WO1995009925A1 - Dosage in vitro destine a detecter les inhibiteurs de biosynthese de proteines et/ou d'arnm - Google Patents
Dosage in vitro destine a detecter les inhibiteurs de biosynthese de proteines et/ou d'arnm Download PDFInfo
- Publication number
- WO1995009925A1 WO1995009925A1 PCT/GB1994/002088 GB9402088W WO9509925A1 WO 1995009925 A1 WO1995009925 A1 WO 1995009925A1 GB 9402088 W GB9402088 W GB 9402088W WO 9509925 A1 WO9509925 A1 WO 9509925A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mixture
- protein
- reporter enzyme
- detecting
- vitro
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
Definitions
- the present invention relates to an in vitro assay for detecting inhibitors of protein and/or mRNA biosynthesis, to the use of this assay in the discovery of novel antibiotics and herbicides and in determining their mode of action and to biologically active inhibitors of protein, particularly bacterial protein, and/or mRNA biosynthesis and herbicides obtained thereby.
- Inhibition of protein and/or mRNA biosynthesis is an interesting biological effect which can have potential applications in, for example, the antibiotic and, as the applicants have found, the agrochemical and in particular the herbicidal fields.
- screens employed for detecting inhibition of protein biosynthesis are low throughput and radiometric and hence unsuitable for the large scale screening of compounds necessary to discover useful biological activity or for detecting leads in these areas.
- the applicants have developed a non-radiometric screen for the high throughput screening of inhibitors of protein and/or mRNA biosynthesis.
- a method of detecting compounds having activity as inhibitors of protein and/or mRNA biosynthesis comprises incubating a mixture of reagents suitable for the DNA-directed synthesis in vitro of a functional reporter enzyme at a detectable rate, both alone as a control and in the presence of a test compound, detecting the functional reporter enzyme produced in the control and test mixtures and comparing the results.
- the test is suitably carried out on a microtitre scale.
- Conditions such as the incubation period for protein synthesis, time of assay, temperature and reagent concentrations etc., can be adapted to ensure that detectable quantities of reporter enzyme are generated in the control test within these time periods most convenient for high throughput screening.
- an incubation period for protein synthesis is suitably less than about 2 hours and preferably about 1 hour.
- Suitable reporter enzymes are those which can be assayed for example by addition of chro ogenic or fluorogenic substrates. These will include ⁇ -galactosidase and ⁇ -glucuronidase. Alternatively firefly luciferase could be used to generate light.
- the mode of detection of the reporter enzyme will depend upon the particular enzyme chosen. For example colorimetric or light generation assay techniques can be employed depending upon the enzyme. Assay chemicals are added either intially to the reaction mixture and/or at suitable points during the method.
- a preferred reaction for use in the method of the invention is based upon that described by G. Zubay supra and further modified by J. Collins supra.
- a mixture of reagents suitable for enzyme synthesis comprises the following components:
- the amino acid mixture is suitably a mixture of the 20 protein L-amino acids in equivalent proportions.
- concentrations of individual amino acids in the mixture can be varied (for example, to investigate whether an inhibitor is likely to act by inhibiting any of the a inoacyl- tRNA synthetases) .
- concentrations of individual amino acids are varied and it is observed whether there is any antidoting effect through increasing the concentration of a specific amino acid. This can be taken to indicate that the particular inhibitor is most likely to be acting as a competitive inhibitor of the corresponding a inoacyl-tRNA synthetase.
- the concentration of amino acids is varied suitably from 0.01 to lOmM. We have found that a reduction of the concentration of amino acid mixture relative to that described by G. Zubay supra is preferred in order to maximise the sensitivity of the test for the detection of protein biosynthesis inhibitors and, in particular, inhibitors of the aminoacyl- tRNA synthetases.
- the plasmid DNA is suitably any convenient plasmid DNA carrying the lac Z gene, such as that commercially available from Promega.
- Suitable S30 extracts are similar to those described by G. Zubay supra, and may include comrnercially available extracts such as those sol by Promega. A method of preparing such an extract is exemplified hereinafter.
- the low molecular weight compound mix can contain nucleosides, salts, buffers, solvents etc., as would be apparent to the biochemist.
- An example of a suitable low molecular weight compound mix is given hereinafter.
- the optimum amount of magnesium ion for the protein synthesising reaction needs to be established and standardised for each new batch of J . coli or S30 extract and of low molecular weight compound mix which is prepared. (Having established the amount of magnesium ion necessary to produce the most ⁇ -D-galactosidase in initial controls, and which is used in a preferred embodiment of the present invention, then this amount is used for all subsequent inhibitor testing until either all of the S30 extract or the low molecular weight component mix is used up).
- a convenient source of magnesium ion is magnesium acetate. In practice, the optimum amount is usually found somewhere between 5 and 30 mM of added magnesium acetate (i.e. concentration added to the protein synthesising reaction).
- the present invention provides a colorimetric in vitro method for detecting inhibitors of transcription (RNA polymerase) and translation (protein synthesis) in an E___ coli extract, which method comprises supplementing the £-. coli extract with tRNA and cofactors required for biosynthesis, priming the mixture with plasmid DNA carrying the lac Z gene, allowing the primed mixture to synthesize ⁇ -D-galactosidase, quantitatively detecting the amount of enzyme made, and determining whether the presence of a potential inhibitor has reduced the ⁇ -D-galactosidase activity relative to controls.
- RNA polymerase RNA polymerase
- translation protein synthesis
- the J . coli strain chosen for making the extract is one which lacks ⁇ -D-galactosidase (e.g. the readily available lac deleted strain MC1061).
- the cofactors include the amino acid mixture and the low molecular weight compound mix comprising the basic chemicals required for protein synthesis in vitro as described above.
- the ⁇ -D-galactosidase is suitably detected and quantitated by adding a chromogenic substrate such as o-nitrophenyl ⁇ -D-galactoside.
- Inhibitors of protein synthesis are detected by measuring the reduction in yellow colour absorbing at 410 nm formed in microtitre wells which contain inhibitor. Control experiments using ⁇ -D-galactosidase (commercially available from Sigma (UK) Ltd) are then necessary to distinguish between genuine inhibitors of mRNA or protein synthesis and molecules which are merely inhibitors of ⁇ -D-galactosidase itself.
- Inhibitors of bacterial RNA poly erase and/or protein biosynthesis include many antibiotics and, because of the close similarity between the protein synthesis machinery of chloroplasts and of bacteria, also herbicides.
- the assay therefore provides a novel means of screening for compounds potentially useful as herbicides or antibiotics or as a means of providing in vitro active leads around which a programme of analogue synthesis could then lead to compounds useful as antibiotics or herbicides.
- a method of screening for compounds potentially useful herbicides comprises incubating a mixture of reagents suitable for the synthesis of a functional reporter enzyme at a detectable rate in vitro, both alone as a control, and in the presence of a test compound, detecting the reporter enzyme produced in the control and test mixtures and comparing the results.
- the assay also useful as an indicator of herbicide and/or antibiotic mode of action.
- the test will indicate whether a lead with an unknown mode of action is or is not an inhibitor of bacterial protein biosynthesis. In many cases, it will be possible to use the test to further narrow down the site of action to a more specific site within transcription/translation. In particular, it is possible to diagnose whether compounds act as specific inhibitors of any one of the aminoacyl-tRNA synthetases. Inhibitors of these are particularly valuable having use as both antibiotics and herbicides such as those described in cope ⁇ ding International Patent Publication No. W093/19599. So, for example, competitive inhibitors of isoleucyl-tRNA synthetase can be readily identified through the specific ability of isoleucine to antagonise the inhibition observed in this test.
- a biologically active inhibitor of bacterial protein and/or mRNA biosynthesis (the transcription and translation of a functional protein gene product) identified using the method according to the present invention.
- the invention also comprises herbicidal compounds consisting of, or derived from such inhibitors, but excluding those of International Patent
- R represents a group of sub-formula (IC) or (ID or (IE) and wherein R is a
- R is a group -R wherein R is an
- R is a group
- R and R are the same or different and each represent an agrochemically acceptable amide-forming radical; stereoiso ers of the compounds of formula (I), (IA) and (IB) and salts of the compound of formula (I), (IA) and (IB) wherein R 2 is COXR 3 , X is 0 and R 3 is hydrogen.
- Tris tris(hydroxymethyl)amino ethane
- H ? 0 Dissolve Tris (tris(hydroxymethyl)amino ethane) in H ? 0 and mix the following reagents in the order listed below.
- the solution was made up in the order above and finally adjusted to pH 8.2 with 5M potassium hydroxide.
- DNA pGe carrying the gene encoding ⁇ -D-galactosidase (commercially available from Promega)
- the bacterial strain E__, coli MC1061 was grown in a rich medium (as described by G. Zubay supra) at 28°C in fermenters run at a working volume of 141. Samples were removed throughout the fermentation to measure the O.D. 550nm.
- the chemicals used were generally of ANALAR (or supplier's equivalent) quality.
- the buffer used throughout the following preparation is lOmM Tris- acetate pH 8.2 containing 14mM magnesium acetate, 60mM potassium acetate and ImM DL-dithiothreitol .
- Test Compounds 1-3 and some known antibiotics were dissolved in appropriate concentrations up to lOOpp in 4% DMSO (dimethyl sulfoxide) or H 2 0. Blank solutions were used as appropriate. Control experiments indicate that DMSO did not interfere at 4% in this solution (i.e. 1% in the protein synthesis reaction) .
- 17 ⁇ l 530 extract lO ⁇ l of inhibitor (or, in controls, 10 ⁇ l of water, or 4% DMSO in H,,0, according to what the inhibitors are dissolved in).
- control experiments The purpose of the control experiments is to check that the inhibitors discovered really are inhibitors of protein biosynthesis and not simply inhibitors of ⁇ -D-galactosidase.
- the assay may also be used as a diagnostic test to determine the exact mode of action of such inhibitors as described above.
- the inhibition of Compound No. 2 is shown to be reversed by L-isoleucine indicating isoleucyl-tRNA synthetase as the site of inhibition.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP94927716A EP0722505A1 (fr) | 1993-10-06 | 1994-09-26 | Dosage in vitro destine a detecter les inhibiteurs de biosynthese de proteines et/ou d'arnm |
JP7510666A JPH09503131A (ja) | 1993-10-06 | 1994-09-26 | 蛋白質および/またはmRNAの生合成の阻害物質を検出するためのインビトロアッセイ法 |
NZ273643A NZ273643A (en) | 1993-10-06 | 1994-09-26 | Assay to detect and evaluate protein and mrna inhibitors |
KR1019960701800A KR960705056A (ko) | 1993-10-06 | 1994-09-26 | 단백질 및/또는 mRNA 생합성 저해제를 검출하는 시험관내 분석방법(IN VITRO ASSAY TO DETECT INHIBITORS OF PROTEIN AND/OR mRNA BIOSYNTHESIS) |
AU77023/94A AU686231B2 (en) | 1993-10-06 | 1994-09-26 | In vitro assay to detect inhibitors of protein and/or MRNA biosynthesis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB939320562A GB9320562D0 (en) | 1993-10-06 | 1993-10-06 | Novel assay and applications |
GB9320562.3 | 1993-10-06 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995009925A1 true WO1995009925A1 (fr) | 1995-04-13 |
Family
ID=10743067
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1994/002088 WO1995009925A1 (fr) | 1993-10-06 | 1994-09-26 | Dosage in vitro destine a detecter les inhibiteurs de biosynthese de proteines et/ou d'arnm |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP0722505A1 (fr) |
JP (1) | JPH09503131A (fr) |
KR (1) | KR960705056A (fr) |
AU (1) | AU686231B2 (fr) |
GB (1) | GB9320562D0 (fr) |
HU (1) | HUT73691A (fr) |
NZ (1) | NZ273643A (fr) |
WO (1) | WO1995009925A1 (fr) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5747338A (en) * | 1996-08-15 | 1998-05-05 | Chiron Corporation | Method and construct for screening for inhibitors of transcriptional activation |
WO1998048274A1 (fr) * | 1997-04-22 | 1998-10-29 | Smithkline Beecham Corporation | Test par fluorescence homogene pour mesurer l'effet des composes sur l'expression d'un gene |
EP0975801A1 (fr) * | 1997-03-27 | 2000-02-02 | Case Western Reserve University | PROCEDES DE DEPISTAGE D'AGENTS ANTIMICROBIENS UTILISANT (aarC) ET DE LEURS COMPOSITIONS |
WO2000034512A1 (fr) * | 1998-12-08 | 2000-06-15 | Proteus (S.A.) | Methode de detection et/ou de quantification d'une fonction connue a partir d'un echantillon d'acides nucleiques |
WO2000034514A1 (fr) * | 1998-12-08 | 2000-06-15 | Proteus (S.A.) | PROCEDE DE DETERMINATION DE L'ACTIVITE D'UNE SUBSTANCE METTANT EN OEUVRE UN TEST FONCTIONNEL $i(IN VITRO) |
EP1043403A1 (fr) * | 1999-04-09 | 2000-10-11 | GPC AG, Genome Pharmaceuticals Corporation | Méthode pour l'identification des composés antibactériens |
WO2000061793A2 (fr) * | 1999-04-09 | 2000-10-19 | Gpc Biotech Ag | Nouvelle methode d'identification de composes antibacteriens |
WO2002046461A2 (fr) * | 2000-12-04 | 2002-06-13 | Isis Innovation Limited | Procede d'identification de modulateurs de transcription |
US6818396B1 (en) | 2000-11-28 | 2004-11-16 | Proteus S.A. | Process for determination of the activity of a substance using an in vitro functional test |
-
1993
- 1993-10-06 GB GB939320562A patent/GB9320562D0/en active Pending
-
1994
- 1994-09-26 KR KR1019960701800A patent/KR960705056A/ko not_active Application Discontinuation
- 1994-09-26 WO PCT/GB1994/002088 patent/WO1995009925A1/fr not_active Application Discontinuation
- 1994-09-26 JP JP7510666A patent/JPH09503131A/ja active Pending
- 1994-09-26 AU AU77023/94A patent/AU686231B2/en not_active Ceased
- 1994-09-26 EP EP94927716A patent/EP0722505A1/fr not_active Withdrawn
- 1994-09-26 NZ NZ273643A patent/NZ273643A/en unknown
- 1994-09-26 HU HU9600859A patent/HUT73691A/hu unknown
Non-Patent Citations (3)
Title |
---|
G. ZUBAY: "In vitro synthesis of protein in microbial systems", ANNUAL REVIEW OF GENETICS, vol. 7, 1973, PALO ALTO, CA, US, pages 267 - 287 * |
J COLLINS: "Cell-free synthesis of proteins coding for mobilisation functions of Co1E1 and transposition functions of Tn3", GENE., vol. 6, 1979, AMSTERDAM NL, pages 29 - 42 * |
MANIATIS ET AL.: "Molecular Cloning", 1989, COLD SPRING HARBOR LABORATORY PRESS * |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5747338A (en) * | 1996-08-15 | 1998-05-05 | Chiron Corporation | Method and construct for screening for inhibitors of transcriptional activation |
EP0975801A4 (fr) * | 1997-03-27 | 2001-10-24 | Univ Case Western Reserve | PROCEDES DE DEPISTAGE D'AGENTS ANTIMICROBIENS UTILISANT (aarC) ET DE LEURS COMPOSITIONS |
EP0975801A1 (fr) * | 1997-03-27 | 2000-02-02 | Case Western Reserve University | PROCEDES DE DEPISTAGE D'AGENTS ANTIMICROBIENS UTILISANT (aarC) ET DE LEURS COMPOSITIONS |
WO1998048274A1 (fr) * | 1997-04-22 | 1998-10-29 | Smithkline Beecham Corporation | Test par fluorescence homogene pour mesurer l'effet des composes sur l'expression d'un gene |
WO2000034512A1 (fr) * | 1998-12-08 | 2000-06-15 | Proteus (S.A.) | Methode de detection et/ou de quantification d'une fonction connue a partir d'un echantillon d'acides nucleiques |
WO2000034514A1 (fr) * | 1998-12-08 | 2000-06-15 | Proteus (S.A.) | PROCEDE DE DETERMINATION DE L'ACTIVITE D'UNE SUBSTANCE METTANT EN OEUVRE UN TEST FONCTIONNEL $i(IN VITRO) |
US8017318B1 (en) | 1998-12-08 | 2011-09-13 | Proteus S.A. | Method for detecting and/or quantifying a known function from a nucleic acid sample |
EP1666612A3 (fr) * | 1998-12-08 | 2006-06-21 | Proteus S.A. | Procédé de détermination de l'activité d'une substance mettant en oeuvre un test fonctionnel in vitro |
WO2000061793A2 (fr) * | 1999-04-09 | 2000-10-19 | Gpc Biotech Ag | Nouvelle methode d'identification de composes antibacteriens |
WO2000061793A3 (fr) * | 1999-04-09 | 2001-01-11 | Gpc Biotech Ag | Nouvelle methode d'identification de composes antibacteriens |
EP1043403A1 (fr) * | 1999-04-09 | 2000-10-11 | GPC AG, Genome Pharmaceuticals Corporation | Méthode pour l'identification des composés antibactériens |
US6818396B1 (en) | 2000-11-28 | 2004-11-16 | Proteus S.A. | Process for determination of the activity of a substance using an in vitro functional test |
WO2002046461A2 (fr) * | 2000-12-04 | 2002-06-13 | Isis Innovation Limited | Procede d'identification de modulateurs de transcription |
WO2002046461A3 (fr) * | 2000-12-04 | 2003-03-13 | Isis Innovation | Procede d'identification de modulateurs de transcription |
Also Published As
Publication number | Publication date |
---|---|
JPH09503131A (ja) | 1997-03-31 |
KR960705056A (ko) | 1996-10-09 |
EP0722505A1 (fr) | 1996-07-24 |
NZ273643A (en) | 1997-12-19 |
AU7702394A (en) | 1995-05-01 |
HU9600859D0 (en) | 1996-05-28 |
GB9320562D0 (en) | 1993-11-24 |
AU686231B2 (en) | 1998-02-05 |
HUT73691A (en) | 1996-09-30 |
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