Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

Abstract

The mechanism by which the androgen receptor (AR) distinguishes between agonist and antagonist ligands is poorly understood. AR antagonists are currently used to treat prostate cancer. However, mutations commonly develop in patients that convert these compounds to agonists. Recently, our laboratory discovered selective androgen receptor modulators, which structurally resemble the nonsteroidal AR antagonists bicalutamide and hydroxyflutamide but act as agonists for the androgen receptor in a tissue-selective manner. To investigate why subtle structural changes to both the ligand and the receptor (i.e. mutations) result in drastic changes in activity, we studied structure-activity relationships for nonsteroidal AR ligands through crystallography and site-directed mutagenesis, comparing bound conformations of R-bicalutamide, hydroxyflutamide, and two previously reported nonsteroidal androgens, S-1 and R-3. These studies provide the first crystallographic evidence of the mechanism by which nonsteroidal ligands interact the that changes to the of and for ligand the AR and that a to the of and the of hydroxyflutamide is bound to the AR we that in AR the a these studies provide for of nonsteroidal AR agonists and The mechanism by which the androgen receptor (AR) distinguishes between agonist and antagonist ligands is poorly understood. AR antagonists are currently used to treat prostate cancer. However, mutations commonly develop in patients that convert these compounds to agonists. Recently, our laboratory discovered selective androgen receptor modulators, which structurally resemble the nonsteroidal AR antagonists bicalutamide and hydroxyflutamide but act as agonists for the androgen receptor in a tissue-selective manner. To investigate why subtle structural changes to both the ligand and the receptor (i.e. mutations) result in drastic changes in activity, we studied structure-activity relationships for nonsteroidal AR ligands through crystallography and site-directed mutagenesis, comparing bound conformations of R-bicalutamide, hydroxyflutamide, and two previously reported nonsteroidal androgens, S-1 and R-3. These studies provide the first crystallographic evidence of the mechanism by which nonsteroidal ligands interact the that changes to the of and for ligand the AR and that a to the of and the of hydroxyflutamide is bound to the AR we that in AR the a these studies provide for of nonsteroidal AR agonists and The androgen receptor (AR) used used is a receptor in of prostate and and in ligands for the AR the and compounds for the AR provide in the of and androgen AR ligands are commonly used in the of prostate cancer. The nonsteroidal and are of and of to the AR that to by the compounds to agonists and a currently prostate that the AR AR mutations for hydroxyflutamide of as that these ligands the AR by the of the that the of the AR ligand in AR bound to a AR mutations to to result in agonist for as as AR antagonists in the are for the of androgen The of nonsteroidal AR agonists by structural of bicalutamide and reported to the bicalutamide as in the AR ligand of these the bound of in the and structurally selective androgen receptor in the AR The of the the of nonsteroidal selective androgen receptor in the by the of in the our to of the AR to to the of the AR bound to agonists that resemble bicalutamide and in we the first evidence as to nonsteroidal ligands are in the AR by changes to the of in the the of bound to the we evidence that a for and in bicalutamide to mutations to and by a AR by and and the in the and the the to the AR and as previously The AR as a in for by in a and by of The for and ligand and The in a and the AR the The in and and a of in the The in a to a and and to and in the in and for the and in and these a and to to and in for The by a in to in AR to a of and a and The and The The used as a for and of for of the and the and of and of in and and to in of the of by The and of a of and of in the to and to and and by of for of the to and of of a the of the to a of a a to for in in as the (i.e. of AR to in in a as receptor of AR bound to agonist ligands is and of and through The AR is by in the of antagonist compounds and that AR is as a result of receptor mutations to the AR that to a to crystallographic evidence for antagonist conformations to the AR and provide as to the in the AR to AR bound to the agonists S-1 and a of The of AR in the and AR of the AR in these as to for the of and by in of and in the the and we that the we of the by for and the that we in the and for in the are in of is The is of the that are the is The is of the that are the in a of in the is of that by the of a to a which a of a in a in to the AR However, agonist for the AR (i.e. The of bound to the AR a bound as in the and the of and by of a a and the the of However, is the in the for The of is to the of and the of is for and the of to the in the is as in the which is bound AR The which is in the the by and as in by the in to as the and which the of the of a in to the and of is in the AR by and in the and AR to but and changes in in AR of AR and the the in the the AR AR the are by that and the of R-3. and R-3. the and the for in the the but is the the S-1 in in the the AR the S-1 to the AR are in S-1 as R-3. the of the S-1 as to the S-1 to the and of and of the to that in the the for the as the of of of the S-1 of in is agonist for the AR that tissue-selective in and a of AR ligands as selective androgen receptor S-1 structurally but a in of the S-1 in the and is that S-1 agonist these structural in the AR The of the and that S-1 to the and of R-3. the S-1 and the to of the by the of S-1 in the AR and The is between the of and by and the of S-1 is to that of in the AR ligand R-bicalutamide, of The of S-1 is the to the of between the and the in in the between and nonsteroidal bound AR is the The of and a to and the nonsteroidal AR ligands S-1 and a and the is in the a and the the agonist of S-1 and S-1 as in the The in is to the of the to as in the S-1 the in the as in the between the and to the of S-1 are by to the in the a in the that in the AR to to the in the the of S-1 to the of by a to the as the is in the and in AR bound to nonsteroidal of ligand in the and the The of a The of is in the the of in to the S-1 bound to the the a is that to the of and the of The the to is in the the the for and by the in a of the for to of the is for of AR ligands the The of the of the of and to as which the The in the and is in the as in the AR to the the to the to between the and a and changes to and in and as of the changes by and S-1 to the the of in the by the which the to to in The is in and the the the in of the to of the S-1 changes in the of in the AR bound to S-1 and the the in the for the of in the the is between the and the of that the of the in the by the of in the of AR which is to to bicalutamide and a in is of the in of in the in that between the and the for the of to the agonist of S-1 in the that the the a in S-1 The compounds and for and as to bicalutamide and which to to AR bicalutamide in mutations to the result of in to to the bicalutamide of the and that the of these ligands the in receptor The that the of S-1 and is in a the AR is bound to agonists The of R-bicalutamide, in of in the the by the in the the in S-1 to a in of S-1 in the agonist AR S-1 in in the as the However, the that is the to the of that the is of S-1 in the is in the and mutations and is as a result of between the of the and the to the between the and the in the for S-1 to the in the The antagonist of to of by the To we the of to in the to in the AR discovered in patients and to bicalutamide to mutations to to in the of to in the the that of is for the antagonist of AR to the AR and resemble the in receptor to agonist ligands The of and in bound to antagonist compounds in which is the is to the AR is is to in the of a and agonist for the AR evidence that ligand the as agonists that the the AR However, our to AR to to to the and S-1 are structurally AR to that AR which agonist to these antagonist and the as to agonists that subtle structural AR is a result of the of AR ligands to a which of the for result is that AR agonists the structural through and which that in the and androgens, and to and the the agonist of the nonsteroidal AR ligands and that in a between S-1 which is in the that a in AR by of a between the of the a and the of in the by to receptor to the of the S-1 of the to and of is to to the However, the of the of in the AR S-1 to and which the of to receptor why S-1 and R-bicalutamide, but the AR in the of is that the the of S-1 and R-bicalutamide, which is of a that to a the to of of compounds the of in AR The of the the of S-1 and to in the of The to that in the between the of and for of S-1 in the AR and in the and AR mutations to that interact the ligand and convert antagonists agonists provide as to antagonists AR The studies are the first to the of ligands AR The of and the of and of that of and the ligand is in agonist in the AR a and is the of the AR which and The of to agonist in the to the result of of which by the the of to in AR to the result of the of the ligand to a that between nonsteroidal ligands and studies are to the of the and AR and the for and AR to S-1 the to and of the structural of the AR the and and of the structural of the AR the of of the of in the and that to the ligand as a result of the of the of R-bicalutamide, which the to The androgen receptor (AR) used used is a receptor in of prostate and and in ligands for the AR the and compounds for the AR provide in the of and androgen AR ligands are commonly used in the of prostate cancer. The nonsteroidal and are of and of to the AR that to by the compounds to agonists and a currently prostate that the AR AR mutations for hydroxyflutamide of as that these ligands the AR by the of the that the of the AR ligand in AR bound to a AR mutations to to result in agonist for as as AR antagonists in the are for the of androgen The of nonsteroidal AR agonists by structural of bicalutamide and androgen receptor hydroxyflutamide ligand androgen receptor hydroxyflutamide ligand To reported to the bicalutamide as in the AR ligand of these the bound of in the and structurally selective androgen receptor in the AR The of the the of nonsteroidal selective androgen receptor in the by the of in the our to of the AR to to the of the AR bound to agonists that resemble bicalutamide and in we the first evidence as to nonsteroidal ligands are in the AR by changes to the of in the the of bound to the we evidence that a for and in bicalutamide to mutations to and by a AR by and and the in the and the the to the AR and as previously The AR as a in for by in a and by of The for and ligand and The in a and the AR the The in and and a of in the The in a to a and and to and in the in and for the and in and these a and to to and in for The by a in to in AR to a of and a and The and The The used as a for and of for of the and the and of and of in and and to in of the of by The and of a of and of in the to and to and and by of for of the to and of of a the of the to a of a a to for in in as the (i.e. and by a AR by and and the in the and the the to the AR and as previously The AR as a in for by in a and by of The for and ligand and The in a and the AR the The in and and a of in the The in a to a and and to and in the in and for the and in and these a and to to and in for The by a in to in AR to a of and a and The and The The used as a for and of for of the and the and of and of in and and to in of the of by The and of a of and of in the to and to and and by of for of the to and of of a the of the to a of a a to for in in as the (i.e. of AR to in in a as receptor of AR bound to agonist ligands is and of and through The AR is by in the of antagonist compounds and that AR is as a result of receptor mutations to the AR that to a to crystallographic evidence for antagonist conformations to the AR and provide as to the in the AR to AR bound to the agonists S-1 and a of The of AR in the and AR of the AR in these as to for the of and by in of and in the the and we that the we of the by for and the that we in the and for in the are in of is The is of the that are the is The is of the that are the in a of in the is of that by the of a to a which a of a in a in to the AR However, agonist for the AR (i.e. The of bound to the AR a bound as in the and the of and by of a a and the the of However, is the in the for The of is to the of and the of is for and the of to the in the is as in the which is bound AR The which is in the the by and as in by the in to as the and which the of the of a in to the and of is in the AR of AR and the the in the the AR AR the are by that and the of R-3. and R-3. the and the for in the the but is the the S-1 in in the the AR the S-1 to the AR are in S-1 as R-3. the of the S-1 as to the S-1 to the and of and of the to that in the the for the as the of of of the S-1 of in is agonist for the AR that tissue-selective in and a of AR ligands as selective androgen receptor S-1 structurally but a in of the S-1 in the and is that S-1 agonist these structural in the AR The of the and that S-1 to the and of R-3. the S-1 and the to of the by the of S-1 in the AR and The is between the of and by and the of S-1 is to that of in the AR ligand R-bicalutamide, of The of S-1 is the to the of between the and the in in the between and nonsteroidal bound AR is the The of and a to and the nonsteroidal AR ligands S-1 and a and the is in the a and the the agonist of S-1 and S-1 as in the The in is to the of the to as in the S-1 the in the as in the between the and to the of S-1 are by to the in the a in the that in the AR to to the in the the of S-1 to the of by a to the as the is in the and in AR bound to nonsteroidal of ligand in the and the The of a The of is in the the of in to the S-1 bound to the the a is that to the of and the of The the to is in the the the for and by the in a of the for to of the is for of AR ligands the The of the of the of and to as which the The in the and is in the as in the AR to the the to the to between the and a and changes to and in and as of the changes by and S-1 to the the of in the by the which the to to in The is in and the the the in of the to of the S-1 changes in the of in the AR bound to S-1 and the the in the for the of in the the is between the and the of that the of the in the by the of in the of AR which is to to bicalutamide and a in is of the in of in the in that between the and the for the of to the agonist of S-1 in the that the the a in S-1 The compounds and for and as to bicalutamide and which to to AR bicalutamide in mutations to the result of in to to the bicalutamide of the and that the of these ligands the in receptor The that the of S-1 and is in a the AR is bound to agonists The of R-bicalutamide, in of in the the by the in the the in S-1 to a in of S-1 in the agonist AR S-1 in in the as the However, the that is the to the of that the is of S-1 in the is in the and mutations and is as a result of between the of the and the to the between the and the in the for S-1 to the in the The antagonist of to of by the To we the of to in the to in the AR discovered in patients and to bicalutamide to mutations to to in the of to in the the that of is for the antagonist of of AR to in in a as receptor of AR bound to agonist ligands is and of and through The AR is by in the of antagonist compounds and that AR is as a result of receptor mutations to the AR that to a to crystallographic evidence for antagonist conformations to the AR and provide as to the in the AR to AR bound to the agonists S-1 and a of The of AR in the and AR of the AR in these as to for the of and by in of and in the the and we that the we of the by for and the that we in the of in the is of that by the of a to a which a of a in a in to the AR However, agonist for the AR (i.e. The of bound to the AR a bound as in the and the of and by of a a and the the of However, is the in the for The of is to the of and the of is for and the of to the in the is as in the which is bound AR The which is in the the by and as in by the in to as the and which the of the of a in to the and of is in the AR of in is agonist for the AR that tissue-selective in and a of AR ligands as selective androgen receptor S-1 structurally but a in of the S-1 in the and is that S-1 agonist these structural in the AR The of the and that S-1 to the and of R-3. the S-1 and the to of the by the of S-1 in the AR and The is between the of and by and the of S-1 is to that of in the AR ligand R-bicalutamide, of The of S-1 is the to the of between the and the in in the between and nonsteroidal bound AR is the The of and a to and the nonsteroidal AR ligands S-1 and a and the is in the a and the the agonist of S-1 and S-1 as in the The in is to the of the to as in the S-1 the in the as in the between the and to the of S-1 are by to the in the a in the that in the AR to to the in the the of S-1 to the of by a to the as the is in the and in AR bound to nonsteroidal to of the is for of AR ligands the The of the of the of and to as which the The in the and is in the as in the AR to the the to the to between the and a and The AR which is to to bicalutamide and a in is of the in of in the in that between the and the for the of to the agonist of S-1 in the that the the a in S-1 The compounds and for and as to bicalutamide and which to to AR bicalutamide in mutations to the result of in to to the bicalutamide of the and that the of these ligands the in receptor The that the of S-1 and is in a the AR is bound to agonists The of R-bicalutamide, in of in the the by the in the the in S-1 to a in of S-1 in the agonist AR S-1 in in the as the However, the that is the to the of that the is The of S-1 in the is in the and mutations and is as a result of between the of the and the to the between the and the in the for S-1 to the in the The antagonist of to of by the To we the of to in the to in the AR discovered in patients and to bicalutamide to mutations to to in the of to in the the that of is for the antagonist of AR to the AR and resemble the in receptor to agonist ligands The of and in bound to antagonist compounds in which is the is to the AR is is to in the of a and agonist for the AR evidence that ligand the as agonists that the the AR However, our to AR to to to the and S-1 are structurally AR to that AR which agonist to these antagonist and the as to agonists that subtle structural AR is a result of the of AR ligands to a which of the for result is that AR agonists the structural through and which that in the and androgens, and to and the the agonist of the nonsteroidal AR ligands and that in a between S-1 which is in the that a in AR by of a between the of the a and the of in the by to receptor to the of the S-1 of the to and of is to to the However, the of the of in the AR S-1 to and which the of to receptor why S-1 and R-bicalutamide, but the AR in the of is that the the of S-1 and R-bicalutamide, which is of a that to a the to of of compounds the of in AR The of the the of S-1 and to in the of The to that in the between the of and for of S-1 in the AR and in the and AR mutations to that interact the ligand and convert antagonists agonists provide as to antagonists AR The studies are the first to the of ligands AR The of and the of and of that of and the ligand is in agonist in the AR a and is the of the AR which and The of to agonist in the to the result of of which by the the of to in AR to the result of the of the ligand to a that between nonsteroidal ligands and studies are to the of the and AR and the for AR to the AR and resemble the in receptor to agonist ligands The of and in bound to antagonist compounds in which is the is to the AR is is to in the of a and agonist for the AR evidence that ligand the as agonists that the the AR However, our to AR to to to the and S-1 are structurally AR to that AR which agonist to these antagonist and the as to agonists that subtle structural AR is a result of the of AR ligands to a which of the for result is that AR agonists the structural through and which that in the and androgens, and to and the the agonist of the nonsteroidal AR ligands and that in a between S-1 which is in the that a in AR by of a between the of the a and the of in the by to receptor to the of the S-1 of the to and of is to to the However, the of the of in the AR S-1 to and which the of to receptor why S-1 and R-bicalutamide, but the AR in the of is that the the of S-1 and R-bicalutamide, which is of a that to a the to of of compounds the of in AR The of the the of S-1 and to in the of The to that in the between the of and for of S-1 in the AR and in the and AR AR mutations to that interact the ligand and convert antagonists agonists provide as to antagonists AR The studies are the first to the of ligands AR The of and the of and of that of and the ligand is in agonist in the AR a and is the of the AR which and The of to agonist in the to the result of of which by the the of to in AR to the result of the of the ligand to a that between nonsteroidal ligands and studies are to the of the and AR and the for