A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues

Background  

Analogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker.     

Comparative investigation of vitamins and their analogues on terminal differentiation, from preadipocytes to adipocytes, of 3T3-L1 cells

1. The effects of 20 kinds of vitamins or their analogues on the growth rate of preadipocytes and the terminal differentiation of preadipocytes to adipocytes was systematically compared in 3T3-L1 cells. 2. The addition of vitamin C markedly increased the growth rate of preadipocytes at over 50 microM. 3. The addition of vitamin K3 slowed down the growth rate at over 0.1 microM. 4. In water soluble vitamins and their analogues tested, the vitamin B6 group and vitamin C significantly stimulated the differentiation, and consequently increased the glycerophosphate dehydrogenase activity and triglyceride accumulation, to a concentration of over 10 microM. 5. Many fat soluble vitamins and their analogues (the vitamin A group, including beta-carotene, the vitamin D group, vitamin E and the vitamin K group) strongly inhibited the adipose conversion of 3T3-L1 cells at microM level.     

Novel synthetic approach to 1alpha,25-dihydroxyvitamin D3 and analogues

A mild and stereoconvergent synthesis of 1alpha,25-dihydroxyvitamin D(3) (calcitriol, 1a) is described. The key step is a cascade process consisting of two consecutive transformations: An initial palladium-catalyzed 6-exo-cyclocarbopalladation of vinyl triflate 4 followed by a Negishi cross-coupling reaction with alkenyl zinc 3. This approach is of interest for the rapid synthesis of a variety of new vitamin D(3) analogues of therapeutic potential, especially those modified at the triene and ring-A. The mildness of the method also allows the preparation of thermal sensitive vitamin D(3) analogues.     

Synthesis and biological evaluation of new 6-s-cis locked 1,2,25-trihydroxyprevitamin D3 analogues

An efficient synthesis of several diastereomers of 2-hydroxy substituted 1alpha,25-dihydroxyprevitamin D3 derivatives was accomplished utilizing a practical route to the A-ring synthon. The biological activity of the analogues was evaluated in vitro. All the synthesized derivatives demonstrated low affinity for the vitamin D receptor and vitamin D-binding protein compared with 1alpha,25-dihydroxyvitamin D3, the natural hormone. 1alpha,2beta,25-trihydroxy-19-nor-pre-D3 was the most potent of the analogues in inhibiting proliferation of MCF-7 cells but requires higher EC50 concentrations than 1alpha,25-dihydroxyvitamin D3.     

A-ring analogs of 1,25-dihydroxyvitamin D(3)

The growing interest in1α,25(OH)(2)D(3), the hormonally active form of vitamin D(3), has prompted numerous efforts to synthesize vitamin D analogs as potential therapeutic agents, and some of these are already on the market and in clinical development. Although most vitamin D preparations developed thus far have focused on side-chain modifications, providing many useful analogues with high potency and selectivity, in recent years, modifications of the A-ring has attracted much attention because it can afford useful analogues exhibiting unique activity profiles as well. In this review we will focus on the current understanding of the relationship between selected modifications in the A-ring of the 1α,25(OH)(2)D(3) molecule, such as epimerization and/or substitution at C-1 and C-3, substitution at C-2, and removal of the 10,19-exocyclic methylene group, and their effect on biological potency and selectivity. Finally, suggestions for the structure-based design of therapeutically valuable A-ring vitamin D analogs will conclude the review.     

Chemoenzymatic synthesis and biological evaluation of C-3 carbamate analogues of 1alpha,25-dihydroxyvitamin D3

The synthesis of new analogues of 1alpha,25-dihydroxyvitamin D3 containing a carbamate function at the A-ring fragment has been described using the cross-coupling approach. The carbamate group was selectively introduced at the C-3 position by regioselective enzymatic alkoxycarbonylation of A-ring enyne 3 and subsequent treatment with ammonia, amines, amino alcohols, and amino acids. Biological studies to evaluate the potency of all five of these carbamate analogues were performed and demonstrated very low binding affinity for the vitamin D receptor compared with 1alpha,25-dihydroxyvitamin D3. Moreover, all the carbamate analogues were less active than 1alpha,25-dihydroxyvitamin D3 in inhibiting cell proliferation or stimulating cell differentiation. Of all the five analogues, the 3-O-carbamoyl-1alpha,25-(OH)2-D3 analogue 10a was the most potent one in vitro. However, all investigated carbamate analogues demonstrated lower calcemic effects in vivo than the parent compound.     

Synthesis and biological evaluations of C-23-modified 26,26,26,27,27,27-F6-vitamin D3 analogues

A convenient synthetic method which could allow flexible modification at C-23 of 26,26,26,27,27,27-hexafluoro-1alpha,25-dihydroxyvitamin D3 (3) has been developed. An effective construction of hexafluoroacetone (HFA) aldol part on the side chain of 10 was achieved by aldol reaction with HFA gas. This route is also attractive as an approach to diverse 26,27-modified vitamin D3 analogues. The preliminary biological activities of 23-modifed 26,27-F6 vitamin D3 analogues are evaluated. The potency of VDR affinities of the C-23-substituted analogues (keto group (4); OH group (5a,5b); fluorine atom (6a,6b); and oxetane ring (7a,7b)) was found to vary depending upon both the nature and stereochemistry of the substituents. In contrast, the HL-60 cell differentiation property was less varied than VDR affinity, and depended upon the nature rather than the stereochemistry of the substituents.     

Synthesis of two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3

Hapten derivatives of 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) were synthesized using the Wittig-Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxylated vitamin D(3) analogues.     

Synthesis and biological activity of 22-oxa CD-ring modified analogues of 1alpha,25-dihydroxyvitamin D3: cis-perhydrindane CE-ring analogues

The synthesis and biological activity of novel CD-ring modified analogues of 22-oxa-1alpha,25-dihydroxyvitamin D(3), lacking the D-ring and featuring a connection between C-12 and C-21 (cis-perhydrindane CE-ring analogues), is described. The synthesis of the CE-ring system follows Meyers' methodology for the preparation of enantiomerically pure hydrinden-2-ones. The analogues show a complete lack of binding affinity for the vitamin D receptor (pig nVDR) and of antiproliferative activity (MCF-7 cells), as compared to calcitriol.     

Synthesis of gamma- and delta-lactones from 1alpha-hydroxy-5,6-trans-vitamin D3 by ring-closing metathesis route and their reduction with metal hydrides

New synthetic pathway towards 19-functionalized derivatives of 1alpha-hydroxy-5,6-trans-vitamin D3 was described. Ring-closing metathesis (RCM) of 1alpha-hydroxy-5,6-trans-vitamin D3 1-omega-alkenoates was a key-step. Hydride reduction of resulting lactones led to the new vitamin D3 analogues.     

23-Keto-25-hydroxyvitamin D3: a vitamin D3 metabolite with high affinity for the 1,25-dihydroxyvitamin D specific cytosol receptor

A new metabolite of 23,25-dihydroxyvitamin D3 has been generated with kidney homogenates prepared from vitamin D treated chicks. The metabolite was purified with three high-performance liquid chromatographic steps and was identified as 23-keto-25-hydroxyvitamin D3 by ultraviolet absorption spectroscopy, mass spectrometry, and chemical reactivity. The R stereoisomer of 23,25-dihydroxyvitamin D3 was 10-fold more effective as an in vitro precursor to 23-keto-25-hydroxyvitamin D3 than was the naturally occurring S stereoisomer. Approximately 500 ng of 23-keto-25-hydroxyvitamin D3 was necessary to produce the same degree of intestinal-calcium transport as 25 ng of vitamin D3--a difference of about 20-fold. 23-Keto-25-hydroxyvitamin D3 was not active at stimulating bone calcium resorption at the doses and times tested. This new vitamin D3 metabolite, however, had greater affinity than 25-hydroxyvitamin D3 to both the rat plasma vitamin D binding protein and the 1,25-dihydroxyvitamin D specific cytosol receptor. Heretofore, only 1 alpha-hydroxylated metabolites of 25-hydroxyvitamin D3 or analogues possessing a pseudo 1 alpha-hydroxy group were known to bind to the 1,25-dihydroxyvitamin D receptor with higher affinity than 25-hydroxyvitamin D3. Ketone formation at the 23 position, therefore, is the first side-chain modification of 25-hydroxyvitamin D3 that results in enhanced binding to the 1,25-dihydroxyvitamin D receptor binding protein.     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1alpha,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1alpha-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 and 22-iodo-1alpha,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1alpha,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.     

The effect of parathyroid hormone and vitamin D3 metabolites on CaBP synthesis by embryonic duodenum, in vitro

Embryonic chick duodena were incubated, in vitro, for two days in the presence of vitamin D analogues and metabolites and the ability of PTH to potentiate the vitamin-D-dependent synthesis of CaBP measured. PTH potentiated CaBP synthesis in the presence of vitamin D3, 25-OH-D3, 1 alpha-OH-D3, DHT, and 1,25(OH)2D3. The synthesis of CaBP was directly related to the log of the meddium PTH concentration over the range 1 x 10(-8) M to 5 x 10(-7) M.     

1,25-Dihydroxyvitamin D3 regulates proliferation of activated T-lymphocyte subsets

The biologically active vitamin D metabolite, 1,25-(OH) 2D3 suppressed phytohaemagglutinin (PHA)-induced lymphocyte proliferation dose-dependently (0.1 nM-100 nM), and decreased the OKT4+/OKT8+ ratio and transferrin-receptor-positive (OKT9+) cells. A possible parallelism between expression of 1,25-(OH) 2D3 receptors and interleukin 2 (IL2)-receptors recognized by anti-Tac antibody was not confirmed in this study. However, the addition of exogenous IL2 abolished the inhibitory effects of 1,25-(OH) 2D3 on PHA-stimulated T-cell proliferation, and the decrease of OKT4+ and OKT9+ T-cell in this population. Among various vitamin D3 analogues examined, 1,25-(OH) 2D3 was the most potent anti-proliferative effect, followed in order by 1,24S-(OH) 2D3, 1 alpha OH D3, 25 OH D3 and 24,25-(OH) 2D3.     

Design and synthesis of novel 1,25-dihydroxyvitamin D3 analogues having a spiro-oxetane fused at the C2 position in the A-ring

Four structurally novel stereoisomeric analogues of 1,25-dihydroxyvitamin D₃ (3a–d) bearing a spiro-oxetane fused at the C2 position of the A-ring have been designed and synthesised in a convergent manner. The requisite A-ring enyne precursors (13a,b) for the vitamin D analogues (3a,b) and (3c,d), respectively, were synthesised from pentaerythritol according to an eleven-step procedure. Preliminary biological evaluation of the analogues using the bovine thymus vitamin D receptor (VDR) suggested that the incorporation of the spiro-oxetane moiety instead of a gem-dimethyl group at the C2 position had a beneficial effect on the VDR affinity.     

2,2-Functionalized analogues of 1alpha,25-dihydroxyvitamin D3, the potent inducers of cell differentiation

All four possible A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D(3) (4) were designed and convergently synthesized. Nine-step conversion of methyl hydroxypivalate 6 provided the desired A-ring enyne synthon (13a,b) in good overall yield. Cross-coupling reaction of the A-ring synthon 13a,b with the CD-ring portion in the presence of palladium catalyst, followed by deprotection, gave the vitamin analogues (4a-d). We also synthesized four stereoisomers of 2,2-ethano-1,25-dihydroxyvitamin D(3) (5), as novel spiro-ring analogues having cyclopropane fused at the C2 position. Biological potencies of the synthesized compounds were assessed in terms of the vitamin D receptor (VDR) binding affinity, as well as the HL-60 cell differentiation-inducing activity. The 2,2-ethano analogue 5a showed a comparable activity to the natural hormone 1, while the 2,2-dimethyl analogue 4a exhibited one-third of the activity of 1 in cell differentiation, with the reduced VDR binding affinity.     

High-throughput system for analyzing ligand-induced cofactor recruitment by vitamin D receptor

A high-throughput screening system for analyzing small molecule-induced coactivator (CoA) recruitment by the vitamin D receptor (VDR) has been developed. The vitamin D-induced protein-protein interactions between VDR and fluorophore (Cy3 or Cy5)-labeled TIF2 or SRC-1 were successfully detected by using a new HCHO fixing method of the protein complex on microplates. The results obtained from this screening of our synthetic vitamin D analogues suggest that the CoA-recruiting activities play an important role in determining the biological activity of various vitamin D analogues and explain the discrepancies between the VDR binding affinity and their biological activity.