HUT73691A - In vitro assay to detect inhibitors of protein and/or mrna biosynthesis - Google Patents

Description

The present invention relates to a method for detecting the inhibitory effect of compounds on protein and / or mRNA biosynthesis by incubating a mixture of reagents capable of producing a detectable amount of a functional reporter enzyme in a DNA-directed synthesis as a control alone and in the presence of a test compound. functional reporter enzyme produced in the presence of the test compound and the results are compared.

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158/1014

PUBLICATION 'η EDITION A

Represented by: Dr. Jalsovszky Györgyné, lawyer

Associate: Dr. László Tóth-Urbán lawyer

158/1014

IN VITRO CIRCUMSTANCES TEST METHOD FOR DETECTING SUBSTANCES TO PROTECT PROTEINS AND / OR MRNA BIOSYNTHESIS

ZENECA LIMITED, London, United Kingdom

Inventor:

HAWKES Timothy Róbert, Bracknell, Berkshire,

Great Britain

Date of filing: 26/09/1994

Priority: 06.10.1993 (9320562.3) Great Britain

International Application Number: PCT / GB94 / 02088

International Publication Number: WO 95/09925

The present invention relates to an in vitro assay for the detection of substances that inhibit protein and / or mRNA biosynthesis. The present invention relates to the use of a method for detecting novel antibiotics and herbicides and determining their mode of action. Finally, the invention relates to biologically active substances which inhibit the biosynthesis of proteins (in particular bacterial proteins) and / or mRNA and the herbicides provided by them.

A non-radiochemical method for studying transcription and translation of DNA under in vivo conditions was first described by G. Zubay [see, e.g., Ann. Port. Genet. 7: 267-287 (1973)]. Modified versions of this method are described by J. Collins (Gene 6: 29-42 (1979)).

The above methods have been applied in research for scientific purposes.

Inhibition of protein and / or mRNA biosynthesis is an important biological effect that could be exploited, for example, in the field of antibiotics and agrochemicals, particularly herbicides. However, screening assays used to detect inhibition of protein biosynthesis have the disadvantage that they are of low power and radiometric, and are therefore not capable of screening for the large number of compounds required to recognize useful bioactivity or to provide guidance in this field.

In our work, we developed a non-radiometric, high-throughput screening method for the detection of inhibitors of protein and / or mRNA biosynthesis.

The present invention therefore relates to a method for detecting the inhibitory effect of compounds on protein and / or mRNA biosynthesis by controlling a mixture of reagents capable of producing a functional reporter enzyme in a DNA-directed synthesis under in vitro control as a control. • · · · · · · ··· · ··· ·

-3- .....

functional reporter enzyme produced in the control assay and in the presence of the test compound, and the results are compared.

The assay is preferably carried out in microsize.

The operating conditions, such as the incubation time required for protein synthesis, measurement time, temperature, reagent concentrations, and so on. with the consideration that the control assay will produce a detectable amount of reporter enzyme over a period of time that allows for a high throughput screening assay, such as preferably an incubation period of less than about 2 hours, preferably about 1 hour, for protein synthesis.

Suitable reporter enzymes include those that can be detected by the addition of chromogenic or fluorogenic substrates. These include, for example, β-galactosidase and β-glucuronidase. Alternatively, firefly luciferase may be used to produce light.

The method of detecting a reporter enzyme will vary depending on the nature of the enzyme. For example, depending on the nature of the enzyme, colorimetric or light-generating methods may be used to detect the enzyme. The enzyme detection chemicals may be added to the reaction mixture at the beginning of the process and / or at appropriate times during the process.

One of the preferred reactions for use in the process of the present invention is that described by G. Zubay, cited above, and modified by J. Collins, cited above.

reaction. The reagent mixture for enzyme synthesis contains the following components:

1) a mixture of amino acids;

2) plasmid DNA encoding the reporter enzyme;

3) S30 extract from a prokaryotic organism without reporter enzyme activity; and

4) a low molecular weight compound mixture containing reagents for in vitro protein synthesis; furthermore

5) magnesium ion.

Preferably, the amino acid mixture may be a mixture comprising the 20 protein-forming L-amino acids in equivalent ratios.

However, the concentration of the individual amino acids in the mixture may be varied (for example, to test whether the particular inhibitor acts by inhibiting any of the aminoacyl tRNA synthetases). In such experiments, we change the concentrations of the individual amino acids and observe whether there is an antidote effect on increasing the concentration of a particular amino acid. Such results suggest that the particular inhibitor is most likely to act as a competitive inhibitor of the appropriate aminoacyl tRNA synthase.

The concentration of amino acids may conveniently be between 0.01 mmol and 10 mmol. In our experience, it is desirable to reduce the concentration of the amino acid mixture as described in G. Zubay, cited above, in order to achieve maximum sensitivity in the detection of substances that inhibit protein biosynthesis, particularly those that inhibit aminoacyl-tRNA synthetases.

• · • ·

Plasmid DNA can be any conventional plasmid DNA carrying the lac Z gene (e.g., commercially available from Promega).

Useful S30 extracts may be similar to those described in G. Zubay, supra; these include commercially available materials (such as those sold by Promega). The preparation of a suitable extract is described in the Examples.

A low-molecular-weight compound mixture containing reagents for in vitro protein synthesis is known to include components known to biochemists such as nucleosides, salts, buffers, solvents, and the like. contain. The composition of a suitable low molecular weight compound mixture is described in the Examples.

The optimum amount of magnesium ions for the protein synthesis reaction should be determined and standardized separately for each new E. coli or S30 extract or for a new low molecular weight compound mixture. (Once the amount of magnesium ion required to produce the highest amount of β-D-galactosidase used in a preferred embodiment of the invention has been determined in the initial controls, this amount will be used in all further screening for inhibitors until initially used. Extract S30 or low molecular weight compound mixture is not completely consumed. Magnesium acetate is preferably used as the source of magnesium ion. In practice, the optimum concentration of magnesium ions is usually achieved when the protein synthesizer.

Magnesium acetate was added to 6 reaction mixtures to a concentration of 5 to 30 mmol.

In a preferred embodiment, the present invention relates to an in vitro colorimetric method for detecting substances that inhibit transcription (RNA polymerase) and translation (protein synthesis) in E. coli extracts by coactivating the E. coli extract with tRNA and cofactors for biosynthesis. supplemented, primed with plasmid DNA carrying the lac Z gene, allowed to synthesize the β-D-galactosidase, quantitatively detect the amount of enzyme formed, and determine whether the presence of a putative inhibitor reduces β- D-galactosidase activity relative to control.

The extract is prepared using an E. coli strain lacking β-D-galactosidase (such as the readily available, lac-free strain MC1061). The cofactors include the low molecular weight compound mixture described above containing the amino acid mixture and the parent compounds for in vitro protein synthesis.

Preferably, β-D-galactosidase is detected and quantitated by the addition of a chromogenic substrate, such as o-nitrophenyl-β-D-galactoside. Protein synthesis inhibitors are detected by measuring the decrease in the intensity of the yellow color absorbed at 410 nm in microtiter wells containing the inhibitor. Then, β-D-galactosidase (marketed by Sigma. · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Ltd. (Great Britain) to control inhibitors that generally inhibit mRNA or protein synthesis from molecules that have only β-D-galactosidase inhibitory activity.

Agents that inhibit bacterial RNA polymerase biosynthesis and / or protein biosynthesis include many antibiotics and, since the mechanism of protein synthesis of chloroplasts and bacteria is very similar to many herbicidal agents. Accordingly, the present invention provides a novel way of screening herbicidal or antibiotic agents from a range of potentially active compounds, and, by using the in vitro activity as a guideline, provides a program for the synthesis of analogs for which compounds useful as antibiotics or herbicides are expected.

The present invention further relates to a method for screening compounds of herbicidal activity from a group of potentially active compounds by incubating a reagent mixture capable of producing a detectable amount of a functional reporter enzyme in vitro as a control alone and in the presence of a test compound. reporter enzyme, and the results are compared.

This test is also suitable for indicating the herbicidal and / or antibiotic mode of action. The study shows whether a substance with an unknown mode of action inhibits bacterial infection.

- 8 protein biosynthesis or not. In many cases, the assay of the present invention is also suitable for narrowing the scope of action, i.e., to determine the more specific field of action of a given substance within transcription / translation. In particular, the method of the invention can determine whether the compounds act as specific inhibitors of any aminoacyl tRNA synthetase. Inhibitors of aminoacyl-tRNA synthetases are particularly valuable because they can be used as antibiotics and herbicides, as described in WO93 / 19599. For example, competitive inhibitors of isoleucyl tRNA synthetase can be readily identified by its specific ability to antagonize the inhibitory effect observed in the assay of the present invention.

The invention also relates to biologically active substances identified by the method of the invention which inhibit bacterial protein biosynthesis and / or mRNA biosynthesis (transcription and translation of a functional protein gene product).

The invention further relates to compounds having herbicidal activity which are the above inhibitors or compounds derived therefrom, WO93 / 19599. Compounds of Formulas I, IA, and IB described in International Publication No. 2

Y represents a group of formula (IC), (ID) or (IE), and

3

R is a group -CO-XR in which

X represents an oxygen atom or a sulfur atom and ♦ ·· ♦ ····································

R ^{3 is} hydrogen or an agriculturally useful ester-forming group, or

R is a group -R 'in which

R ^{4 is} optionally substituted aryl or heterocyclic group, or

R ^{2 is} a -CO-NR ⁵ R ⁶ group in which

R ⁵ and R ⁶ are the same or different and each represents an agriculturally useful amide-forming group - except for their stereoisomers and salts of compounds of formula I, IA and IB containing R ^{2 -} COOH.

The following examples illustrate the process of the invention.

. example

The following materials were prepared or prepared:

1. Low Molecular Weight Compound Mixture (Usually used in the purest possible form reagents):

Tris (tris (hydroxymethyl) aminomethane) was dissolved in water and mixed with the following reagents in the order indicated:

440 mmol Tris buffer 13.7 mmol DL-dithiothreitol 9.50 mmol adenosine 5'-triphosphate (disodium salt, isolated from horse muscle) 5.50mmol cytidine 5'-triphosphate (Tris-salt, type VI)

* ······"· ·· ·· • · · · · " · • ·· · · ··· " • · · · ······ · ·· ·· · the

5.50 mmol

5.50 mmol

0.21 mol guanosine 5'-triphosphate (tris salt, type VI) uridine 5'-triphosphate (tris salt, type VI) pyruvic acid phosphoenol ester [mono- (cyclohexylammonium) salt]

0.375 mL / mL% poly (ethylene glycol) 6000

0.1 ml stock solution / ml folinic acid (freshly prepared aqueous stock solution at 2.7 mg / ml)

2.50 mmol adenosine 3 ', 5'-ring monophosphate

62.4 μΐ stock solution / ml transfer ribonucleic acid (lyophilized from type XXI, E. coli strain W., available from Sigma Ltd., United Kingdom). Freshly prepared aqueous solution of 17.4 mg / ml. 0.28 mol ammonium acetate 0.56 mol potassium acetate 19 mmol Calcium acetate

The reagents were added in the above order and finally the pH of the resulting solution was adjusted to 8.2 with 5M potassium hydroxide solution.

2. Amino acid mixture:

The mixture consists of 20 protein-forming L-amino acids each

2.5 mmol.

3. Lac-Z Plasmid DNA:

DNA pGem (marketed by Promega) carrying the gene encoding β-D-galactosidase.

4. Magnesium acetate:

The purest material available (about 0.5 M stock solution).

5. S30 Extract:

-11 ··· ···· «·· • · · · · · · · · · • • • · · · · · · ·

This material was prepared as described in G. Zubay, cited above. The method will be described in more detail below.

6. Growth of bacterial cells:

The E. coli strain MC1061 was grown on a rich medium at 28 ° C in 14 L of a useful volume fermenter, as described in G. Zubay, supra. During the fermentation, the sample was periodically sampled and the optical density of the mixture was measured at 550 nm.

After 11.5 hours, the cells were harvested. The culture temperature was reduced to 20 ° C and the first 6 liters of culture was collected in pre-cooled centrifuge containers. The remainder of the culture was transferred to flasks and the flasks were stored on ice. Both cultures thus separated were centrifuged in a Sorvall GS3 6x500 rotor centrifuge at 5000 rpm for 5 minutes at 5 ° C. The cell pellets were rapidly frozen in liquid nitrogen, weighed and stored at -80 ° C.

7. Preparation of Fractured Cell (S30) Extract:

Normally, chemicals of ANALAR quality (or equivalent quality materials from the distributor) were used for the process. In the following procedure, the buffer used in each case was 10 mM Tris-acetate buffer (pH 8.2) containing 14 mM magnesium acetate, 60 mM potassium acetate and 1 mM DL-dithiothreitite.

g of E. coli cell pellet obtained above was resuspended in 500 ml of the above formulation containing 7 mmol of 2-mercaptoethanol in the high season. The cell suspension • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · whatever · mean

The centrifuge was centrifuged on a Sorvall GSA 6x250 rotor centrifuge at 12,000 rpm for 5 minutes at 4 ° C. The supernatant was discarded and resuspended and centrifuged again. The resulting pellet was resuspended in 1.25 ml of buffer solution per gram of cell material and lysed on a French Pressure cell disrupter at 5.5 x ^{10 4} kPa. Immediately afterwards, 10 μΐ of 0.1 M dithiothreitol solution was added to the extract. The extract was centrifuged in a Sorvall 8x50 rotor centrifuge at 18,000 rpm for 30 minutes at 4 ° C. 80% of the supernatant was carefully separated and the centrifugation and extract extraction steps described above were repeated.

9.2 mmol of magnesium acetate, 13.4 mmol of adenosine 5'-triphosphate (disodium salt, isolated from equine isostasis), phosphonoenol ester of pyruvic acid [mono- (cyclohexylammonium) salt], 4.4 mmol of DL-dithio- treite, 4.3 units of fresh pyruvic acid kinase (EC 2.7.1.40, type Sigma III, isolated from rabbit muscle), and 0.29 M Tris buffer containing 20 protein-forming L-amino acids each in 0.132-0.132 mmol. The pH of the solution was adjusted to 8.2 and the solution was added to the supernatant obtained above (0.3 ml of buffer was added to 1 ml of supernatant). The resulting mixture was incubated for 80 minutes in a 37 ° C water bath with shaking under slow shaking.

Samples of the extract (approximately 20 ml) were dialyzed against 4 volumes of 1 liter of cold buffer for 4 hours at 4 ° C. The product thus obtained is

The S30 extract was then frozen and stored in the form of beads in liquid nitrogen. For use, aliquots of the extract were thawed.

8. Inhibitors:

Compounds of formulas (1), (2) and (3), as well as some known antibiotics, were dissolved in water or 4% dimethylsulfoxide at appropriate concentrations (up to 100 ppm). For blind measurements, suitable solvents were used. The results of the control tests show that dimethyl sulfoxide at a concentration of 4% in solution (corresponding to a concentration of 1% in the reaction mixture of protein synthesis) does not affect the results of the test.

9. Substrate solution:

O-Nitrophenyl-β-D-galactopyranoside (1.82 mmol) in 0.1 M phosphate buffer (sodium salt), pH 7.3 containing 0.1 mol of 2-mercaptoethanol.

10. Sodium carbonate:

1.0 M sodium carbonate solution.

The following tests were performed using the materials listed:

Protein synthesis reaction:

The inhibitor to be tested, the amino acid mixture, the S30 extract, the plasmid DNA and the low molecular weight compound mixture were mixed in the following ratio in a well of a 96-well microtiter plate:

μΐ low molecular weight compound mixture

2 μΐ amino acid mixture 1.5-3.0 μg lac Z plasmid DNA 17 μΐ S30 extract 10 μΐ inhibitor (or in control studies, depending on the solvent of the inhibitor, 10 μ megfelelően of water or 4% aqueous dimethylsulfoxide) Magnesium acetate an appropriate quantity as determined above

The volume of the mixtures in each well was made up to 42 μΙ with water. To initiate the reaction, the low molecular weight compound mixture was added last and the contents of the well were mixed; or all components were weighed and pre-mixed under ice-cooling, and the reaction was then started by raising the temperature to 37 ° C. The mixture was allowed to react at room temperature for 1 hour.

Enzyme Testing:

Next, 200 μΐ of a solution of o-nitrophenyl-β-D-galactoside in buffer was added to start the determination of the enzyme produced. After 1-3 hours, 100 μΐ of sodium carbonate solution was added and the absorbance of the mixture was measured at 410 nm against a reference wavelength of 570 nm using a Dynatech 7000 microtiter plate reader.

Results:

The results are reported in Table I below. The concentration given in Table I for the test compounds is the concentration measured in the initial reaction mixture for protein synthesis. % Inhibition to control values (no inhibitor)

• ···· ···· -15 ·· · «t« * · · · · · · · · · · · · · · • ♦ · «· values assays containing ··) relative to the blank values (background absorbance in assays without plasmid DNA) was calculated using the following formula:

sample value - blind value% inhibition = 1 ---------------------------- x 100 control value - blind value

Table I

inhibitor Concentration, μιηόΐ % inhibition chloramphenicol 48 100 Streptomycin 48 66 Compound of Formula (1) 48 100 Compound of Formula (1) 1 22 Compound of Formula (1) 0, 5 7 Compound (2) 24 43 Compound of Formula (3) 2, 3 18

Checking example

The purpose of the control studies is to establish that compounds that are inhibitory are indeed inhibitors of protein biosynthesis and not only inhibitors of β-galactosidase.

When tested for potential inhibitors, the vast majority of compounds which are typical inhibitors of peak concentrations of 20-40 ppm actually inhibit transcription or translation. Occasionally, however, compounds that are directly β ·· ·> may also appear to be of high potency

·· · · · r * · · · · · «*.:. ... ·: .. ·

They inhibit -16-D-galactosidase. The latter inhibitors can be readily screened by employing the β-D-galactosidase enzyme (E. coli grade IV material, available from Sigma Ltd., United Kingdom) in place of the starting mixture in the protein synthesis reaction in the assays described above. (The diluted enzyme should be used in an amount that produces a change in absorbance at 410 nm of about 0.5 to 1.0 after 1 hour of reaction; this amount is readily determined experimentally.)

Example 2

The assay of the invention can also be used for diagnostic purposes to determine the exact mode of action of the inhibitors. In this example, it was found that the inhibitory effect of the compound of formula (2) is suppressed by L-isoleucine, indicating that the compound exerts its inhibitory effect on isoleucyl-tRNA synthetase.

Table 2

inhibitor Concentration μιηόΐ % inhibition Isoleucine mmol (2) formula compound 24 43 0.119 (2) formula compound 24 4 1.16

-17 ···· «··· ·«

Claims

Claims (10)

Claims CLAIMS 1. A method for detecting the inhibitory effect of compounds on protein and / or mRNA biosynthesis, comprising incubating a reagent mixture capable of producing detectable amounts of a functional reporter enzyme in vitro, as a control alone and in the presence of a test compound, in a control assay, and functional reporter enzyme, and the results are compared. 2. A method for screening herbicidal and / or antibiotic compounds from a group of potentially active compounds, which comprises incubating a reagent mixture as a control in itself and in the presence of the test compound for the production of a detectable amount of a functional reporter enzyme. functional reporter enzyme produced in the presence of compound 5, and the results are compared. 3. A method for determining the mode of action of herbicides and antibiotics, comprising incubating a reagent mixture capable of producing a detectable amount of a functional reporter enzyme under control in itself and in the presence of the test compound, detecting the functional reporter enzyme produced in the control assay and in the presence of the test compound. , and the results are compared. -18 • * · · ··· ··· · ·· 4. A method according to any one of claims 1 to 3, characterized in that a reagent mixture is used which comprises (1) an amino acid mixture, (2) plasmid DNA encoding a functional reporter enzyme, (3) an S30 extract from a prokaryotic organism without reporter enzyme activity, and (4) a mixture of low molecular weight compounds containing reagents for in vitro protein synthesis and (5) magnesium ions. 5. The method of claim 4, wherein the plasmid DNA carrying the lac Z gene is used and an extract of S30 from an E. coli strain lacking β-D-galactosidase is used. 6. The method of claim 5, wherein the E. coli strain is laceless MC1061. Method according to any one of the preceding claims, characterized in that the detection is carried out by a colorimetric or light-generating test method. 8. In vitro colorimetric assay for the detection of substances that inhibit the transcription (RNA polymerase) and / or translation of a functional protein gene product in E. coli extracts, characterized in that the E. coli extract is supplemented with tRNA and cofactors necessary for biosynthesis. , priming the mixture with plasmid DNA carrying the lac Z gene, allowing the primed mixture to synthesize β-D-galactosidase, quantifying the amount of enzyme formed, and determining whether the presence of a putative inhibitor reduces β-D-galactosidase activity relative to the control. Biologically active substances as defined by the method of any of the preceding claims, which inhibit bacterial protein and / or mRNA biosynthesis (transcription and translation). 10. Herbicidal inhibitors as defined in claim 9 or compounds derived therefrom, compounds of formulas (I), (IA) and (IB) - in which: Y represents a group of formula (IC), (ID) or (IE), and 2 3 R is a group -CO-XR in which X represents an oxygen atom or a sulfur atom, and R ^{3 is} hydrogen or an agriculturally useful ester-forming group, or 2 u R is a group -R in which R represents an optionally substituted aryl or heterocyclic group, or R ^{2 is} a group -CO ~ NR ^{4 5} R ^{6 in} which R ⁵ and R ⁶ are the same or different and each represents an agriculturally useful amide-forming group - except for their stereoisomers and salts of the compounds of formula (I), (IA) and (IB) wherein R ² is -COOH.