Cloning, expression and functional characterization of type 1 and type 2 steroid 5 alpha-reductases from Cynomolgus monkey: comparisons with human and rat isoenzymes.
The Cynomolgus monkey may provide an alternative pharmacological model in which to evaluate the efficacy of novel inhibitors of the two known human steroid 5 alpha-reductase (SR) isoenzymes. To evaluate the suitability of this species at the level of the molecular targets, a Cynomolgus monkey prostate cDNA library was prepared and screened using human SR type 1 and 2 cDNAs as hybridization probes. Two distinct cDNA sequences were isolated encoding the monkey type 1 and 2 SR isoenzymes. These sequences share 93 and 95% amino acid sequence identity with their human enzyme counterparts, respectively. Difference in monkey type 1 SR, however, was found within the contiguous four amino acids corresponding to the regions in the human and rat sequences that have been proposed previously to influence steroid and inhibitor affinities. Subsequently, both monkey cDNAs were individually expressed in a mammalian cell (CHO) line. Enzyme activities of both monkey SRs were localized to the membrane fractions of CHO cell extracts. Like the human and rat enzymes, the monkey type 1 and type 2 SRs were most active at neutral and low pH, respectively. The results of inhibition studies with over 30 known SR inhibitors, including epristeride, 4MA, and finasteride, indicate that the monkey SR isoenzymes are functionally more similar to the human than the rat homologues. The results from initial velocity and inhibition studies as functions of pH with the human and monkey type 2 SRs also compare favorably. These results, together, suggest that the monkey SR isoenzymes are structurally and functionally comparable on a molecular level to their respective human counterparts, supporting the relevance and use of the Cynomolgus monkey as a pharmacological model for in vivo evaluation of SR inhibitors.
Levy
M A
MA
Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Brandt
M
M
Sheedy
K M
KM
Holt
D A
DA
Heaslip
J I
JI
Trill
J J
JJ
Ryan
P J
PJ
Morris
R A
RA
Garrison
L M
LM
Bergsma
D J
DJ
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D058891:5-alpha Reductase Inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000235:genetics*; D008252:Macaca fascicularis; D008297:Male; D008969:Molecular Sequence Data; D011467:Prostate / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000378:metabolism*
D058891:5-alpha Reductase Inhibitors; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013256:Steroids; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase