Isolated GRP94 ligand binding domain polypeptide and nucleic acid encoding same, crystalline form of same, and screening methods employing same
AssigneeDuke University
InventorsDaniel T. Gewirth, Christopher V. Nicchitta
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Abstract
An isolated GRP94 ligand binding domain polypeptide, a three-dimensional crystal structure of the same, and methods of using the same to design modulators of Hsp90 proteins.
Claims62
A substantially pure GRP94 ligand binding domain polypeptide in crystalline form.
The polypeptide of claim 4, wherein the GRP94 polypeptide 1 has the amino acid sequence of SEQ ID NO:6.
A method for determining the three-dimensional structure of a crystallized GRP94 ligand binding domain polypeptide to a resolution of about 1.8 Å or better, the method comprising:
- (a) crystallizing a GRP94 ligand binding domain polypeptide;
- and
- (b) analyzing the GRP94 ligand binding domain polypeptide to determine the three-dimensional structure of the crystallized GRP94 ligand binding domain polypeptide, whereby the three-dimensional structure of a crystallized GRP94 ligand binding domain polypeptide is determined to a resolution of about 1.8 Å or better.
A method of generating a crystallized GRP94 ligand binding domain polypeptide, the method comprising:
- (a) incubating a solution comprising a GRP94 ligand binding domain with a reservoir;
- and
- (b) crystallizing the GRP94 ligand binding domain polypeptide using the hanging drop method, whereby a crystallized GRP94 ligand binding domain polypeptide is generated.
A method of designing a modulator of an Hsp90 protein, the method comprising:
- (a) designing a potential modulator of an Hsp90 protein that will make interactions with amino acids in the ligand binding site of the Hsp90 protein based upon the atomic structure coordinates of a GRP94 ligand binding domain polypeptide;
- (b) synthesizing the modulator;
- and
- (c) determining whether the potential modulator modulates the activity of the Hsp90 protein, whereby a modulator of an Hsp90 protein is designed.
A method of designing a modulator that selectively modulates the activity of a GRP94 polypeptide, the method comprising:
- (a) obtaining a crystalline form of a GRP94 ligand binding domain polypeptide;
- (b) determining the three-dimensional structure of the crystalline form of the GRP94 ligand binding domain polypeptide;
- and
- (c) synthesizing a modulator based on the three-dimensional structure of the crystalline form of the GRP94 ligand binding domain polypeptide, whereby a modulator that selectively modulates the activity of a GRP94 polypeptide is designed.
A method for identifying a GRP94 modulator, the method comprising:
- (a) providing atomic coordinates of a GRP94 ligand binding domain to a computerized modeling system;
- and
- (b) modeling ligands that fit spatially into the binding pocket of the GRP94 ligand binding domain to thereby identify a GRP94 modulator.
A method of identifying modulator that selectively modulates the activity of a GRP94 polypeptide compared to other Hsp90 polypeptides, the method comprising:
- (a) providing atomic coordinates of a GRP94 ligand binding domain polypeptide to a computerized modeling system;
- and
- (b) modeling a ligand that fits into the binding pocket of a GRP94 ligand binding domain and that interacts with conformationally constrained residues of a GRP94 ligand binding domain polypeptide conserved among Hsp90 subtypes, whereby a modulator that selectively modulates the activity of a GRP94 polypeptide compared to other Hsp90 polypeptides is identified.
A method of identifying an Hsp90 modulator that selectively modulates the biological activity of one Hsp90 polypeptide compared to GRP94, the method comprising:
- (a) providing an atomic structure coordinate set describing a GRP94 ligand binding domain polypeptide and at least one other atomic structure coordinate set describing an Hsp90 ligand binding domain, each ligand binding domain comprising a ligand binding site;
- (b) comparing the atomic structure coordinate sets to identify at least one diference between the sets;
- (c) designing a candidate ligand predicted to interact with the difference of step (b);
- (d) synthesizing the candidate ligand;
- and
- (e) testing the synthesized candidate ligand for an ability to selectively modulate an Hsp90 as compared to GRP94, whereby an Hsp90 modulator that selectively modulates the biological activity of the Hsp90 compared to GRP94 is identified.
A method of designing a modulator of a GRP94 polypeptide, the method comprising:
- (a) selecting a candidate GRP94 ligand;
- (b) determining which amino acid or amino acids of a GRP94 polypeptide interact with the ligand using a three-dimensional model of a crystallized a GRP94 ligand binding domain polypeptide;
- (c) identifying in a biological assay for GRP94 activity a degree to which the ligand modulates the activity of the GRP94 polypeptide;
- (d) selecting a chemical modification of the ligand wherein the interaction between the amino acids of the GRP94 polypeptide and the ligand is predicted to be modulated by the chemical modification;
- (e) synthesizing a chemical compound with the selected chemical modification to form a modified ligand;
- (f) contacting the modified ligand with the GRP94 polypeptide;
- (g) identifying in a biological assay for GRP94 activity a degree to which the modified ligand modulates the biological activity of the GRP94 polypeptide;
- and
- (h) comparing the biological activity of the GRP94 polypeptide in the presence of modified ligand with the biological activity of the GRP94 polypeptide in the presence of the unmodified ligand, whereby a modulator of a GRP94 polypeptide is designed.
An assay method for identifying a compound that inhibits binding of a ligand to a GRP94 polypeptide, the assay method comprising:
- (a) designing a test inhibitor compound based on the three dimensional atomic coordinates of a GRP94 ligand binding domain polypeptide;
- (b) incubating a GRP94 polypeptide with a ligand in the presence of a test inhibitor compound;
- (c) determining an amount of ligand that is bound to the GRP94 polypeptide, wherein decreased binding of ligand to the GRP94 protein in the presence of the test inhibitor compound relative to binding of ligand in the absence of the test inhibitor compound is indicative of inhibition;
- and
- (d) identifying the test compound as an inhibitor of ligand binding if decreased ligand binding is observed, whereby a compound that inhibits binding of a ligand to a GRP94 polypeptide is identified.
A method of screening a plurality of compounds for a modulator of a GRP94 ligand binding domain polypeptide, the method comprising:
- (a) providing a library of test samples;
- (b) contacting a GRP94 ligand binding domain polypeptide with each test sample;
- (c) detecting an interaction between a test sample and the GRP94 ligand binding domain polypeptide;
- (d) identifying a test sample that interacts with the GRP94 ligand binding domain polypeptide;
- and
- (e) isolating a test sample that interacts with the GRP94 ligand binding domain polypeptide, whereby a plurality of compounds is screened for a modulator of a GRP94 ligand binding domain polypeptide.
Dependencies are inferred from the claim text.