IT9020670A1 - PROTEINS INHIBIT RIBOSOMES AND THEIR DERIVATIVES - Google Patents

Description

Description of the industrial invention having as title: PROTEINS INHIBITING THE RIBOSOMES AND THEIR DERIVATIVES

The present invention relates to inactivating proteins and their sulphhydrylate derivatives useful for the preparation of immunotoxins

Several proteins isolated from various species of higher plants and from fungi are known, whose cytotoxicity is linked to their ability to catalytically inactivate the 60 S ribosomal subunits of eukaryotic ribosomes. Said proteins, indicated for the sake of brevity with the abbreviation RIP (Ribosome inhibiting proteins), can be single-chain proteins (RIP type 1) or double-chain proteins (RIP type 2), in which the so-called A-chain possesses the enzymatic and the other, (B) has the properties of a specific lectin for galactose. Type 1 RIPs are more common, occurring in different parts of many and perhaps all plants, including seeds, roots, leaves and latexes, sometimes in more than one form, possibly isoforms. RIPs have unusual N-glycosidase activity, and break the N-gliooside bond of 28S rRNA adenine 4324 by introducing a lesion that makes it possible for aniline to break RNA at a site adjacent to the sarcin-induced rupture. This renders riboscms unable to bind to elongation factors 1 or 2, resulting in a halt in protein synthesis. Despite the numerous similarities in their structure and in their apparently identical enzymatic activity, RIPs have different effects on ribosortium isolated from different organisms (from plants, protozoa and metazoans).

Interest in RIPs is growing because they have been used as components of '' immunotoxins ", hybrid molecules including a toxic function linked to an antibody. It is hoped that immunotoxins are able to eliminate harmful cells, such as neoplastic, immunocompetent cells. and parasite.

Further interest in RIPs stems from their antiviral activity. All the RIPs examined have or inhibited the infectivity of plant and animal viruses. The first identified type 1 RIP, namely the phytolac antiviral protein (PAP), was initially purified as an antiviral protein. This property of RIPs was attributed to easier penetration into infected cells with consequent inactivation of their ribosans and arrest of viral multiplication. Recently, however, a type 1 RIP, trichosanthin, has been shown to inhibit HIV replication through a mechanism apparently independent of the effect on ribosons.

The availability of different RIPs is particularly desirable for the preparation of both immunotoxins and conjugates active against cells resistant to some RIPs, and to avoid the problems deriving from neutralization due to the immunological reaction after administration and from the inactivation of some RIPs during conjugation procedure.

New proteins have now been found which inactivate the ribosans obtained from the plants Citrullus colocynthis, Lychnis chalcedonica, Manihot palmata and Phytolacca americana.

The activity of the proteins of the invention consists in a powerful inhibition of protein synthesis, mainly in non-cellular-structured systems ("cell-free"); although not very toxic towards the majority of mammalian cells, they can be converted into highly cytotoxic agents by conjugation - with suitable chemical agents - to suitable carrier agents ("aptomers"), for example monoclonal antibodies. If these carrier agents are capable of specifically recognizing tumor cells (for example their specific antigens), the RIPs object of the invention can then be used for the selective destruction of the latter.

Therefore, the invention also includes derivatives of proteins extracted from the plants mentioned above, in which sulfhydryl groups have been introduced, represented by the formula (I).

These derivatives show inhibitory activity the synthesis of proteins equal to or slightly lower than that of the starting RIPs and therefore, being both the synthesis intermediates of the immunotoxins and the inhibitory agents that the latter release to the cell, they are useful for the preparation of antibody conjugates. -toxins. These derivatives are obtained using bifunctional reagents, such as, for example 3, 3-dithiobispropionimidate (Wang & Richards reagent) or one of the following reagents:

1) N-succinimidyl-3 [2-pyrididylthio] propionate

2) 2-Iminothiolane (Traut's reagent):

3) S-acetyl-mercapto-succinic anhydride (SAMSA);

The RIPs of the invention will be indicated with the following names:

Colocina 1 and 2, from Citrullus colocynthis; Lychnina from Lychnis chalcedonica; Mapalmina from Manihot palmata; PAP-R from the roots of Phytolacca americana. All proteins have an average molecular weight of about 30,000 and an alkaline isoelectric point. Colocine 2 and mapalmine sinus glycoproteins.

The proteins of the invention, the characteristics of which are reported below, are obtained with conventional methods for the isolation of proteins from vegetables. The proteins of the invention are therefore obtainable by a process which includes:

a) grinding and extraction of seeds, leaves or roots;

b) centrifugation and / or filtration of the extract;

c) chromatography on cross-linked dextran or dialysis of the suratant of step b)

d) chromatography and ion exchange and / or gel filtrations in suitable sequences;

e) final purification by dialysis, lyophilization, chromatography, precipitation or other conventional techniques.

The proteins thus obtained can then be functionalized by means of the aforementioned sulphidrilating reactants, according to known techniques. The derivatives obtained, in turn, can be used for the preparation of immunotoxins that can be used in therapy, for example for the treatment of tumor forms, in which case they will be administered parenterally in doses ranging from 1 to 100 mg of immunoconjugate, using suitable pharmaceutical formulations.

The following non-limiting examples further illustrate the invention.

EXAMPLE 1

Preparation of crude extracts.

The seeds are chopped wet, in suitable grinders, and sent for extraction; the leaves and roots, crushed into small pieces, are - before extraction - subjected to centrifugation with filtration,

recovering the juice and starting the residue for extraction. The extraction

it is carried out with a 5 mW buffer in sodium phosphate, 0.14 M in sodium chloride, pH 7.4—7.5 under stirring for 12 hours and at a temperature of 4—10 ° C.

It is clarified by centrifugation; in the case of the leaves and roots, the respective juices, obtained as mentioned above, are added to the clarified and it is cleared by centrifugation at a high number of g. The final clarified solutions ("crude extracts") are moved on to the next step. These extracts are brought to pH between 4.2 and 4.8, for example at pH 4.5, with acetic acid; after standing, a precipitate is obtained which is centrifuged and eliminated. The clear suratants ("acid extracts") are loaded onto a column of S-Sepharose, equilibrated with 10 mM sodium acetate buffer, pH 4.5. After washing with the same buffer and then with 5 mM sodium phosphate, pH 7.5, until the effluent is neutral, it is eluted with phosphate buffer made 1 M in sodium chloride.

The clear eluates are saturated with harmonious sulphate; after standing, the precipitates are isolated by centrifugation.

The solids are dissolved in 5 mM phosphate buffer, pH 7.0; the solutions, clarified by centrifugation, are sent to the phase of isolation of the single RIPs ("leachate from S-Sepharose"). Alternatively, this final extract can be prepared by subjecting the crude extracts to dialysis for at least 24 hours, at 4 ° C, against 50-500 volumes of 5 nM phosphate buffer, pH 6.5, with at least two total changes. Any precipitate formed during dialysis is removed by centrifugation and the clear extract is sent to the isolation procedure. During the stages of: "crude extract", "acid extract", "leachate from S-Sepharose", total protein content, specific and total activity determinations are carried out, described below for each RIP.

EXAMPLE 2

Preparation of colocine 1 and 2, mapalmine, lycnin and PAP-R

The extracts from S-Sepharose obtained from seeds, or roots of the plants indicated above, were subjected to a series of chromatographies on the materials indicated in the following Tables. The fractions from the protein synthesis inhibition chromatographs. The active fractions were collected, dialysed in water center and lyophilized. The following tables show the results obtained and the stationary phases used for the various proteins.

Table 1A

Table 1B

The protein eluted from CM-Sepharose at 8.5 m S / cm was applied The protein eluted from CM-Sepharose at 10.0 m S / cm was applied

Table 1C

EXAMPLE 3

Physico-chemical characterization of the proteins of Example 2

The proteins described in Example 2 were analyzed by the following methods:

a) Protein content of the extractive solutions:

results obtained by the methods:

- by Lowry (J. Biol- Chenu 193, 265, 1951) (standard: bovine albumin serum),

- by Kalb & Bernlhor (Anal. Blochem. 82, 362, 1977),

- spectrophotometric, using the absorption at 280 nm of the purified protein, with a molar extinction coefficient of 0.8. b) Relative molecular mass (Mr):

determined for:

- polyacrylamide gel electrophoresis (Nature 227, 680, 1970) with the following markers (the relative Mr in brackets) :. cytochrome c (12

300), myoglobin (17200), carbonic anhydrase (30000), ovalbumin

(45,000), bovine serum albumin (66 250), ovotransferrin (76,000-78,000);

- gel-filtration on Sephacryl S-200 (95 x 1.6 cm columns)

equilibrated with 20 mM sodium phosphate buffer, pH 7.5, containing 0.3 M NaCl; elution rate 8 ml / hour at 20 ° C; calibration; (in brackets the relative Mr): dextran blue serum albumin (66250), ovalbumin (45000), chymotrypsinogen A (25000), ribonuclease A (13700).

c) Isoelectric point, amino acid composition, composition in amino sugars and neutral sugars: determined as described in Biochem. J.

240, 659, 1986.

The results are reported in the following Table 2.

TABLE 2

Table 2. Physico-chemical properties of the proteins of the invention.

For the composition in amino acids and sugars, the molecular weights determined by electrophoresis on SDS-gel were considered. Values for amino acids obtained from hydrolysis at 24, 48 and 72 hours were extrapolated to time zero.

EXAMPLE 4

Biological characterization

The inhibition by the proteins of the invention of protein synthesis in non-cellular systems was measured with a rabbit reticulocyte lysate by defining as the activity unit the amount of protein necessary to inhibit protein synthesis by 50% in 1 ml of mixture of lysate reaction of rabbit reticulocytes. The method described above was followed.

The IC50 was calculated by linear regression analysis.

The effect on cellular protein synthesis was evaluated in parallel with that of PAP-S. TG cells (human ovarian duct carcinoma, JAR cells (human choriocarcinoma), human fibroblasts, NB100 cells (human neuroblastoma cell line), HeLa, BEWO and human trophablasts were maintained as monolayer cultures in KPMI1640 medium supplemented with non-essential amino acids, antibiotics and 10% fetal calf serum. Cells were inseminated into 2 cm well plates, 0.5 to 1 per 10 <5> cells / well. The following day the medium was replaced by serum-free RPMI1640 medium containing the appropriate amount of protein to be tested After 18 hours the protein synthesis was measured by incubating the cells for two hours in RPMI1640 medium free from serum and leucine to which 0.1 μCi of [14c] -leucine / ml was added. The radioactivity incorporated in the protein was determined as described in Biochem. J. 176, 278-1978.

Toxicity

The toxicity of the purified protein was evaluated in female Swiss mice weighing 27-32 g, fed ad libitum. The protein, dissolved in 0.9% NaCl, was injected i.p. at 6 different dose levels ranging from 20 to 0.31 mg / kg of body weight with a ratio of 2 between doses, in groups of 6 animals for each dose. Acute and sub-acute LD50s were calculated at 48 hours and 14 days, respectively, by the linear regression method. The organs of the dead animals were subjected to autopsy. The results are reported in the following Tables 3 and 4.

TABLE 3

Table 3. Effect on protein synthesis.

TABLE 4

Toxicity i.p. in the mouse

The resistance of the proteins of the invention to modification with reagents such as 2-iminothiolane and dimethyl-3,3 '-dithiobispropionimidate (SPDP) was also studied. No loss of activity was noted after modifying PAP-R with SPDP.

Claims (7)

Claims 1. A ribosan inactivating protein having the following characteristics: - molecular weight per gel filtration: 20400 - molecular weight for electrophoresis: 26300 - isoelectric point: at 9.5 - extinction at 280 nm: 6.3 - amino acid composition for PM: 26300 - total content in neutral sugars: 0.40% - opposition in sugars (moles / moles of protein) for MW: 26300 sugars moles / moles RIP 2. A ribosome-inactivating protein having the following characteristics: - molecular weight per gel filtration: 19500 - molecular weight for electrophoresis: 26300 - isoelectric point: ≥ 9.5 - extinction at 280 nm: 7 - amino acid composition for PM: 26300 - total content in neutral sugars: 1.59% - composition in sugars (moles / moles of protein) by MW: 26300 sugars moles / moles.RIP 3. A ribosome inactivating protein having the following characteristics: - molecular weight per gel filtration: 20000 - molecular weight for electrophoresis: 26600 - isoelectric point: ≥ 9.5 - extinction at 280 nm: 7.3 - amino acid composition for PM: 26600 - total content in neutral sugars: 0.31% - composition in sugars (moles / moles of protein) by MW: 26600 sugars moles / moles RIP 4. A ribosome-inactivating protein having the following characteristics: - molecular weight per gel filtration: 26900 - molecular weight for electrophoresis: 32300 - isoelectric point: ≥ 9.5 - extinction at 280 nm: 7.1 - amino acid composition for PM: 32300 - total content in neutral sugars: 5.99% - composition in sugars (moles / mole protein) for EM: 32300 sugars moles / mole RIP 5. A ribosome-inactivating protein having the following characteristics: - molecular weight per gel filtration: 25000 - molecular weight for electrophoresis: 29800 - isoelectric point: ≥ 9.5 - extinction at 280 nm: 8 - amino acid composition for PM: 29800 - total content of neutral sugars: traces 6. Protein derivatives of claims 1-5 of formula (I) with bifunctional reactants. 7. Derivatives according to claim 6, wherein the bifunctional reactants are selected from 2-iminothiolane, N-succinimidyl-3-L2-pyridyl diethio] propionate, S-acetylmercapto-succinic anhydride.