Hi -
A) To fully address this question, you could break it into several
pieces. (1) Does the drug bind to cyclins D and E
(2) Does binding of the drug reduce binding to CDK's in vitro/in cells
(3) Does the drug produce the expected sequela for it's proposed
mechanism
1) To determine if the drug binds to cyclins D and E the proteins
could be immobilized and exposed to labeled drug. Retained label
would indicate binding - this could be done either in a column or
another immobile substrate (ie ELISA plate). Alternatively, if the
drug can be immobilized it could be used as bait to bind and retain
the protein.
Similar techniques are used in this paper:
http://www.retroconference.org/2002/Posters/13803.pdf
2) To determine if the drug reduces binding to CDK's in cells, I would
suggest immobilizing the cyclin and demonstrating binding to the CDK's
of interest (probably 2, 4, 6). Pretreatment of the immobilized
protein with the drug might then be expected to prevent subsequent
binding of the cyclin. Additionally, if the binding is competitive,
one could add the drug to the immobilized cyclin - cdk complex and
demonstrate dissociation of the complex with the drug (this would also
allow one to calculate kinetic binding parameters).
Similar techniques are used here:
http://www.retroconference.org/2002/Posters/13803.pdf
Yeast two-hybrid binding studies could also be performed using the
cyclin and cdk proteins as binding partners with and without the drug.
In the absence of the drug, it would be expected that the cyclin and
cdk proteins would bind, in the presence of the drug the proteins
should not bind.
A description of the yeast two-hybrid process:
http://www.devbio.com/chap05/link0518.shtml
To determine if the drug reduces binding in cells, I would first
suggest immunoprecipitating the cyclins and the CDK's separately and
checking for the precipitation of bound partner protein (ie a cyclin
IP would be expected to bring down cdk2 for instance, and a cdk4 IP
would also be expected to bring down cyclin). Once the method for
detecting binding of the proteins is demonstrated in cells, one could
treat the cells with drug and again look for binding between the
cyclins and the CDK's. One would expect the binding to decrease if
the drug works as advertised.
A description of immunoprecipitations:
http://www.protocol-online.org/prot/Molecular_Biology/Protein/Immunoprecipitation/
(3) Binding of the cyclin proteins to CDK's activates the CDK,
therefore if this drug is working as proposed, there should be a
measureable decrease in the activity of the CDK's. Cyclin A is
downstream from cyclins D and E, therefore any interference at the D/E
stage would be expected to produce a decrease in the amount and
activity of downstream cyclins such as cyclin A.
Additionally, as the activity of these cyclin-CDK complexes is
required for continued mitosis, it would be expected that the cells
would stop dividing upon treatment.
CDK's, cyclins, the cell cycle:
http://www.web-books.com/MoBio/Free/Ch8A.htm
To further demonstrate that the effect of the drug is at the level of
cyclin D/E-CDK, constituitively active (ie do not require binding to
cyclin to be active) mutants of the cdks could be transfected into the
cells - these mutants would be expected to be insensitive to the
effects of the drug thus demonstrating that the disruption caused by
the drug occurs at the cyclin D/E level.
A description of a similar approach can be found here:
http://www.hhmi.org/research/investigators/maller.html
Please let me know if you require further explanation. |
