WO2004090543A1 - Assay method - Google Patents
Assay method Download PDFInfo
- Publication number
- WO2004090543A1 WO2004090543A1 PCT/GB2004/001502 GB2004001502W WO2004090543A1 WO 2004090543 A1 WO2004090543 A1 WO 2004090543A1 GB 2004001502 W GB2004001502 W GB 2004001502W WO 2004090543 A1 WO2004090543 A1 WO 2004090543A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lincosamide
- ribosomal
- peptidyl transferase
- assay method
- fluorescence
- Prior art date
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- 238000003556 assay Methods 0.000 title claims abstract description 18
- 108090000279 Peptidyltransferases Proteins 0.000 claims abstract description 22
- 150000001875 compounds Chemical class 0.000 claims abstract description 20
- 210000003705 ribosome Anatomy 0.000 claims abstract description 15
- 230000003115 biocidal effect Effects 0.000 claims abstract description 5
- 238000000198 fluorescence anisotropy Methods 0.000 claims description 7
- 150000003839 salts Chemical group 0.000 claims description 4
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 claims description 3
- 229960002227 clindamycin Drugs 0.000 claims description 3
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 claims description 3
- 238000002875 fluorescence polarization Methods 0.000 claims description 3
- 229960005287 lincomycin Drugs 0.000 claims description 3
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 210000004708 ribosome subunit Anatomy 0.000 claims description 3
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 3
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- YMZMTOFQCVHHFB-UHFFFAOYSA-N 5-carboxytetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(C(O)=O)C=C1C([O-])=O YMZMTOFQCVHHFB-UHFFFAOYSA-N 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 238000013537 high throughput screening Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- -1 BODIPY®-FL Substances 0.000 description 3
- 241000191965 Staphylococcus carnosus Species 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 150000004675 formic acid derivatives Chemical class 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 238000001243 protein synthesis Methods 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- WWZKQHOCKIZLMA-UHFFFAOYSA-N Caprylic acid Natural products CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 2
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- GONOPSZTUGRENK-UHFFFAOYSA-N benzyl(trichloro)silane Chemical compound Cl[Si](Cl)(Cl)CC1=CC=CC=C1 GONOPSZTUGRENK-UHFFFAOYSA-N 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000000752 ionisation method Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 229940041028 lincosamides Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N n-hexanoic acid Natural products CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- VWFRSNKRTNUMET-UHFFFAOYSA-N 2-[3-(dimethylamino)-6-dimethylazaniumylidenexanthen-9-yl]-5-(2,5-dioxopyrrolidin-1-yl)oxycarbonylbenzoate Chemical compound C=12C=CC(=[N+](C)C)C=C2OC2=CC(N(C)C)=CC=C2C=1C(C(=C1)C([O-])=O)=CC=C1C(=O)ON1C(=O)CCC1=O VWFRSNKRTNUMET-UHFFFAOYSA-N 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- UOZODPSAJZTQNH-UHFFFAOYSA-N Paromomycin II Natural products NC1C(O)C(O)C(CN)OC1OC1C(O)C(OC2C(C(N)CC(N)C2O)OC2C(C(O)C(O)C(CO)O2)N)OC1CO UOZODPSAJZTQNH-UHFFFAOYSA-N 0.000 description 1
- 108010034396 Streptogramins Proteins 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 238000002820 assay format Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000003835 ketolide antibiotic agent Substances 0.000 description 1
- 239000003041 laboratory chemical Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 229960001914 paromomycin Drugs 0.000 description 1
- UOZODPSAJZTQNH-LSWIJEOBSA-N paromomycin Chemical compound N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO UOZODPSAJZTQNH-LSWIJEOBSA-N 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940041030 streptogramins Drugs 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
Definitions
- This invention relates to an assay method for determining whether a test compound binds to the peptidyl transferase centre of the ribosome, more particularly the ribosome of a bacterium.
- ribosome In the cell, protein synthesis takes place on the ribosome.
- the ribosome is a large macromolecular assembly of several RNA molecules, and more than fifty proteins. It consists of two subunits: 30S and 50S. Both contain several functional sites for interaction with a large number of different ligands during protein synthesis. The most important, on which protein assembly takes place, is the peptidyl transferase centre of the 50S subunit.
- a large number of anti-infective antibiotics act by inhibition of protein synthesis, by direct binding with high affinity to specific sites on the ribosome.
- HTS high throughput screening
- Such an assay would be useful for screening, especially HTS or ultra HTS (uHTS) of large compound libraries as potential antibiotics.
- Ribosomal peptidyl transferase is a target for several classes of natural and semi synthetic antibiotics, such as macrolides, lincosamides, streptogramins, chloramphenicol and ketolides. It would, therefore, be desirable to have a robust assay capable of detecting the binding of a test compound to the peptidyl transferase centre of the ribosome.
- the present invention provides an assay method for determining whether a test compound binds to the ribosomal peptidyl transferase centre, which comprises determining a fluorescence characteristic of a pair of reporter molecules, namely (i) a ribosomal element which includes the ribosomal peptidyl transferase centre and (ii) a fluorophore-tagged lincosamide antibiotic, in the presence and absence of the test compound, a change in the fluorescence characteristic being taken as indication binding of the test compound to the peptidyl transferase centre.
- the assay of the invention is suitable for use with any part of the ribosome which includes the peptidyl transferase centre, for example a whole ribosome or the 50S ribosomal subunit.
- the fluorophore-tagged lincosamide antibiotic is conveniently a fluorophore- tagged clindamycin or lincomycin-derived molecule, but there are other known members of the lincosamide family which are suitable including, for example, celestecetin.
- Suitable fluorophore tags are TAMRA and BODIPY ® TMR-X, but again there are many known fluorophore alternatives available as standard laboratory chemicals, including BODIPY ® -FL, Alexa Fluor ® , Cy3, Cy5 and other different types of commercially available fluorophores.
- TAMRA is the common acronym for either 5-carboxytetramethylrhodamine, its 6-carboxy position isomer, or a mixture of the 5- and 6-position isomers.
- BODIPY ® TMR-X is the acronym for 6-((4,4-difluoro-1 ,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a- diaza-s-indacene-2-propionyl)amino) hexanoic acid.
- TAMRA-lincosamide having either of the following structures (I) or (II) or a mixture thereof
- BODIPY ® TMR-X-lincosamide having structure (III), and salts thereof
- the fluorescence characteristic determined in the assay of the invention is preferably fluorescence anisotropy or fluorescence polarisation, but fluorescence intensity can also be used.
- Solvent B 95% acetonitrile + 5% solvent A + 0.08% v/v formic acid. UV detection was performed at 230, 254 and 270 nm. Mass spectrometry was carried out on a HP1100 MSD, series A instrument. The ionisation method was positive ion electrospray and the molecular weight scan range was 150-1000.
- Preparative LC-MS was performed on a Waters FractionLynx MS autopurification system using a Luna 5 ⁇ m, C18(2), 100 mm x 21.2 mm i.d. column from Phenomenex at ambient temperature.
- Solvent A water + 0.08% v/v formic acid.
- Solvent B 95% methanol-water + 0.08% v/v formic acid.
- Flow rate 20 cm 3 min -1 .
- the instrument incorporated a photo diode array detector (210-400 nm) and a MicroMass ZQ mass spectrometer.
- the ionisation method was positive ion electrospray and the molecular weight scan range was 150-1000. Fraction collection was triggered by detection of the selected mass ion.
- Displacement of the probe from the ribosome is thus associated with a reduction in anisotropy.
- Compounds that bind to the peptidyl transferase centre displace the fluorophore-tagged lincosamide, and the concomitant decrease in fluorescence anisotropy is determined.
- test compound 10 nM TAMRA-lincosamide or BODIPY ® TMR-X-lincosamide and 2 ⁇ M S. carnosus or S. aureus ribosomes in buffer (20 mM Tris-HCI pH 7.6, 30 mM KCI, 70 mM NH 4 CI, 6 mM MgCI 2 , 100 ⁇ g/ml bovine serum albumin).
- Chloramphenicol, erythromycin, lincomycin and clindamycin are known to bind in the peptidyl transferase centre region of the 50S ribosomal subunit and are competitors in the assay.
- Puromycin also binds to the peptidyl transferase centre and leads to an increase in the fluorescence polarization of the TAMRA- lincosamide or BODIPY ® TMR-X-lincosamide (not shown on a Figure).
- Paromomycin and streptomycin, two ribosome-binding antibiotics that act on sites different from the peptidyl transferase centre show no activity.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
An assay method for determining whether a test compound binds to the ribosomal peptidyl transferase centre, which comprises determining a fluorescence characteristic of a pair of reporter molecules, namely (i) a ribosomal element which includes the ribosomal peptidyl transferase centre and (ii) a fluorophore-tagged lincosamide antibiotic, in the presence and absence of the test compound, a change in the fluorescence characteristic being taken as indication binding of the test compound to the peptidyl transferase centre.
Description
FLUORESCENT ASSAY FOR COMPOUNDS BINDING TO THE PEPTIDYL TRANSFERASE CENTER OF THE RIBOSOME
This invention relates to an assay method for determining whether a test compound binds to the peptidyl transferase centre of the ribosome, more particularly the ribosome of a bacterium.
Background to the Invention
In the cell, protein synthesis takes place on the ribosome. The ribosome is a large macromolecular assembly of several RNA molecules, and more than fifty proteins. It consists of two subunits: 30S and 50S. Both contain several functional sites for interaction with a large number of different ligands during protein synthesis. The most important, on which protein assembly takes place, is the peptidyl transferase centre of the 50S subunit.
A large number of anti-infective antibiotics act by inhibition of protein synthesis, by direct binding with high affinity to specific sites on the ribosome. There is, therefore, a need for a simple, inexpensive, straightforward, homogeneous and miniaturised high throughput screening (HTS) assay for ribosomal inhibitors. Such an assay would be useful for screening, especially HTS or ultra HTS (uHTS) of large compound libraries as potential antibiotics. Ribosomal peptidyl transferase is a target for several classes of natural and semi synthetic antibiotics, such as macrolides, lincosamides, streptogramins, chloramphenicol and ketolides. It would, therefore, be desirable to have a robust assay capable of detecting the binding of a test compound to the peptidyl transferase centre of the ribosome.
Detailed Description of the Invention
The present invention provides an assay method for determining whether a test compound binds to the ribosomal peptidyl transferase centre, which comprises determining a fluorescence characteristic of a pair of reporter molecules, namely (i) a ribosomal element which includes the ribosomal peptidyl transferase centre and (ii) a fluorophore-tagged lincosamide antibiotic,
in the presence and absence of the test compound, a change in the fluorescence characteristic being taken as indication binding of the test compound to the peptidyl transferase centre.
The assay of the invention is suitable for use with any part of the ribosome which includes the peptidyl transferase centre, for example a whole ribosome or the 50S ribosomal subunit.
The fluorophore-tagged lincosamide antibiotic is conveniently a fluorophore- tagged clindamycin or lincomycin-derived molecule, but there are other known members of the lincosamide family which are suitable including, for example, celestecetin. Suitable fluorophore tags are TAMRA and BODIPY® TMR-X, but again there are many known fluorophore alternatives available as standard laboratory chemicals, including BODIPY®-FL, Alexa Fluor®, Cy3, Cy5 and other different types of commercially available fluorophores. TAMRA is the common acronym for either 5-carboxytetramethylrhodamine, its 6-carboxy position isomer, or a mixture of the 5- and 6-position isomers. BODIPY® TMR-X is the acronym for 6-((4,4-difluoro-1 ,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a- diaza-s-indacene-2-propionyl)amino) hexanoic acid.
Thus, TAMRA-lincosamide, having either of the following structures (I) or (II) or a mixture thereof, and BODIPY® TMR-X-lincosamide, having structure (III), and salts thereof, are novel entities in their own right, and form part of the present invention:
(I) (II)
(HI)
The fluorescence characteristic determined in the assay of the invention is preferably fluorescence anisotropy or fluorescence polarisation, but fluorescence intensity can also be used.
The following non-limiting Examples describe the synthesis of two fluorophore- tagged lincosamides (structures I and III), either of which are suitable for use as the fluorescent reporter molecule in the assay of the invention. Also described is the use of each reporter in one assay format of the invention.
In Examples 1 and 2, purification and characterisation of compounds were performed using the following techniques.
1H NMR spectra were recorded on a Bruker DPX400 instrument. Chemical shifts are quoted in parts per million, and are referenced to the residual solvent peak. The following abbreviations are used: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad. Coupling constants are quoted to the nearest 0.5 Hz.
Analytical liquid chromatography-mass spectrometrγ (LC-MS) was performed on an HP1100 instrument, using a Luna 3 μm, C18(2), 30 mm x 4.6 mm i.d. column from Phenomenex at ambient temperature. Gradient elution was performed as follows: 0-0.25 min, 5% solvent B, flow rate 2 cm3min"1; 0.25-2.5 min, 95% B, 2 cm3min"1; 2.5-2.55 min, 95% B, 3 cm3 min"1; 2.55-3.6 min, 95% B, 3 cm3 min"1; 3.6-3.65 min, 95% B, 2 cm3 min"1; 3.65-3.7 min, 5% B, 2 cm3 min"1; 3.7-3.75 min, 5% B, 2 cm3 min"1. Solvent A: 10 mM aqueous ammonium acetate + 0.08% v/v formic acid. Solvent B: 95% acetonitrile + 5% solvent A + 0.08% v/v formic acid. UV detection was performed at 230, 254 and 270 nm. Mass spectrometry was carried out on a HP1100 MSD, series A instrument. The ionisation method was positive ion electrospray and the molecular weight scan range was 150-1000.
Preparative LC-MS was performed on a Waters FractionLynx MS autopurification system using a Luna 5 μm, C18(2), 100 mm x 21.2 mm i.d. column from Phenomenex at ambient temperature. Solvent A: water + 0.08% v/v formic acid. Solvent B: 95% methanol-water + 0.08% v/v formic acid. Flow rate: 20 cm3 min-1. The instrument incorporated a photo diode array detector (210-400 nm) and a MicroMass ZQ mass spectrometer. The ionisation method was positive ion electrospray and the molecular weight scan range was 150-1000. Fraction collection was triggered by detection of the selected mass ion.
Example 1
7-(/?)-Amino-7-deoxylincomycin (IV) was prepared using the protocol of Sztaricskai, Omura and their co-workers.1,2 5-Carboxytetramethylrhodamine (5-TAMRA) lincosamide (I) (herein called TAMRA-lincosamide) was synthesised in the form of its formate salt from 7-( :?)-amino-7-deoxylincomycin (IV) in accordance with Scheme 1.
5-TAMRA lincosamide (I)
Scheme 1
7-( ?)-7-(3-Carboxy-4-{9-[(6-dimethylaminoxanthen-3-ylidene) dimethylammonium]}benzenecarbamoyl)-7-deoxylincomycin, diformate salt (5-TAMRA lincosamide) (I)
To a solution of 7-(r?)-amino-7-deoxylincomycin (IV)1'2 (0.015 g, 0.038 mmol) and triethylamine (0.010 cm3, 0.076 mmol) in anhydrous dimethyl formamide (2 cm3) was added 5-carboxytetramethylrhodamine, succinimidyl ester (5- TAMRA, SE) (V) (0.020 g, 0.038 mmol). After 18 h, the reaction mixture was evaporated, and the residue subjected to preparative LC-MS under a gradient elution (0 min, 5% solvent B; 0.5 min, 30% B; 7.0 min, 60% B; 7.5 min, 95% B; 9.5 min, 95% B; 10.0 min, 5% B). The eluted material (retention time 5.89 min), obtained as a purple solid (0.010 g, 31%), was identified as the title compound
(i); δH (400 MHz; d4-MeOH) 8.53 (2 H, s), 8.49 (1 H, d, J 2 Hz), 8.00 (1 H, dd, J 8 and 2 Hz), 7.35 (1 H, d, J8 Hz), 7.25 (1 H, d, J 9.5 Hz), 7.24 (1 H, d, J 9.5 Hz),
7.01 (2 H, dd, J 9.5 and 2.5 Hz), 6.92 (2 H, d, J 2.5 Hz), 5.29 (1 H, d, J5.5 Hz), 4.58-4.48 (2 H, m), 4.32 (1 H, d, J7.5 Hz), 4.12 (1 H, dd, J 10 and 5.5 Hz),
4.02 (1 H, d, J3 Hz), 3.56 (1 H, dd, J 10 and 3 Hz), 3.28 (12 H, s), 3.30-3.23 (1 H, m), 3.11 (1 H, dd, J 10.5 and 4 Hz), 2.47 (3 H, s), 2.32-2.21 (1 H, m), 2.14 (3 H, s), 2.17-2.03 (2 H, m), 1.90-1.80 (1 H, m), 1.42-1.23 (4 H, m), 1.30 (3 H, d, J6.5 Hz) and 0.91 (3 H, t, J 7 Hz),
m/z410 (ιVf2+, 100%), retention time 1.77 min.
Example 2
BODIPY® TMR-X lincosamide (III) in the form of its formate salt was synthesised from 7-(A?)-amino-7-deoxylincomycin (IV) in accordance with Scheme 2.
BODIPY® TMR-X lincosamide (III)
Scheme 2
7-( ?)-7-({6-[4,4-Difluoro-153-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a- diaza-s-indacene-2-propionyl]amino}hexanecarbamoyl)-7- deoxylincomycin, formate salt (BODIPY® TMR-X lincosamide) (III)
To a solution of 7-(/?)-amino-7-deoxylincomycin (IV) (0.0133 g, 0.033 mmol) and triethylamine (0.010 cm3, 0.076 mmol) in anhydrous dimethyl formamide cm3) was added 6-(4,4-difluoro-1 ,3-dimethyl-5-(4-methoxyphenyl)-4-bora-
3a,4a-diaza-s-indacene-2-propionyl)amino) hexanoic acid, succinimidyl ester (BODIPY® TMR-X, SE) (VI) (0.020 g, 0.033 mmol). After 18 h, the reaction mixture was evaporated, and the residue subjected to preparative LC-MS under a gradient elution (0 min, 5% solvent B; 0.5 min, 60% B; 7.0 min, 95% B; 7.5 min, 95% B; 9.5 min, 95% B; 10.0 min, 5% B). The eluted material (retention time 6.78 min), obtained as a purple solid (0.011 g, 30%), was identified as the title compound (III); δH (400 MHz; d4-MeOH) 8.50 (1 H, s), 7.87 (2 H, d, J 9 Hz), 7.42 (1 H, s), 7.07 (1 H, d, J4 Hz), 6.97 (2 H, d, J 9 Hz), 6.60 (1 H, d, J4 Hz), 5.22 (1 H, d, J5.5 Hz), 4.30 (1 H, dd, J7.5 and 6.5 Hz), 4.23-4.17 (1 H, m), 4.14 (1 H, d, J 7.5 Hz), 4.07 (1 H, dd, J 10 and 5.5 Hz), 3.92 (1 H, d, J3 Hz), 3.85 (3 H, s), 3.50 (1 H, dd, J 10 and 3 Hz), 3.25 (1 H, dd, J8.5 and 6 Hz), 3.13-3.07 (3 H, m), 2.75 (2 H, t, J7.5 Hz), 2.50 (3 H, s), 2.43 (3 H, s), 2.33 (2 H, t, J 7.5 Hz), 2.25 (3 H, s), 2.30-2.17 (1 H, m), 2.14 (1 H, dd, J 10 and 8.5 Hz), 2.07 (3 H, s), 2.10-1.98 (3 H, m), 1.87-1.81 (1 H, m), 1.56-1.49 (2 H, m), 1.46-1.40 (2 H, m), 1.38- 1.32 (4 H, m), 1.21-1.18 (2 H, m), 1.09 (3 H, d, J 6.5 Hz) and 0.91 (3 H, t, J 6.5 Hz), mlz 899 (MH+, 70%) and 440 [(/W-F)2+, 100], retention time 2.13 min.
Example 3
Interference assay with TAMRA-lincosamide or BODIPY® TMR-X- lincosamide reporter and Staphylococcus carnosus or Staphylococcus aureus ribosomes
The interaction between fluor tagged-lincosamide and Staphylococcus carnosus or Staphylococcus aureus ribosomes was monitored by measuring the change in fluorescence anisotropy A [A = (///- l±)/( /// + 2 Ix), where /)/ and l±are time-independent steady state values for fluorescent intensity polarized parallel and perpendicular]. In solution, a fluorescent probe (low molecular weight ligand, MW <1 kDa) is generally rotationally free with low anisotropy. When bound to the ribosome (macromolecular target, MW >2.2 MDa), due to the huge mass change, it becomes rotationally constrained and anisotropy is
high. Displacement of the probe from the ribosome is thus associated with a reduction in anisotropy. Compounds that bind to the peptidyl transferase centre displace the fluorophore-tagged lincosamide, and the concomitant decrease in fluorescence anisotropy is determined. Some compounds, when bound to the peptidyl transferase centre, cause an increase in the interaction between tagged lincosamide and the ribosome and the concomitant increase in fluorescence anisotropy is determined.
Measurements were made on a Packard Fusion plate reader. In the titrations, increasing amounts of test compound were added to a solution of 10 nM TAMRA-lincosamide or BODIPY® TMR-X-lincosamide and 2 μM S. carnosus or S. aureus ribosomes in buffer (20 mM Tris-HCI pH 7.6, 30 mM KCI, 70 mM NH4CI, 6 mM MgCI2, 100 μg/ml bovine serum albumin). For each titration point, a fluorescence anisotropy measurement was acquired using an excitation wavelength of 535 nm with the excitation slits set to 25 nm and an emission wavelength of 580 nm with the emission slits set to 15nm. Compounds binding to the peptidyl transferase centre interfere with the binding of TAMRA-lincosamide or BODIPY® TMR-X-lincosamide and either the concomitant decrease or increase in fluorescence anisotropy is determined. Typical data are shown on Figure 1.
Chloramphenicol, erythromycin, lincomycin and clindamycin are known to bind in the peptidyl transferase centre region of the 50S ribosomal subunit and are competitors in the assay. Puromycin also binds to the peptidyl transferase centre and leads to an increase in the fluorescence polarization of the TAMRA- lincosamide or BODIPY® TMR-X-lincosamide (not shown on a Figure). Paromomycin and streptomycin, two ribosome-binding antibiotics that act on sites different from the peptidyl transferase centre, show no activity.
Hence, interference with the fluorescent label was only observed for those antibiotics known to bind to the region of the peptidyl transferase centre.
References
1. F. Sztaricskai, Z. Dinya, M. M. Puskas, G. Batta, R. Masuma and S. Omura, J. Antibiotics, 1996, 49, 941-943.
2. F. Sztaricskai, G. Batta, Z. Dinya, M. Hornyak, E. Roth, R. Masuma and S. Omura, J. Antibiotics, 1999, 52, 1050-1055.
Claims
1. An assay method for determining whether a test compound binds to the ribosomal peptidyl transferase centre, which comprises determining a fluorescence characteristic of a pair of reporter molecules, namely (i) a ribosomal element which includes the ribosomal peptidyl transferase centre and (ii) a fluorophore-tagged lincosamide antibiotic, in the presence and absence of the test compound, a change in the fluorescence characteristic being taken as indication binding of the test compound to the peptidyl transferase centre.
2. An assay method as claimed in claim 1 , wherein the ribosomal element which includes the peptidyl transferase centre is a whole ribosome or a 50S ribosomal subunit.
3. An assay method as claimed in claim 1 or claim 2 wherein the fluorophore-tagged lincosamide antibiotic is a fluorophore-tagged clindamycin, lincomycin or other lincosamide-derived molecule.
4. An assay method as claimed in any of the previous claims wherein the fluorophore tag is a TAMRA or BODIPY® TMR-X fluorescent tag.
5. An assay method as claimed in any of the preceding claims wherein the fluorescence characteristic determined is fluorescence anisotropy, fluorescence polarisation or fluorescence intensity.
6. TAMRA-lincosamide of formula (I) or formula (II) or a mixture thereof, optionally in salt form: 
BODIPY® TMR-X lincosamide of formula (III), optionally in salt form:
(III)
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|---|---|---|---|
| GB0308018.1 | 2003-04-07 | ||
| GB0308018A GB0308018D0 (en) | 2003-04-07 | 2003-04-07 | Assay method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007012319A1 (en) * | 2005-07-29 | 2007-02-01 | Sigma-Aldrich Production Gmbh | Lc/ms blends containing ionising additives |
| US8609364B2 (en) | 2009-05-07 | 2013-12-17 | bioM{tilde over (e)}rieux, Inc. | Methods for antimicrobial resistance determination |
| CN104374922A (en) * | 2014-11-26 | 2015-02-25 | 绍兴康知生物科技有限公司 | Method for preparing protein for detecting chloramphenicol |
-
2003
- 2003-04-07 GB GB0308018A patent/GB0308018D0/en not_active Ceased
-
2004
- 2004-04-06 WO PCT/GB2004/001502 patent/WO2004090543A1/en active Application Filing
Non-Patent Citations (2)
| Title |
|---|
| BRANDT-RAUF P ET AL: "FLUORESCENT ASSAY FOR ESTIMATING THE BINDING OF ERYTHROMYCIN DERIVATIVES TO RIBOSOMES", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 14, no. 1, 1978, pages 88 - 94, XP000923144, ISSN: 0066-4804 * |
| SZTARICSKAI FERENC ET AL: "Structural modification of the lincomycin antibiotic", JOURNAL OF ANTIBIOTICS (TOKYO), vol. 52, no. 11, November 1999 (1999-11-01), pages 1050 - 1055, XP009033414, ISSN: 0021-8820 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007012319A1 (en) * | 2005-07-29 | 2007-02-01 | Sigma-Aldrich Production Gmbh | Lc/ms blends containing ionising additives |
| US8609364B2 (en) | 2009-05-07 | 2013-12-17 | bioM{tilde over (e)}rieux, Inc. | Methods for antimicrobial resistance determination |
| US9175329B2 (en) | 2009-05-07 | 2015-11-03 | Bio Merieux, Inc. | Methods for antimicrobial resistance determination |
| US10988792B2 (en) | 2009-05-07 | 2021-04-27 | Biomerieux, Inc. | Methods for antimicrobial resistance determination |
| CN104374922A (en) * | 2014-11-26 | 2015-02-25 | 绍兴康知生物科技有限公司 | Method for preparing protein for detecting chloramphenicol |
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