Methods of identifying and designing cell surface receptor inhibitors
OwnerM. Arnaout, Thilo Stehle +1inventors
InventorsM. Arnaout, Thilo Stehle, Simon Goodman
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Abstract
The methods described herein provide for ways of modulating, specifically inhibiting, integrin activation. The methods include identifying compounds such as naturally occurring and non-naturally occurring polypeptides and small molecules which bind to the βA domain of an integrin thus mimicking the contact between the CD loop with βA domain. Contacting the βA domain in this manner locks the integrin such that it is unable to be activated.
Claims37
A method for evaluating the ability of a compound to associate with a molecule or molecular complex comprising a non-ligand binding site of an integrin βA domain, the method comprising:
- (a) employing computational means to perform a fitting operation between the compound and β tail domain (βTD) contact region on the strand-F/α7 loop of an unliganded integrin (e.g., αVβ3);
- and
- (b) analyzing the results of the fitting operation to quantify said association potential.
A method for identifying a candidate selective modulator of the activity of an integrin, the method comprising:
- (a) modeling test compounds that fit spatially and preferentially into a βA domain non-ligand binding site of an integrin of interest using an atomic structural model of the integrin βA domain, wherein the atomic structural model is generated using amino acid sequence comprising C663VVRFQYYE671D672S673S674G675 KSEYVVEEPEC687 or a fragment thereof, or K618KFDREPYMTENTCNR633YCRD or fragment thereof;
- (b) screening test compounds in a biological assay for integrin activation characterized by binding of a test compound to the βA domain non-ligand binding site of the integrin;
- and
- (c) identifying a test compound that selectively modulates the activity of the integrin, and optionally,
- (d) screening an identified test compound in a biological assay for its ability to prevent interaction of the βA domain and the βTD domain by binding of the identified test compound to the βA domain non-ligand binding site of the integrin, and
- (e) identifying the screened test compound as a compound capable of selectively modulating the activity of an integrin from the pool of test compounds. In various embodiments:
- the modulating comprises inhibiting the integrin activation and the modulating comprises inhibiting ligand binding to the integrin.
A method of identifying a candidate inhibitor of the activity of an integrin, the method comprising:
- (a) introducing into a suitable computer program information defining a non-ligand binding site of an integrin βA domain, the information comprising a conformation defined by the coordinate atoms as in Table 1 or Table 2 or both as defined in FIGS. 9 and 10, wherein the program displays the three-dimensional structure thereof;
- (b) creating a three dimensional structure of a test compound in the computer program;
- (c) superimposing the model of the test compound on the model of the non-ligand binding site of the integrin βA domain;
- and
- (d) assessing whether the test compound model fits spatially into the non-ligand binding site.
A method for identifying a candidate integrin modulating compound, the method comprising:
- (a) generating a three-dimensional structure of the βTD contact region of strand-F/α7 loop of an βA domain of a non-ligand bound integrin;
- (b) employing the three dimensional structure to design or select the candidate integrin modulating compound, and
- (c) identifying said candidate integrin modulating compound by the data obtained by steps (a) and (b).
A method for identifying a candidate integrin modulating compound, the method comprising:
- (a) expressing recombinant integrin fragments containing βA domain and βTD domains;
- and
- (b) employing the protein-protein interaction of these two domains to in screening assays to identify modulators of the βA domain and βTD domain interaction.
A method of inhibiting activation of an integrin the method comprising contacting a compound with an integrin thereby locking the βA domain structure of the integrin into a non-activatable form.
A method of identifying an integrin modulator comprising:
- (a) selecting a potential inhibitor by performing rational drug design with the three-dimensional structural coordinates of Table 1 or Table 2 or both as define in FIGS. 9 and 10, wherein selecting is performed in conjunction with computer modeling;
- (b) contacting the potential inhibitor with an integrin domain;
- and
- (c) detecting the ability of the potential inhibitor for inhibiting the integrin.
A pharmaceutical compositions comprising such compounds and a pharmaceutically acceptable carrier.
A method for modulating, inhibiting or stimulating binding of ligands or associated proteins to integrins by modifying the interaction of integrin beta-A domain (βA) with the beta-tail domain (βTD).
A method for evaluating the ability of a compound to associate with a molecule or molecular complex comprising a non-ligand binding site of an integrin βA domain, the method comprising:
- (a) constructing a computer model of the binding site defined by structure coordinates wherein the root mean square deviation between the structure coordinates and the structure coordinates of the amino acids of Table 1 or Table 2 according to FIGS. 9 and 10 is not more than about 4.0 Å;
- (b) selecting a compound to be evaluated by a method selected from the group consisting of (i) assembling molecular fragments into the compound, (ii) selecting a compound from a small molecule database, (iii) de novo ligand design of the compound, and (iv) modifying a known inhibitor, or a portion thereof, of a protein kinase;
- (c) employing computational means to perform a fitting program operation between computer models of the compound to be evaluated and the binding site in order to provide an energy-minimized configuration of the compound in the binding site;
- and
- (d) evaluating the results of the fitting operation to quantify the association between the said compound and the binding site, whereby evaluating the ability of the compound to associate with the binding site.
A method for identifying an activator or inhibitor of an integrin a non-ligand binding site of an integrin βA domain, the method comprising:
- (a) constructing a computer model of the binding site defined by structure coordinates wherein the root mean square deviation between the structure coordinates and the structure coordinates of the amino acids of Table 1 or Table 2 according to FIGS. 9 and 10 is not more than about 4.0 Å;
- (b) selecting a compound to be evaluated as a potential activator or inhibitor by a method selected from the group consisting of (i) assembling molecular fragments into the compound, (ii) selecting the compound from a small molecule database, (iii) de novo ligand design of the compound, and (iv) modifying a known inhibitor, or a portion thereof, of an integrin;
- (c) employing computational means to perform a fitting program operation between computer models of the compound to be evaluated and the binding site in order to provide an energy-minimized configuration of the compound in the binding site;
- (d) evaluating the results of the fitting operation to quantity the association between the compound and the binding site model, whereby evaluating the ability of the the compound to associate with the said binding pocket;
- (e) providing the compound;
- and
- (f) contacting the compound with the molecule to determine the ability of the compound to activate or inhibit the molecule.
Dependencies are inferred from the claim text.