Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-11-25
2001-04-10
Celsa, Bennett (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S501000, C436S517000, C436S518000, C436S519000, C436S173000
Reexamination Certificate
active
06214561
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for detecting at least one compound (a ligand) in a compound library using at least one other compound (a receptor) that binds to the ligand.
BACKGROUND OF THE INVENTION
Such methods are common in the biochemical laboratory praxis.
A compound in the sense of the present invention can be a molecule, an aggregate or a complex composed of many like or dislike molecules and/or atoms.
A ligand that binds to a receptor, and therefore shows binding affinity for this receptor, is called a biologically active compound.
Compound libraries which are also called combinatorial libraries are synthetic or natural mixtures that contain a number of different compounds. In most cases, identity and quantity of particular compounds in the library are not known, the procedure, however, that led to these mixtures is often known. Such compound libraries can either consist of one sort of compounds, e.g. peptides or organic synthetic compounds, or they can comprise several different components, e.g. plant extracts. Also, complex components such as whole viruses, bacteria or cells can be part of a compound library.
Especially in drug research compound libraries play an important role when compounds are to be identified from complex mixtures for drug targets or when new biologically active substances have to be identified in such complex mixtures.
Such investigations are called “screening of compound libraries”. The screening, i.e. the investigation whether an interesting compound or compound with potentially interesting properties is part of a compound library, is performed using a receptor. A receptor is the fishing pole, that allows to fish the ligand out of the compound library.
Many different methods to screen compound libraries have been developed in pharmaceutical research and development as well as in biochemical and medical basic research. Common methods are e.g. the ELISA-test, the RIA-test, the affinity chromatography or different well known blotting methods such as Western-, Northern- or Southern-Blot.
All these methods have in common that with the help of the receptor the ligand in the compound library is spatially separated from the other compounds in the library. The spatial separation can take place during or after binding of the receptor to the ligand. The spatial separation is achieved by immobilizing one of the partner (either the receptor or the components of the compound library) to a solid support. Such supports are mostly microtiterplates, chromatography resins, beads, or filter papers.
In many cases it is also necessary to label one of the molecules in the study by using radioactive or non-radioactive isotopes in order to detect the receptor-ligand complex. Alternatively, a specific detection tool, e.g. an antibody, can be used to detect the receptor-ligand complex.
Affinity chromatography is widely used for screening of compound libraries by utilizing a receptor that is immobilized on a chromatography resin, which in turn is used to produce a chromatography column. If a compound library, which contains a ligand for the immobilized receptor, is passed over this column the ligand specifically binds to the immobilized receptor and by this process can be separated from the other compounds in the library. Subsequent detection of the ligand is achieved by elution of the ligand from the column and specific detection reactions.
Detection reactions can be based on detection of a ligand with a specific antibody, or if the ligand was labeled, on detection of the label.
In many cases it is not possible to label the compounds of a library, and also in many cases no specific antibodies exist. This is especially true if a new ligand in a compound library has to be identified.
In such cases a chemical analysis has to be performed, e.g. sequencing reactions that at present time are only applicable to nucleic acids and peptides.
A ligand that has been separated from the compound library with the help of a receptor can also be analyzed by mass spectrometry. In the recent past this method was increasingly used to analyze compound libraries.
An example for such a method is described in the publication “Matrix-assisted laser desorption ionization for a rapid determination of the sequences of biologically active peptides isolated from support-bound combinatorial peptide libraries” of Youngquist et al., 1994, Rapid Communications in Mass Spectrometry, No. 8, Pages 77-81.
Here a special mass spectrometry technique, the “matrix-assisted laser desorption ionization” (MALDI) was used to isolate such peptides from the synthetic peptide library, that are potentially useful to inhibit infections with the HI-virus.
In that procedure the peptide library was automatically synthesized on a solid support in form of inert plastic beads carrying of random peptide sequences. The peptides were immobilized on beads. Biological activity was tested by using a monoclonal antibody, that was directed against a surface protein of the HI-virus. Beads that were bound to the antibody were detected by an enzyme-conjugated staining method known from ELISA and by immunoblotting techniques. Colored beads were isolated and peptides were cleaved off. These peptides were analyzed with MALDI, which allows a direct sequencing and, therefore, identification of the peptides.
Each step of this method shows fundamental disadvantages that are also characteristic for the other methods known for detecting of specific ligands in compound libraries. All these methods are based on the principle that the receptor in a complex with the ligand is spatially separated from the other components of the compound library. The spatial separation requires either immobilization of the ligand as described in the publication mentioned above or immobilization of the receptor, e.g. in the affinity chromatography where the receptor is bound to a solid support.
This immobilization represents a considerable problem because biologically active receptors are often inactivated by immobilization.
The receptor binding sites for ligands are usually located on the surface. Therefore, these binding sites can participate in the immobilization reaction and it is impossible to control which sites of the receptor are used for the immobilization. In fact, in many cases the receptor binding site is directly coupled to the support and, thus, the binding site is no longer accessible for the ligand.
Another problem occuring in the immobilization of particularly protein receptors is denaturation, i.e. a conformational change of a receptor, that leads to loss of binding affinity.
It cannot be predicted whether all compounds in an immobilized library are equally well immobilized and which of the compounds are still active after immobilization. This is risky, because biologically active compounds present in the library may not be immobilized and therefore would not be detected with this process. In such a case the detection reaction is negative even though a bioactive ligand was present in the compound library.
Known processes have another major disadvantage in that they are complicated and lengthy. After immobilization of the ligand or the receptor many washing steps are necessary to remove non-immobilized molecules and only thereafter can the compound library be incubated with the receptor. Washing steps are again necessary after incubation to remove unbound material. Subsequently, complicated analytical procedures such as immunostaining or, after separation of the analyte from the solid support, mass spectrometric analysis has to be performed.
Immunostaining methods usually require specific antibodies against the ligand. This is impossible if new ligands are to be found and difficult if ligands are to be investigated that are not proteins.
An alternative to immunostaining is the radioactive or non-radioactive labelling of compounds in the library. Only nucleic acid libraries can be labelled without problems. For all other classes of compounds either no labelling technique is known or the labelling technique has major disadva
Meyer Bernd
Peters Thomas
Celsa Bennett
Garcia Maurie E.
Hamrick Claude A. S.
Oppenheimer Wolff & Donnelly LLP
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