US 2009/0023158 A1 · Compositions and Methods for Identifying Substrate Specificity of Inhibitors of Gamma Secretase

Abstract

The invention provides assays and methods for determining the substrate specificity of gamma secretase inhibitors and for identifying substrate-selective (and substrate isoform-selective) inhibitors of gamma secretase. The invention provides assays and methods for determining whether a compound inhibits gamma secretase in a site specific or substrate specific manner. The invention provides isolated polypeptide sequences comprising modified gamma secretase substrates, and polynucleotide sequences encoding the polypeptide sequences. The invention also provides compounds that inhibit gamma secretase, pharmaceutical compositions comprising such compounds, and methods of treating Alzheimer's disease using such compounds.

Claims32

Dependent claims

A method for determining whether a compound inhibits gamma secretase in a substrate specific matter comprising:

(a) contacting a first gamma secretase substrate comprising a gamma cleavage site with the compound and gamma secretase under conditions that allow for gamma secretase activity;
(b) separately contacting a second gamma secretase substrate comprising a gamma cleavage site with the compound and gamma secretase under conditions that allow for gamma secretase activity;
(c) determining the amount of gamma secretase activity at the gamma cleavage site of the first substrate and the second substrate;
(d) comparing the amounts of gamma secretase activity at the gamma cleavage site from step (a) with the amount of gamma secretase activity at the gamma cleavage site from step (b) and determining that the compound inhibits gamma secretase in a substrate specific manner when the amount of gamma secretase activity at the gamma cleavage site from step (a) is different from step (b).

1 more element

A method for determining whether a compound selectively inhibits gamma secretase activity at a first gamma secretase substrate relative to a second gamma secretase substrate, comprising:

(a) contacting a first transfected cell culture with the compound at various concentrations under conditions that allow for gamma secretase activity;
(b) contacting a second transfected cell culture with the compound at various concentrations under conditions that allow for gamma secretase activity;
(c) measuring ICD produced by each of the first and second transfected cell cultures at each of the various compound concentrations to generate a first dose response curve of the effect of the compound on the first transfected cell culture and a second dose response curve of the effect of the compound on the second transfected cell culture;
and
(d) comparing the first and second dose response curves,
wherein:
the first transfected cell culture is transfected with a first polynucleotide encoding a first polypeptide comprising a juxtamembrane domain sequence (JMD1) and a transmembrane domain sequence (TMD1) of the formula [JMD1][TMD1], wherein [JMD1] is from a first gamma secretase substrate;
and
the second transfected cell culture is transfected with a second polynucleotide encoding a second polypeptide comprising a juxtamembrane domain sequence (JMD2) and a transmembrane domain sequence (TMD1) of the formula [JMD2][TMD1], wherein [JMD2] is from a second gamma secretase substrate and the TMD1 of the first and second polypeptides is the same.

6 more elements

A method for determining whether a compound selectively inhibits gamma secretase activity of a first gamma secretase substrate relative to a second gamma secretase substrate, comprising:

(a) contacting a first transfected cell culture with the compound at various concentrations under conditions that allow for gamma secretase activity;
(b) contacting a second transfected cell culture with the compound at various concentrations under conditions that allow for gamma secretase activity;
(c) measuring ICD produced by each of the first and second transfected cell cultures at each of the various compound concentrations to generate a first dose response curve of the effect of the compound on the first transfected cell culture and a second dose response curve of the effect of the compound on the second transfected cell culture;
and
(d) comparing the first and second dose response curves,
wherein:
the first transfected cell culture is transfected with a polynucleotide encoding a first polypeptide comprising Formula II:
[JMDΔC4]-X1-X2-X3-X4-[TMD]
wherein
[JMDΔC4] comprises the amino acid sequence of a juxtamembrane domain (JMD) sequence of a gamma secretase substrate, wherein the JMD lacks the four C-terminal peptides;
[TMD] comprises a transmembrane domain sequence of a gamma secretase substrate;
and
X1-X2-X3-X4 are independently selected from any amino acid;
and
the second transfected cell culture is transfected with a second polynucleotide encoding a second polypeptide comprising Formula II:
[JMDΔC4]-X1-X2-X3-X4-[TMD]
wherein X1-X2-X3-X4 are independently selected from any amino acid.

14 more elements

Dependencies are inferred from the claim text.

Compositions and Methods for Identifying Substrate Specificity of Inhibitors of Gamma Secretase

Life of this patent

Compositions and Methods for Identifying Substrate Specificity of Inhibitors of Gamma Secretase

Life of this patent

Abstract

Claims32