Screening of selective inhibitors of 1α,25-dihydroxyvitamin D3 24-hydroxylase using recombinant human enzyme expressed in Escherichia coli.

High-level heterologous expression of human 1α,25-dihydroxyvitamin D(3) 24-hydroxylase (CYP24A1) in Escherichia coli was attained via a fusion construct by appending the mature CYP24A1 without the leader sequence to the maltose binding protein (MBP). Facile purification was achieved efficiently through affinity chromatography and afforded fully functional enzyme of near homogeneity, with a k(cat) of 0.12 min(-1) and a K(M) of 0.19 µM toward 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. A convenient and reliable cell-free assay was established and used to screen vitamin D analogues with potential inhibitory properties toward CYP24A1. Some of the compounds exhibited potent inhibition with K(I) values as low as 0.021 µM. Furthermore, TS17 and CPA1 exhibited superior specificity toward CYP24A1 over 25-hydroxyvitamin D(3) 1α-hydroxylase (CYP27B1), with selectivities of 39 and 80, respectively. Addition of TS17 or CPA1 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D(3) in the medium. Their activities in vitamin D receptor (VDR) binding, CYP24A1 transcription, and HL-60 cell differentiation were evaluated as well.

Removal of the 20-methyl group from 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD) selectively eliminates bone calcium mobilization activity.

The 18-nor (7), 21-nor (8) and 18,21-dinor (9) analogs of (20S)-1alpha,25-dihydroxy-2-methylene-19-norvitamin D(3) (6, 2MD) were prepared by convergent syntheses. The known phosphine oxide 10 was coupled by the Wittig-Horner process with the corresponding C,D-fragments (13-15), obtained by a multi-step procedure from commercial vitamin D(2). The goal of our studies was to examine the influence of removal of the methyl groups located at carbons 13 and 20 on the biological potency of 2MD in the hope of finding analogs with improved therapeutic profiles. Replacement of the 20-methyl with hydrogen in 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD) did not affect binding to the rat vitamin D receptor and had little effect on transcription activity and on HL-60 differentiation. However, the mobilization of calcium from bone was largely eliminated while intestinal calcium transport remained strong. Curiously, removal of both the C-13-methyl and 20-methyl restored slightly the bone calcium mobilizing activity. Thus, the 21-nor analog of 2MD may provide a potent analog with a greater margin of safety than 2MD.

Synthesis and biological properties of 2-methylene-19-nor-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactones--weak agonists.

In a continuing effort to explore the 2-methylene-1alpha-hydroxy-19-norvitamin D(3) class of pharmacologically important vitamin D compounds, two novel 2-methylene-19-nor-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactones, GC-3 and HLV, were synthesized and biologically tested. Based on reports of similarly structured molecules, it was hypothesized that these compounds might act as antagonists, at least in vitro. The pathway designed to synthesize these compounds was based on two key steps: first, the Lythgoe-type Wittig-Horner coupling of Windaus-Grundmann-type ketone 18, with phosphine oxide 15, followed, later in the synthesis, by the Zn-mediated Reformatsky-type allylation of aldehyde 20 with methylbromomethylacrylate 8. Our biological data show that neither compound has antagonistic activity but acts as weak agonists in vitro and in vivo.