kirilowii
US5162502A (en) Modified trichokirin and their preparation for use in conjugates as inhibitors of protein synthesis
US4672053A (en) Immunoaffinity purification of phytolaccin proteins and their use in treating Herpes Simplex virus type II
IT9020670A1 (en) PROTEINS INHIBIT RIBOSOMES AND THEIR DERIVATIVES
Endo et al. Mechanisms of action of ribotoxins
EP1435981A2 (en) Anti-tumor activity from reptile serum

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 19910911

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB IT LI LU NL SE

XX Miscellaneous (additional remarks)

Free format text: VERFAHREN ABGESCHLOSSEN INFOLGE VERBINDUNG MIT 90105725.7/0390040 (EUROPAEISCHE ANMELDENUMMER/VEROEFFENTLICHUNGSNUMMER) VOM 04.06.92.