Screening of Selective Inhibitors of 1α,25-Dihydroxyvitamin D₃ 24-Hydroxylase Using Recombinant Human Enzyme Expressed in Escherichia coli

Abstract

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.

Rat CYP24A1 acts on 20-hydroxyvitamin D3 producing hydroxylated products with increased biological activity

“…We have addressed these questions in the current study using purified rat CYP24A1 expressed in E. coli. The rat enzyme was chosen for these studies because it is easier to express and purify than the less stable human enzyme [33], plus we have used rodents to test the toxicity and other effects of 20(OH)D 3 [22, 23] where potential metabolism by CYP24A1 is of importance. The rat and human enzymes share 90% amino acid sequence identity [34] and both metabolize 25(OH)D 3 and 1,25(OH) 2 D 3 predominantly by the C24 oxidation pathway, therefore data for 20(OH)D 3 metabolism for the rat enzyme should provide a useful model for 20(OH)D 3 metabolism by human CYP24A1.…”

Novel, Selective Vitamin D Analog Suppresses Parathyroid Hormone in Uremic Animals and Postmenopausal Women

“…2) and potently suppresses serum PTH [24]. This property and its slow rate of metabolism by CYP24A1 [36] immediately suggested it as a candidate for the treatment of secondary hyperparathyroidism of renal failure.…”

Novel styryl-indoles as small molecule inhibitors of 25-hydroxyvitamin D-24-hydroxylase (CYP24A1): Synthesis and biological evaluation

“…Inhibition assay of CYP27B1 was performed in a similar way to the CYP24A1 assay as previously described [23], and the resulting cell-free assay system applied in the screening of the compounds to measure Ki and IC 50 (Table 2).…”

“…Inhibition of CYP24A1 was carried out as described previously [23]. Inhibition assay of CYP27B1 was performed in a similar way to that of CYP24A1 as previously described [23].…”

“…Inhibition assay of CYP27B1 was performed in a similar way to that of CYP24A1 as previously described [23]. The concentration of substrate 25(OH)D 3 was 2.5 μM and K M for CYP27B1 was 0.48 μM.…”