Compounds and methods for modulating non-transcriptional effects of steroid hormones

Abstract

This invention provides new methods and compounds for controlling the intracellular and physiological effects of steroid hormones, including but not limited to estrogen, through modulation of the interaction of such hormone receptors with phosphatidylinositol-3 kinase. Compounds and methods for controlling the activation of endothelial nitric oxide synthase are also disclosed.

What is claimed is:  
     
         1 . A method of modulating the physiological effects induced by binding of a steroid hormone to a steroid hormone receptor, wherein said effects are mediated by non-transcriptional, non-MAP kinase intracellular messengers, said method comprising contacting said cell with an agonist or antagonist of the steroid hormone receptor, or with a compound sufficient to enhance or attenuate activation of said intracellular messengers by said steroid hormone receptor induced by binding of said steroid hormone to said steroid hormone receptor.  
     
     
         2 . The method according to  claim 1  wherein said intracellular messenger is phosphatidylinositol-3 kinase.  
     
     
         3 . The method according to  claim 2  which comprises contacting said cell with a ligand in a sufficient amount to induce activation or deactivation of said kinase.  
     
     
         4 . The method according to  claim 3  wherein said hormone is estrogen.  
     
     
         5 . The method according to  claim 4  which comprises contacting said cell with an estrogen receptor agonist or antagonist.  
     
     
         6 . The method according to  claim 4  which comprises contacting said cell with a phosphoinositide kinase activator or inhibitor.  
     
     
         7 . A method of modulating a disease in a human or non-human animal associated with activation of phosphoinositide 3-kinase by binding of a steroid hormone to a steroid hormone receptor which comprises administering to said human or non-human animal (a) an inhibitor of the binding of said steroid hormone to its receptor; (b) an inhibitor of the activation of said phosphoinositide 3-kinase by said steroid hormone receptor; (c) an inhibitor of said phosphoinositide 3-kinase; or (d) combinations of (a), (b), and (c).  
     
     
         8 . The method according to  claim 2  wherein said phosphoinositide 3-kinase activates the Akt kinase wherein said method comprises contacting said cell with an agonist or antagonist of said Akt kinase, or contacting said cell with a compound sufficient to disrupt activation of said Akt kinase by said phosphoinositide 3-kinase.  
     
     
         9 . A method of modulating a disease in a human or non-human animal associated with activation of Akt kinase by binding of a steroid hormone to a steroid hormone receptor which comprises administering to said human or non-human animal (a) an inhibitor of the binding of said steroid hormone to its receptor; (b) an inhibitor of the activation of phosphoinositide 3-kinase by said steroid hormone receptor; (c) an inhibitor of phosphoinositide 3-kinase; (d) an inhibitor of the activation of said Akt kinase; (e) an inhibitor of Akt kinase; or (f) combinations of (a)-(e).  
     
     
         10 . The method according to  claim 8  wherein said Akt kinase induces activation of nitric oxide synthase, wherein said method comprises contacting said cell with a compound to activate or inhibit the activity of said nitric oxide synthase or to enhance or inhibit activation of said nitric oxide synthase by said Akt kinase.  
     
     
         11 . A method of modulating a disease in a human or non-human animal associated with activation nitric oxide synthase by binding of a steroid hormone to a steroid hormone receptor which comprises administering to said human or non-human animal (a) an inhibitor of the binding of said steroid hormone to its receptor; (b) an inhibitor of the activation of phosphoinositide 3-kinase by said steroid hormone receptor; (c) an inhibitor of phosphoinositide 3-kinase; (d) an inhibitor of the activation of Akt kinase; (e) an inhibitor of Akt kinase; (f) an inhibitor of the activation of said nitric oxide synthase; (g) an inhibitor of said nitric oxide synthase; or (h) combinations of (a)-(g).  
     
     
         12 . A method of identifying a compound which modulates the non-transcriptional, non-MAP-kinase induced effects of a steroid hormone which comprises contacting cells with a range of concentrations of a test compound, and measuring the effect of each concentration of said test compound on (a) the concentration of intracellular nitric oxide concentration, (b) the activity of nitric oxide synthase, (c) the activity of Akt kinase, (d) the activity of phosphatidylinositol-3-kinase, (e) the interaction of a steroid hormone receptors and phosphatidylinositol-3-kinase, or (f) combinations of the measurements defined in (a)-(e).  
     
     
         13 . The method according to  claim 12  wherein said steroid hormone is estrogen.  
     
     
         14 . The method according to  claim 13  which comprises contacting cells with estrogen or an estrogen analog in the presence of a range of concentrations of a test compound.  
     
     
         15 . A method for modulating a disease condition affected by binding of a steroid hormone with a steroid hormone receptor which comprises contacting cells in a human or non-human animal with a physiologically effective amount of a compound identified by means of the method of  claim 12 .  
     
     
         16 . The method according to  claim 15  wherein said disease condition is selected from the group consisting of cardiovascular disease, cancer, osteoporosis, Alzheimer's disease, obesity, and diabetes.  
     
     
         17 . The method according to  claim 16  which comprises contacting a cell with a sufficient quantity of said compound to induce an increase in endothelial nitric oxide synthase activity.  
     
     
         18 . The method according to  claim 16  which comprises contacting a cell with a sufficient quantity of estrogen or an estrogen analog to induce binding of the estrogen receptor with phosphatidylinositol-3 kinase.  
     
     
         19 . The method according to  claim 15  wherein said cell is a constituent of a tissue selected from the group consisting of cardiovascular, neuronal, skeletal, muscular, and reproductive tissue.  
     
     
         20 . A method of modulating a disease affected by binding of steroid hormone to steroid hormone receptor which comprises treating a human or non-human patient in need thereof with an effective amount of a compound sufficient to elicit a change in the intracellular concentration of phosphatidylinositol-3 kinase or nitric oxide, or to alter the activation state of phosphoinositide 3-kinase, Akt kinase, nitric oxide synthase, or combinations thereof.  
     
     
         21 . A method of modulating the physiological effect of a steroid hormone effected through the level of intracellular nitric oxide synthase activity which comprises contacting a human or non-human patient in need thereof with an agonist or antagonist of phosphatidylinositol-3 kinase.  
     
     
         22 . The method according to  claim 1  wherein said physiological effect to be modulated is the relaxation state of the vasculature of a human or non-human patient.  
     
     
         23 . A method for identifying compounds effective in the alteration of the relaxation state of the vasculature which comprises: 
 (a) isolating and growing endothelial cells in culture;    (b) contacting said cells with a test compound;    (c) measuring the level of activity of endothelial nitric oxide synthase in the presence of said test compound as compared with the level of activity of endothelial nitric oxide synthase in the absence of said test compound.    
     
     
         24 . A compound which specifically modifies the interaction of the estrogen receptor with phosphatidylinositol-kinase.  
     
     
         25 . A compound which specifically modifies wortmannin-sensitive endothelial nitric oxide synthase activation induced by binding of a steroid hormone to its receptor.  
     
     
         26 . A method for modifying wortmannin-sensitive endothelial nitric oxide synthase activation in a cell which comprises contacting said cell with a steroid hormone, a steroid hormone agonist, a steroid hormone antagonist analog, a PI3-kinase activator, a PI3-kinase inhibitor, an Akt activator, an Akt inhibitor, and combinations thereof.  
     
     
         27 . The method according to  claim 26  wherein said compound is estrogen, an estrogen receptor antagonist, or an estrogen receptor agonist.  
     
     
         28 . A method for modulating PI3-kinase effects on metabolic functions in different tissues and cell types selected from cellular uptake of glucose, regulation of the membrane translocation of the glucose transporter, GLUT4, control of glycogen synthesis and lipolytic process, regulation of growth factor-induced cell growth and proliferation, and cellular apoptotic processes, which comprises contacting a cell with a steroid hormone receptor agonist or antagonist.  
     
     
         29 . The method according to  claim 28  wherein said steroid hormone is estrogen, an estrogen receptor agonist, or an estrogen receptor antagonist.  
     
     
         30 . The method according to  claim 29  for treating vascular and non-vascular diseases.  
     
     
         31 . A method of modulating the physiological effects induced by binding of a steroid hormone to a steroid hormone receptor, wherein said effects are mediated by non-transcriptional, non-MAP kinase intracellular messengers, said effects excluding nuclear transcriptional events, said method comprising contacting said cell with an agonist or antagonist of the steroid hormone receptor, or with a compound sufficient to enhance or attenuate activation of said intracellular messengers by said steroid hormone receptor induced by binding of said steroid hormone to said steroid hormone receptor.  
     
     
         32 . The method according to  claim 31  wherein said intracellular messenger is phosphatidylinositol-3 kinase.  
     
     
         33 . The method according to  claim 32  which comprises contacting said cell with a ligand in a sufficient amount to induce activation or deactivation of said kinase.  
     
     
         34 . The method according to  claim 33  wherein said hormone is estrogen.  
     
     
         35 . The method according to  claim 34  which comprises contacting said cell with an estrogen receptor agonist or antagonist.  
     
     
         36 . The method according to  claim 34  which comprises contacting said cell with a phosphoinositide kinase activator or inhibitor.  
     
     
         37 . A method of modulating a disease in a human or non-human animal associated with binding of a steroid hormone to a steroid hormone receptor, wherein said effects are mediated by non-transcriptional, non-MAP kinase intracellular messengers, said effects excluding nuclear transcriptional events, and wherein said disease is associated with activation of phosphoinositide 3-kinase by binding of a steroid hormone to a steroid hormone receptor which comprises administering to said human or non-human animal (a) an inhibitor or activator of the binding of said steroid hormone to its receptor; (b) an inhibitor or activator of the activation of said phosphoinositide 3-kinase by said steroid hormone receptor; (c) an inhibitor or activator of said phosphoinositide 3-kinase; or (d) combinations of (a), (b), and (c).  
     
     
         38 . The method according to  claim 32  wherein said phosphoinositide 3-kinase activates the Akt kinase wherein said method comprises contacting said cell with an agonist or antagonist of said Akt kinase, or contacting said cell with a compound sufficient to disrupt activation of said Akt kinase by said phosphoinositide 3-kinase.  
     
     
         39 . A method of modulating a disease in a human or non-human animal associated with binding of a steroid hormone to a steroid hormone receptor, wherein said effects are mediated by non-transcriptional, non-MAP kinase intracellular messengers, said effects excluding nuclear transcriptional events, and wherein said disease is associated with activation of Akt kinase by binding of a steroid hormone to a steroid hormone receptor which comprises administering to said human or non-human animal (a) an inhibitor or activator of the binding of said steroid hormone to its receptor; (b) an inhibitor or activator of the activation of phosphoinositide 3-kinase by said steroid hormone receptor; (c) an inhibitor or activator of phosphoinositide 3-kinase; (d) an inhibitor or activator of the activation of said Akt kinase; (e) an inhibitor or activator of Akt kinase; or (f) combinations of (a)-(e).  
     
     
         40 . The method according to  claim 38  wherein said Akt kinase induces activation of nitric oxide synthase, wherein said method comprises contacting said cell with a compound to activate or inhibit the activity of said nitric oxide synthase or to enhance or inhibit activation of said nitric oxide synthase by said Akt kinase.  
     
     
         41 . A method of modulating a disease in a human or non-human animal associated with binding of a steroid hormone to a steroid hormone receptor, wherein said effects are mediated by non-transcriptional, non-MAP kinase intracellular messengers, said effects excluding nuclear transcriptional events, and wherein said disease is associated with activation nitric oxide synthase by binding of a steroid hormone to a steroid hormone receptor which comprises administering to said human or non-human animal (a) an inhibitor or activator of the binding of said steroid hormone to its receptor; (b) an inhibitor or activator of the activation of phosphoinositide 3-kinase by said steroid hormone receptor; (c) an inhibitor or activator of phosphoinositide 3-kinase; (d) an inhibitor or activator of the activation of Akt kinase; (e) an inhibitor or activator of Akt kinase; (f) an inhibitor or activator of the activation of said nitric oxide synthase; (g) an inhibitor or activator of said nitric oxide synthase; or (h) combinations of (a)-(g).  
     
     
         42 . A method of identifying a compound which modulates the non-transcriptional, non-MAP-kinase induced effects of a steroid hormone which comprises contacting cells with a range of concentrations of a test compound, and measuring the effect of each concentration of said test compound on (a) the concentration of intracellular nitric oxide concentration, (b) the activity of nitric oxide synthase, (c) the activity of Akt kinase, (d) the activity of phosphatidylinositol-3-kinase, (e) the interaction of a steroid hormone receptors and phosphatidylinositol-3-kinase, or (f) combinations of the measurements defined in (a)-(e), and selecting a compound which either has no modulatory effect on said non-transcriptional, non-MAP kinase induced effects, acting exclusively through transcriptionally mediated means, or which acts exclusively through non-transcriptional, non-MAP kinase mediated means, or which acts through any known blend of said means.  
     
     
         43 . The method according to  claim 42  wherein said steroid hormone is estrogen.  
     
     
         44 . The method according to  claim 43  which comprises contacting cells with estrogen or an estrogen analog in the presence of a range of concentrations of a test compound.  
     
     
         45 . A method for modulating a disease condition affected by binding of a steroid hormone with a steroid hormone receptor which comprises contacting cells in a human or non-human animal with a physiologically effective amount of a compound identified by means of the method of  claim 42 .  
     
     
         46 . The method according to  claim 45  wherein said disease condition is selected from the group consisting of cardiovascular disease, cancer, osteoporosis, Alzheimer's disease, obesity, and diabetes.  
     
     
         47 . The method according to  claim 46  which comprises contacting a cell with a sufficient quantity of said compound to induce an increase in endothelial nitric oxide synthase activity.  
     
     
         48 . The method according to  claim 46  which comprises contacting a cell with a sufficient quantity of estrogen or an estrogen analog to induce binding of the estrogen receptor with phosphatidylinositol-3 kinase.  
     
     
         49 . The method according to  claim 45  wherein said cell is a constituent of a tissue selected from the group consisting of cardiovascular, neuronal, skeletal, muscular, and reproductive tissue.