Novel steroid 5alpha-reductase

Abstract

The invention provides an isolated, novel steroid 5α-reductase enzyme termed SRD5AIII. The protein has an estimated molecular weight of 37 kDa and is capable of converting testosterone to dihydrotestosterone at a pH of about 7.0. Also provided is a method for identifying inhibitors of SRD5AIII by contacting SRD5AIII with a test compound and measuring the activity of the enzyme. A reduced activity relative to a control indicates that the test compound is an inhibitor of SRD5AIII. A method is also provided for detecting androgen stimulated prostate cancer or recurrent prostate cancer in an individual. The method comprises obtaining a prostate biopsy from an individual and determining the level of expression of SRD5AIII gene or protein relative to a normal control. An increased expression of SRD5AIII relative to the control is indicative of androgen stimulated prostate cancer or recurrent prostate cancer.

1 . An isolated steroid 5α-reductase enzyme which has an estimated molecular weight of 37 kDa from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) analysis.  
     
     
         2 . The enzyme of  claim 1 , wherein the enzyme has the amino acid sequence of SEQ ID NO:2 or an amino acid sequence at least 95% identical to the sequence of SEQ ID NO:2.  
     
     
         3 . The enzyme of  claim 1 , wherein the enzyme is capable of catalyzing the conversion of testosterone to dihydrotestosterone (DHT) at pH 7.0.  
     
     
         4 . The enzyme of  claim 3 , wherein the specific activity of the enzyme for catalyzing the conversion of testosterone to DHT is 15.6 pmol/mg protein/hour.  
     
     
         5 . The enzyme of  claim 1 , wherein the enzyme is expressed from an expression vector comprising a polynucleotide encoding the amino acid sequence of the enzyme.  
     
     
         6 . The enzyme of  claim 5 , wherein the polynucleotide comprises the sequence of SEQ ID NO:1 from nucleotide 93 to 1049.  
     
     
         7 . The enzyme of  claim 5 , wherein the enzyme has the amino acid sequence of SEQ ID NO:2.  
     
     
         8 . The enzyme of  claim 5 , wherein the expression vector is a recombinant adenovirus.  
     
     
         9 . A method for identifying an inhibitor of the enzyme of  claim 1  comprising the steps of: 
 a) contacting the enzyme with a test compound in;    b) measuring the activity of the enzyme; and    c) comparing the activity of the enzyme to a control, wherein the activity of the control is measured in the absence of the test compound;    wherein reduced activity in step b) relative to the control of c) is indicative that the test compound is an inhibitor of the enzyme.    
     
     
         9 . The method of  claim 9 , wherein the enzyme has the amino acid sequence of SEQ ID NO:2.  
     
     
         10 . The method of  claim 9 , wherein the enzyme is expressed from an expression vector comprising a polynucleotide encoding the amino acid sequence of the enzyme.  
     
     
         11 . The method of  claim 10 , wherein the polynucleotide comprises the sequence of SEQ ID NO:1 from nucleotide 93 to 1049.  
     
     
         12 . The method of  claim 9 , wherein the rate of conversion of testosterone to DHT is measured at pH 7.0.  
     
     
         13 . The method of  claim 9 , wherein the activity of the enzyme is the rate of conversion of testosterone to dihydrotestosterone (DHT).  
     
     
         14 . The method of  claim 9 , wherein the activity of the enzyme is the rate of conversion of testosterone to or androstanedione.  
     
     
         15 . A method of detecting androgen stimulated prostate cancer or recurrent prostate cancer in an individual comprising the steps of: 
 a) obtaining a biopsy sample of prostate from the individual;    b) determining the level of expression of SRD5AIII gene or protein;    c) comparing the level of expression to that from a normal control,    wherein an increased expression relative to the normal control is indicative of androgen stimulated prostate cancer or recurrent prostate cancer.    
     
     
         16 . The method of  claim 15 , wherein step b) further comprises normalizing the expression of SRD5AIII to the level of expression of SRD5AI or SRD5AII.  
     
     
         17 . The method of  claim 15 , wherein in step b) the level of SRD5AIII MRNA is determined.  
     
     
         18 . The method of  claim 17 , wherein the level of mRNA is determined by reverse transcriptase polymerase chain reaction (RTPCR).  
     
     
         19 . The method of  claim 18 , wherein the RTPCR is performed using a first primer having the sequence of SEQ ID NO:9 and a second primer having the sequence of SEQ ID NO:10.  
     
     
         20 . The method of  claim 13 , wherein in step b) the level of SRD5AIII protein is determined.