Characterization of a novel analogue of 1alpha,25(OH)(2)-vitamin D(3) with two side chains: interaction with its nuclear receptor and cellular actions.
The hormone 1alpha,25(OH)(2)-vitamin D(3) (125D) binds to its nuclear receptor (VDR) to stimulate gene transcription activity. Inversion of configuration at C-20 of the side chain to generate 20-epi-1alpha,25(OH)(2)D(3) (20E-125D) increases transcription 200-5000-fold over 125D with its 20-normal (20N) side chain. This enhancement has been attributed to the VDR ligand-binding domain (LBD) having different contact sites for 20N and 20E side chains that generate different VDR conformations. We synthesized 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3) (Gemini) with two six-carbon side chains (both 20N and 20E orientations). Energy minimization calculations indicate the Gemini side chain possesses significantly more energy minima than either 125D or 20E-125D (2346, 207, and 127 minima, respectively). We compared activities of 125D, 20E-125D, and Gemini, respectively, in several assays: binding to wild-type (100%, 147%, and 38%) and C-terminal-truncated mutant VDR; transcriptional activity (of the transfected osteopontin promoter in ROS 17/2.8 cells: ED(50) 10, 0.005, and 1.0 nM); mediation of conformational changes in VDR assessed by protease clipping (major trypsin-resistant fragment of 34, 34, and 28 kDa). For inhibition of cellular clonal growth of human leukemia (HL-60) and breast cancer (MCF7) cell lines, the ED(50)(125D)/ED(50)(Gem) was respectively 380 and 316. We conclude that while Gemini readily binds to the VDR and generates unique conformational changes, none of them is able to permit a superior gene transcription activity despite the presence of a 20E side chain.
Norman
A W
AW
Departments of Biochemistry and Chemistry and Division of Biomedical Sciences, University of California, Riverside, California 92521, USA. Norman@ucrac1,ucr.edu
Manchand
P S
PS
Uskokovic
M R
MR
Okamura
W H
WH
Takeuchi
J A
JA
Bishop
J E
JE
Hisatake
J I
JI
Koeffler
H P
HP
Peleg
S
S
D000818:Animals; D001667:Binding, Competitive; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002645:Chickens; D002999:Clone Cells; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D053495:Osteopontin; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D012795:Sialoglycoproteins / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
C411462:1,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3); D008024:Ligands; D018167:Receptors, Calcitriol; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D013937:Thymidine Kinase; D002117:Calcitriol