Process for determination of the activity of a substance using an in vitro functional test

• US 6,818,396 B1
• Filed: 11/28/2000
• Issued: 11/16/2004
• Est. Priority Date: 11/28/2000
• Status: Expired due to Fees

• Current Assignee: Proteus Corporation
• Original Assignee: Proteus Corporation
• Inventors: Bloch, Jean François, Lefevre, Fabrice, Dautel, Sandrine, Dupret, Daniel, Masson, Jean-Michel
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/722,587
• Time in Patent Office: 1,449 Days
• Field of Search: 435/6, 435/183, 435/91.1, 435/91.2, 436/89, 536/23.1, 424/88
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/161 gag-pol, e.g. p55, p24/25, p17/18, p.7, p6, p66/68, p51/52, p31/34, p32, p40
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

The invention concerns a method for determining the activity of a substance using a functional test, characterized in that it consists in detecting and/or measuring the variation of a known function corresponding to one or several proteins produced in vitro in the presence and in the absence of said substances or to the substance in the presence or in the absence of one or several proteins produces in vitro.

Claims

1. A method of screening a substance able to modify the known function of one or more proteins comprising the steps of:

(a) producing in vitro said proteins

(b) measuring and/or detecting variation of the known function in the presence and in the absence of the substance which is screened.

2. The method of claim 1, further comprising the following steps:

a) preparing at least one nucleic acid molecule comprising the gene(s) coding for one or several proteins and the control elements necessary for the transcription and the translation of said gene(s),

b) transcribing the nucleic acid molecule(s) prepared at step (a),

c) translating in vitro the transcript(s) prepared at step (b),

d) detecting and/or measuring the variation of a known function corresponding to the proteins produced at step (c) in the presence and in the absence of said substance or to the substance in the presence and in the absence of the proteins produced at step (c).

3. The method of claim 2, wherein preparation of one or several nucleic acid molecules of step (a) consists of placing the gene(s) coding for said protein(s) under the control:

of a 5′

promoter for transcription and/or of a ribosome binding site upstream of said gene(s) for translation.

4. The method of claim 3, comprising detecting and/or measuring variation of the known function of the protein(s) produced at step (c).

5. The method of claim 4, wherein step (a) comprises preparation of the nucleic acid molecule(s) by amplifying the gene(s) coding for said protein(s).

6. The method of claim 5, wherein step (a) comprises preparing the nucleic acid molecule(s) by PCR or NASBA type amplifying of the gene(s) coding for said protein(s), with the aid of one or several pairs of primers, each one comprising:

(a) some sequence hybridizing upstream of one or several nucleic acid molecules comprising gene(s) coding for said protein(s), and of an RNA polymerase promoter for the sense primer, and

(b) some sequence hybridizing downstream of one or several nucleic acid molecules comprising gene(s) coding for said protein(s) for the antisense primer.

7. The method of claim 2, wherein after step (d), it is verified that said substance does not inhibit one of steps (a) to (c).

8. A kit for the implementation of the method of claim 2, wherein said kit comprises:

means for revealing the function, an RNA polymerase, nucleotide sequences for the preparation of the nucleic acid molecules comprising the gene(s) permitting the expression of protein(s) corresponding to the detected and/or quantified function, four triphosphate nucleotides, and mixtures necessary for said preparation, transcription and translation steps.

9. The kit of claim 8, wherein said kit further comprises:

(a) products necessary for the preparation of the nucleic acid molecules comprising the gene(s) permitting the expression of the protein(s) corresponding to the detected and/or quantified function, and/or

(b) a support containing;

means for revealing a function, an RNA polymerase, four triphosphate nucleotides, and mixtures necessary for said transcription and translation steps.

10. The method of claim 6, wherein, for the sense primer, the sequence hybridizing upstream of one or several nucleic acid molecules further comprises genes coding for a ribosome binding site.

11. The method of claim 6, wherein, for the antisense primer, the sequence hybrizing downstream of one or several nucleic acid molecules further comprises genes coding for an RNA polymerase terminator.

12. The method of claim 4, wherein said function corresponds to a collection of target proteins of which the genes coding for these proteins are located on the same DNA fragment as in the case of an operon, or at different places of the DNA.

13. The method of claim 12, wherein step (a) comprises preparing a nucleic acid molecule comprising genes (the operon) coding for the proteins, 5′

of the collection of said genes (from the operon) a DNA polymerase promoter, and for each of said genes its natural ribosome binding site.

14. The method of claim 13, wherein the ribosome binding site of each one of the genes is its natural ribosome binding site and, at step (c), a translation extract is prepared starting from the organism that the target gene(s) come from.

15. The method of claim 12, wherein step (a) comprises preparing one or several nucleic acid molecules comprising the genes coding for the proteins, 5′

of each of said genes an RNA polymerase promoter and a ribosome binding site.

16. The method of claim 15, wherein the ribosome binding site is the natural site of each one of the genes.

17. The method of claim 1, wherein said proteins are different mutants of a protein or different mutants of a collection of proteins.

18. The method of claim 2, wherein one of several reporter molecules are added at one of steps (a), (b), (c) or (d) permitting detecting and/or measuring of the activity of the protein(s) produced at step (c) or of the substance.

19. The method of claim 2, wherein the reporter molecule is a molecule capable of directly or indirectly revealing the activity of one or several of said proteins or of said substance.

20. The method of claim 19, wherein the reporter molecule is a protein that is produced during step (c) conjointly with said protein(s).

21. The method of claim 20, wherein the gene coding for the reporter molecule is placed under the control of transcription and translation regulation sequences corresponding to those of the gene(s) coding for said protein(s), such that the reporter gene is co-expressed with said gene(s).

22. The method of claim 18, wherein said protein or one of said proteins produced at step (c) is also a reporter molecule.

23. The method of claim 2, further comprising introducing said substance before, after and/or during the transcription and/or translation of steps (b) and/or (c) and/or of detecting and/or of measuring of the variation of at least one known functional step (d).

24. The method of claim 1 wherein, said substance comprises polynucleotides, 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, antiviruses, inhibitors, or stimulants.