Methods of identifying modulators of the estrogen receptor
First Claim
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1. A method of selecting for an agent which binds to the estrogen receptor but which dissipates a transmembrane pH gradient to a lesser extent than does tamoxifen, wherein the method comprises:
- (a) choosing an agent which binds to the estrogen receptor, (b) choosing a vesicle and conditions wherein the pH inside the vesicle differs from the pH outside the vesicle, whereby a transmembrane pH gradient is formed, (c) employing conditions under which tamoxifen dissipates the transmembrane pH gradient in the vesicle of step (b), and, in the absence of tamoxifen, contacting the agent of step (a) with the vesicle of step (b), (d) measuring any change in the magnitude of the transmembrane pH gradient of the vesicle when contacted with the agent, and comparing the change measured to that seen when the vesicle is separately contacted with tamoxifen, (e) selecting for the agent when the comparison of step (d) indicates that the agent dissipates the transmembrane pH gradient to a lesser extent than does tamoxifen.
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Abstract
The present invention discloses the mechanism of tamoxifen mediated inhibition of acidification. This mechanism information is then used to design methods of identifying compounds that will have the pharmaceutical effect of tamoxifen without the corresponding side-effects.
27 Citations
26 Claims
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1. A method of selecting for an agent which binds to the estrogen receptor but which dissipates a transmembrane pH gradient to a lesser extent than does tamoxifen, wherein the method comprises:
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(a) choosing an agent which binds to the estrogen receptor, (b) choosing a vesicle and conditions wherein the pH inside the vesicle differs from the pH outside the vesicle, whereby a transmembrane pH gradient is formed, (c) employing conditions under which tamoxifen dissipates the transmembrane pH gradient in the vesicle of step (b), and, in the absence of tamoxifen, contacting the agent of step (a) with the vesicle of step (b), (d) measuring any change in the magnitude of the transmembrane pH gradient of the vesicle when contacted with the agent, and comparing the change measured to that seen when the vesicle is separately contacted with tamoxifen, (e) selecting for the agent when the comparison of step (d) indicates that the agent dissipates the transmembrane pH gradient to a lesser extent than does tamoxifen. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
(f) measuring a surface membrane charge of the vesicle before contact with either the agent or tamoxifen, (g) measuring a surface membrane charge of the vesicle after contact with either tamoxifen or the agent, and comparing the results, (h) selecting for the agent if the agent meets the conditions of step (e) and if the agent alters the surface membrane charge of the vesicle to a lesser extent than does tamoxifen.
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11. A method of selecting for an agent which binds the estrogen receptor but which alters a surface membrane charge to a lesser extent than does tamoxifen, comprising:
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(a) choosing an agent which binds to the estrogen receptor, (b) selecting a vesicle and conditions under which tamoxifen is known to alter a surface membrane charge of the vesicle, (c) in the absence of tamoxifen, contacting the agent of step (a) with the vesicle under the conditions of step (b), (d) measuring any alteration in the surface membrane charge of the vesicle upon contact with the agent, and comparing the measured alteration to that seen when the vesicle is separately contacted with tamoxifen, (e) selecting an agent when the alteration in the surface membrane charge in the presence of the agent is less than the alteration in the presence of tamoxifen. - View Dependent Claims (12, 13)
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14. A method of selecting for an agent which is an antagonist of the estrogen receptor but which dissipates a transmembrane pH gradient to a lesser extent than does tamoxifen, wherein the method comprises:
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(a) choosing an agent which is an antagonist of the estrogen receptor, (b) choosing a vesicle and conditions wherein the pH inside the vesicle differs from the pH outside the vesicle, whereby a transmembrane pH gradient is formed, (c) employing conditions under which tamoxifen dissipates the transmembrane pH gradient in the vesicle of step (b), and in the absence of tamoxifen, contacting the agent of step (a) with the vesicle of step (b), (d) measuring any change in the magnitude of the transmembrane pH gradient of the vesicle when contacted with the agent, and comparing the change measured to that seen when the vesicle is separately contacted with tamoxifen, (e) selecting for the agent when the comparison of step (d) indicates that the agent dissipates the transmembrane pH gradient to a lesser extent than does tamoxifen. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23)
(f) measuring a surface membrane charge of the vesicle before contact with either the agent or tamoxifen, (g) measuring a surface membrane charge of the vesicle after contact with either tamoxifen or the agent, and comparing the results, (h) selecting for the agent if the agent meets the conditions of step (e) and if the agent alters the surface membrane charge of the vesicle to a lesser extent than does tamoxifen.
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24. A method of selecting for an agent is an antagonist of the estrogen receptor but which alters a surface membrane charge to a lesser extent than does tamoxifen, comprising:
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(a) choosing an agent which is an antagonist of the estrogen receptor, (b) selecting a vesicle and conditions under which tamoxifen is known to alter a surface membrane charge of the vesicle, (c) in the absence of tamoxifen, contacting the agent of step (a) with the vesicle under the conditions of step (b), (d) measuring any alteration in the surface membrane charge of the vesicle upon contact with the agent, and comparing the measured alteration to that seen when the vesicle is separately contacted with tamoxifen, (e) selecting an agent when the alteration in the surface membrane charge in the presence of the agent is less than the alteration in the presence of tamoxifen. - View Dependent Claims (25, 26)
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Specification