1974 Current Medicinal Chemistry, 2010, 17, 1974-1994

Phytoecdysteroids and Vitamin D Analogues – Similarities in Structure

and Mode of Action
Noémi Tóth1, Attila Hunyadi1, Mária Báthori1 and Ern Zádor*,2
1
Institute of Pharmacognosy, Faculty of Pharmacy, University of Szeged, H-6720 Szeged, Eötvös utca 6., Hungary
2
Institute of Biochemistry, Faculty of Medicine, University of Szeged, H-6720 Szeged, Dóm tér 9., Hungary
Abstract: Phytoecdysteroids are plant steroids with identical or analogue structures to the molting hormone in arthropods.
The ecdysteroids exert several beneficial effects on mammals, from which the most cited and deeply examined one is the
increase of muscle size and strength. This shows similarities with the mode of action of the androgenic steroids but the
ecdysteroids do not bind to the cytoplasmic/nuclear receptor of the mammalian steroids. These findings led to the hy-
pothesis that ecdysteroids possibly bind to membrane bound receptors and they are likely to influence signal transduction
pathways. Probably because of their closely related chemical structures, ecdysteroids exert some similar effects in verte-
brates to those of the hormone 1,25-dihydroxyvitamin D3 (1,25D) which is produced in the kidney from 25-
hydroxyvitamin D3, after being converted in the liver from Vitamin D3.
1,25D generates biological responses via both genomic and rapid, nongenomic mechanisms. Structure-activity relation-
ship studies with different Vitamin D analogues could open the possibility to show that the two ways of action (genomic
and nongenomic) can be influenced separately.
The connection between the Vitamin D status and muscle function is already well-described in clinical studies, and sev-
eral efforts have been made to evaluate the effect of Vitamin D deficiency or supplementation on muscle morphological
changes and the underlying molecular mechanisms.
This paper aims to summarize the main structural commonalities between the ecdysteroids, 1,25D and other Vitamin D
analogues. The similarities in their effects and pathways that might be involved in the mechanism of action of these com-
pounds will also be discussed.
Keywords: Ecdysteroid, vitamin D analogues, vitamin D receptor, alternative ligand binding pocket, conformational ensemble
model.

INTRODUCTION vitamin D analogues and the ecdysteroids as regards chemi-

Ecdysteroids act as allobiotic compounds in warm-
blooded animals, but the exact mechanism of their action has
not yet been elucidated. The effect of ecdysteroids on mam-
mals and birds is mostly anabolic, and very similar to those
of anabolic steroids. However, ecdysteroids are unable to
compete with agonists of mammalian steroid receptors in an
in vitro receptor assay, which clearly demonstrates the lack
of their specific binding. On the other hand, sporadic reports
have appeared on the ecdysteroid-induced stimulation of
signal transduction pathways. The same pathways are fre-
quently used for the rapid, non-genomic (NG) response to
steroid hormones and this has led to the suggestion that ec-
dysteroids use a mode of action that is similar to the rapid
response of mammalian steroids [1]. The rapid NG action of
steroid hormones is less well characterized than the slower
genomic (G) effect, but recent research has placed emphasis
on the conformational changes of steroid receptors and pro-
vided a clue towards the explanation of the rapid response to
certain agonists, and potentially the allobiotic effects of ec-
dysteroids. Among the mammalian steroids, the vitamin D
hormone-derived calcitriol (1,25D, 1) is the most similar to
the ecdysteroids. This raises the possibility that the ecdyster-
oid effects on mammals can be explained by a mechanism
similar to the rapid response to 1 and its analogues. This pa-
per provides an overview of the similarities between the
*Address correspondence to this author at the Department of Biochemistry,
University of Szeged, H-6720 Szeged, Dóm tér 9., Hungary;
Tel: +36/62/545-096; Fax: +36/62/545-097;
E-mail: erno@bioch.szote.u-szeged.hu
cal structure, receptors and the mode of action, especially in
the skeletal muscle, where the effect of both steroid groups is
being increasingly well recognized.
The Biosynthesis of Vitamin D Hormone and Phytoec-
dysteroids
Vitamin D analogues and phytoecdysteroids may display
similarities in chemical structures but they are different in
natural origin.
Vitamin D3 is produced photochemically in the skin from
7-dehydrocholesterol and/or is consumed in the diet. This is
a precursor which itself is inert as concerns endocrine ef-
fects, but undergoes a two-step metabolic conversion: it is
first metabolized to 25-hydroxyvitamin D3 (25D, 3) in the
liver, which is then transported by the plasma vitamin D-
binding protein to the kidney, where it is converted into
1,25-dihydroxyvitamin D3 (calcitriol, 1,25D, 1) by 1
-
hydroxylase. The production of this secosteroid hormone is
stereospecific and the resulting compound is released into
the circulation [2-5]. Besides the kidney, 3 is also conveyed
to other organs and tissues that have a relatively low 1
-
hydroxylase activity, which results in the local synthesis of
in a minor amount of 1 [6].
1 has been shown to be present in unconjugated form in
various organs of Solanum glaucophyllum and is synthesized
in this plant under cultured conditions [7]. Interestingly, dur-
ing this conversion, ring B is opened independently of UV
light, possibly by an enzymatic reaction [8]. As the overall
rate of biosynthesis of this phytocalcitriol is limited by the

0929-8673/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.

Phytoecdysteroids and Vitamin D Analogues Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1975

21
20 22
24
18
H 23 26
12 17 25
11 13
9
16 OH
14 15
8 27

H
7 H
6 OH 19
9 OH
19
5 1 10
4 10
3
H
3 1
2
HO OH HO

1,25D (1
,25-dihydroxyvitamin D3; 1) 1,25-preD (1
,25-dihydroxyprevitamin D3; 2)

A-ring modified analogs:

H
OH
25D (25-dihydroxyvitamin D3; 3): R1 = R2 = H, R3 =
OH
H 3d-1,25D (3-deoxy-1
,25-dihydroxyvitamin D3; 4): R1 = OH, R2 = R3 = H
3-epi-1,25D (3-epi-1,25-dihydroxyvitamin D3; 5): R1 =
OH, R2 = H, R3 = OH
3epi-1,25D D3 (3-epi-1
,25-dihydroxyvitamin ; 6): R1 = R3 = OH, R2 = H
1,25D (1,25-dihydroxyvitamin D3; 7): R1 = R3 =
OH, R2 = H
3
2
1 2
Me-1,25D (2
-methyl-1
,25-dihydroxyvitamin D3; 8): R1 = OH, R2 =
CH3, R3 =
OH
R3 R1 2Me-1,25D (2-methyl-1
,25-dihydroxyvitamin D3; 9): R1 = OH, R2 = CH3, R3 =
OH
2Me-1,25D (2-methyl-1,25-dihydroxyvitamin D3; 10): R1 = R3 =
OH, R2 = CH3
R2
2Me-3-epi-1,25D (2-methyl-3-epi-1,25-dihydroxyvitamin D3; 11): R1 =
OH, R2 = CH3, R3 = OH
2Me-3-epi-1
,25D (2-methyl-3-epi-1
,25-dihydroxyvitamin D3; 12): R1 = OH, R2 = CH3, R3 = OH
19-nor-1,25D (19-nor-1
,25-dihydroxyvitamin D3; 13): R1 = OH, R2 = H, R3 =
OH

HO 20

H
OH

19-nor-Gemini (21-(3-hydroxy-3-methyl-butyl)-19-nor-1
,25-dihydroxyvitamin D3; 14)

10
3 1
2
HO OH

B-ring modified analogs:

20 20

H H
OH OH
9 9

H H
OH 19
OH
19 19
1
3 10
10 3
3 1 1
HO 2
HO OH HO OH

1,25T (1
,25-dihydroxytachysterol3; 15) 1,25-cis-isoT (1
,25-dihydroxy-cis-isotachysterol3; 16) 1,25-trans-isoT (1
,25-dihydroxy-trans-isotachysterol3; 17)

H H
OH OH
H OH CD3 OH
OH 19 R2 9 14
OH
1 D
1
1 R1 H D
3 3
H H HO
3 HO
HO
1,25L (1
,25-dihydroxylumisterol3; 19): R1 = H, R2 =
CH3 1,25-d5-preD (1
,25-dihydroxy-pentadeuterio-previtamin D3; 22)
1,25(OH)2-7-DHC (1
,25-dihydroxy-7-dehydrocholesterol3; 18) 1,25P (1
,25-dihydroxypyrocalciferol3; 20): R1 =
H, R2 =
CH3
1,25-isoP (1
,25-dihydroxy-isopyrocalciferol3; 21): R1 = H, R2 = CH3
1976 Current Medicinal Chemistry, 2010 Vol. 17, No. 18 Tóth et al.

(Fig. 1). Contd…..

Side chain modified analogs:
R1 R4
20 R5
23
25
H 24
R2 R3 R6
20-epi-1,25D (20-epi-1
,25-dihydroxyvitamin D3; 23): R1 = CH3, R2 = R3 = R4 = R5 = H, R6 = OH
H 1,24R-D (1
,24R-dihydroxyvitamin D3; 24): R1 =
CH3, R2 = R3 = H, R4 = OH, R5 = H, R6 = H
1,24S-D (1
,24S-dihydroxyvitamin D3; 25): R1 =
CH3, R2 = R3 = H, R4 = H, R5 = OH, R6 = H
1,24R,25-D (1
,24R,25-trihydroxyvitamin D3; 26): R1 =
CH3, R2 = R3 = H, R4 = OH, R5 = H, R6 = OH
1,24S,25-D (1
,24S,25-trihydroxyvitamin D3; 27): R1 =
CH3, R2 = R3 = H, R4 = H, R5 = OH, R6 = OH
24-oxo-1,25D (24-oxo-1
,25-dihydroxyvitamin D3; 28) R1 =
CH3, R2 = R3 = H, (R4 + R5) = O, R6 = OH
3 1
1,23R,25D (1
,23R,25-trihydroxyvitamin D3; 29): R1 =
CH3, R2 = OH, R3 = H, R4 = R5 = H, R6 = OH
HO OH
1,23S,25-D (1
,23S,25-trihydroxyvitamin D3; 30): R1 =
CH3, R2 = H, R3 = OH, R4 = R5 = H, R6 = OH

20
23 R1
20
23 OH H 25
O R2
H 25
O
O
O H
H

3 1

3 1 HO OH
HO OH
23S,25R-lac (23S,25R-1
,25-dihydroxyvitamin D3-23,26-lactone; 32): R1 = OH, R2 = CH3
23R,25S-lac (23R,25S-1
,25-dihydroxyvitamin D3-23,26-lactone; 31) 23S-lac [(23S)-25-dehydro-1
-hydroxyvitamin D3-23,26-lactone; 34]: (R1 + R2) = CH2

HO 20

H
OH

3 1
HO OH

Gemini (21-(3-hydroxy-3-methyl-butyl)-1
,25-dihydroxyvitamin D3; 33)

Other analogs not used in structure activity relationship determination:

R2

H
R3

3 1
HO R1

1D (1
-hydroxyvitamin D3; 35): R1 = OH, R2 = H, R3 = H

24,25D (24,25-dihydroxyvitamin D3 36): R1 = H, R2 = OH, R3 = OH

Fig. (1). The two isoforms of 1,25D (1-2), and chemical structures of the vitamin D analogs discussed (3-36).

25- and 1
-hydroxylating enzymes, upregulation of their
activity might open up the way for the large-scale biotechno-
logical production of 1 [7].
Relatively little is known about the biosynthesis of phy-
toecdysteroids. Unlike insects, plants are capable of the bio-
synthesis of phytoecdysteroids from mevalonic acid or via
sterols [9]. The last steps of the biosynthetic routes probably
differ in the various plants. Ecdysone (48) is converted to 20-
hydroxyecdysone (20E, 37) by 20-hydroxylation involving
the microsomal enzyme cytochrome P450 in ferns [10],
spinach [11] and Ajuga reptans [12]. It has recently been
reported that 20-hydroxylase in spinach is more efficient in
using 2-deoxyecdysone (45) than 48 as a substrate. On the
Phytoecdysteroids and Vitamin D Analogues
other hand, C-2-hydroxylase which produces 48 from 45 is
much more active in using 2-deoxy-20-hydroxyecdysone (2-
deoxy 20E, 44) than 45 as substrate. This suggests that C-20-
hydroxylation precedes C-2-hydroxylation, at least in spin-
ach, unlike the situation in insects, where 48 is converted
into the physiologically more active 37. The great differ-
ences support the independent origin of ecdysteroid biosyn-
thetic pathways in plants and insects [13].
STRUCTURE AND CONFORMATIONAL FLEXI-
BILITY OF THE VITAMIN D ANALOGUES AND
ECDYSTEROIDS
Steroid hormones are classified chemically on the basis
of the traditional presence of a structure involving four rings,
A, B, C and D, which is related to cyclopentanoperhydro-
phenanthrene. Most of the steroid hormones bear either a
truncated side-chain (cortisol, aldosterone or progesterone)
or no side-chain (estradiol and testosterone), but their rings
are fully intact. 1 is a structurally unique steroid hormone
because it possesses not only the complete 25-
hydroxycholesterol side-chain, but also a seco-B-triene struc-
ture, indicating an open ring B (the C-9-C-10 bond is bro-
ken). These structural differences render the seco-B-steroid 1
considerably more flexible conformationally. This flexibility
occurs as a result of the following structural features [14]:
• the opened ring B exhibits 360º rotation around the C-
6-C-7 bond, forming a 6-s-trans (vitamin D form) or
a 6-s-cis (previtamin D form) conformation;
• ring A can undergo a chair-chair interconversion typi-
cal of cyclohexane, which changes the orientation of
the axial 1
- and equatorial 3-hydroxyl groups (
-
chair) to equatorial/axial (-chair);
• the side-chain in 1 exhibits rotation around the 5 sin-
gle C-C bonds (Fig. 1).
Apart from the more than 35 naturally occuring biologi-
cally active and inactive metabolites [15], at least 400 ana-
logues of 1 have been synthesized chemically [16]. The ana-
logues are modified: (a) in ring A, through the addition or
removal of functional groups, epimerization of the 1- and 3-
OH groups, 19-nor or oxidized derivatives; (b) in ring B,
through locking of the 6-s-cis or 6-s-trans position, epimeri-
zation of C-9 and C-10; (c) in rings C/D through, 14-
epimerization; or (d) in the side-chain, through the addition
or removal of functional groups, epimerization, locking of
the single C-C bonds or addition of an extra side-chain as in
Fig. (1).
OH OH
HO OH
OH
HO
H
O OH
20E oxo form
Fig. (2). Tautomer interconversion of 20E (37).
Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1977
Ecdysteroids comprise a class of polyhydroxylated ster-
oids. They are generally characterized by a basic skeleton
containing 27-29 carbon atoms with a long sterol alkyl side-
chain on C-17 and the presence of a 7-en-6-one chromophore
group in its ring B. In view of these features, the naturally
occurring hormone 37 and most of the ecdysteroids can be
classified among the conformationally flexible steroids, be-
cause the side-chain rotates freely around the 5 single C-C
bonds. However, ecdysteroids with 19, 21 or 24 carbon at-
oms may also be formed from C-27 ecdysteroids by cleavage
of the sterol side-chain. Moreover, the conjugated oxo func-
tion at C-6 has been considered to undergo a tautomer inter-
conversion, forming a 5,7-diene structure [17,18] similar to
6-s-cis locked vitamin D analogues Fig. (2). Characteristic
OH groups are present in positions 3- and 14
-, but further
hydroxylation is often observed at C-1, 2, 5, 11, 20, 22, 25,
26 or 27. Other known features include additional double
bonds or oxo groups, and glucosylation, esterification or
etherification of certain OH groups [1]. Ecdysteroids dis-
cussed in this paper are depicted in Fig. (3).
THE VITAMIN D RECEPTOR (VDR)
The VDR, a member of the nuclear receptor (NR) super-
family, is regulated by 1. NRs are transcription factors that
regulate the expressions of genes containing NR-specific
responsive elements [19-22]. The NRs share a modular do-
main structure, which includes, from the N-terminus to the
C-terminus, the modulatory A/B domain, the DNA-binding
domain (DBD; the C domain), the hinge D domain, the
ligand-binding domain (LBD; the E domain) and a variable F
domain. Sequence-specific binding to ‘responsive elements’
in target genes is mediated by the DBD. The LBD mediates
ligand activation, ligand-independent repression and dimeri-
zation [20]. In the NR nomenclature, the VDR belongs in the
NR1I subfamily, which also comprises the constitutive an-
drostane receptor (CAR) and the pregnane X receptor (PXR).
Together with the NR1H receptors, the ecdysone receptor
(EcR), the liver X receptor (LXR) and the farnesoid X recep-
tor (FXR) heterodimerize after ligand binding with their
ubiquitous partner, RXR or its invertebrate orthologue, USP
[23,24].
The various effects of 1 on cell signalling are possibly
exerted via membrane receptors [25] or through the theoreti-
cal conformation of the membrane-bound VDR NR [26].
The most appealing rapid response to 1, the rapid Ca2+ and
phosphate uptake in the chicken intestine, turned out to be
dependent on a membrane protein referred to as 1,25D3-
OH OH
HO OH
OH
HO
20E enol form
1978 Current Medicinal Chemistry, 2010 Vol. 17, No. 18 Tóth et al.

R6 R7
R9

R5
R1
R8 R10
R2

OH

R3
R4
O

Ecdysteroid R1 R2 R3 R4 R5 R6 R7 R8 R9 R10

20E (37) H OH OH H H OH OH H H OH
20E 2,3,22-triacetate (38) H OAc OAc H H OH OAc H H OH
20E H OAc OAc H H OH OAc H H OAc
2,3,22,25-tetraacetate (39)
20E 2,3-acetonide (40) H 2 H H OH OH H H OH
O
3
20E 2,3;20,22-diacetonide (41) H H H H H OH
O
O O

20 22

20E 22-benzoate (42) H OH OH H H OH OBz H H OH

24(28)-Dehydromakisterone A (43) H OH OH H H OH OH H =CH2 OH
2-Deoxy-20E (44) H H OH H H OH OH H H OH
2-Deoxy-ecdysone (45) H H OH H H H OH H H OH
Ajugasterone C (46) H OH OH H OH OH OH H H H
Cyasterone (47) H OH OH H H OH OH
O
25

23
O

Ecdysone (48) H OH OH H H H OH H H OH
Integristeone A (49) OH OH OH H H OH OH H H OH
Muristerone A (50) H OH OH OH OH OH OH H H H
Polypodine B (51) H OH OH OH H OH OH H H OH
Ponasterone A (52) H OH OH H H OH OH H H H
Rubrosterone (53) H OH OH H H O
17

Silenoside A (54) H OH OH H H OH O-gal H H OH

Turkesterone (55) H OH OH H OH OH OH H H OH
Viticosterone E (56) H OH OH H H OH OH H H OAc

Fig. (3). The chemical structures of ecdysteroids cited in this paper (37-56).

MARRS. 1,25D3-MARRS differs from the VDR in antibody
and antisense inhibition [27]. Interestingly, in the rat intes-
tine the rapid response is mediated by both 1,25D3-MARRS
and the cell surface VDR [28], suggesting that the VDR is a
more advanced type receptor [29]. However, this novel re-
ceptor has not yet been demonstrated in skeletal muscle, and
the effect of 1 on muscle signalling is therefore more likely
to be exerted through the membrane-localized VDR.
The VDR conformation depends on the nature of the vi-
tamin D sterol (VDS) binding to it. The ensemble model of
the VDS-VDR might explain how the different VDSs pref-
erably influence the NG response through the membrane-
bound NR [26]. This concept may explain the wide range of
effects in the responses of VDSs produced by plants and
other organisms [26].
Crystal Structure of the VDR LBD Bound to 1 (Genomic
Pocket, GP)
The tertiary structure of the VDR LBD is similar to that
of other NR LBDs bound to their ligands, bearing a three-
Phytoecdysteroids and Vitamin D Analogues Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1979

stranded  sheet and 12  helices (H1-H12), forming a three-

layer sandwich completely encompassing the ligand in a
hydrophobic core [26]. The first solved VDR crystal struc-
ture was that of a human VDR LBD mutant, from which 50
residues were removed in order to improve the crystal
growth. The deletion, carried out in the region connecting H1
to H3, did not alter the structure of the LBD or the transacti-
vation capacity of the protein [30,31]. Small helices H2 and
H3n connect helices H1 and H3. The tip of the  sheet before
H3 is shifted outwards and enlarges the binding cavity of 1,
stabilized by hydrogen-bonds (H-bonds) with residues of the
connecting loop between H2 and H3n. Helix H6 is shifted to
the surface, also causing enlargement of the ligand-binding
cavity [30]. The position of activation helix H12 is deter-
mined by the coactivator binding and the transactivation. In
the VDR complexed with 1, it can be found in a transcrip-
tionally agonist position, making direct van der Waals con-
tacts with the ligand. The helix position is stabilized by hy-
drophobic contacts (residues of H3, H5 and H11) and polar
interactions (H3 and H4). Some of the residues stabilizing
H12 are also in direct contact with the ligand, suggesting a
ligand mediating positioning of the activation helix [30].
Ring A of 1 complexed to the VDR GP has a -chair con-
formation with equatorial and axial orientation of the 1-OH
and 3-OH groups, respectively. The conjugated triene sys-
tem, connecting ring A to rings C and D adopts an almost
trans conformation. The torsion angle of the C-6-C-7 single
bond deviates by 30º from planar geometry, resulting in the
curved shape of the ligand bound to the GP. The ligand bind-
ing cavity is large (697 ) and rather hydrophobic, where the
ligand is arranged with ring A pointing to H3 and H5 and the
25-OH at the end of the side-chain close to H7 and H11. The
distances between 25-OH and the ring A OH groups are 13 Fig. (4). VDR LBD complexed to 1,25D (1) based on [30]. -
 (1-OH) and 15  (3-OH). The OH groups on ring A form Helices and the –OH groups of 1,25D (1) are indicated. The dashed
H-bonds to the H3 and H5 residues. The conjugated triene oval shows the approximate location of the hypothetical VDR AP
system fits tightly into a hydrophobic channel formed by based on [26].
loop H5-, 1 and the residues of H3. The C-18-methyl
group on ring C points towards H3. The side-chain has an branes [36] and in T tubules of cardiac myocytes [37]. Ana-
extended conformation that is almost parallel with the C-13- logues, which are poor agonists for G effects and full ago-
C-18 bond [30,31]. Fig. (4) depicts the VDR LBD structure nists of NG responses, such as 19, have been synthesized
bound to 1. [38] and docked by in silico modelling in VDR [39]. The
Homology docking of 37 to an EcR LBD constructed by ligand forms strong H-bonds with residues in H3 and H5 via
taking the crystal structures of human RAR and VDR GP as its 3-OH group, and water-mediated H-bonds with a residue
templates resulted in a favourable interaction between the of H5 via its 1-OH and with a residue of the -sheet loop
LBD and 37, though the orientation of 37 was inverted in the via its 25-OH, suggesting an alternative ligand binding
two distinct cavities [32]. It was later confirmed in an X-ray pocket (AP) in the VDR which partially overlaps with the
crystallographic experiment that the ecdysteroid analogue GP. Interestingly, the residues of H3 and H5, which form
ponasterone A (50) in its cognate receptor adopts a reversed bonds with 3-OH of 1 analogues, are the same as that mak-
orientation as compared with 1 in the VDR GP. However, ing contact with 1-OH of the analogues in the GP, and thus
the long and thin L-shaped cavity of the EcR-LBD in the the overlapping territory is responsible for the accommoda-
complex with 50 is quite similar to that of the VDR GP tion of ring A in the different analogues [40]. The X-ray
complexed to 1. Analogues 1 and 50 also reveal a similar crystal structures of the apo- and holo-NRs indicate that the
positioning of their respective aliphatic side-chain (at posi- ligands might enter the GP via two distinct routes: through
tion 17 of ring D) in their cognate receptors [33]. the H2/ sheet region [41] or through the H11/H12 region,
the mechanism of which is defined as the mouse-trap model
Alternative Ligand Binding Pocket (AP) [42]. Accordingly, the unligated NR exists with an open
H11/H12 conformation, allowing the ligand to enter, but
Like other steroid hormones 1 exhibits rapid, NG effects upon ligand binding it recloses to form a transcriptionally
(reviewed in [34] for vertebrate hormones and for inverte- active conformation, completely encapsulating the ligand. It
brate ecdysone (46) in [35]). Experiments have supported the has been suggested that the ligand enters the VDR AP via the
hypothesis that these responses are mediated by the VDR H2/ sheet region instead of H11/H12 region [39], by a
itself, which is present in calveolae-enriched plasma mem-
1980 Current Medicinal Chemistry, 2010 Vol. 17, No. 18
mechanism analogous to a flickering gate [43], because of
the smaller enthalpy of activation needed for entry through
this portal as compared with the enthalpy required for the
repositioning of H11/12 [44,45]. Interestingly, this region
was earlier proposed as a portal to GP of the thyroid receptor
 (TR) [41] and an energetically preferred exit route for
retinoic acid from the RAR GP [46] and 1 analogue ligands
from the VDR GP [47]. The orientation of the hypothetical
AP is shown in Fig. (4). Similarly to VDR, both EcR [33]
and FXR [48] exhibit an apparent ability to bind different
ligands in distinct and partially overlapping binding pockets,
while in silico modelling results indicate that ER
also
seems to have an additional AP [39,49]. The modelling data
suggest that the GP and AP in combination form a single
LBD channel if H12 is in a closed position [26].
When 19, a 6-s-cis locked analogue of 1, is docked to
AP, the terminal end of the side-chain is solvent-exposed.
Docking of the 6-s-cis conformation of 1 to the AP maintains
a -chair conformation for ring A, and its 1
-OH is able to
make H-bond contacts with the same residues of H1 and
loop H5- as in the case of the analogue 19. Docking of 1
with an
-chair conformation of ring A, the seco ring B
adopts a 6-s-trans conformation. As deduced from their total
potential energies, the
-chair, 6-s-trans conformation is
favoured because it has increased intramolecular stability but
lacks the two additional H-bonds in the AP, which are
formed by the -chair 6-s-cis conformation. Finally, it was
concluded that the AP shows no preference for 6-s-cis or 6-s-
trans sterols, which is in contrast with the GP -chair, 6-s-
trans selectivity [39].
Conformational Ensemble Model
On the basis of these results, a conformational ensemble
model has been proposed [41], on the analogy of the protein
ensemble model described by Bursavich in 2002. In this
model, the unbound receptor macromolecules are present in
the cytoplasm in multiple conformations, in equilibrium ac-
cording to the law of standard statistical Boltzman distribu-
tion [50]. In the case of vitamin D, both the VDR and 1 exist
in multiple conformations and are able to create different
ensembles of stable ligand/receptor complexes responsible
for the different types of physiological signalling. In other
words, the ligands do not induce a conformational change in
the receptor protein, but rather serve to shift the pre-existing
equilibrium of the VDR conformers [43]. The affinity of 1
analogues for the GP and AP differ, and the occupancies of
the two LBDs are influenced by factors such as the ligand
chemistry, the concentrations of ligand and protein (the mass
action effect), and the gating properties of the two receptor
portals. The AP is more hydrophilic, preferring ligands with
a more linear shape, but accessible for more different con-
formational isomers as compared with the GP [26]. The sug-
gested model postulates that the ligand occupancy of the AP
is kinetically favoured, while the occupancy of the GP is
thermodynamically favoured for most of the 1 analogues
[39,43]. An additional refinement was recently proposed on
the basis of this model, depending on the ‘side-chain-first’ or
‘ring A-first’ entry of the 1 analogues into the AP. When the
ligand enters side-chain-first, it is properly oriented to move
towards the GP, while the ring A-first entry may lead to
more stable interactions forming in the AP, which is kineti-
Tóth et al.
cally favoured and energetically more stable and thought to
initiate NG responses via a shifting in the H12 equilibrium
towards a transiently open, transcriptionally antagonist con-
formation [26].
THE EFFECT OF VITAMIN D ON SKELETAL
MUSCLE
1 interacts with its own receptor and perhaps with other
receptors in the autocrine and paracrine environments
[25,51]. The VDR is expressed in a number of different cells
and tissues [6]. Some of these tissues can be organized into
physiological systems, as in the intestine, the bone, the para-
thyroid gland and the kidney during regulation of the cal-
cium homeostasis. One of the additional and traditionally
less considered physiological systems is the muscle [6]: the
skeletal muscle constitutes about half of the human body
mass and its significance is increasingly recognized in the
prevention and cure of health problems.
The connection between vitamin D and the skeletal mus-
cle was established long ago in a range of clinical studies
[reviewed in 52]. An insufficiency of vitamin D is paralleled
by a large variety of muscle dysfunctions [53,54], and the
supplementation of vitamin D and its derivatives leads
mostly to beneficial effects [55,56]. The positive effect of
vitamin D on muscle adaptation to exercise has been demon-
strated, but several other factors, such as the serum level of
3, the parathyroid hormone (PTH) and calcium may also
contribute to the picture [57]. One of the applied aspects of
this field is improvement of the tenderness of beef by feed-
ing vitamin D to cattle [58]. The main question in the inter-
pretation of these results was whether vitamin D acts directly
on the muscle. Identification of the VDR in various stages of
muscle development provided a firm basis for the direct ef-
fect [59-61], implying that skeletal muscle might be a
physiological target for 1.
Deletion of the VDR gene in mice created an animal
model of vitamin D-dependent rickets of type II (type I rick-
ets is related to defects of 1 production). VDR -/- mice ex-
hibited normal development in early neonatal life, but after
weaning they displayed abnormal muscle development, re-
duced and variable muscle fibre size (cross-sectional area,
CSA) and a deregulated expression of developmentally im-
portant genes as compared with the wild-type control [62].
The expression of the MyoD family of transcriptional factors
was also significantly altered relative to the tendencies de-
scribed in normal muscle development: myf-5 and myogenin
failed to decline in 3-weeks old VDR -/- mice as in the con-
trols. The expression of the embryonic, neonatal and type II
myosin heavy chain isoforms (MyHC) – markers of muscle
development and differentiation – also remained at a higher
than normal level in VDR-deficient mice. Treatment with 1
downregulated the above parameters in a wild-type mouse
cell line, suggesting a negative correlation with the hormone
level [62].
Genomic Effects
1 exerts its primary action on a ligand-gated NR acting
also as a transcription factor. The VDR has been identified in
nuclei of human skeletal muscle by immunostaining [63]
Phytoecdysteroids and Vitamin D Analogues
demonstrating the presence of the 1 NR. After transportation
to the cell, 1 binds to this receptor, and the activated receptor
enters the nucleus and results in changes in mRNA transcrip-
tion, protein translation, cell cycle arrest and subsequently
differentiation [64]. The active VDR forms a heterodimer
with an orphan steroid receptor, the retinoic receptor (RxR).
This heterodimerization facilitates the association of the Zn-
finger domains of the receptor with DNA and activates tran-
scription. The other interaction of VDR occurs with PGC-1

(peroxisome proliferator-activated receptor- coactivator-1
)
[65]. PGC-1
has a key role in mitochondrial biogenesis
therefore in muscle development and differentiation; when it
is ectopically expressed in skeletal and heart muscle, it coor-
dinates the gene expression toward mitochondrial formation
[66].
This genomic pathway is considered a slow effect of vi-
tamin D because it takes from hours to days. The genomic
pathway controls calcium uptake, trans-membrane phosphate
transport, the phospholipid metabolism, cell proliferation and
differentiation in muscle [52], but there is a more rapid effect
of the vitamin D hormone, in the time range from seconds to
minutes, which can not be explained by new gene transcrip-
tions.
Non-Genomic Effects
The most characteristic example of the rapid response is
the enhanced calcium transport through chicken intestinal
cells to the circulatory system by the physiological concen-
tration of 1. This occurs in 4-5 minutes in the ex vivo system
of the perfused chicken intestine, and is termed transcal-
tachia [67]. A number of other examples indicate similar
responses in the skeletal muscle; these rapid events can not
be prevented by inhibitors of transcription, and most likely
involve signal transduction events [68-70].
The complex effect of 1 on signalling pathways is well
demonstrated in ex vivo muscle slices of young (3 months)
and old (24 months) rats [71,72]. A nanomolar concentration
of the hormone stimulated the biphasic transient release of
diacylglycerol (DAG) from the hydrolysis of phospholipids
[71,72]. It is important that the hormonal nature of the effect
was confirmed, because close derivatives of vitamin D, 3, 35
and 36 did not increase the muscle inositol triphosphate (IP3)
level. DAG was first liberated within 15 seconds, in parallel
with IP3 from the hydrolysis of phosphoinositides by phos-
pholipase C (PLC). The second wave of DAG production
resulted in 2 minutes from 1 stimulation of phospholipase D
(PLD) activity. PLD hydrolyses phosphatidylcholine (PC)
and generates phosphatidic acid, which is converted to DAG
by a phosphatidate phosphatase. The stimulations of both
PLC and PLD activities were dependent on extracellular
calcium, inhibited by calcium channel blockers and further
stimulated by calcium ionophores. Activators of G-protein-
mediated signals mimicked the effect of 1 on these phosphol-
ipases, but inhibitors suppressed it. Analogue 1 also stimu-
lated the activity of protein kinase C (PKC), this effect prov-
ing synergistic with that of the phorbol ester TPA. The 1-
stimulated PC hydrolysis resulting in DAG formation there-
fore occurred through PLD activation in a mechanism in-
volving calcium, PKC and G proteins [71]. Preceding the
stimulation of PLD activity, the phosphatidylinositol hydro-
Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1981
lysing PLC activity was a pertussis toxin-sensitive G-
protein-dependent mechanism in rat muscles [72]. Facchi-
netti et al. [72] also demonstrated that basal IP3 and DAG
levels were higher in the muscles of aged (24 months) rats
than in those of young rats; the second messenger-generating
effect of 1 was therefore reduced in the aged animals. The
pertussis toxin sensitivity of IP3 production stimulation was
also lost in aged rats, indicating the altered effect of the hor-
mone [72].
Aging had a detrimental effect on the activation of PKC
isoforms by 1 [73]. The increases in the total and membrane
PKC activities on hormone administration (10-9 M) were
blunted in the muscles of old rats: the expressions of the
, 
and  isoforms were greatly reduced, but those of the , 
and  isoforms were less related to age. After exposure for 1
minutes to 1, the amounts of PKC
and  increased in the
membranes, and reverse translocation (from the membranes
to the cytosol) was observed for PKC in young, but not in
old animals. This shows that aging impacts on both calcium-
dependent (
and ) and calcium-independent (, ,  and )
PKC isoforms and these are selectively regulated by 1 in the
skeletal muscle [73].
Analogue 1 increased the phosphorylation and activity of
p38 MAPK (mitogen-activated protein kinase) within 1 min-
utes in the C2C12 mouse myoblast cell line [74]. The imme-
diate upstream protein kinases were also phosphorylated,
suggesting their involvement in MAPK activation. The ubiq-
uitous cytosolic tyrosine kinase c-Scr was dephosphorylated
and inhibitors implied its participation in the hormonal acti-
vation of p38. The hormone also increased the phosphoryla-
tion of mitogen-activated protein kinase activating protein
kinase 2 (MAPKAP-kinase 2) and the subsequent phos-
phorylation of heat shock protein 27 (HSP27) in a p38 kinase
activation-dependent manner. However, 1 also stimulated the
phosphorylation of c-jun N-terminal protein kinases (JNK
)
within the same interval, which suggests a parallel contribu-
tion of the two pathways in the hormonal regulation of cell
proliferation and differentiation in muscle [74].
Analogue 1 is effective in improving the insulin resis-
tance of muscle [75]. Free fatty acid (FFA) treatment re-
duced the myotube diameter and insulin sensitivity of a
mouse muscle cell culture. Besides the insulin-mediated glu-
cose uptake, the tyrosine phosphorylation of insulin receptor
substrate 1 (IRS-1) and the phosphorylation of Akt/protein
kinase B were decreased, whereas the serine phosphorylation
of IRS-1 and the phosphorylation of JNK were increased.
The supplementation of 1 resulted in an improvement of the
FFA-induced inhibition of glucose uptake in a time and
dose-dependent manner. In parallel with this, the tyrosine
phosphorylation of IRS-1 and the phosphorylation of
Akt/protein kinase B increased, while the serine phosphory-
lation of IRS-1 and the phosphorylation of JNK decreased.
Treatment with 1 also restored the FFA-reduced diameter of
the myotubes. This shows that the link between a vitamin D
deficiency and insulin resistance involves the changed activ-
ity of important signal routes [75,76]. The same signal routes
are influenced by ecdysteroids [77-79] and 37 was found to
decrease the level of hyperglycaemia and the body weight in
diet-induced obesity of mice [79].
After a longer time, stimulation of signal transduction
pathways by 1 also results in the activation of transcription
1982 Current Medicinal Chemistry, 2010 Vol. 17, No. 18 Tóth et al.

and the induction of early genes, such as c-fos. Treatment
with 1 for 1-1.5 hour led to phosphorylation of the transcrip-
tion factor CREB in a MAPK-dependent manner in the
C2C12 cell line and this resulted in induced c-fos expression.
A similar phosphorylation of the Elk-1 transcription factor
was seen after 17
-estradiol administration and this also in-
duced c-fos in a MAPK-dependent way [80]. This suggests
that a longer administration of 1 might result in gene activity
changes via signalling pathways. However, a similar effect
has not been reported yet for ecdysteroids in C2C12 cells.
Calcium is a central second messenger in the regulation
of gene expression in the skeletal muscle, and its intracellu-
lar level is influenced by steroid action [81]. The process is
controlled by depletion of the internal calcium stores, which
opens plasma membrane calcium channels (store-operated
channels, SOCs) and results in capacitive calcium entry
(CCE) into the cytoplasm [82]. The TRPC (transient receptor
potential channels) family involves candidates for SOCs
[82]. 1 stimulates calcium release in chicken muscle cells
from inner stores and the calcium influx through voltage-
dependent and store-operated channels. TRPC3, an ex-
pressed membrane channel in muscle cells, is involved in
this process, together with the VDR; the transfection of the
cells with antisense oligonucleotide either for TRPC3 or for
the VDR prevented the Ca2+ and Mn2+ influx. TRPC3 and
VDR were coprecipitated under denaturating conditions,
raising the possibility that 1 modulates CCE by influencing
the interplay between TRPC3 and the VDR [83]. A further
study from the same research group confirmed the similar
effects of 1 and thapsigargin (an inhibitor of Ca2+ ATPase
pumps that refills the sarcoplasmic/endoplasmic reticulum
with Ca2+) [84]. The research group also demonstrates the
participation of other (INAD-scaffold) proteins in the VDR-
TRPC3 association in muscle cells [83].
STRUCTURE-ACTIVITY RELATIONSHIP
Vitamin D Analogues
The relationship between the different analogues of 1 and
their G and NG effects have been assayed several times by
means of in vivo, ex vivo and in vitro methods (Table 1)
through measurement of the median inhibitory concentra-
tions and median effective concentrations, the partial trypsin
digest footprint / protease sensitivity assay (PSA) and with in
silico modelling with the methods outlined in [39].
In most cases, the results from the different studies did
not reveal any correlation, and it is a challenging task to
evaluate any structure-activity relationship. However, with
the application of the conformational ensemble model [39],
the deviating functional results could be explained in terms
of the relative VDR GP and AP and by the stability and frac-
tional occupancy of the different ligands [39,40,85,95]. The
results of different experiments are presented in Table 2.

Table 1. Description of Functional Experiments Performed with 1,25D (1) Analogues In Vitro and Ex Vivo Used for the Determi-
nation of Structure Activity Relationship

Assay Cell or organ References

Genomic assay systems

Decrease in PTH secretion bPTC [85]
VDRE-driven rat osteocalcin gene expression linked to hGH produc- COS-1 [86]
tion
VDRE-driven gene expression linked to hGH production COS-7 [38]
VDRE-driven alkaline phosphatase gene expression CV-1 [39]
VDRE-driven mouse osteopontin gene expression G-361 [86]
VDRE-driven CYP24 gene expression HaCaT [87]
Induction of cell differentiation (induction of 4-nitroblue tetra- HL60 [38], [88]
zolium/NBT production, induction of the expression of CD11b protein)
Inhibition of proliferation (inhibition of [3H]thymidine incorporation) MCF-7 [38]
Induction of cell proliferation (induction of human osteocalcin) MG-63 [38]
VDRE-driven 24-hydroxylase gene expression osteosarcoma cell line, [89]
HL60 [90]
VDRE-driven rat osteopontin gene expression ROS 17/2.8 [38]
Nongenomic assay systems
Induction of cell differentiation (reducing of 4-nitroblue tetra- NB4 [91]
zolium/NBT production)
Transcaltachia chick duodenum [92]
Cl- channel opening ROS 17/2.8 [93]
45
Ca2+ uptake ROS 17/2.8 [94]
Phytoecdysteroids and Vitamin D Analogues Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1983

Table 2. The Activity of 1,25D (1) Analogues in Different In Vitro, Ex Vivo, In Vivo and In Silico Systems. The Data are Based on
the Following References: [26,39,40,85,95-97]

In vitro, ex vivo, in vivo experiments In silico modeling

A-ring
/ PIE in GP PIE in AP
PSA foot- Functional ex- A-ring chair / B-
Ligand RCI (%)a chair
print b periment results ring orientationd (kcal/mol) e (kcal/mol)f
ratioc
/trans -107.9 -92.7
Full agonist of G
1,25D (1) 100 c1>>>c3 and NG responses 45:55
/trans - -91.6
/cis - -93.7
A-ring modified analogues
/trans -91 no binding
25D (3) ~0.15 c3>c1>>c2 NG agonist 60:40
/trans -89 -86
/cis - -78
/trans -98 -84
3d-1,25D (4) ~6.0 c1 Potent G agonist 35:65
/trans -96 -83
/cis - -85
/trans -98 -91
3-epi-1,25D (5) ~0.2 weak c1 Poor G agonist -
/trans -100 -89
/cis - -94
/trans -105 -89
3-epi-1,25D (6) ~24 c1 G agonist 80:20
/trans -95 -91
/cis - -94
/trans -100 -82
1,25D (7) ~1 weak c1 NG antagonist 10:90
/trans -94 -95
/cis - -80
2
Me-1,25D (8) ~400 - G agonist - - ~ -110.8 -
/trans -108 -92
2Me-1,25D (9) ~13 - NG agonist -
/trans -96 -89
/cis - -83
/trans -102 -86
2Me-1,25(OH)2D3
(10)
- NG antagonist 8:92
/trans -105 -92
/cis - -81
/trans -97 -89
2Me-3-epi-
1,25(OH)2D3 (11)
- NG antagonist 30:70
/trans -101 no binding
/cis - -91
/trans -107 -90
2Me-3-epi-1,25D (12) - NG antagonist 40:60
/trans -94 -
/cis - -96
19-nor-1,25D (13) ~9 - G and NG agonist - - -107.3 -
19-nor-Gemini (14) ~6 - G and NG agonist - - -129 -
B-ring modified analogues
1,25-trans-T (15) ~0.005 - NG agonist - - - -
1,25-cis-isoT (16) ~0.4 - NG agonist - - - -
1,25-trans-iso-T (17) ~1 - None - - - -
1,25(OH)2-7-DHC (18) ~0.1 - NG agonist 0:100 - - -
1,25L (19) ~1 c1>>>c3 NG agonist 0:100 /cis -104 -89
1,25P (20) ~0.2 - NG agonist 0:100 - - -
1,25-iso-P (21) ~0.3 - NG agonist 0:100 - - -
1,25-d5-preD (22) ~10 - NG agonist - - - -
1984 Current Medicinal Chemistry, 2010 Vol. 17, No. 18 Tóth et al.

(Table 2). Contd…..

In vitro, ex vivo, in vivo experiments In silico modeling

A-ring
/ PIE in GP PIE in AP
PSA foot- Functional ex- A-ring chair / B-
Ligand RCI (%)a chair
print b periment results ring orientationd (kcal/mol) e (kcal/mol)f
ratioc

Side chain modified analogues

20-epi-1,25D (23) ~196 c1>>>c2 G superagonist 45:55 - -109 -90
G agonist (de-
1,24R-D (24) ~94 - pending on assay - - -101.3 -
system)
G agonist (de-
1,24S-D (25) ~9 - pending on assay - -107.2
system)
/trans -111 -97
1,24R,25D (26) ~39 c1>c3 G agonist -
/trans - -98.1
/cis - -98.7
/trans -112.6 -100
G agonist (de-
1,24S,25D (27) ~4 c3>>c1 pending on assay -
/trans - -98.9
system)
/cis - -101.4
/trans -110.4 -95.6
G agonist (de-
24-oxo-1,25D (28) ~98 - pending on assay -
/trans - -95.1
system)
/cis - -94
/trans -115 -104.3
G agonist (de-
1,23R,25D (29) ~11 c3>c1 pending on assay -
/trans - -103.3
system)
/cis - -104.2
/trans -117.4 -103.1
G agonist (de-
1,23S,25D (30) ~12 c3>>>c1 pending on assay -
/trans - -102.8
system)
/cis - -103.6
/trans -112.5 -100.3
23R,25S-lac (31) ~12 - G agonist -
/trans - -100
/cis - -98.7
/trans -109.7 -100.7
23S,25R-lac (32) ~0.5 c3>>c1 Poor G agonist -
/trans - -100.5
/cis - -104.9
/trans -130.3 -106.7
Gemini (33) ~40 c3>>c1 G superagonist -
/trans - -106.2
/cis - -109.4
/trans -94.6 -
23S-lac (34) ~5 c1>>>c3 G antagonist -
/trans - -83.4
/cis - -88.6
a 3
Relative competitive index: relative ability of the various analogues to compete with [ H]-1,25D in vitro for the chick intestinal VDR. The RCI value for 1,25D is set to 100% by
definition [98].
b
Partial trypsin digest / Protease sensitivity assay (PSA) a method for assaying VDR H12 conformation and dynamics in solution and yields insight into the population distribution of
apo/holo-VDR isomers under steady-state conditions. The observed population distribution (PSA footprint) is depending on the ligand chemistry. Three conformers are defined: c1,
the ligands are stabilizing closed (transcription agonist) H12 conformation, c2, the ligands are stabilizing partially closed H12 conformation (observed by a class of VDR genomic
antagonist) and c3, the ligands are stabilizing open (inactive) H12 conformation, associated with binding to AP [26,85]. PSA footprint detailed here only at saturating ligand concen-
tration.
c
The
/ chair ratios were determined under equilibrium conditions from the three bond (3J)NMR coupling constants in CDCl3 (dielectric = 4.8) [99-101].
d
A-ring chair conformations and seco-B-ring conformation observed in the end of the calculation.
e,f
Potential interaction energies calculated for the lowest energy complex in GP and AP, respectively. The complexes are hypothetical projections, may not present the actual lowest
energy structures. This value reflects the potential non-bond interactions formed between the ligand and VDR residues. It does not reflect the total binding energy, therefore only
docking the same ligand with different conformations, epimers and constitutional isomers can be directly compared [85].
Phytoecdysteroids and Vitamin D Analogues
DIFFERENCIES IN THE ACTION OF VITAMIN D
ANALOGUES DUE TO STRUCTURAL MODIFICA-
TIONS
G Agonists
1. Ring A Modifications
Epimerization or Removal of OH Groups; Addition of 2
-
Methyl Group (Analogues 4, 5, 6 and 8)
In ex vivo genomic function assays, 1 and 6 exhibited
PTH-decreasing ability in bPTC cells, while 5 increased the
hormone secretion [95]. Analogue 6 affected a noteworthy
PTH decrease comparable to that of 1, but failed to induce
HL60 cell differentiation and VDRE-driven 24-hydroxylase
mRNA production, which are also propagated in response to
1 G signalling [87,91,102]. Removal of the 3-OH group (4)
did not affect the transactivating ability of the sterol in the
CV-1 cell assay [95]. 2
-CH3 substitution (8) 1 enhanced the
affinity of the sterol for the VDR (RCI = ~400%), but almost
diminished its apoptosis-stimulating efficiency (G effect)
[96,103-105]. Docking results for the VDR GP indicate that
a H-bond at position 3 is missing, not only in the case of 4
(without a 3-OH group) but, for steric reasons, also in the
case of 6 (3
-OH) [95]. Of the ring A diastereomers, 6 and 5
were able to form stable complexes in all three ring A/B-
combinations with the AP, which was similar to the situation
for 1, but 6 forms a more stable complex with the GP and
has a predominantly G effect (functional tests and PSA foot-
print), while 5 is only a poor G agonist (PSA footprint, RCI)
[95] and is expected to activate only AP-connected NG sig-
nalling cascades [95,106]. The 2
-CH3 group in 8 does not
allow the formation of low-energy complexes in AP for ei-
ther the 6-s-cis or the 6-s-trans -chair sterol conformations,
which might explain its extremely high RCI value [85,96].
A hypercalcaemic response can be induced by pharma-
cological doses of 1, which limits the in vivo administration
of higher concentrations of the hormone. This phenomenon
was not observed for the ring A diastereomers in chicken
[107]; this response is linked to the increased stability of 1 in
the GP and/or the reduced stability in the AP [95].
Removal of C-19 (Analogues 13 and 14)
Removal of C-19 resulted in a 5-10-fold reduction in RCI
and in no effect on the VDR transactivation potency in COS-
1 and CV1 cells, but it decreased the induction of in vivo
hypercalcaemia [86]. The docking results suggested that the
removal of C-19 decreases the van der Wals attractive forces
of the ligand, resulting in a slightly decreased stability in the
GP. In silico modelling also demonstrated that the removal
of C-19 reduced the energy barrier to rotation around the C-
6-C-7 bond. This would increase the ring A transitions be-
tween the surfaces below and above rings CD [108].
2. Side-Chain Modifications
It has already been shown that, of the natural side-chain
metabolites, only 26 is as efficient G transactivator as 1,
while the VDR agonist activity of the other side-chain me-
tabolites (23-25 and 27-33) varies significantly in a cell or
assay-specific manner (HL60, G-361 and HaCaT cell assays)
[84,109,110]. Analogues 23 and 33 are well-characterized G
superagonists [111-113].
Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1985
Addition of a 24-OH Group (Analogues 24, 25, 26 and 27)
On docking of the constitutional isomers 24 and 25 to the
GP, H-bond contacts identical to those in the case of 1 were
formed, while RCI values were varying greatly (9% and 94%
for 24 and 25, respectively). However, docking of the same
analogues to the AP showed that, while 25 is able to form a
favourable complex with a residue of the AP, 24 failed to
make this contact, thereby reduced its stability in the AP.
These results suggest that the concentration of free 25 mole-
cules is reduced by binding to the AP, while the unfavour-
able AP binding of 24 allows this analogue to undergo 94%
binding to the VDR GP [85]. An additional OH group on C-
25 further reduces RCI values of the analogues [26 (~39%)
and 27 (~4%)]. This is again inconsistent with the observed
increase in GP PI E, which can be attributed to the higher
number of attractive non-bonding interactions. On the other
hand, their increased stability in the AP might explain their
reduced VDR affinity [85].
Oxidation of the 24-OH Group (Analogue 28)
Oxidation of the 24-OH group (28) restored the RCI to
the level for 1. When docked to AP, it was determined that
the 24-oxo group is not able to form the same favourable
non-bonding contacts as observed for 24-OH analogues [85].
Addition of a 23-OH Group (Analogues 29 and 30)
The C-23 metabolite 30 and a synthetic analogue 29 have
similar RCI values (12% and 11%) and PSA profiles relative
to analogues containing a 24-OH group, but they form more
stabilizing H-bonds in the GP and more non-bonding con-
tacts in the AP than C-24 metabolites or 1 itself [85].
Lactone Formation (Analogues 31 and 32)
Further oxidization may produce a lactone moiety on the
side-chain (natural 32, synthetic 31). The 23R epimer (31)
has a higher RCI than that of the natural 23S epimer (32)
(~12% and ~0.05%, respectively). Moreover, 31 is a better
VDR G agonist than the 23S metabolite, but its activity is
still far from that of 1 [114]. When these lactones were
docked to the VDR AP, 32 formed a slightly more stable
complex than their epimer, while 31 was more favourable in
GP [85].
Double Side-Chain (Analogue 33)
Analogue 33, also named Gemini, which has two identi-
cal side-chains and turned out to be a strong agonist of the
VDR genomic function [111]. Interestingly, a 33-VDR crys-
tal structure indicates that the H11/H12 helices are stabilized
in the same position as in case of 1 or other superagonists.
Furthermore, the VDR LBD adapts to the 25% size increase
in 33 relative to 1, forming a new channel extending the
original pocket [115,116]. In contrast, the RCI of this com-
pound is only ~40% and it seems to stabilize c3, the tran-
scriptionally inactive conformation of VDR. The increased
AP stability of 33 results in a lower amount of free molecule
available for the GP (decreased RCI and PSA results), but
the complex formed is more stable and longer-lasting than
that of the GP-1 and its analogues. This might mean that the
33-mediated G events are triggered by a lower fractional
occupancy of the GP [85,87,109-111].
1986 Current Medicinal Chemistry, 2010 Vol. 17, No. 18
G Antagonists
1. Side-Chain Modification
Lactone Formation (Analogue 34)
Although analogue 34 has a reduced RCI, it was found to
stabilize a higher population of the transcriptionally active c1
PSA conformation than that of the inactive c3. Interestingly,
34 is considered a VDR G response antagonist. Indeed, its
inhibitory effect has been demonstrated on HL60 cell differ-
entiation and VDRE-driven 24-hydroxylase gene expression
in HL60 and human osteosarcoma cells [90,117,118].
NG Agonist
1. Ring A Modifications
Epimerization or Removal of OH Groups; Addition of a 2

Methyl Group (Analogues 3 and 9)
Removal of the 3-OH (3) or addition of an equatorial 2-
methyl group (9) results in a low RCI [95,96,104,105] and a
higher population of c3 VDR H12 conformers relative to 1
[95]. This suggests that 3 should rather display NG action.
Indeed, 3 has a dramatically reduced transactivating potency
in the CV-1 cell assay (G effect) [95], but the Cl- channel
activating potency in ROS 17/2.8 cells, and also the differen-
tiation-inducing potency of 9 in NB4 cells, are maintained at
the same level as for 1 (NG effect) [39,96,104,105]. In its
preferred
-chair ring A conformation, 3 is able to form an
energetically stable complex in the AP, which is more or less
comparable with the potential energy observed in the GP
[95]. However, the GP prefers the -chair conformation,
meaning that 3 preferably binds AP, as confirmed by its PSA
footprint and high Cl- channel-activating efficiency [39]. The
equatorial 2-methyl group in 9 also forms favourable hy-
drophobic contacts in AP [85,96].
Removal of C-19 (Analogues 13 and 14)
As the AP accepts more vitamin D analogue conformers
than the GP, it might be expected that 19-nor analogues (13
and 14) will show a higher affinity for the AP and induce
increased NG signalling [85].
2. Ring B Modifications
Locking the Rotatable C-6-C-7 Bond (Analogues 15 and
16, 17-22)
The RCI values for all the analogues were dramatically
decreased after locking of the rotatable C-6-C-7 bond or
when the seco-ring B was closed [~10% (22) - ~0.005%
(15)]. PSA analysis, available only for analogue 19, indicates
that it stabilizes the c1 conformation and weakly the c3 con-
formation. None of the 6-s-cis or 6-s-trans locked analogues
were able to exert a G effect in G assays, whereas NG bio-
logical systems were responsive to the group of 6-s-cis
locked analogues {transcaltachia in the isolated perfused
chick duodenum (the rapid hormonal stimulation of intestinal
calcium absorption), 45Ca2+ uptake in ROS 17/2.8 cells [38]}.
Not all the analogues were as effective as 1 in NG assays,
which suggests that the subtle differences in the shape of the
molecule stemming from the different orientations of C-9
and C-10 can alter their effects. The 6-s-trans locked ana-
logues did not display a noteworthy effect in NG assays [38].
In the case of 22, it was shown by kinetic investigations that
Tóth et al.
the presence of deuterium atoms suppresses the rearrange-
ment from the previtamin to the vitamin form, and thus 22
can function as a 6-s-cis locked analogue [97].
NG Antagonists
1. Ring A Modifications
Removal of OH Group; Addition of a 2
-Methyl Group
(Analogues 7 and 10-12)
Analogue 7 exhibited PTH-decreasing ability only at
higher concentration [95]; it had no effect on HL60 cell dif-
ferentiation [87], but it antagonized 1-induced NB4 cell dif-
ferentiation. In ROS 17/2.8 cells, 7 resulted in Cl- channel
opening [39] dedicated to vitamin D NG signalling cascades
[92]. 2-CH3 analogues (10, 11 and 12) displayed antagonis-
tic effects on the NB4 cell line [96]. Analogues 7, 10, 11 and
12 favour -chair conformation and their PIE is generally
lower in the GP than in the AP. They are not able to form
even weak complexes in the AP, so their free concentration
is increased relative to that of other analogues, which might
facilitate their GP occupancy. Since the GP partially overlaps
with the AP, ligands occupying the GP sterically block the
AP, thereby becoming potent antagonists of the NG effects
of 1, but without producing G responses, probably because
of an unfavoured stereochemistry [95,96].
As a conclusion from the surveyed 1 analogues, the frac-
tional occupancy of the VDR AP can be increased by (a)
removal of the 1-OH group, (b) locking the 6-s-cis ring B
conformation, (c) introducing side-chain OH groups on C-23
and C-24, where S isomers are preferred, (d) reducing the
energy barrier of rotation about the C-6-C-7 bond (removal
of C-19), or (e) addition of a 2/2
-CH3 group. This latter
option has not been completely evaluated yet, as it is highly
dependent on the ring A and side-chain stereochemistry. A
general overview of the effects exerted by the chemical
modifications is depicted in Fig. (5).
Ecdysteroids
Interestingly, ecdysteroids exert physiological effects on
mammals, even though EcR is not present in them. They do
not bind to mammalian AR, ER and PR receptors [1], so
their action might be mediated via secondary messenger sys-
tems [119-121]. The relationship of the structures and ana-
bolic activities of the ecdysteroids was recently reviewed by
our group; for a more detailed description, see [1].
As concerns the anabolic activity, only three in vivo ani-
mal experiments have been reported on the weight gain of
pubertal or impubertal castrated and intact rats [122], [14C]-
labeled amino acid incorporation into the liver proteins of
mice [123], and the hypoazotaemic effect (the ability to
lower the blood levels of urea and residual nitrogen, which is
directly related to the rate of protein synthesis) in male rats
[124]. A recent study in vitro reported on the activities of
four ecdysteroids (37, 51, 52 and 55) on the C2C12 cell line,
where the rate of incorporation of [3H]leucine into myotubes
was determined at different ecdysteroid concentrations [125].
The most important findings of these studies, as regards
the coherent structure-activity relationship data in compari-
son with the structure and corresponding activities of 37,
were as follows:
Phytoecdysteroids and Vitamin D Analogues Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1987

Addition of 24-OH or oxo group favours GP

Epimerization of C-20 or binding, but genomic effects are dependent
introduction of a second side on assay system
chain highly increases genomic
effects

20
18 24 23,25-lactone formation
H 23 favours GP binding, but
25 decreases genomic effect
17
9 14
OH
8

H
Addition of 23-OH favors
7
6 GP binding, but genomic
effects are dependent on
19 assay system
5
3 1
2
HO OH
Removal of C-19 maintaines the activity in ex vivo
genomic assays, might have higher affinity to AP

Removal of 3-OH does not alter
GP binding or genomic effects
Epimerization of 3-OH favours GP
binding, but decreases genomic
effect
2
methylation favours GP Removal of 1-OH favours AP
binding, but decreases genomic binding
effect Epimerization of 1-OH favours
2 methylation favours AP GP binding, but decreases
binding and displays genomic effect
nongenomic effects

Epimerization of 1,3-OH and 2 methylation

favours GP binding and antagonize
non-genomic effects

6-s-cis locked analogs favour AP

binding and display nongenomic
effects, dependent on the
stereochemistry of C-9, C-10
H
OH 19
9 OH
1
10 H
3
HO

Fig. (5). Summary of structural modification based activity changes of vitamin D analogues.

1. Ring A modifications acid incorporation into the mouse liver [123] and the blood
Addition or Removal of –OH Group levels of urea and residual nitrogen in rats [124].
An additional OH group on C-1 (49) decreases the ana- 2. Ring C modifications
bolic activity in vivo [122-124]. Hydroxylation at C-5 (51) Addition of an OH Group
exerted a negative effect on the protein metabolism, as
An additional
-OH on C-11 (55) increased the anabolic
measured by the urea and residual nitrogen in the blood, activity in all of the discussed studies [122-124]. Higher
[124] and also lowered the rate of amino acid incorporation
anabolic activities of 46 and 55 were also observed in other
into mouse liver proteins [123]. Moreover, in vitro experi-
in vivo experiments, as reviewed in [120]. Interestingly, un-
ments with analogue 51 demonstrated a decreased rate of
der in vitro conditions 11-hydroxylated turkesterone (55)
incorporation of [3H]leucine into the myotubes [125]. Re-
showed somewhat less activity, even in higher concentra-
moval of the 2-OH group (44 and 45) resulted in a decreased
tions, than 37, which is not in line with the in vivo result
weight gain and altered residual nitrogen and urea levels in [125].
the blood in rats [122,124], and decreased amino acid uptake
by the mouse liver [123]. Studies of 50 and 52 with the RKO cell line revealed an
unexpected antiapoptotic effect, independently of the ec-
Conjugation of 2,3-OH Groups
dysone-inducible system. Incubated with RKO cells, and
37 lost most of its anabolic activity following conjuga- apoptosis inducer ligands hFasL or TRIAL, both of these
tion with a 2,3-acetonide group (40), as measured via amino ecdysteroids were able to decrease the rate of apoptosis and
1988 Current Medicinal Chemistry, 2010 Vol. 17, No. 18
consequent cell death. Muristerone A (50), with the same
structure as 52 but with an extra 11-OH group, exhibited a
significantly stronger apoptosis-reducing capacity, suggest-
ing that this functional group might play a role in the activity
in this system as well [78].
3. Side-Chain Modifications
Removal of the Entire Side- Chain
Loss of the side-chain, as in the case of 53, increased the
protein built into mouse liver with a higher efficiency than
that of 37 [126].
Lactone Formation
A compound with a closed side-chain (47) was found to
increase the in vivo liver protein uptake in mice, and posi-
tively influenced the protein balance of rats with a higher
rate than that of 37 [123, 124].
Alteration of 20-OH or 22-OH Groups
The presence of a 20,22-diol group in the ecdysteroid
molecule is important for the anabolic activity. Loss of the
20-OH group (45 and 48) negatively altered the activity of
the ecdysteroids relative to 37 in all the in vivo experiments
[87-89]; and the acetylation (38, 39) [122,124] or benzoyla-
tion (42) of the 22-OH group decreased the anabolic activity
[123,124]. Conjugation of 37 with a 20,22-acetonide group,
together with a 2,3-acetonide group, likewise resulted in a
sharp decrease in the rate of amino acid incorporation into
the mouse liver [123].
However, conjugation with a more polar sugar moiety in
the case of analogue 54 had a positive influence on the
weight gain, amino acid incorporation and protein metabo-
lism in vivo [122-124].
Alkyl Substitution at C-24
Compound 43, with an additional methylene group on C-
24 has a somewhat lower, but more or less comparable ana-
bolic activity to that of 37 [122,123].
Removal or Conjugation of the 25-OH Group
Under in vitro conditions, the loss of the OH group from
C-25 (52) resulted in a lower activity [125]. Acetylation of
the 25-OH group (56) also sharply decreased the activity of
37 [122-124].
Important structural elements which lead to an enhanced
anabolic activity are: a) the presence of free 2- or 3-OH on
ring A; b) the presence of 11
-OH on ring B (depending on
the assay system); c) loss of the side-chain, or side-chain
lactone formation; d) if the entire side-chain is present, the
presence of a free 20-, 22- and 25-OH or 22-O-glycoside.
Although there are very few similarities between those
structural changes of vitamin D analogues and ecdysteroids
that are necessary to enhance the NG response or to increase
the anabolic activity, some can still be listed. The 3ß-OH and
25-OH groups are important elements, and the lack of 1-OH
is beneficial in both cases. With the closure of the seco-ring
B (abolishing the main difference between the two steroid
groups), binding to the AP and the potency to induce an NG
response are increased.
Tóth et al.
EFFECTS OF ECDYSTEROIDS ON MUSCLE
The anabolic effect of ecdysteroids on muscle size has
been demonstrated in a wide range of animals, including
quails, rats, sheep and pigs. This effect resembles that of the
anabolic steroids, but no ecdysteroid binding has been dem-
onstrated in mammalian or vertebrate cells. Further studies
on this subject were assessed in a recent review [1]. The
morphological effect of 37 has been described on fast and
slow type muscles in the rat [119]. 37 uniformly increased
the size of the developing fibres in the regenerating soleus
muscle. In contrast, the differentiated fast and slow type fi-
bres increased in size in a muscle-specific manner in re-
sponse to 37. The increase in size of the differentiated fibres
was modified according to the distance from the treatment
and the presence of regenerating muscle in the animal. This
suggested that 37 interacts with the paracrine and endocrine
systems and probably influences signalling pathways during
its anabolic effect on muscle [119]. In a recent study, phyto-
ecdysteroids, applied as various ecdysteroids and extracts of
ecdysteroid-containing plants, increased the in vitro protein
synthesis by 20% in mouse myotubes and human primary
myotubes [125]. The gain in protein synthesis was abolished
when the PI3K inhibitor was administered to the mouse
myotubes together with 37. This was consistent with the hy-
pothesis that (37) increases protein synthesis [126] via the
PI3K pathway, which is a general signalling route for the
enhancement of translation on ribosomes [127]. Interest-
ingly, the mode of action of 37 on translation in the mouse
liver was demonstrated some 25 years ago [128]. The grip
strength of rats was significantly increased by the admini-
stration of 37 (50 mg/kg for 28 days) in a dose comparable to
that of methandrostenolone, an anabolic steroid (10 mg/kg
for 28 days). A similar improvement in grip strength resulted
from feeding with a 1 g/kg spinach extract containing 3% 37
[125]. The naturally occurring phytoecdysteroid therefore
resulted in a true anabolic effect on the grip muscles via
stimulation of a signalling pathway that is also possibly in-
volved in the NG effect of the VDR [6].
Common Features of Vitamin D and Ecdysteroid Ana-
logues
Despite the obvious differences, some important struc-
tural similarities can still be recognized: a) among the steroid
hormones, only these two groups bear an entire cholesterol
side-chain; b) both 1 and the vast majority of ecdysteroids
contain a 3-OH group and c) considering the capability of
the ecdysteroids for tautomeric interconversion, the conju-
gated double-bond system formed in ring B also occurs in
the 6-s-cis-locked vitamin D analogues (18-21).
An early study demonstrated the stimulatory effect of
steroids on shell regeneration in an aquatic gastropod [129],
which might be an orthologue to calciferol-dependent bone
mineralization in vertebrates. Later, a significant number of
studies were performed through direct comparison of the
effects of vitamin D analogues and ecdysteroids, but most of
them were not published in detail in English. These reports
dealt with the early-phase, NG effects of two hydroxysterols.
The early (0-2 h) action of 37 in the enterocytes of the thin
intestine of chicken with experimental D-hypovitaminosis
revealed changes in the biosynthesis of nucleic acids and
Phytoecdysteroids and Vitamin D Analogues Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1989

proteins, accompanied by normalized Ca2+ translocation increased the AP fractional occupancy in the vitamin D ana-
[130]. The administration of 37 and an extract from Ser- logues, (d) their “cis-locked” structures, which also increased
ratula coronata containing 37 normalized the decreased the AP fractional occupancy in the vitamin D analogues. If
ATP, creatine phosphate and carnosine levels and the in- this holds true, then, besides the fields of invertebrate biol-
creased Ca2+ content of the skeletal muscle of rats with ogy, inducible eukaryotic gene expression systems and pesti-
experimental D-hypovitaminosis [131]. Compounds 37 and cide research, the ecdysteroids might gain a further impor-
1 were reported to activate the phospholipase A2, C and D in tance as agonists of the vitamin D NG signalling pathway,
brain and heart cells, and therefore to enhance the phosphoi- which would provide ecdysteroid research with a significant
nositol cycle and possibly other signal transduction pathways and completely new direction.
[132,133]. Further papers from the same laboratory de-
scribed the similar influence of the above two hormones on ACKNOWLEDGEMENT
the changes in cyclic purine nucleotides [134], the activity of
guanylate cyclase [135], and the levels of arachidonic acid The authors gratefully thank Dr. Iván Bélai for his gener-
and its oxidative derivatives [136]. It was recently found that ous help in the preparation of the manuscript.
37 protects the mitochondrial permeability transition pores
(MPTPs) against free radicals [137]. Opening of the MPTPs
ABBREVIATIONS
lets out Ca2+ and preapoptotic factors into the cytoplasm.
Compound 37 probably mimicks the scavenging action of 1 1,25D3-MARRS = 1
,25-dihydroxyvitamin D3-membrane
on oxygen and nitrogen free radical species, therefore pre- associated, rapid response steroid bind-
venting the mitochondrial aging. The free radical scavenging ing receptor
roles of both 37 and 1 have been suggested in protection
against lipid peroxidation [138-140], streptozotocin-induced 20E = 20-hydroxyecdysone
diabetes of type I [141] and alterations in the liver chromatin Akt/PKB = protein kinase B
in D-hypovitaminosis [142]. These studies were carried out
on the brain and heart, and sometimes included the liver, but AP = alternative ligand binding pocket
it may be worthwhile to try similar approaches in the skeletal ATP = adenosine triphosphate
muscle, which is a major target of insulin and it is also af-
fected by nitrosative and oxidative stress in constraints bPTC = bovine parathyroid cell line
[143,144]. CAR = constitutive androstane receptor
CCE = capacitive calcium entry
CONCLUSION AND FUTURE PROSPECTS
c-fos = cellular proto-oncogene, belonging to
Although few direct comparisons have been reported on the immediate early gene family of tran-
the NG effects of ecdysteroids and vitamin D analogues on scription factors
mammals, a number of concordant observations can none- COS-1 = SV40 transformed African green mon-
theless be made. For example, both vitamin D analogues key kidney cell line
[145] and 37 activate nuclear p53 in keratinocytes resulting
in protection of skin against UV irradiation1 [146], and in the COS-7 = SV40 transformed African green mon-
skeletal muscle both 37 and vitamin D are selectively able to key kidney cell line
increase the size of the fast glycolytic fibres [119,147]. CREB = cyclic adenosine monophosphate
Moreover, ecdysteroids also seem to act via PI3K/Akt, (cAMP) responsive element binding
PKC/MAPK signalling cascades in some in vitro systems
[78], similarly to vitamin D analogues [148]. These similari- CSA = cross-sectional area
ties give rise to the suspicion that the “mystic” mechanism of c-Scr = cytosolic tyrosine kinase
action of ecdysteroids on mammalian systems is mediated by
CV-1 = African green monkey kidney cell line
the vitamin D NG signalling pathways, or, in other words,
that the ecdysteroids might be able to bind to the VDR AP. DAG = diacylglycerol
Only one attempt is known to have been made in an effort to
DBD = DNA binding domain
support this hypothesis, where docking of the 6-enol form of
37 to the VDR AP resulted in a gold score value acceptable DNA = deoxyribonucleic acid
for the indication of a strong ligand-protein interaction1. This EcR = ecdysone receptor
might be explained by (a) their common steroid structure
with an entire side-chain, (b) the presence of the 3-OH elk-1 = transcription factor
group, which was necessary in binding to the AP in the case ER
= estrogen receptor

of vitamin D analogues, (c) the lack of a 1-OH group, which
FFA = free fatty acid
1
FXR = farnesoid X receptor
Presentation held at XVIIth International Ecdysone-Workshop 2008, July 20-24, Ulm,
Germany. Meybeck, A.; Yang, C.-R.; Zhang, Y.-J.; Salmon, M.; Belot, N.; Toussaint, G = genomic
O.; Wurtz, J.-M.; Moras, D.; Ho, R.; Raharivelomanana, P. Ecdysteroids from plants
prevent UV induced premature senescence of human skin fibroblasts. Available on- G-361 = human melanoma cell line
line:
http://www.uni-ulm.de/biologie1-ecdysone2008/cms2/files/ecdysone2008_book.pdf GP = genomic ligand binding pocket
2008, pp. 76.
1990 Current Medicinal Chemistry, 2010 Vol. 17, No. 18 Tóth et al.

hGH = human growth hormone VDR = vitamin D receptor

HaCaT = human keratinocyte cell line VDRE = vitamin D responsive element
HL60 = human promyelocytic leukaemia cell VDS = vitamin D sterol
line
Hsp27 = heat shock protein 27 REFERENCES

INAD = inactivation-no-afterpotential D [1] Bathori, M.; Toth, N.; Hunyadi, A.; Marki, A.; Zador, E.
Phytoecdysteroids and anabolic-androgenic steroids--structure and
IP3 = inositol triphosphate effects on humans. Curr. Med. Chem., 2008, 15, 75-91.
[2] Henry, H. L.; Norman, A. W. Studies on calciferol metabolism. IX.
IRS-1 = insulin receptor substrate-1 Renal 25-hydroxy-vitamin D3-1 hydroxylase. Involvement of
cytochrome P-450 and other properties. J. Biol. Chem., 1974, 249,
JNK = c-jun N-terminal protein kinases
7529-7535.
LBD = ligand binding domain [3] Bikle, D. D.; Rasmussen, H. The ionic control of 1,25-
dihydroxyvitamin D3 production in isolated chick renal tubules. J.
LXR = liver X receptor Clin. Invest., 1975, 55, 292-298.
[4] Goltzman, D.; Miao, D.; Panda, D. K.; Hendy, G. N. Effects of
MAPK = mitogen-activated protein kinase calcium and of the Vitamin D system on skeletal and calcium
MAPKAP = MAP kinase-activated protein kinase homeostasis: lessons from genetic models. J. Steroid Biochem.
Mol. Biol., 2004, 89-90, 485-489.
MCF-7 = human breast adenosarcoma cell line [5] Chlon, T. M.; Taffany, D. A.; Welsh, J.; Rowling, M. J. Retinoids
modulate expression of the endocytic partners megalin, cubilin, and
MG-63 = human osteosarcoma cell line disabled-2 and uptake of vitamin D-binding protein in human
mammary cells. J. Nutr., 2008, 138, 1323-1328.
MPTP = mitochondrial permeability transition [6] Norman, A. W. From vitamin D to hormone D: fundamentals of the
pore vitamin D endocrine system essential for good health. Am. J. Clin.
Nutr., 2008, 88, 491S-499S.
mRNA = messenger ribonucleic acid [7] Curino, A.; Milanesi, L.; Benassati, S; Skliar, M.; Boland R. Effect
myf-s = myogenic regulatory factor of culture conditions on the synthesis of vitamin D(3) metabolites
in Solanum glaucophyllum grown in vitro. Phytochemistry. 2001,
MyHC = myosine heavy chain 58, 81-89.
[8] Curino, A.; Skliar, M.; Boland, R. Identification of 7-
MyoD = myogenic regulatory factor dehydrocholesterol, vitamin D3, 25(OH)-vitamin D3 and
1,25(OH)2-vitamin D3 in Solanum glaucophyllum cultures grown
NB4 = human promyelocytic cell line in absence of light. Biochim. Biophys. Acta, 1998, 1425, 485-492.
NG = non-genomic [9] Adler, J. H.; Grebenok, R. J. Occurrence, biosynthesis, and putative
role of ecdysteroids in plants. Crit. Rev. Biochem. Mol. Biol., 1999,
NR = nuclear receptor 34, 253-264.
[10] Canals, D.; Irurre-Santilari, J.; Casas, J. The first cytochrome P450
PSA = protease sensitivity assay (trypsin digest in ferns. Evidence for its involvement in phytoecdysteroid
footprint) biosynthesis in Polypodium vulgare. FEBS J., 2005, 272, 4817-
4825.
PC = phosphatidylcholine [11] Grebenok, R. J.; Galbraith, D. W.; Benveniste, I.; Feyereisen, R.
PGC-1
= peroxisome proliferator-activated recep- Ecdysone 20-monooxygenase, a cytochrome P450 enzyme from
spinach, Spinacia oleracea. Phytochemistry, 1996, 42, 927-933.
tor- coactivator-1
[12] Alekseeva, L. I. Ecdysone 20-monooxygenase activity of
PI3K = phosphoinositide 3-kinase cytochrome P450 in plants and cell culture of Ajuga reptans L.
Prikl. Biokhim. Mikrobiol., 2004, 40, 159-164.
PIE = potential interaction energies [13] Bakrim, A.; Guittard, E.; Maria, A.; De Virville, J. D.; Lafont, R.;
Takvorian, N. Phytoecdysteroid C2-hydroxylase is microsomal in
PKC = protein kinase C spinach, Spinacia oleracea L. Arch. Insect Biochem. Physiol, 2009,
72, 210-219.
PLC = phospholipase C [14] Okamura, W. H.; Midland, M. M.; Hammond, M. W.; Abd, R. N.;
PLD = phospholipase D Dormanen, M. C.; Nemere, I.; Norman, A. W. Chemistry and
conformation of vitamin D molecules. J. Steroid Biochem. Mol.
PTH = parathyroid hormone Biol., 1995, 53, 603-613.
[15] Henry, H. L. Vitamin D hydroxylases. J. Cell Biochem., 1992, 49,
PXR = pregnane X receptor 4-9.
[16] Bouillon, R.; Okamura, W. H.; Norman, A. W. Structure-function
RAR = retinoic acid receptor relationships in the vitamin D endocrine system. Endocr. Rev.,
RCI = relative competitive index 1995, 16, 200-257.
[17] Pis, J.; Vaisar, T. H/D isotopic exchange in the fast atom
ROS 17/2.8 = rat osteogenic sarcoma cell line bombardment of ecdysteroids. J. Mass Spectrometry, 1997, 32,
1050-1056.
RXR = retinoid X receptor [18] Vaisar, T.; Pis, J. Cyclic boronates in the mass-spectrometry of
ecdysteroids. Rapid Commun. Mass-Spectrometry, 1993, 7, 46-52.
SOCS = store-operated channels [19] Evans, R. M. The steroid and thyroid hormone receptor
TR = thyroid receptor  superfamily. Science, 1988, 240, 889-895.
[20] Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schutz,
TRPC = transient receptor potential channels G.; Umesono, K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon,
P.; Evans, R. M. The nuclear receptor superfamily: the second
USP = ultraspiracle decade. Cell, 1995, 83, 835-839.
Phytoecdysteroids and Vitamin D Analogues
[21] Sutton, A. L.; Macdonald, P. N. Vitamin D: more than a "bone-a-
fide" hormone. Mol. Endocrinol., 2003, 17, 777-791.
[22] Weatherman, R. V.; Fletterick, R. J.; Scanlan, T. S. Nuclear-
receptor ligands and ligand-binding domains. Annu. Rev. Biochem.,
1999, 68, 559-581.
[23] Zhang, X. K.; Lehmann, J.; Hoffmann, B.; Dawson, M. I.;
Cameron, J.; Graupner, G.; Hermann, T.; Tran, P.; Pfahl, M.
Homodimer formation of retinoid X receptor induced by 9-cis
retinoic acid. Nature, 1992, 358, 587-591.
[24] Billas, I. M.; Moras, D. Ligand-binding pocket of the ecdysone
receptor. Vitam. Horm., 2005, 73, 101-129.
[25] Nemere, I.; Hintze, K. Novel hormone "receptors". J. Cell
Biochem., 2008, 103, 401-407.
[26] Mizwicki, M. T.; Norman, A. W. The vitamin D sterol-vitamin D
receptor ensemble model offers unique insights into both genomic
and rapid-response signaling. Sci. Signal., 2009, 2, re4.
[27] Nemere, I.; Farach-Carson, M. C.; Rohe, B.; Sterling, T. M.;
Norman, A. W.; Boyan, B. D.; Safford, S. E. Ribozyme
knockdown functionally links a 1,25(OH)2D3 membrane binding
protein (1,25D3-MARRS) and phosphate uptake in intestinal cells.
Proc. Natl. Acad. Sci. U. S. A, 2004, 101, 7392-7397.
[28] Nemere, I. The 1,25D3-MARRS protein: contribution to steroid
stimulated calcium uptake in chicks and rats. Steroids, 2005, 70,
455-457.
[29] Nemere, I.; Hintze, K. Novel hormone "receptors". J. Cell
Biochem., 2008, 103, 401-407.
[30] Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.; Moras, D. The
crystal structure of the nuclear receptor for vitamin D bound to its
natural ligand. Mol. Cell, 2000, 5, 173-179.
[31] Rochel, N.; Tocchini-Valentini, G.; Egea, P. F.; Juntunen, K.;
Garnier, J. M.; Vihko, P.; Moras, D. Functional and structural
characterization of the insertion region in the ligand binding
domain of the vitamin D nuclear receptor. Eur. J. Biochem., 2001,
268, 971-979.
[32] Wurtz, J. M.; Guillot, B.; Fagart, J.; Tietjen, K.; Schindler, M. A
new model for 20-hydroxyecdysone and dibenzoylhydrazine
binding: A homology modeling and docking approach. Protein
Science, 2000, 9, 1073-1084.
[33] Billas, I. M.; Iwema, T.; Garnier, J. M.; Mitschler, A.; Rochel, N.;
Moras, D. Structural adaptability in the ligand-binding pocket of
the ecdysone hormone receptor. Nature, 2003, 426, 91-96.
[34] Losel, R. M.; Falkenstein, E.; Feuring, M.; Schultz, A.; Tillmann,
H. C.; Rossol-Haseroth, K.; Wehling, M. Nongenomic steroid
action: controversies, questions, and answers. Physiol. Rev., 2003,
83, 965-1016.
[35] Schlattner, U.; Vafopoulou, X.; Steel, C. G.; Hormann, R. E.;
Lezzi, M. Non-genomic ecdysone effects and the invertebrate
nuclear steroid hormone receptor EcR--new role for an "old"
receptor? Mol. Cell Endocrinol., 2006, 247, 64-72.
[36] Huhtakangas, J. A.; Olivera, C. J.; Bishop, J. E.; Zanello, L. P.;
Norman, A. W. The vitamin D receptor is present in caveolae-
enriched plasma membranes and binds 1 alpha,25(OH)2-vitamin
D3 in vivo and in vitro. Mol. Endocrinol., 2004, 18, 2660-2671.
[37] Tishkoff, D. X.; Nibbelink, K. A.; Holmberg, K. H.; Dandu, L.;
Simpson, R. U. Functional vitamin D receptor (VDR) in the t-
tubules of cardiac myocytes: VDR knockout cardiomyocyte
contractility. Endocrinology, 2008, 149, 558-564.
[38] Norman, A. W.; Okamura, W. H.; Hammond, M. W.; Bishop, J. E.;
Dormanen, M. C.; Bouillon, R.; van, B. H.; Ridall, A. L.; Daane,
E.; Khoury, R.; Farach-Carson, M. C. Comparison of 6-s-cis- and
6-s-trans-locked analogs of 1alpha,25-dihydroxyvitamin D3
indicates that the 6-s-cis conformation is preferred for rapid
nongenomic biological responses and that neither 6-s-cis- nor 6-s-
trans-locked analogs are preferred for genomic biological
responses. Mol. Endocrinol., 1997, 11, 1518-1531.
[39] Mizwicki, M. T.; Keidel, D.; Bula, C. M.; Bishop, J. E.; Zanello, L.
P.; Wurtz, J. M.; Moras, D.; Norman, A. W. Identification of an
alternative ligand-binding pocket in the nuclear vitamin D receptor
and its functional importance in 1alpha,25(OH)2-vitamin D3
signaling. Proc. Natl. Acad. Sci. U. S. A, 2004, 101, 12876-12881.
[40] Mizwicki, M. T.; Bishop, J. E.; Norman, A. W. Applications of the
Vitamin D sterol-Vitamin D receptor (VDR) conformational
ensemble model. Steroids, 2005, 70, 464-471.
Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1991
[41] Wagner, R. L.; Apriletti, J. W.; McGrath, M. E.; West, B. L.;
Baxter, J. D.; Fletterick, R. J. A structural role for hormone in the
thyroid hormone receptor. Nature, 1995, 378, 690-697.
[42] Renaud, J. P.; Rochel, N.; Ruff, M.; Vivat, V.; Chambon, P.;
Gronemeyer, H.; Moras, D. Crystal structure of the RAR-gamma
ligand-binding domain bound to all-trans retinoic acid. Nature,
1995, 378, 681-689.
[43] Norman, A. W.; Mizwicki, M. T.; Norman, D. P. Steroid-hormone
rapid actions, membrane receptors and a conformational ensemble
model. Nat. Rev. Drug Discov., 2004, 3, 27-41.
[44] Egea, P. F.; Mitschler, A.; Rochel, N.; Ruff, M.; Chambon, P.;
Moras, D. Crystal structure of the human RXRalpha ligand-binding
domain bound to its natural ligand: 9-cis retinoic acid. EMBO J.,
2000, 19, 2592-2601.
[45] Egea, P. F.; Klaholz, B. P.; Moras, D. Ligand-protein interactions
in nuclear receptors of hormones. FEBS Lett., 2000, 476, 62-67.
[46] Carlsson, P.; Burendahl, S.; Nilsson, L. Unbinding of retinoic acid
from the retinoic acid receptor by random expulsion molecular
dynamics. Biophys. J., 2006, 91, 3151-3161.
[47] Perakyla, M. Ligand unbinding pathways from the vitamin D
receptor studied by molecular dynamics simulations. Eur. Biophys.
J., 2009, 38, 185-198.
[48] Soisson, S. M.; Parthasarathy, G.; Adams, A. D.; Sahoo, S.; Sitlani,
A.; Sparrow, C.; Cui, J.; Becker, J. W. Identification of a potent
synthetic FXR agonist with an unexpected mode of binding and
activation. Proc. Natl. Acad. Sci. U. S. A, 2008, 105, 5337-5342.
[49] van Hoorn, W. P. Identification of a second binding site in the
estrogen receptor. J. Med. Chem., 2002, 45, 584-589.
[50] Bursavich, M. G.; Rich, D. H. Designing non-peptide
peptidomimetics in the 21st century: inhibitors targeting
conformational ensembles. J. Med. Chem., 2002, 45, 541-558.
[51] Dreier, R.; Gunther, B. K.; Mainz, T.; Nemere, I.; Bruckner, P.
Terminal differentiation of chick embryo chondrocytes requires
shedding of a cell surface protein that binds 1,25-dihydroxyvitamin
D3. J. Biol. Chem., 2008, 283, 1104-1112
[52] Ceglia, L. Vitamin D and skeletal muscle tissue and function. Mol.
Aspects Med., 2008, 29, 407-414.
[53] Boland, R. Role of vitamin D in skeletal muscle function. Endocr.
Rev., 1986, 7, 434-448.
[54] Ritz, E.; Boland, R.; Kreusser, W. Effects of vitamin D and para-
thyroid hormone on muscle: potential role in uremic myopathy.
Am. J. Clin. Nutr., 1980, 33, 1522-1529.
[55] Ekbom, K.; Hed, R.; Kirstein, L.; Astroem, K. E. Weakness of
proximal limb muscles, probably due to myopathy after partial
gastrectomy. Preliminary report. Acta Med. Scand., 1964, 176, 493-
496.
[56] Smith, R.; Stern, G. Myopathy, osteomalacia and hyperparathyroid-
ism. Brain, 1967, 90, 593-602.
[57] Pfeifer, M.; Begerow, B.; Minne, H. W. Vitamin D and muscle
function. Osteoporos. Int., 2002, 13, 187-194.
[58] Montgomery, J. L.; King, M. B.; Gentry, J. G.; Barham, A. R.;
Barham, B. L.; Hilton, G. G.; Blanton, J. R., Jr.; Horst, R. L.; Ga-
lyean, M. L.; Morrow, K. J., Jr.; Wester, D. B.; Miller, M. F. Sup-
plemental vitamin D3 concentration and biological type of steers.
II. Tenderness, quality, and residues of beef. J. Anim. Sci., 2004,
82, 2092-2104.
[59] Simpson, R. U.; Thomas, G. A.; Arnold, A. J. Identification of
1,25-dihydroxyvitamin D3 receptors and activities in muscle. J.
Biol. Chem., 1985, 260, 8882-8891.
[60] Costa, E. M.; Blau, H. M.; Feldman, D. 1,25-dihydroxyvitamin D3
receptors and hormonal responses in cloned human skeletal muscle
cells. Endocrinology, 1986, 119, 2214-2220.
[61] Zanello, S. B.; Collins, E. D.; Marinissen, M. J.; Norman, A. W.;
Boland, R. L. Vitamin D receptor expression in chicken muscle tis-
sue and cultured myoblasts. Horm. Metab. Res., 1997, 29, 231-236.
[62] Endo, I.; Inoue, D.; Mitsui, T.; Umaki, Y.; Akaike, M.; Yoshizawa,
T.; Kato, S.; Matsumoto, T. Deletion of vitamin D receptor gene in
mice results in abnormal skeletal muscle development with deregu-
lated expression of myoregulatory transcription factors. Endocri-
nology, 2003, 144, 5138-5144.
[63] Bischoff, H. A.; Borchers, M.; Gudat, F.; Duermueller, U.; Theiler,
R.; Stahelin, H. B.; Dick, W. In situ detection of 1,25-
dihydroxyvitamin D3 receptor in human skeletal muscle tissue.
Histochem. J., 2001, 33, 19-24.
1992 Current Medicinal Chemistry, 2010 Vol. 17, No. 18
[64] Eelen, G.; Gysemans, C.; Verlinden, L.; Vanoirbeek, E.; De Clercq,
P. Van Haver, D.; Mathieu, C.; Bouillon, R.; Verstuyf, A. Mecha-
nism and potential of the growth-inhibitory actions of vitamin D
and analogs. Curr. Med. Chem. 2007, 14, 1893-910.
[65] Savkur, R. S.; Bramlett, K. S.; Stayrook, K. R.; Nagpal, S.; Burris,
T. P. Coactivation of the human vitamin D receptor by the perox-
isome proliferator-activated receptor gamma coactivator-1 alpha.
Mol. Pharmacol., 2005, 68, 511-517.
[66] Wu, Z.; Puigserver, P.; Andersson, U.; Zhang, C.; Adelmant, G.;
Mootha, V.; Troy, A.; Cinti, S.; Lowell, B.; Scarpulla, R. C.;
Spiegelman, B. M. Mechanisms controlling mitochondrial biogene-
sis and respiration through the thermogenic coactivator PGC-1.
Cell, 1999, 98, 115-124.
[67] Nemere, I.; Yoshimoto, Y.; Norman, A. W. Calcium transport in
perfused duodena from normal chicks: enhancement within four-
teen minutes of exposure to 1,25-dihydroxyvitamin D3. Endocri-
nology, 1984, 115, 1476-1483.
[68] de Boland, A. R.; Nemere, I. Rapid actions of vitamin D com-
pounds. J. Cell Biochem., 1992, 49, 32-36.
[69] Nemere, I.; Zhou, L. X.; Norman, A. W. Nontranscriptional effects
of steroid hormones. Receptor, 1993, 3, 277-291.
[70] Baran, D. T.; Sorensen, A. M. Rapid actions of 1 alpha-25-
dihydroxyvitamin D3 physiologic role. Proc. Soc. Exp. Biol. Med.,
1994, 207, 175-179.
[71] Facchinetti, M. M.; Boland, R.; de Boland, A. R. Calcitriol trans-
membrane signalling: regulation of rat muscle phospholipase D ac-
tivity. J. Lipid Res., 1998, 39, 197-204.
[72] Facchinetti, M. M.; Boland, R.; de Boland, A. R. Age-related loss
of calcitriol stimulation of phosphoinositide hydrolysis in rat skele-
tal muscle. Mol. Cell Endocrinol., 1998, 136, 131-138.
[73] Facchinetti, M. M.; de Boland, A. R. Effect of ageing on the ex-
pression of protein kinase C and its activation by 1,25(OH)2-
vitamin D3 in rat skeletal muscle. Cell Signal., 1999, 11, 39-44.
[74] Buitrago, C. G.; Ronda, A. C.; de Boland, A. R.; Boland, R. MAP
kinases p38 and JNK are activated by the steroid hormone
1alpha,25(OH)2-vitamin D3 in the C2C12 muscle cell line. J. Cell
Biochem., 2006, 97, 698-708.
[75] Zhou, Q. G.; Hou, F. F.; Guo, Z. J.; Liang, M.; Wang, G. B.;
Zhang, X. 1,25-Dihydroxyvitamin D improved the free fatty-acid-
induced insulin resistance in cultured C2C12 cells. Diabetes Metab.
Res. Rev., 2008, 24, 459-464.
[76] Mathieu, C.; Gysemans, C.; Giulietti, A.; Bouillon, R. Vitamin D
and diabetes. Diabetologia, 2005, 48, 1247-1257.
[77] Sahach, V. F.; Korkach, I.; Kotsiuruba, A. V.; Rudyk, O. V.; Vav-
ilova, H. L. Mitochondrial permeability transition pore opening in-
hibition by ecdysterone in heart mitochondria of aging rats. Fiziol.
Zh., 2008, 54, 3-10.
[78] Oehme, I.; Bosser, S.; Zornig, M. Agonists of an ecdysone-
inducible mammalian expression system inhibit Fas Ligand- and
TRAIL-induced apoptosis in the human colon carcinoma cell line
RKO. Cell Death. Differ., 2006, 13, 189-201.
[79] Kizelsztein, P.; Govorko, D.; Komarnytsky, S.; Evans, A.; Wang,
Z.; Cefalu, WT.; Raskin, I. 20-Hydroxyecdysone decreases weight
and hyperglycemia in a diet-induced obesity mice model. Am. J.
Physiol. Endocrinol. Metab., 2009, 296, E433-439.
[80] Ronda, A. C.; Buitrago, C.; Colicheo, A.; de Boland, A. R.; Rol-
dan, E.; Boland, R. Activation of MAPKs by 1alpha,25(OH)2-
Vitamin D3 and 17beta-estradiol in skeletal muscle cells leads to
phosphorylation of Elk-1 and CREB transcription factors. J. Ster-
oid Biochem. Mol. Biol., 2007, 103, 462-466.
[81] Estrada, M.; Espinosa, A.; Gibson, C. J.; Uhlen, P.; Jaimovich, E.
Capacitative calcium entry in testosterone-induced intracellular
calcium oscillations in myotubes. J. Endocrinol., 2005, 184, 371-
379.
[82] Parekh, A. B.; Putney, J. W., Jr. Store-operated calcium channels.
Physiol. Rev., 2005, 85, 757-810.
[83] Santillan, G.; Baldi, C.; Katz, S.; Vazquez, G.; Boland, R. Evidence
that TRPC3 is a molecular component of the 1alpha,25(OH)2D3-
activated capacitative calcium entry (CCE) in muscle and os-
teoblast cells. J. Steroid Biochem. Mol. Biol., 2004, 89-90, 291-
295.
[84] Santillan, G.; Katz, S.; Vazquez, G.; Boland, R. L. TRPC3-like
protein and vitamin D receptor mediate 1alpha,25(OH)2D3-
Tóth et al.
induced SOC influx in muscle cells. Int. J. Biochem. Cell Biol.,
2004, 36, 1910-1918.
[85] Mizwicki, M. T.; Bula, C. M.; Bishop, J. E.; Norman, A. W. New
insights into Vitamin D sterol-VDR proteolysis, allostery,
structure-function from the perspective of a conformational
ensemble model. J. Steroid Biochem. Mol. Biol., 2007, 103, 243-
262.
[86] Olivera, C. J.; Bula, C. M.; Bishop, J. E.; Adorini, L.; Manchand,
P.; Uskokovic, M. R.; Norman, A. W. Characterization of five 19-
nor-analogs of 1alpha,25(OH)2-Vitamin D3 with 20-cyclopropyl-
modified side-chains: implications for ligand binding and calcemic
properties. J. Steroid Biochem. Mol. Biol., 2004, 89-90, 99-106.
[87] Harant, H.; Spinner, D.; Reddy, G. S.; Lindley, I. J. Natural
metabolites of 1alpha,25-dihydroxyvitamin D(3) retain biologic
activity mediated through the vitamin D receptor. J. Cell Biochem.,
2000, 78, 112-120.
[88] Rao, D. S.; Campbell, M. J.; Koeffler, H. P.; Ishizuka, S.;
Uskokovic, M. R.; Spagnuolo, P.; Reddy, G. S. Metabolism of
1alpha,25-dihydroxyvitamin D(3) in human promyelocytic
leukemia (HL-60) cells: in vitro biological activities of the natural
metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-
60 cells. Steroids, 2001, 66, 423-431.
[89] Ozono, K.; Saito, M.; Miura, D.; Michigami, T.; Nakajima, S.;
Ishizuka, S. Analysis of the molecular mechanism for the
antagonistic action of a novel 1alpha,25-dihydroxyvitamin D(3)
analogue toward vitamin D receptor function. J. Biol. Chem., 1999,
274, 32376-32381.
[90] Ishizuka, S.; Miura, D.; Eguchi, H.; Ozono, K.; Chokki, M.;
Kamimura, T.; Norman, A. W. Antagonistic action of novel
1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs on 25-
hydroxyvitamin-D(3)-24-hydroxylase gene expression induced by
1alpha,25-dihydroxy-vitamin D(3) in human promyelocytic
leukemia (HL-60) cells. Arch. Biochem. Biophys., 2000, 380, 92-
102.
[91] Miura, D.; Manabe, K.; Gao, Q.; Norman, A. W.; Ishizuka, S.
1alpha,25-dihydroxyvitamin D(3)-26,23-lactone analogs antago-
nize differentiation of human leukemia cells (HL-60 cells) but not
of human acute promyelocytic leukemia cells (NB4 cells). FEBS
Lett., 1999, 460, 297-302.
[92] Yoshimoto, Y.; Nemere, I.; Norman, A. W. Hypercalcemia inhibits
the rapid stimulatory effect on calcium transport in perfused
duodena from normal chicks mediated in vitro by 1,25-
dihydroxyvitamin D3. Endocrinology, 1986, 118, 2300-2304.
[93] Zanello, L. P.; Norman, A. W. Stimulation by 1alpha,25(OH)2-
vitamin D3 of whole cell chloride currents in osteoblastic ROS
17/2.8 cells. A structure-function study. J. Biol. Chem., 1997, 272,
22617-22622.
[94] Khoury, R.; Ridall, A. L.; Norman, A. W.; Farach-Carson, M. C.
Target gene activation by 1,25-dihydroxyvitamin D3 in
osteosarcoma cells is independent of calcium influx.
Endocrinology, 1994, 135, 2446-2453.
[95] Mizwicki, M. T.; Bula, C. M.; Bishop, J. E.; Norman, A. W. A
perspective on how the Vitamin D sterol/Vitamin D receptor
(VDR) conformational ensemble model can potentially be used to
understand the structure-function results of A-ring modified
Vitamin D sterols. J. Steroid Biochem. Mol. Biol., 2005, 97, 69-82.
[96] Miura, D.; Norman, A. W.; Mizwicki, M. T.; Fujishima, T.; Konno,
K.; Kittaka, A.; Takayama, H.; Ishizuka, S. The antagonism
between 2-methyl-1,25-dihydroxyvitamin D3 and 2-methyl-20-epi-
1,25-dihydroxyvitamin D3 in non-genomic pathway-mediated
biological responses induced by 1alpha,25-dihydroxyvitamin D3
assessed by NB4 cell differentiation. J. Steroid Biochem. Mol.
Biol., 2005, 94, 469-479.
[97] Norman, A. W.; Okamura, W. H.; Farach-Carson, M. C.;
Allewaert, K.; Branisteanu, D.; Nemere, I.; Muralidharan, K. R.;
Bouillon, R. Structure-function studies of 1,25-dihydroxyvitamin
D3 and the vitamin D endocrine system. 1,25-dihydroxy-
pentadeuterio-previtamin D3 (as a 6-s-cis analog) stimulates
nongenomic but not genomic biological responses. J. Biol. Chem.,
1993, 268, 13811-13819.
[98] Wecksler, W. R.; Norman, A. W. An hydroxylapatite batch assay
for the quantitation of 1alpha,25-dihydroxyvitamin D3-receptor
complexes. Anal. Biochem., 1979, 92, 314-323.
Phytoecdysteroids and Vitamin D Analogues
[99] Sheves, M.; Friedman, N.; Mazur, Y. Conformational equilibria in
vitamin D. Synthesis of 1beta-hydroxyvitamin D3. J. Org. Chem.,
1977, 42, 3597-3599.
[100] Sheves, M.; Berman, E.; Freeman, D.; Mazur, Y. Conformational
equilibria in vitamins D. The synthesis of 1alpha-hydroxy-3-
epivitamin D3 (1alpha-hydroxy-3alpha-cholecalciferol). J. Chem.
Soc. , Chem. Commun., 1975, 511, 643-644.
[101] Konno, K.; Fujishima, T.; Maki, S.; Liu, Z.; Miura, D.; Chokki, M.;
Ishizuka, S.; Yamaguchi, K.; Kan, Y.; Kurihara, M.; Miyata, N.;
Smith, C.; DeLuca, H. F.; Takayama, H. Synthesis, biological
evaluation, and conformational analysis of A-ring diastereomers of
2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: unique
activity profiles depending on the stereochemistry of the A-ring
and at C-20. J. Med. Chem., 2000, 43, 4247-4265.
[102] Kamao, M.; Tatematsu, S.; Hatakeyama, S.; Sakaki, T.; Sawada,
N.; Inouye, K.; Ozono, K.; Kubodera, N.; Reddy, G. S.; Okano, T.
C-3 epimerization of vitamin D3 metabolites and further
metabolism of C-3 epimers: 25-hydroxyvitamin D3 is metabolized
to 3-epi-25-hydroxyvitamin D3 and subsequently metabolized
through C-1alpha or C-24 hydroxylation. J. Biol. Chem., 2004, 279,
15897-15907.
[103] Konno, K.; Fujishima, T.; Maki, S.; Liu, Z.; Miura, D.; Chokki, M.;
Ishizuka, S.; Yamaguchi, K.; Kan, Y.; Kurihara, M.; Miyata, N.;
Smith, C.; DeLuca, H. F.; Takayama, H. Synthesis, biological
evaluation, and conformational analysis of A-ring diastereomers of
2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: unique
activity profiles depending on the stereochemistry of the A-ring
and at C-20. J. Med. Chem., 2000, 43, 4247-4265.
[104] Saito, N.; Matsunaga, T.; Fujishima, T.; Anzai, M.; Saito, H.;
Takenouchi, K.; Miura, D.; Ishizuka, S.; Takayama, H.; Kittaka, A.
Remarkable effect of 2[small alpha]-modification on the VDR
antagonistic activity of 1small alpha-hydroxyvitamin D3-26,23-
lactones. Org. Biomol. Chem., 2003, 1, 4396-4402.
[105] Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.; Miura, D.; Chokki, M.;
Takayama, H. A novel and practical route to A-ring enyne synthon
for 1 alpha,25-dihydroxyvitamin D3 analogs: synthesis of A-ring
diastereomers of 1 alpha,25-dihydroxyvitamin D2 and 3-methyl-
1,25-dihydroxyvitamin D3. Bioorg. Med. Chem. Lett., 1998, 8,
151-156.
[106] Brown, A. J.; Ritter, C.; Slatopolsky, E.; Muralidharan, K. R.;
Okamura, W. H.; Reddy, G. S. 1Alpha,25-dihydroxy-3-epi-vitamin
D3, a natural metabolite of 1alpha,25-dihydroxyvitamin D3, is a
potent suppressor of parathyroid hormone secretion. J. Cell
Biochem., 1999, 73, 106-113.
[107] Norman, A. W.; Bouillon, R.; Farach-Carson, M. C.; Bishop, J. E.;
Zhou, L. X.; Nemere, I.; Zhao, J.; Muralidharan, K. R.; Okamura,
W. H. Demonstration that 1 beta,25-dihydroxyvitamin D3 is an
antagonist of the nongenomic but not genomic biological responses
and biological profile of the three A-ring diastereomers of 1
alpha,25-dihydroxyvitamin D3. J. Biol. Chem., 1993, 268, 20022-
20030.
[108] Mizwicki, M. T.; Norman, A. W. Two key proteins of the vitamin
D endocrine system come into crystal clear focus: comparison of
the X-ray structures of the nuclear receptor for 1alpha,25(OH)2
vitamin D3, the plasma vitamin D binding protein, and their
ligands. J. Bone Miner. Res., 2003, 18, 795-806.
[109] Rao, D. S.; Campbell, M. J.; Koeffler, H. P.; Ishizuka, S.;
Uskokovic, M. R.; Spagnuolo, P.; Reddy, G. S. Metabolism of
1alpha,25-dihydroxyvitamin D(3) in human promyelocytic
leukemia (HL-60) cells: in vitro biological activities of the natural
metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-
60 cells. Steroids, 2001, 66, 423-431.
[110] Ishizuka, S.; Sumitani, K.; Hiura, K.; Kawata, T.; Okawa, M.;
Hakeda, Y.; Kumegawa, M. Biological activity assessment of 1
alpha,25-dihydroxyvitamin D3-26,23-lactone and its intermediate
metabolites in vivo and in vitro. Endocrinology, 1990, 127, 695-
701.
[111] Norman, A. W.; Manchand, P. S.; Uskokovic, M. R.; Okamura, W.
H.; Takeuchi, J. A.; Bishop, J. E.; Hisatake, J. I.; Koeffler, H. P.;
Peleg, S. 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. J. Med. Chem., 2000,
43, 2719-2730.
Current Medicinal Chemistry, 2010 Vol. 17, No. 18 1993
[112] Peleg, S.; Sastry, M.; Collins, E. D.; Bishop, J. E.; Norman, A. W.
Distinct conformational changes induced by 20-epi analogues of 1
alpha,25-dihydroxyvitamin D3 are associated with enhanced
activation of the vitamin D receptor. J. Biol. Chem., 1995, 270,
10551-10558.
[113] Binderup, L.; Latini, S.; Binderup, E.; Bretting, C.; Calverley, M.;
Hansen, K. 20-epi-vitamin D3 analogues: a novel class of potent
regulators of cell growth and immune responses. Biochem.
Pharmacol., 1991, 42, 1569-1575.
[114] Ishizuka, S.; Norman, A. W. The difference of biological activity
among four diastereoisomers of 1 alpha,25-dihydroxychol-
ecalciferol-26,23-lactone. J. Steroid Biochem., 1986, 25, 505-510.
[115] Ciesielski, F.; Rochel, N.; Mitschler, A.; Kouzmenko, A.; Moras,
D. Structural investigation of the ligand binding domain of the
zebrafish VDR in complexes with 1alpha,25(OH)2D3 and Gemini:
purification, crystallization and preliminary X-ray diffraction
analysis. J. Steroid Biochem. Mol. Biol., 2004, 89-90, 55-59.
[116] Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. M.; Mitschler, A.;
Moras, D. Crystal structures of the vitamin D receptor complexed
to superagonist 20-epi ligands. Proc. Natl. Acad. Sci. U. S. A, 2001,
98, 5491-5496.
[117] Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.; Norman, A.
W.; Ishizuka, S. Antagonistic action of novel 1alpha,25-
dihydroxyvitamin D3-26, 23-lactone analogs on differentiation of
human leukemia cells (HL-60) induced by 1alpha,25-
dihydroxyvitamin D3. J. Biol. Chem., 1999, 274, 16392-16399.
[118] Ozono, K.; Saito, M.; Miura, D.; Michigami, T.; Nakajima, S.;
Ishizuka, S. Analysis of the molecular mechanism for the
antagonistic action of a novel 1alpha,25-dihydroxyvitamin D(3)
analogue toward vitamin D receptor function. J. Biol. Chem., 1999,
274, 32376-32381.
[119] Toth, N.; Szabo, A.; Kacsala, P.; Heger, J.; Zador, E. 20-
Hydroxyecdysone increases fiber size in a muscle-specific fashion
in rat. Phytomedicine., 2008, 15, 691-698.
[120] Lafont, R.; Dinan, L. Practical uses for ecdysteroids in mammals
including humans: an update. J. Insect Sci., 2003, 3, 7.
[121] Syrov, N. V.; Saatov, Z.; Sagdullaev, Sh. Sh.; Mamatkhanov, A. U.
Study of the Structure-Anabolic Activity Relationship for
Phytoecdysteroids Extracted from some Plants of Central Asia.
Pharm. Chem. J., 2001, 35, 667-671.
[122] Syrov, V. N. Comparative experimental investigations of the
anabolic activity of ecdysteroids and steranabols. Pharm. Chem. J.,
2000, 34, 193-197.
[123] Syrov, V. N.; Khushbaktova, Z. A. Experimental study of
pharmacotherapeutic effect of phytoecdisteroids and nerobol in
toxic liver damage. Eksp. Klin. Farmakol., 2001, 64, 56-58.
[124] Saatov, Z.; Agzamkhodzhaeva, D. A.; Syrov, V. N. Distribution of
phytoecdysteroids in plants of Uzbekistan and the possibility of
using drugs based on them in neurological practice. Chemistry of
Natural Compounds, 1999, 35, 186-191.
[125] Gorelick-Feldman, J.; Maclean, D.; Ilic, N.; Poulev, A.; Lila, M.
A.; Cheng, D.; Raskin, I. Phytoecdysteroids increase protein
synthesis in skeletal muscle cells. J. Agric. Food Chem., 2008, 56,
3532-3537.
[126] Otaka, T.; Uchiyama, M.; Okui, S.; Takemoto, T.; Hikino, H.
Stimulatory effect of insect-metamorphosing steroids from
Achyranthes and Cyathula on protein synthesis in mouse liver.
Chem. Pharm. Bull. (Tokyo), 1968, 16, 2426-2429.
[127] Guttridge, D. C. Signaling pathways weigh in on decisions to make
or break skeletal muscle. Curr. Opin. Clin. Nutr. Metab Care,
2004, 7, 443-450.
[128] Syrov, V. N. Mechanism of the anabolic action of phytoecdister-
oids in mammals. Nauchnye. Doki. Vyss. Shkoly. Biol. Nauki, 1984,
11, 16-20.
[129] Whitehead, D. L. Steroids enhance shell regeneration in an aquatic
gastropod (Biomphalaria glabrata). Comp. Biochem. Physiology C,
1977, 58, 137-41.
[130] Kotsiuruba, A. V.; Akhmed, I.; Tarakanov, S. S; Kholodova, Iu. D.
Mechanism of action of ecdysterone in chickens with D-
hypovitaminosis. Early phase of effect. Ukr.Biokhim.Zh. 1993, 65,
83-90.
[131] Kholodova, Iu. D.; Tugai, V. A.; Zimina, V. P. Effect of vitamin
D3 and 20-hydroxyecdysone on the content of ATP, creatine phos-
1994 Current Medicinal Chemistry, 2010 Vol. 17, No. 18
phate, carnosine and Ca2+ in skeletal muscles. Ukr.Biokhim. Zh.,
1997, 69, 3-9.
[132] Kotsiuruba, A. V.; Bukhanevich, O. M.; Mehed', O. F.; Tarakanov,
S. S.; Berdishev, A. H.; Tuhanova, A. V. The C(27)-steroid hor-
mones ecdysterone and calcitriol activate the phosphoinositol cycle
in its membrane phase. Ukr. Biokhim. Zh., 1999, 71, 27-32.
[133] Kotsiuruba, A. V.; Bukhanevich, O. M.; Tarakanov, S. S.; Tu-
hanova, A. V.; Berdyshev, A. H.; Mehed', O. F. The C(27)-steroid
hormones calcitriol and ecdysterone in the early phase of its action
activates hydrolysis of phosphatidylcholines in target animal tis-
sues. Ukr. Biokhim. Zh., 1998, 70, 37-44.
[134] Kotsiuruba, A. V.; Bukhanevych, O. M.; Tarakanov, S. S.; Kholo-
dova, I. Modulation of intracellular pools of cyclic purine nucleo-
tides by biologically active oxysterol-ecdysterone and vitamin D3.
Ukr. Biokhim. Zh., 1993, 65, 76-83.
[135] Kotsiuruba, A. V.; Bukhanevich, O. M.; Tuhanova, A. V.; Taraka-
nov, S. S.; Berdyshev, A. H. Mechanisms of the early effect of bio-
logically active hydroxysterols: calcitriol and ecdysterone. Modula-
tion of systems which generate low molecular weight activators of
guanylate cyclase. Ukr. Biokhim. Zh., 1995, 67, 58-64.
[136] Kotsiuruba, A. V.; Bukhanevich, O. M.; Tuhanova, A. V.; Taraka-
nov, S. S. Mechanisms of the early effect of biologically active hy-
droxysterols: calcitriol and ecdysterone. Modulation of intracellular
pools of arachidonic acid and products of its oxidative metabolism.
Ukr. Biokhim. Zh., 1995, 67, 45-52.
[137] Sahach, V. F.; Korkach, Iu. P.; Kotsiuruba, A. V.; Rudyk, O. V.;
Vavilova, H. L. Mitochondrial permeability transition pore opening
inhibition by ecdysterone in heart mitochondria of aging rats. Fiz-
iol. Zh., 2008, 54, 3-10.
[138] Kuzmenko, A. I.; Morozova, R. P.; Nikolenko, I. A.; Korniets, G.
V.; Kholodova, Yu. D. Effects of vitamin D3 and ecdysterone on
free-radical lipid peroxidation. Biokhimiya (Moscow), 1997, 62,
609-612.
[139] Kuzmenko, A. I.; Morozova, R. P.; Nikolenko, I. A.; Korniets, G.
V.; Kholodova, Yu. D. Effects of vitamin D3 and ecdysterone on
free-radical lipid peroxidation. Biokhimiya (Moscow,) 1997, 62,
609-612.
Received: December 26, 2009 Revised: March 28, 2010 Accepted: March 29, 2010
Tóth et al.
[140] Kuzmenko, A. I.; Morozonv, R. P.; Nikolenko, I. A.; Donchenko,
G. V. Vitamin D3 and 20-hydroxyecdysone - inhibiting agents of
lipid free radical oxidation in D-hypovitaminosis. Ukr. Biokhim.
Zh., 2001, 73, 44-50.
[141] Sahach, V. F.; Korkach, I.; Kotsiuruba, A. V.; Prysiazhna, O. D.
The inhibition of oxidative and nitrosative stresses by ecdysterone
as the mechanisms of its cardio- and vasoprotective action in ex-
perimental diabetes type I. Fiziol. Zh., 2008, 54, 46-54.
[142] Levitsky, E. L.; Kholodova, Yu. D.; Gubski, Yu. I.; Primak, R. G.;
Chabanny, V. N.; Kindruk, N. L.; Mozzhukhina, T. G.;
Lenchevskaya, L. K.; Mironova, V. N. et al. Biochemical charac-
teristics of rat liver fractionated chromatin under experimental D-
hypovitaminosis and after treatment by steroidal préparations. Ukr.
Biokhim. Zh., 1993, 65, 28-36.
[143] Kaneki, M.; Shimizu, N.; Yamada, D.; Chang, K. Nitrosative stress
and pathogenesis of insulin resistance. Antioxid. Redox. Signal.,
2007, 9, 319-329.
[144] Bonetto, A.; Penna, F.; Muscaritoli, M.; Minero, V. G.; Fanelli, F.
R.; Baccino, F. M.; Costelli, P. Are antioxidants useful for treating
skeletal muscle atrophy? Free Radic. Biol. Med., 2009, 47, 906-
916.
[145] Gupta, R.; Dixon, K. M.; Deo, S. S.; Holliday, C. J.; Slater, M.;
Halliday, G. M.; Reeve, V. E.; Mason, R. S. Photoprotection by
1,25 dihydroxyvitamin D3 is associated with an increase in p53 and
a decrease in nitric oxide products. J. Invest Dermatol., 2007, 127,
707-715.
[146] Dixon, K. M.; Deo, S. S.; Norman, A. W.; Bishop, J. E.; Halliday,
G. M.; Reeve, V. E.; Mason, R. S. In vivo relevance for
photoprotection by the vitamin D rapid response pathway. J.
Steroid Biochem. Mol. Biol., 2007, 103, 451-456.
[147] Sato, Y.; Iwamoto, J.; Kanoko, T.; Satoh, K. Low-dose vitamin D
prevents muscular atrophy and reduces falls and hip fractures in
women after stroke: a randomized controlled trial. Cerebrovasc.
Dis., 2005, 20, 187-192.
[148] Norman, A. W. Minireview: vitamin D receptor: new assignments
for an already busy receptor. Endocrinology, 2006, 147, 5542-
5548.