Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-26
2002-12-10
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S024300, C436S501000
Reexamination Certificate
active
06492116
ABSTRACT:
The present invention relates to an assay for testing inhibitors of the interaction between proteins p53 and hdm2.
The protein encoded by the human double minute 2 gene, hdm2, forms a complex with the tumor suppressor gene product p53 both in vitro and in vivo. In some human cancers hdm2 is overexpressed and binds most of the cellular p53. Formation of this complex is favoring nucleoplasmic transformation since the complexed p53 looses the tumor suppressor activity. Compounds which prevent the interaction between p53 and hdm2 will release p53, promoting its tumor suppression activity within these cancerous cells. Similar results could also be obtained with animal cancer cells, e. g. in mouse. The mouse homologue to hdm2 is mdm2.
To search for inhibitors of p53 -hdm2 interaction, a high throughput primary binding assay, for example ELISA, can be used to select compounds and to initiate a medicinal chemistry program. However, assays which can be used for such a primary screening of p53-hdm2 binding have the disadvantage that artefacts may occur, i. e. wrong positive reaction can be obtained because of artefactual results due to the chemical properties of the tested substances.
In addition, compounds which inhibit the interaction between p53 and hdm2 can also alter p53 specific DNA binding, which is a totally undesired effect because DNA binding is a prerequisite of p53 tumor suppressor activity.
For example, p53 is sensible to several chemical agents that inhibit its activity. The main criteria of activity of p53 is the DNA binding which reveals that the protein is properly folded and not aggregated or unfolded. Compounds which like metal chelators precipitate p53 might be considered as true inhibitors of the p53 -hdm2 interaction in a classical binding assay because the precipitated p53 cannot form complexes.
Therefore, additional testing of the impact of the substance on p53-DNA binding is important because compounds inhibiting p53-DNA binding are not good candidates for therapeutic uses. However, in a high through put assay it is not possible to test whether a compound which inhibits p53-hdm2 interaction prevents p53 specific DNA binding or disturbs the p53 conformation so that p53 can no more fulfill the desired biological function.
To avoid these two problems and to start a chemistry program based on more relevant lead compounds, the use of a good confirming assay is crucial.
A confirming assay according to the present invention could, for example, be a gel shift assay. A gel shift of a p53-DNA complex in an agarose gel after incubation with adeno-virus E1B protein is described in Yew et al. [
Genes & Dev.
8, 190-202, 1994].
In Wang et al. [
PNAS
91, 2230-2234, 1994] the detection of the binding of HB virus X protein to p53 by measuring the inhibition of p53-DNA binding is described. However, none of the prior art publications describes an assay in which the DNA binding property of p53 remains if p53 is complexed with a double minute 2 protein.
So far, all the in vitro assays described in the literature to study the interaction between p53 and hdm2 are immunoprecipitation assays for testing the binding of hdm2 to p53 [
Leng et al. , Oncogene
10:1275(1995)]. None of these assays simultaneously show that hdm2 binds to p53 and does not disturb its specific DNA binding.
In the present invention it was surprisingly found that p53-DNA binding is maintained after complex formation with hdm2 and that it is possible to measure in one and the same reliable assay the effect of a substance on both the p53-hdm2 and p53-DNA binding.
OBJECT OF THE INVENTION
It is the object of the invention to provide a reliable test method for compounds which inhibit the formation of complexes between hdm2 and p53 but which do not inhibit binding of DNA to p53 or disturb the p53 conformation so that p53 can no more fulfill the desired biological function.
SUMMARY OF THE INVENTION
The present invention concerns a new assay which allows the identification to compounds which inhibit the formation of complexes between a product of the double minute 2 gene (“dm2”), for example human hdm2 or mouse mdm2, and p53 but not between p53 and DNA. Both the complex formation of labeled DNA, C-terminally truncated p53 and dm2 and disruption of dm2 from the labeled DNA-p53 complex by an inhibitor of the p53-dm2 interaction can be detected by a gel shift assay procedure. This assay permits the selection of compounds which, besides their inhibitory property, do not alter p53 specific DNA binding and do not disturb p53 conformation required for DNA binding or formation of active tetramer.
The invention further concerns a test kit for testing the effect of a substance on the binding of a dm2 protein to p53, comprising (a) a p53 or functional equivalent thereof having DNA-binding, oligomerisation and hdm2-binding properties, (b) a hdm2 or functional equivalent thereof having the p53 binding domain, and (c) a DNA sequence specifically binding to the p53 binding domain.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns a test method for a substance inhibiting the formation of a complex between p53 and a product of the double minute 2 gene (“dm2”), for example human (h)dm2 or mouse (m)dm2, while not inhibiting the formation of a complex between p53 and DNA. The method comprises measuring complex formation in a mixture of p53, dm2 and DNA binding to p53 in the presence and in the absence of a substance to be tested. In the presence of the desired property of the tested substance, a complex between p53 and DNA is formed (“double complex”), while in the absence of the desired property either a complex between p53, DNA and dm2 (“triple complex”; if no inhibiting activity is present) or no complex (if the tested compound inhibits both the dm2-p53 and p53-DNA complex formation or if the tested substance destroys the p53 conformation so that it is no more DNA binding) is formed. While any method being able to discriminate between the different conditions (triple complex, double complex, no complex) is suitable for performing the present assay, in a preferred embodiment of the invention the assay performed is a gel shift assay.
Thus, the test system essentially comprises a p53, a dm2, and DNA. While the use of the human proteins or active variants thereof is most preferred, the invention is not limited to the use of the human proteins. The corresponding proteins from other species can also be used, e. g. from mouse. However, it is preferred that both the p53 and dm2 protein used in the assay originate from the same species.
For performing the present invention, a p53 protein must be used which both is able to bind DNA and dm2.
p53 according to the present invention can be a recombinant form of p53 or purified from the original organism. It is, however, not necessary to use a full length p53 for performing the present invention. Accordingly, the p53 form used herein also means any useful variant or fragment of p53, preferentially of human p53. The features of such a useful variant or fragment are clear from the description hereinafter.
For DNA binding, p53 must be able to form tetrameric complexes. Consequently, for DNA binding both an active p53 DNA binding domain (e. g. residues 102-292 of p53) and a p53 functional oligomerisation domain (e. g. residues 325-356 of p53) must be present in the p53 form used in the present invention. For improving DNA binding properties of p53, the protein can be activated by interaction with a specific antibody (for example the monoclonal antibody Pab421 known in the art which binds to the amino acid stretch between amino acid 372 and 380 of the human p53), phosphorylation by kinases (casein kinase 11 phosphorylating Ser392 of human p53 or protein kinase C phosphorylating Ser 370 and Ser 375 of human p53) or, more preferably, truncation of its C-terminus (deletion of maximal 38 amino acids of the C-terminus of the natural p53 sequence). An example for the latter is p53D30, i. e. natural p53 lacking the C-terminal 30 amino acids, used in the Examples he
Chène Patrick
Hochkeppel Heinz-Kurt
Cancer Research Campain Technology Ltd.
Fredman Jeffrey
Knobbe Martens Olson & Bear LLP
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