Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-28
2004-11-16
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S183000, C435S091100, C435S091200
Reexamination Certificate
active
06818396
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the filed of research of substances capable of modifying a function corresponding to a protein or a collection of proteins implicated in a biological process. This type of work, also designated screening, today constitutes an empirical mode of research but particularly for forming new substances, used in numerous laboratories. Among the numerous fields of application of these screening strategies, there can be cited the following examples:
Pharmaceutical laboratories set up libraries of molecules, and research those capable of inhibiting or of slowing down the activity of enzymes implicated in the development of genetic or infectious bacterial or viral diseases.
Following the intensive use of antibiotics, the speed of the appearance of new resistances is currently more rapid than that of the discovery of new antibiotics. Certain hospital laboratories therefore are looking for, always starting from banks of very specific molecules, new antibiotics or new beta-lactamase inhibitors.
Work is equally being carried out to find inhibitors of microorganism multiplication implicated in the biological corrosion of pipes or of containers used in industrial processes.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention has for its object a process for the determination of the activity in vitro of one or several substances using a functional test consisting of detecting and/or measuring a variation of at least one known function corresponding either to one or several proteins produced in vitro in the presence or absence of said substance, or of the substance in the presence or in the absence of proteins produced in vitro. The process of the invention will therefore be designated hereinafter, screening process.
Said known function preferably corresponds to proteins expressed in vitro also called target proteins. The process of the invention therefore aims to determine the activity of the substance tested based on a known function corresponding to one or several protein(s) produced in vitro.
However, in another embodiment of the process of the invention, the known function corresponds to the substance(s) tested. In this case, the process of the invention permits determining if the known activity of this substance is modified by one or several protein product(s) in vitro.
By function is understood the activity of one or several specific proteins still called target proteins of at least one organisms or of at least a process, and that it is detected and/or quantified according to the present invention by the by means of a functional test. Preferably, the target protein is an enzyme. This enzyme can correspond among other things to reverse transcriptase or to the aspartic acid proteinase of the Aids virus, to the
Mycobacterium tuberculosis
RecA intein, to an antibiotic resistance protein, or also to a collection of enzymes implicated in a metabolic pathway.
By known function is understood the function that is analyzed according to the process of the invention and which can be detected and/or measured by a functional test.
In a particular case of the process of the invention, function is understood as the activity of one or more substances that are detected and/or measured by a functional test.
Variation of function is understood as the difference of the activity of the measured function in the presence or absence of substance. In a second embodiment of the process of the invention, the variation in function corresponds to the activity difference of the substance in the presence of or in the absence of protein expressed in vitro.
The functional test can make use of one or multiple reporter molecules. The reporter molecule can correspond to any molecule capable of directly or indirectly revealing the activity of one or multiple target proteins, and can include a nucleic acid molecule, a protein, a peptide, such as an antibody or a mixture of specific antibodies capable of revealing the activity of a target protein, or such as a substrate or a cascade of substrates, of which one is that of a target protein.
The function can correspond to an enzymatic activity or to an affinity. In the scope of the demonstration of a variation of function corresponding to an enzymatic activity, any type of specific functional test can be contemplated by a person skilled in the art to demonstrate this variation. A person skilled in the art for example can make reference to works such as Methods In Enzymology or Annual Review of Biochemistry, in which a large number of enzyme measuring and substrate preparation methods have been described. In the case of the demonstration of a variation of function corresponding to the affinity for example of an antigen for an antibody, of a protein for DNA, of a receptor for a ligand etc . . . . The demonstration of a variation of function can be carried out for example by tests such as labeled ligand fixation by an isotope or by an enzyme or by a fluorophore, by an immunological detection using antibodies labeled by a metal or by an enzyme or by a fluorophore.
By organism is understood any type of organism or microorganism, such as viruses, bacteria, algae, fungi, or any product containing synthetic or natural nucleic acids permitting the expression of one or more proteins advantageously coding for a function.
By process is understood the development of a disease, of an infection or of cells for example cancers or contaminants or bacterial resistance mechanisms. These processes can take place in a host or in an industrial process (agribusiness, paper treatment, textile).
By polynucleotide substances is understood peptides, proteins, ions, molecules or natural or synthetic chemical compositions, hormones, aromatic compounds, antibodies, antibody fragments, genes, cellular receptors, amino acids, glycopeptides, lipids, glycolipids, sugars, polysaccharides, etc . . . , capable of modifying the activity of one or more functions of an organism or of a process. These listed substances will be capable of corresponding to one or more series heads. Included is any agent capable of modifying the function or functions such as antiviral, inhibitors, stimulants, physico-chemical, radiation or thermal treatments.
The process of the invention is therefore applicable more particularly to the screening of substances capable of modifying the activity of one or more target proteins or function, implicated in a biological process thanks to the in vitro expression of said protein or proteins. The process of the invention permits the screening of multiple substances simultaneously, notably when it relates to substances liable to interact in order to express a function or to modify a function. Consequently, substance must be understood as singular as well as plural of the process of the invention described below.
Similarly, the use subsequently of the term “function” in the singular will equally cover the term “functions” in the plural and vice versa, except when it is explicitly indicated that it relates to an embodiment of the method of the invention for plural functions.
Thus, the process of the invention permits in a preferred aspect of the invention the screening of substances capable of modifying the activity of a single target protein expressed in vitro having for example an enzymatic activity, such as an amylase, a polymerase, a protease, or capable of modifying an observable property of this protein, such as for example a “DNA-binding” activity, an antigenic activity, or also capable of modifying the activity or a property of a variant of this target protein, such as for example a thermostable amylase, a more processive polymerase, a protease resistance to an anti-protease, or a “DNA binding” protein having a stronger affinity for DNA. But the process of the invention also permits the screening of substances capable of modifying the activity of a collection of proteins expressed in vitro. In this case it is the collection of proteins that make up the target of the substances to test. This collection of expressed proteins can
Bloch Jean François
Dautel Sandrine
Dupret Daniel
Lefevre Fabrice
Masson Jean-Michel
Hunton & Williams LLP
Ketter James
Proteus S.A.
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