Compound screening methods

• US 6,540,996 B1
• Filed: 04/14/2000
• Issued: 04/01/2003
• Est. Priority Date: 04/15/1999
• Status: Expired due to Fees

• Current Assignee: deVGen NV (China National Chemical Corporation)
• Original Assignee: deVGen NV (China National Chemical Corporation)
• Inventors: Zwaal, Richard, Groenen, José, Bogaert, Thierry
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): ANGELL, JON E
• Application Number:
  US09/549,872
• Time in Patent Office: 1,082 Days
• Field of Search: 435/69.1, 800/8, 800/3, 424/93.4
• US Class Current: 424/93.21
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0333 Genetically modified invertebrates, e.g. transgenic, polyploid
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: G01N 2333/43534 from Caenorhabditis
• CPC Class Codes: G01N 2333/4712 Muscle proteins, e.g. myosin, actin, protein
• CPC Class Codes: G01N 33/5085 of invertebrates
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/6887 from muscle, cartilage or connective tissue
• CPC Class Codes: G01N 33/9406 Neurotransmitters

Abstract

The invention provides methods of screening for compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA), using the nematode worm C. elegans.

Claims

1. A method of identifying compounds which enhance or up-regulate the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises:

contacting C. elegans which exhibit reduced SERCA ATPase activity compared to wild type C. elegans in one or more cell types or tissues with a compound under test; and

detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity in the one or more cell types or tissues which exhibit reduced SERCA activity in the absence of the compound.

2. A method as claimed in claim 1 wherein the C. elegans have been treated with a SERCA inhibitor to reduce the activity of SERCA in one or more cell types or tissues prior to contact with the compound under test.

3. A method as claimed in claim 2 wherein the SERCA inhibitor is thapsigargin.

4. A method as claimed in claim 1 wherein the C. elegans have been treated with antisense or double-stranded RNA to specifically reduce the expression of SERCA in one or more cell types or tissues prior to contact with the compound under test.

5. A method as claimed in claim 1 wherein the C. elegans is a mutant C. elegans which exhibits reduced SERCA calcium ATPase activity in one or more cell types or tissues.

6. A method as claimed in claim 5 wherein the C. elegans is a mutant C. elegans which exhibits reduced expression of SERCA in one or more cell types or tissues.

7. A method as claimed in any one of claims 1 to 6 wherein the C. elegans exhibit reduced SERCA activity in the muscles of the pharynx, as compared to wild type C. elegans and the step of detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity comprises detecting a change in the pharynx pumping efficiency of the C. elegans in the presence of the compound under test.

8. A method as claimed in claim 7 wherein the C. elegans further contain a transgene comprising a promoter which directs gene expression in the muscles of the C. elegans pharynx operatively linked to nucleic acid encoding an apoaequorin protein.

9. A method as claimed in claim 8 wherein the promoter is the C. elegans myo-2 promoter or the C. elegans SERCA promoter.

10. A method as claimed in claim 8 wherein the step of detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity comprises comparing the level of apoaequorin luminescence in the absence of the compound under test and the level of apoaequorin luminescence in the presence of the compound under test.

11. A method as claimed in any one of claims 1 to 6 wherein the C. elegans exhibit reduced SERCA activity in the muscles of the vulva, as compared to wild type C. elegans.

12. A method as claimed in claim 11 wherein the step of detecting a phenotypic, biochemical or behavioural change indicating a reversion towards wild type SERCA activity comprises detecting a change in the egg laying behaviour of the C. elegans in the presence of the compound under test.

13. A method as claimed in claim 11 wherein the step of detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity comprises detecting a change in the amount of progeny produced by the C. elegans.

14. A method as claimed in claim 11 wherein the C. elegans further contain a transgene comprising a promoter which directs gene expression in the muscles of the C. elegans vulva operatively linked to nucleic acid encoding an apoaequorin protein.

15. A method as claimed in claim 14 wherein the step of detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity comprises comparing the level of apoaequorin luminescence in the absence of the compound under test and the level of apoaequorin luminescence in the presence of the compound under test.

16. A method as claimed in any one of claims 1 to 6 wherein the C. elegans exhibit reduced SERCA activity in the anal repressor and/or the anal sphincter and the step of detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity comprises detecting a change in the defecation behaviour of the C. elegans in the presence of the compound under test.

17. A method as claimed in any one of claims 1 to 6 wherein the step of detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity comprises comparing the growth rate of the C. elegans in the absence of the compound under test and the growth rate of the C. elegans in the presence of the compound under test.

18. A method as claimed in any one of claims 1 to 6 wherein the step of detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity comprises comparing the turbidity of the C. elegans in culture in the absence of the compound under test and the turbidity of the C. elegans in culture in the presence of the compound under test.

19. A method as claimed in any one of claims 1 to 6 wherein the step of detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity comprises detecting a change in the movement behaviour of the C. elegans.