Protein crystal of human cytochrome p450 aromatase and uses thereof

Abstract

The present invention relates to a protein crystal of at least one binding site of a human aromatase. The present invention also relates to a fully processed human cytochrome P450 aromatase and a protein crystal thereof. The present invention further relates to methods of making and using the aromatase and the protein crystal thereof.

1 . A crystal comprising at least one ligand binding site of a human aromatase, wherein said human aromatase comprises an amino acid sequence of SEQ ID NO:1. 
     
     
         2 . A crystal according to  claim 1 , wherein the ligand binding site is selected from the group consisting of an active/substrate-binding heme distal site, a heme proximal/P450 reductase coupling site, and both the active/substrate-binding heme distal site and the heme proximal/P450 reductase coupling site. 
     
     
         3 . A crystal according to  claim 2 , wherein said active/substrate-binding heme distal site comprises amino acid residues Arg115 through Phe147, Ile217 through Leu228, Leu301 through Ser314, Pro368 through Arg375, and Ile474 through His480 of SEQ ID NO:1. 
     
     
         4 . A crystal according to  claim 2 , wherein said heme proximal/P450 reductase coupling site comprises amino acid residues Arg145 through Met149, Glu357 through Met364, and Pro423 through Met447 of SEQ ID NO:1. 
     
     
         5 . A crystal according to  claim 1  further comprising an active site access channel comprising amino acid residues Asp186 through Arg193, Gln218 through Leu228, Pro308 through Phe317, Pro368 through Leu372, and Gln472 through Lys485 of SEQ ID NO:1. 
     
     
         6 . A crystal according to  claim 1 , wherein the crystal comprises a space group of P3 2 21 and unit cell parameters of a=b=140.2 Å, c=119.3 Å, α=β=90°, and γ=120°. 
     
     
         7 . A crystal according to  claim 1 , wherein the crystal comprises a three-dimensional structure described by atomic coordinates that substantially conform to atomic coordinates comprising:
 coordinates 553 through 821, 1359 through 1459, 2062 through 2161, 2611 through 2673, and 3468 through 3535 as set forth in Appendix A;   coordinates 1112 through 1179, 1367 through 1459, 2113 through 2191, 2611 through 2647, and 3450 through 3565 as set forth in Appendix A; and/or   coordinates 793 through 840, 2508 through 2578, and 3057 through 3257 as set forth in Appendix A.   
     
     
         8 . A crystal according to  claim 1 , wherein the crystal comprises a three-dimensional structure described by atomic coordinates that substantially conform to atomic coordinates corresponding to SEQ ID NO:1 as set forth in Appendix A. 
     
     
         9 . A crystal according to  claim 1  further comprising a ligand in complex with the human aromatase, wherein at least a portion of said ligand is:
 bound to at least a portion of the at least one ligand binding site, wherein the ligand binding site comprises an active site of the human aromatase;   bound to the human aromatase at a location so as to block an access channel to the at least one ligand binding site, wherein the ligand is either bound to at least a portion of the ligand binding site or not bound to at least a portion of the ligand binding site;   bound to the human aromatase at a location so as to block coupling of a cytochrome P450 reductase to the human aromatase; and/or   covalently linked to a catalytic region of the human aromatase, wherein said catalytic region comprises Ala306, Asp309, and/or Thr310 of SEQ ID NO:1, or a heme Fe associated with the human aromatase.   
     
     
         10 . The crystal according to  claim 9 , wherein said ligand is selected from the group consisting of an androgenic substrate of human aromatase, an androgenic substrate intermediate of human aromatase, and a competitive inhibitor of human aromatase. 
     
     
         11 . The crystal according to  claim 10 , wherein the androgenic substrate of human aromatase is selected from the group consisting of androstenedione, testosterone, 16α-hydroxytestosterone, and analogs or derivatives thereof. 
     
     
         12 . The crystal according to  claim 10 , wherein the androgenic substrate intermediate of human aromatase is selected from the group consisting of 19-hydroxyandrostenedione, 19-aldoandrostenedione, 19-hydroxytestosterone, and analogs or derivatives thereof. 
     
     
         13 . The crystal according to  claim 10 , wherein the competitive inhibitor of human aromatase is selected from the group consisting of exemestane, 7,8-benzoflavone, apigenin, chrysin, letrozole, anastrazole, formestane, fadrozole, aminoglutethimide, and analogs or derivatives thereof. 
     
     
         14 . The crystal according to  claim 9 , wherein the active site of the human aromatase comprises amino acid residues selected from the group consisting of Arg115, Ile133, Phe134, Phe221, Trp224, Ala306, Asp309, Thr310, Val370, Val373, Met374, and Leu477 of SEQ ID NO:1. 
     
     
         15 . The crystal according to  claim 9 , wherein the active site comprises a catalytic cleft comprising amino acid residues selected from the group consisting of Ile133, Phe134, Ile305, Ala306, Asp309, Thr310, Val370, Leu372, Val373, Met374, Leu477, and Ser478 of SEQ ID NO:1. 
     
     
         16 . The crystal according to  claim 10 , wherein the active site further comprises amino acid residues selected from the group consisting of Arg192, Gln218, Gln225, Leu228, Pro308, Met311, and Glu483 of SEQ ID NO:1. 
     
     
         17 . The crystal according to  claim 9 , wherein the active site comprises three-dimensional regions selected from the group consisting of:
 an I-helix comprising Ile305, Ala306, Asp309, and/or Thr310 of SEQ ID NO:1;   a B-C loop comprising Ile133 and Phe134 of SEQ ID NO:1;   a K-helix-β3-loop comprising Val370, Leu372, and Val373 of SEQ ID NO:1;   a β3 segment comprising Met374 of SEQ ID NO:1; and   a β8-β9 loop comprising Leu477 and Ser378 of SEQ ID NO:1.   
     
     
         18 . The crystal according to  claim 9 , wherein the access channel comprises an interior protein border comprising at least amino acid residues Arg192, Asp309, Ser478, and Glu483 of SEQ ID NO:1. 
     
     
         19 . The crystal according to  claim 9 , wherein the ligand is an androstenedione bound to at least a portion of the active site of the human aromatase, and wherein the crystal is sufficiently pure to determine atomic coordinates of the complex by X-ray diffraction to a resolution of 2.90 Å or better than 2.90 Å. 
     
     
         20 . The crystal according to  claim 9 , wherein the ligand is a transition state analog compound that is covalently linked to the human aromatase. 
     
     
         21 . The crystal according to  claim 1 , wherein the crystal is of a fully processed human aromatase of SEQ ID NO:1. 
     
     
         22 . A composition comprising the crystal according to  claim 1 . 
     
     
         23 . A composition comprising the crystal according to  claim 9 . 
     
     
         24 . An isolated human cytochrome P450 aromatase, wherein said aromatase is isolated from human placental tissue and is effective in maintaining catalytic activity during crystallization. 
     
     
         25 . An isolated aromatase according to  claim 24 , wherein said aromatase comprises an amino acid sequence according to SEQ ID NO:1 or an active mutant or variant thereof. 
     
     
         26 . A method for crystallizing a human aromatase, the method comprising:
 providing an isolated human aromatase according to  claim 24 ;   incubating the isolated human aromatase in a detergent solution under conditions effective to stabilize the human aromatase for crystallization, wherein said detergent solution comprises a detergent selected from the group consisting of n-dodecyl-β-D-maltopyranoside (BDM) and n-nonyl-β-D-maltopyranoside (BNM); and   crystallizing the stabilized human aromatase in a crystallization solution comprising dithiothreitol (DTT) and a ligand under conditions effective to yield a crystallized human aromatase in complex with the ligand.   
     
     
         27 . The method according to  claim 26 , wherein said DTT is present during crystallization at a concentration of between about 10 mM and 20 mM. 
     
     
         28 . The method according to  claim 26 , wherein the method is carried out at a temperature ranging from about 3° C. to about 6° C., preferably at about 4° C., and in the presence of a protectant that is effective to maintain the aromatase in its natural fold. 
     
     
         29 . The method according to  claim 26  further comprising solubilization of the ligand in a polyethylene glycol solution prior to said incubating step, thereby increasing yield of the crystallized aromatase-ligand complex. 
     
     
         30 . The method according to  claim 26 , wherein said aromatase comprises an amino acid sequence according to SEQ ID NO:1 or an active mutant or variant thereof. 
     
     
         31 . A crystallized human aromatase provided by the method of  claim 26 . 
     
     
         32 . A method for designing or screening for a drug for use in treating estrogen-dependent breast cancer or for use in inhibiting estrogen biosynthesis, the method comprising:
 obtaining a three-dimensional representation of at least one ligand binding site of a human aromatase;   superimposing at least one candidate ligand compound on said three-dimensional representation of the ligand binding site;   evaluating the binding of said at least one candidate compound and said ligand binding site; and   selecting a compound that spatially fits said ligand binding site.   
     
     
         33 . The method according to  claim 32 , wherein said three-dimensional representation of the at least one ligand binding site of the human aromatase is determined from a crystal or co-crystal comprising the human aromatase. 
     
     
         34 . The method according to  claim 32 , wherein the human aromatase comprises an amino acid sequence of SEQ ID NO:1. 
     
     
         35 . The method according to  claim 32 , wherein the three-dimensional representation is described by atomic coordinates that substantially conform to atomic coordinates comprising:
 coordinates 553 through 821, 1359 through 1459, 2062 through 2161, 2611 through 2673, and 3468 through 3535 as set forth in Appendix A;   coordinates 1112 through 1179, 1367 through 1459, 2113 through 2191, 2611 through 2647, and 3450 through 3565 as set forth in Appendix A; and/or   coordinates 793 through 840, 2508 through 2578, and 3057 through 3257 as set forth in Appendix A.   
     
     
         36 . The method according to  claim 32 , wherein the three-dimensional representation is described by atomic coordinates that substantially conform to atomic coordinates corresponding to SEQ ID NO:1 as set forth in Appendix A. 
     
     
         37 . The method according to  claim 32 , wherein the at least one ligand binding site is selected from the group consisting of:
 at least a portion of an active site of the human aromatase;   a location of the human aromatase that is proximate to an access channel to said active site;   at least a portion of a reductase coupling site of the human aromatase; and   a catalytic region of the human aromatase to which a transition state analog compound can bind.   
     
     
         38 . The method according to  claim 37 , wherein the active site is an active/substrate-binding heme distal site comprising amino acid residues Arg115 through Phe147, Ile217 through Leu228, Leu301 through Ser314, Pro368 through Arg375, and Ile474 through His480 of SEQ ID NO:1. 
     
     
         39 . The method according to  claim 37 , wherein the active site is a heme proximal/P450 reductase coupling site comprising amino acid residues Arg145 through Met149, Glu357 through Met364, and Pro423 through Met447 of SEQ ID NO:1. 
     
     
         40 . The method according to  claim 37 , wherein the access channel to the active site comprises amino acid residues Asp186 through Arg193, Gln218 through Leu228, Pro308 through Phe317, Pro368 through Leu372, and Gln472 through Lys485 of SEQ ID NO:1. 
     
     
         41 . The method according to  claim 32 , wherein the ligand binding site is specific to a ligand selected from the group consisting of an androgenic substrate of human aromatase, an androgenic substrate reaction intermediate of human aromatase, and a competitive inhibitor of human aromatase. 
     
     
         42 . The method according to  claim 41 , wherein the androgenic substrate of human aromatase is selected from the group consisting of androstenedione, testosterone, 16α-hydroxytestosterone, and analogs or derivatives thereof. 
     
     
         43 . The method according to  claim 41 , wherein the androgenic substrate intermediate of human aromatase is selected from the group consisting of 19-hydroxyandrostenedione, 19-aldoandrostenedione, 19-hydroxytestosterone, and analogs or derivatives thereof. 
     
     
         44 . The method according to  claim 41 , wherein the competitive inhibitor of human aromatase is selected from the group consisting of exemestane, 7,8-benzoflavone, apigenin, chrysin, letrozole, anastrazole, and analogs or derivatives thereof. 
     
     
         45 . The method according to  claim 32  further comprising:
 obtaining or synthesizing said selected compound; and   contacting said selected compound to the at least one ligand binding site of the human aromatase to determine the ability of said selected compound to interact with the at least one ligand binding site of the human aromatase.   
     
     
         46 . The method of  claim 32  further comprising:
 obtaining or synthesizing said selected compound;   forming a complex of the at least one ligand binding site of the human aromatase and said selected compound; and   analyzing said complex to determine the ability of said selected compound to interact with the at least one ligand binding site of the human aromatase.   
     
     
         47 . The method according to  claim 37 , wherein the active site of the human aromatase comprises amino acid residues selected from the group consisting of Arg115, Ile133, Phe134, Phe221, Trp224, Ala306, Asp309, Thr310, Val370, Val373, Met374, and Leu477 of SEQ ID NO:1. 
     
     
         48 . The method according to  claim 37 , wherein the active site comprises a catalytic cleft comprising amino acid residues selected from the group consisting of Ile133, Phe134, Ile305, Ala306, Asp309, Thr310, Val370, Leu372, Val373, Met374, Leu477, and Ser478 of SEQ ID NO:1. 
     
     
         49 . The method according to  claim 48 , wherein the active site further comprises amino acid residues selected from the group consisting of Arg192, Gln218, Gln225, Leu228, Pro308, Met311, and Glu483 of SEQ ID NO:1. 
     
     
         50 . The method according to  claim 37 , wherein the active site comprises three-dimensional regions selected from the group consisting of:
 an I-helix comprising Ile305, Ala306, Asp309, and/or Thr310 of SEQ ID NO:1;   a B-C loop comprising Ile133 and Phe134 of SEQ ID NO:1;   a K-helix-β3-loop comprising Val370, Leu372, and Val373 of SEQ ID NO:1;   a β3 segment comprising Met374 of SEQ ID NO:1; and   a β8-β9 loop comprising Leu477 and Ser378 of SEQ ID NO:1.   
     
     
         51 . The method according to  claim 37 , wherein the access channel comprises an interior protein border comprising at least amino acid residues Arg192, Asp309, Ser478, and Glu483 of SEQ ID NO:1. 
     
     
         52 . The method according to  claim 37 , wherein the ligand is an androstenedione bound to at least a portion of the active site of the human aromatase, and wherein the crystal is sufficiently pure to determine atomic coordinates of the complex by X-ray diffraction to a resolution of 2.90 Å or better than 2.90 Å. 
     
     
         53 . The method according to  claim 32 , wherein the ligand is a transition state analog compound that is covalently linked to the human aromatase. 
     
     
         54 . The method according to  claim 37 , wherein the crystal is of a fully processed human aromatase of SEQ ID NO:1. 
     
     
         55 . A method for screening for a novel drug that inhibits aromatase activity in humans, the method comprising:
 selecting a candidate compound by performing rational drug design using a three-dimensional structure determined from the crystal of  claim 1 ;   contacting the candidate compound with at least one ligand binding site of a human aromatase or a functional equivalent of said ligand binding site; and   detecting the binding potential of the candidate compound for said ligand binding site or said functional equivalent thereof,   wherein the candidate compound is selected based on its having a greater affinity for said ligand binding site or said functional equivalent thereof than that of a known drug.   
     
     
         56 . A method for designing a candidate drug that interferes with an activity of a human aromatase, the method comprising:
 providing a three-dimensional crystal structure of the isolated human cytochrome P450 aromatase according to  claim 24  in complex with a ligand, wherein said ligand is bound to at least a portion of a ligand binding site of the human aromatase; and   designing a compound predicted to bind the human aromatase configured in the human aromatase and ligand complex.   
     
     
         57 . A method for designing a compound that interferes with an activity of a human aromatase, the method comprising:
 providing on a digital computer a three-dimensional structure of at least one ligand binding site of the isolated human cytochrome P450 aromatase according to  claim 24 ; and   using software comprised by the digital computer to design a compound that is predicted to bind to at least a portion of the at least one ligand binding site of the human aromatase.   
     
     
         58 . The method according to  claim 57  further comprising:
 synthesizing the compound; and   evaluating the compound for an ability to interfere with an activity of the human aromatase.   
     
     
         59 . The method according to  claim 58 , wherein said evaluating comprises assaying the compound for anti-proliferative activity. 
     
     
         60 . The method according to  claim 59 , wherein anti-proliferative activity comprises inhibiting growth of a breast cancer cell line under an estrogenic stimulus. 
     
     
         61 . The method according to  claim 60 , wherein the breast cancer cell line comprises MCF-7. 
     
     
         62 . A high throughput enzymatic assay method for screening candidate compounds that inhibit human aromatase, the method comprising:
 testing a plurality of candidate compounds for human aromatase binding activity, wherein each compound is tested by the method of  claim 32 .   
     
     
         63 . The method according to  claim 62 , wherein said testing comprises evaluating binding affinity of the compound by direct measurement of the association constant using a isothermal titration calorimeter. 
     
     
         64 . The method according to  claim 62 , wherein said testing comprises evaluating anti-proliferative activity of the compound as an aromatase inhibitor in a breast cancer cell line expressing aromatase and an estrogen receptor. 
     
     
         65 . The method according to  claim 62 , wherein said testing comprises determining X-ray crystallographic structures of at least one reaction intermediate of estrogen biosynthesis by initiating hydroxylation reaction in a crystal with X-ray photoelectrons and following the shift in the Soret band with a micro-spectrophotometer suitable for in situ measurement in protein crystals. 
     
     
         66 . A three-dimensional computer image of the three-dimensional structure of human aromatase-ligand complex, wherein the human aromatase comprises the isolated human cytochrome P450 aromatase according to  claim 24  and has a three-dimensional structure that substantially conforms to the three-dimensional atomic coordinates of Appendix A. 
     
     
         67 . A computer-readable medium encoded with a set of three-dimensional atomic coordinates of the crystal according to  claim 7 , wherein, by using a graphical display software program, the three-dimensional atomic coordinates of the crystal create an electronic file that can be visualized on a computer capable of representing said electronic file as a three-dimensional image.