WO1997032897A1 - Novel radioactive peptides useful for screening endothelin-converting enzyme inhibitors - Google Patents

Abstract

Products of formula (IA), wherein X is particularly a (2,3)3H-propionyl radical, the use thereof in an endothelin-converting enzyme inhibitor identification test, and the rapid determination of the inhibitory activity of such compounds, are disclosed. When systematically applied, the test enables a large number of assays to be carried out.

Description

RADIOACTIVE PEPTIDES USEFUL FOR THE SCREENING OF ENDOTHELIN CONVERSION ENZYME INHIBITORS

Since 1980, it has been known that the dilation and vasoconstriction factors are produced by endothelial cells in response to stimulation which can be produced for example by interleukin-1, thrombin, TGF-β, the phorbol ester, l 'angiotensin II, vasopressin or hypoxia-anoxia, to act on smooth muscle cells.

In 1988, Mr. YANAGISAWA discovered endothelin-1 (ET-1) which is a bicyclic peptide of 21 amino acids secreted by vascular endothelial cells. ET-l has a strong vasoconstrictive activity at concentrations lower than a nanomole on the coronary arteries in-vitro and increases the blood pressure after intravenous injection in vivo. ET-1 is a member of a family of structurally close hormones which are endothelin-2 and endothelin-3. The cloning of the pig gene made it possible to predict the sequence of a 212 amino acid preproendothelin which is successively cleaved twice first in proendothelin then in big-endothelin (b-ET-1) before being converted in active endothelin. The sequence of b-ET-1 is recalled in Figure 1 below.

ET-1 has been shown to be a vasoconstrictor 100 times more active than b-ET-1.

The last stage of ET-1 formation is carried out via an endothelin converting enzyme (ECE): ECE specifically cleaves the Trp ²¹ -Val ²² bond from b-ET-1 (1-38) either between amino acids 21 and 22 of b-ET-l which contains 38 amino acids, thus generating two fragments, one, N-terminal, ET-l (1-21) and the other, C-terminal, CTF (22-38).

Many enzymes have been described as having ECE activity: the two main ones are on the one hand an aspartyl protease or cathepsin-like which is active at acid pH and inhibited by pepstatin A and on the other hand an active metalloprotease at neutral pH and inhibited by phosphoramidon. B-ET-1 and ET-1 have been detected in the supernatant of endothelial cell culture and in plasma whereas they exist only at very low concentration in the intracellular medium, suggesting that the conversion of b- ET-l in ET-l could occur in the extracellular medium. Other work would tend to prove that the majority of the conversion would take place inside the cell. The existence of ECE cleaving b-ET-1 is attested by experiments demonstrating in vivo that the administration of b-ET-1 is immediately accompanied by an increase in the amount of plasma ET-1. However, it has not been possible to convert b-ET-l to ET-l in vitro in plasma. There is general agreement that the physiological enzyme appears to be metalloprotease.

One of the keys to the treatment of certain pathologies could be a treatment by inhibition of ECE: one can thus cite the treatment of congestive heart failure, myocardial infarction, reperfusion disorders or lesions, l angina pectoris, vascular spasms, vasospasm following cerebral hemorrhage, coronary spasms, peripheral vascular spasms, gastric ulcer induced by non-steroidal anti-inflammatory agents, nephrotoxicity induced by cyclosporine, kidney failure, the athéro¬ sclerosis, some forms of hypertension such as in particular pulmonary hypertension, asthma treatment, 1 ^• endothoxémie, Raynaud's disease, certain désor- dres diabetics, certain secretory endothelin tumors as well as the prevention of post-angioplasty restenosis, cardiac and vascular fibrosis.

Thus having a good screening test seemed crucial with regard to the development of these ECE inhibitors.

In addition, progress in identifying physiological ECE and its purification has been limited by the absence of an effective test allowing detection at fast, selective and sensitive.

The known tests using b-ET-1 and the analysis of the peptide fragment obtained from b-ET-1 by HPLC, RIA or SPA which are techniques commonly used are either very slow or not very specific, which makes them unsuitable for screening for an inhibitor.

HPLC requires a large amount of b-ET-1 and the enzyme. The RIA lacks selectivity for the recognition of ET-l in the presence of b-ET-l. AB FAWZI (as indicated in the publication Fawzi AB, Clemen RM and Wright DL (1994) Anal. Biochem. 222, 342-350), has developed a test based on the conversion of i-labeled b-ET 125 to ¹²⁵ I-ET and the quantitative determination of ¹²⁵ I-ET produced by fixation on the endothelin receptor. The authors do not use this test for inhibitor screening but for the identification of the enzyme in the tissues that contain it.

T. WARNER (as indicated in the publication Warner TD, Budzik JP, Mitchell JA, Juang ZJ., And Murad F. (1992) 20 (Sup 12): S19-S21), has developed a selective biotest based on ET-1-induced cGMP increase in kidney epithelial cells LLC-PK1. But this test requires a lot of time and is too elaborate for screening.

It has now been developed and it is the object of the present invention a new, simple and rapid test which is selective for the screening of EEC inhibitors.

During the implementation of the test, object of the present invention, a product of formula (I):

XC- ^ - ^ 2- ^Ile -He-Trp-Val-Z ₃ -Thr-Pro-Glu-His-Val-Val- || 21 22

0 (I)

Pro-Tyr-Gly-Leu-Gly-Ser-Z ₄ -Z ₅ -Z ₆ -COOH

in which X represents a hydrocarbon radical comprising at least one radioactive atom, Z _{1 #} Z ₂ , Z ₄ , Z ₅ and Z ₆ identical or different represent amino acids and Z ₃ represents either the amino acid Asn, or the paranitro- Phe, is cleaved by the ECE into a product of formula (II):

XCZ ₁ -Z ₂ -Ile-Ile-Trp-COOH

It (ID 0

in which X, Z- ^ and Z ₂ have the meanings indicated above, in the presence of the product P for which it is desired to determine the inhibitory activity of ECE: this inhibitory activity of ECE will therefore be all the greater that the quantity of product of formula (II) formed, determined by counting the radioactivity, will be lower.

The test of the present invention is therefore based on the use of new peptides of formula (I) in which the N-terminal part is acylated, which has at least three advantages: protect from cleavage by aminopeptidases, strengthen the hydrophobicity of the fragment N-terminal which will be generated by hydrolysis and allow radioactive labeling by acylation providing at least one radioactive atom and in particular by tritiated propionylation.

A subject of the present invention is therefore the products of formula (I):

XCZ ₁ -Z ₂ -Ile-Ile-Trp-Val-Z ₃ -Thr-Pro-Glu-His-Val-Val-

II

O (I)

Pro-Tyr-Gly-Leu-Gly-Ser-Z ₄ -Z ₅ -Z ₆ -COOH

in which X, Z _{1 #} Z ₂ , Z ₃ , Z ₄ , Z _j and Zg have the meanings indicated above.

Z-ι_, Z ₂ , Z ₄ , Z _ζ and Zg are chosen from known amino acids such as for example those belonging to the big-endothelin sequence. Z ₃ represents either the amino acid Asn or paranitrophenylalanine (commercial). In particular, the products of formula (I) are such that X represents a hydrocarbon radical comprising at least one radioactive atom,

Z ₁ and Z ₂ are such that: either Z ₁ and z ₂ represent a single bond, or z ± represents a single bond and Z ₂ represents the amino acid Asp, or Z ₁ and Z ₂ respectively represent the amino acids Leu and Asp,

Z ₃ represents either the amino acid Asn or paranitro-Phe, and Z ₄ , Z ₅ and Zg are such that: either Z ₄ , Z ₅ and Zg represent a single bond, or Z ₅ and Zg represent a single bond and Z _{4 represents} the amino acid Pro, or Zg represents a single bond and Z ₄ and Z ₅ represent respectively the amino acids Pro and Arg, or Z ₄ , Z ₅ and Z ₆ represent respectively the amino acids Pro , Arg and Ser.

The present invention more specifically relates to the products of formula (I) as defined above, corresponding to formula (I _A ):

X-C-Ile-Ile-Trp-Val-Asn-Thr-Pro-Glu-His-Val-Val-

0 (I _A )

Pro-Tyr-Gly-Leu-Gly-Ser-COOH

in which X represents a hydrocarbon radical comprising at least one radioactive atom.

Such products of formula (I) can also be written:

X-C-b-ET-1 (19-35)

In the radical X-C, the hydrocarbon radical that

II O

represents X can be a linear or branched alkyl radical containing at most 4 carbon atoms such as the radical methyl, ethyl, propyl or butyl and preferably ethyl or a phenyl radical.

X can thus contain one or more radioactive atoms, in particular tritium substituted with one or more hydrogen atoms of the alkyl or phenyl radical that X represents.

The present invention particularly relates to the products of formula (I) as defined above, in which the radical X comprises at least one tritium atom. X-C can in particular represent the propionyl radical,

II o which may contain from 1 to 5 atoms of tritium ³ H and in particular 4 or 2 atoms of tritium such as the radical (2,3) ³ H-propionyl.

A more particular subject of the present invention is the products of formula (I) as defined above, in which XC- represents the (2,3) ³ H-propionyl radical.

He O

We thus define the product of formula (I) which can be written:

((2,3) ³ H) -propionyl-b-ET-1 (19-35) or ((2,3) ³ H) -prop-b-ET-1 (19-35).

The present invention also relates to a process for preparing the products of formula (I), as defined above, characterized in that a product of formula (III) is subjected:

Z ₁ -Z ₂ -Ile-Ile-Trp-Val-Z ₃ -Thr-Pro-Glu-His-Val-Val- Pro-Tyr-Gly-Leu-Gly-Ser-Z ₄ -Z ₅ -Z ₆ - COOH (III)

in which Z _lt Z ₂ , Z ₃ , Z ₄ , Z ₅ and Zg have the meanings indicated above, to an acylation reaction with a reactive derivative of the carboxylic acid of formula X-COOH in which X has the meaning indicated above.

The reactive carboxylic acid derivative of formula: X- COOR

in which R represents for example the succinimidyl radical, a norbornene radical or else any esterification compound of the carboxy radical, can be found commercially, such as in particular N-succinimidyl- (2,3) ³ H -propionate, or also prepared according to the usual methods known to those skilled in the art. The radical X can thus contain one or more tritium atoms. Mention may be made, for example, of the X-COOR compounds, of the N-hydroxy-5-norbornene- (2,3) -dicarboximide-propionate compound formed from N-hydroxy-5-norbornene- (2,3) acid. ) -dicar- boximide and propionic acid, which can thus, depending on the labeling of propionic acid, contain 1 to 5 tritium atoms and in particular 4 or 2.

The acylation reaction of the product of formula (III) into the product of formula (I) can be carried out under the usual conditions known to those skilled in the art.

B-ET-1 (19-35) used as starting material can be prepared according to the usual methods such as in particular by an automatic solid phase synthesizer such as the model, for example, 431A from Applied Biosystems using the technology Fmoc, as described in the reference: ATHERTON, E. and SHEPPARD, RC (1989) Solid phase peptide synthesis: a pratical approach, IRL Press, Oxford. Such a process is illustrated below in point I of the experiential part.

The present invention particularly relates to the use of a product of formula (I), as defined above, for the identification of inhibitors of the endothelin converting enzyme.

The present invention more particularly relates to the use as defined above of a radioactive product of formula (I) as defined above, characterized in that the enzyme is brought into contact conversion of endothelin, a product P for which it is desired to determine the inhibitory activity of the endo¬ thelin converting enzyme, and the product of formula (I) as defined above above ; and after incubation, the cleavage reaction is stopped, the product formed of formula (II) is selectively extracted:

XCZ ₁ -Z ₂ -Ile-Ile-Trp-COOH

He (ID

in which X, Z _χ and Z ₂ have the meanings indicated above, and determines by counting the inhibitory activity of the product P to be tested.

The product P as defined above to which the test defined above is applied to determine its ECE inhibiting activity can be of various nature, without limitation, and for example, can be chosen from phosphoni - mides, phosphamides and derivatives of these products, or even metalloproteinase inhibitors such as thiol derivatives, carboxylates or hydroxamates. Mention may also be made, as product P, of phosphoramidone (public domain) which is called here PI and N- (phenylethylphosphonyl) -Leu-Trp (TAKEDA) which is called here P2, whose inhibitory activities of the ECE are known and to which the test of the present invention can be applied, as is shown below in point II of the experimental part.

ECE can be obtained, for example, according to the preparations and purification from rat lung as indicated in the reference: Takahashi M., Matsushita Y., Iijima Y. and Tanzawa K. (1993) J. Biol . Chem. 268, 21394-21398.

A very particular subject of the present invention is the use as defined above, characterized in that the solvent product used to stop the cleavage reaction and selectively extract the product of formula (II), as defined above is l ¹ ethyl acetate.

A more particular subject of the present invention is the use as defined above caracté¬ ized in that a product of formula (I) is used such as defined above in which XC- represents

II o preferably a ³ H-propionyl radical and in particular (2,3) ³ H-propionyl.

This gives the product of formula (II) as defined above in which X-C- represents the radical

II o ³ H-propionyle and which can be written ³ H-prop-b-ET-1 (19-21). The test as defined above may in particular have as starting material the product of formula (I):

³ H-prop-b-ET-l (19-35)

to obtain the product of formula (II):

³ H-prop-b-ET-l (19-21), the amount of which is determined by counting its radioactivity. A subject of the present invention is thus the test for the identification of inhibitors of the endo¬ thelin converting enzyme, characterized in that this test comprises a product of formula (I) as defined above .

Such a test which makes it possible to identify an ECE inhibitor by an assessment that is both qualitative and quantitative of the inhibitory effect of this product, is described and illustrated below in point II of the experimental part. It can be noted that the sensitivity of the test as defined above is increased when in the products of formula (I) as defined above, Z ₃ represents paranitro-Phe and this thanks to an increase in the rate of degradation of the substrate.

The subject of the present invention is therefore, as new industrial products: - the products of formula (III):

Z ₁ -Z ₂ -Ile-Ile-Trp-Val-Z ₃ -Thr-Pro-Glu-His-Val-Val- Pro-Tyr-Gly-Leu-Gly-Ser-Z ₄ -Z ₅ -Z ₆ - COOH (III) - the products of formula (II):

XCZ ₁ -Z ₂ -Ile-Ile-Trp-COOH (II)

among which preferably the product of formula (II) in which XC- represents the radical (2, 3) ³ H-propionyl,

in which formulas, Z _{1 #} Z ₂ , Z ₃ , Z ₄ , Z ₅ , Zg and X have the meanings indicated above.

The following examples of preparation of a product of formula (I) and implementation of the test illustrate the invention without, however, limiting it.

EXPERIMENTAL PART

I - Preparation of the tritiated peptide ((2,3) ³ H) -propionyl-b-ET-1

19-35) a - Synthesis of ((2.3) ³ H) -oropionyl-b-ET-1 (19-35)

N-succinimidyl- (2, 3- H) -propionate (Amersham code

TRK.556) is in solution in 5 ml of toluene at 1 mCi / ml,

99 mCi / mmol ==> 5 mCi and 50 nmoles.

The toluene is evaporated until a 10 μl solution is obtained and then 25 μl of a DMSO solution containing

0.2 mg of b-ET-l (19-35), b-ET-l (19-35) (MW = 2014.2) having been previously dried on potash overnight. It is continued to evaporate for 10 min and then the solution is stirred under a stream of nitrogen for 15 min in order to remove the residual toluene. Then stir gently for 4 days.

When ready to purify, add 225 μl of phosphate buffer pH 6.5 and 50 μl of acetonitrile then stir

10 mins. b - Purification of ((2.3) ³ H) -propionyl-b-ET-l (19-35) The radioactive solution obtained above in a) is separated into 2 injections of 150 μl on a column of

Nucleosil C _1Q (150 x 4.6 mm).

Elution is carried out with a flow rate of 0.8 ml / min per a gradient from 0 to 20% acetonitrile in 20 min then 20 to

35% in 50 minutes.

Analyzes :

The analysis of the fractions is carried out by counting the tritium on the liquid scintillation counter (1 μl in 5 ml of HiSafe3 scintillant) for 60 seconds.

The radioactive fractions are combined and fractionated into 200 μl samples in silicone-coated eppendorf tubes which can be stored at -80 ° C or -20 ° C. Characteristics of the product obtained: dpm = 1 067 274 560 corresponding to 0.5 mCi or 18.5 MBq.

Radioactive yield: 10%.

Specific activity: (99 x 10) / 30.5 = 32.5 Ci / mmol.

II - Endothelin converting enzyme (ECE) inhibitor identification test

10 μl of ECE or 1 to 2 μg of purified ECE are pre-incubated for 30 minutes at 37 ° C., in 400 μl of 50 mM Tristaleate buffer pH = 6.5, 20 μl of chloride sodium 5M and

5 μl of product P whose inhibitory activity is to be tested, in solution at different concentrations between 1 μM and 100 mM (i.e. final concentrations of product P between

10 nM and 1 mM).

The reaction is initiated by adding 10 μl of

((2,3) ³ H) -propionyl-b-ET-1 (19-35) prepared as indicated above in I, at the final concentration of 1.8.10 ^"12 M.

After an hour of incubation at 37 ° C., the reaction is stopped by adding 600 μl of ethyl acetate and the

((2,3) ³ H) -propionyl-b-ET-1 (19-21) is extracted by mechanical stirring for 2 minutes. 300 μl of the organic phase are taken and 5 ml of scintillating liquid are added and the radioactivity is counted for

1 minute on the liquid scintillation counter.

Each measurement is carried out in triplicate except for the ECE control (enzyme control, without product of which one wants to test the inhibiting activity of the ECE), for which the measurement is carried out in sextuplet.

the percentage inhibition is calculated by doing the report: product tested - enzyme blank control - blank The blank is made from the solution obtained without enzyme. The table below gives the results obtained by using known inhibitors as P products, namely PI which is phosphoramidon and P2 which is N- (phenylethylphosphonyl) -Leu-Trp (TAKEDA), the inhibitory activities of which we want to test from ECE. From this table and by plotting the graph of the percentage of inhibition relative to the inhibitor concentration (nM), the IC ₅₀ is calculated which therefore corresponds to the concentration causing a 50% inhibition of the ECE. The inhibition constant (Kl) of a product can be obtained by using the value of the constant

Michaelis K _M which is equal to 12.8 μM for the substrate la or X = (2,3) ³ H-propionyle.

The quantified results obtained are indicated in the table below:

Conclusion: the present test therefore makes it possible to identify inhibitors of EEC because the results obtained confirm the activity of the known products P ^ ^{and p} 2 *

Claims

1) Products of formula (I): XCZ ₁ -Z ₂ -Ile-Ile-Trp-Val-Z ₃ -Thr-Pro-Glu-His-Val-Val- II o (I) Pro-Tyr-Gly-Leu-Gly-Ser-Z ₄ -Z ₅ -Zg-COOH in which X represents a hydrocarbon radical comprising at least one radioactive atom, Z _{1 #} Z ₂ , Z ₄ , Z ₅ and Zg which are identical or different represent amino acids and Z ₃ represents either the amino acid Asn or paranitro-Phe . 2) Products of formula (I) as defined in claim 1 in which X represents a hydrocarbon radical comprising at least one radioactive atom, Z ₁ and Z ₂ are such that: either Z ^ _ and Z ₂ represent a simple bond, either Z ^ represents a single bond and Z ₂ represents the amino acid Asp, or Z ^ - and Z ₂ represent the amino acids Leu and Asp respectively, Z ₃ represents either the amino acid Asn or paranitro-Phe , and Z ₄ , Z _c and Zg are such that: either Z ₄ , Z ₅ and Zg represent a single bond, or Z ₅ and Zg represent a single bond and Z _{4 represents} the amino acid Pro, or Zg represents a single bond and Z ₄ and Z ₅ represent respectively the amino acids Pro and Arg, ie Z ₄ , Z ₅ and Zg represent respectively the amino acids Pro, Arg and Ser. 3) Products of formula (I) as defined in claim 1 or 2, corresponding to formula (I _A ): X- C- Ile- Ile - Trp-Val -Asn-Thr- Pro-Glu-His -Val -Val - 0 (I _A ) Pro - Tyr- Gly - Leu - Gly - Ser - COOH in which X represents a hydrocarbon radical comprising at least one radioactive atom. 4) Products of formula (I) as defined in any one of claims 1 to 3 in which the radical X comprises at least one tritium atom. 5) Products of formula (I) as defined in any one of claims 1 to 4 in which X-C- II o represents the (2,3) ³ H-propionyl radical. 6) Process for preparing the products of formula (I), as defined in any one of claims 1 to 5, characterized in that one submits a product of formula (III): Z ₁ -Z ₂ -Ile-Ile-Trp-Val-Z ₃ -Thr-Pro-Glu-His-Val-Val- Pro-Tyr-Gly-Leu-Gly-Ser-Z ₄ -Z ₅ -Z ₆ - COOH (III) in which Z _{1 #} Z ₂ , Z ₃ , Z ₄ , Z ₅ and Zg have the meanings indicated in claim 1, to an acylation reaction with a reactive derivative of the carboxylic acid of formula X-COOH in which X has the meaning given in claim 1. 7) Use of a product of formula (I), as defined in any one of claims 1 to 5 for the identification of inhibitors of the endothelin converting enzyme. 8) Use according to claim 7 of a radioactive product of formula (I) as defined in any one of claims 1 to 5, characterized in that the endothelin converting enzyme is brought into contact , a product P for which the inhibitory activity of the endothelin converting enzyme is to be determined, and the product of formula (I) as defined above; and after incubation, the cleavage reaction is stopped, the product formed of formula (II) is selectively extracted: XCZ ₁ -Z ₂ -Ile-Ile-Trp-COOH He (II) 0 wherein X, Z _j _ and Z ₂ have the meanings indicated in Claim 1, and determines by counting the inhibitory activity of the product P to be tested. 9) Use according to claim 8, characterized in that the solvent product used to stop the cleavage reaction and selectively extract the product of formula (II), as defined in claim 8 is ethyl acetate. 10) Use according to claim 8 or 9, characterized in that one uses a product of formula (I) as defined in any one of claims 1 to 5 in which X-C- preferably represents a radical II o ³ H-propionyl and in particular (2,3) ³ H-propionyl. 11) Test for identifying inhibitors of the endothelin converting enzyme, characterized in that this test comprises a product of formula (I) as defined in one of claims 1 to 5. 12) As new industrial products: - products of formula (III): Z ₁ -Z ₂ -Ile-Ile-Trp-Val-Z ₃ -Thr-Pro-Glu-His-Val-Val- Pro-Tyr-Gly-Leu-Gly-Ser-Z ₄ -Z ₅ -Z ₆ - COOH (III) the products of formula (II): XCZ ₁ -Z ₂ -Ile-Ile-Trp-COOH (II) II o among which preferably the product of formula (II) in which XC- represents the radical (2,3) - ³ H-propionyl, II o in which formulas, Z _{1 #} Z ₂ , Z ₃ , Z ₄ , Z ₅ , Zg and X have the meanings indicated in one of claims 1 to 5.