Current
Topics in Medicinal Chemistry
ISSN: 1568-0266
Current Topics
in Medicinal Chemistry
Volume 6, Number 12, 2006
Contents
Vitamin D and its Binding Protein: Challenges
and Opportunities for Drug Research
Guest Editor: Dr. Hubert Maehr
Editorial Pp. 1227-1228
Ligand Binding Domain of Vitamin D Receptors Pp.
1229--1241
Natacha Rochel and Dino Moras
[Abstract]
Detailed Molecular Understanding of Agonistic
and Antagonistic Vitamin D Receptor Ligands Pp. 1243-1253
Carsten Carlberg and Ferdinand Molnár
[Abstract]
Ligand Recognition by Vitamin D Receptor: Total
Alanine Scanning Mutational Analysis of the Residues Lining
the Ligand Binding Pocket of Vitamin D Receptor Pp.
1255-1265
Sachiko Yamada and Keiko Yamamoto
[Abstract]
Vitamin D Receptor Signaling of Monocytic Differentiation
in Human Leukemia Cells: Role of MAPK Pathways in Transcription
Factor Activation Pp. 1267-1271
G.P. Studzinski, E. Garay, R. Patel, J. Zhang and X. Wang
[Abstract]
Recent Results on A-Ring Modification of 1α,25
Dihydroxyvitamin D3: Design and Synthesis of VDR-Agonists
and Antagonists with High Biological Activity Pp.
1273-1288
Nozomi Saito, Shinobu Honzawa, and Atsushi Kittaka
[Abstract]
C-20 Cyclopropyl Vitamin D3 Analogs
Pp. 1289-1296
Milan R. Uskokovic, Percy Manchand, Stanislaw Marczak,
Hubert Maehr, Pawel Jankowski, Luciano Adorini and G. Satyanarayana
Reddy
[Abstract]
Vitamin D Receptor Agonists, Cancer and the Immune
System: An Intricate Relationship Pp. 1297-1301
Luciano Adorini, Kenn C. Daniel and Giuseppe Penna
[Abstract]
Vitamin D Receptor Agonists: Opportunities and
Challenges in Drug Discovery Pp. 1303-1316
Tadakatsu Takahashi and Kazumi Morikawa
[Abstract]
Abstracts
[Back to top]
Editorial
Vitamin D and its Binding Protein: Challenges and Opportunities
for Drug Research
The recognition that Vitamin D exerts its activity through
a nuclear mechanism led to the discovery of the binding protein,
the vitamin-D receptor or VDR. As a consequence of this agonist
binding in the ligand-binding domain (LBD), the VDR changes
its topology and, after translocation from the cytoplasm to
the nucleus, permits the VDR to function as a ligand-activated
transcription factor by virtue of its association with the
response elements (VDREs) in some 50 vitamin-D regulated genes.
This binding phenomenon to DNA at the VDRE sites is a complex
process and occurs by heterodimerization with the retinoid
X-receptor (RXR) and recruitment of a complex machinery of
co-modulators and co-activators. The hormonally active form
of vitamin D, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3),
is produced by a sequence of steps commencing with a photochemically
induced conrotatory, electrocyclic ring opening of 7-dehydrocholesterol
in the skin leading to previtamin D3 which is subject
to a thermal, antarafacial [1,7]-hydrogen shift, followed
by 25-hydroxylation in the liver to generate 25(OH)D3,
and 1α -hydroxylation,
preferentially in the kidney. The VDR is present in multitudinous
tissues, including many cancer cells, some with the capability
to 1α -hydroxylate
25(OH)D3 to create the active 1,25(OH)2D3
and hence enabling locally initiated cellular proliferation
and differentiation processes. While today’s picture
of the genomic responses, mediated by 1,25(OH)2D3,
is somewhat coherent, the imputed non-genomic ‘rapid
responses’ are more enigmatic.
The medicinal chemist who seeks to intervene therapeutically
in vitamin-D controlled gene transcription activity has reduced
this intricate scenario to a working hypothesis based on a
few facts and lemmas: Similar to other hormonal nuclear receptors,
the VDR can modify gene expression by hormone-initiated signal
transduction; 1,25(OH)2D3 induces co-localization
of the VDR and RXR, the receptor activation unit, to the VDRE
gene promoter rendering 1,25(OH)2D3
an essential component for gene expression. Cloning of the
VDR and the availability of the VDR LBD crystal structure
has not only illuminated the role of the receptor and its
ligand in transcription regulation but also, to some extent,
the physico-chemical requirements of the ligand. Add to this
the dynamic behavior of the LBD with the resulting variations
of transcriptional activity, the known endocrinal steering
mechanism for the required cellular hormone levels through
complex CYP-mediated metabolic pathway, and the chemical,
biological and even clinical properties of vitamin D analogs
previously prepared, and one arrives at an intriguing and
exciting prospect to view vitamin D and its analogs as multifaceted
tools implicated in a myriad of established and conjectured
biological activities with a set of predefined preferences
of molecular features. And therein are anchored the opportunities
and challenges for the medicinal chemist. 1,25(OH)2D3
can suppress several growth factor and cytokine genes thus
inhibiting the release of IL2, GM-CSF, interferon-γ
and PTH, for example. It regulates mineral homeostasis, cellular
proliferation and differentiation, and autoimmune activities.
The disease targets, envisioned for vitamin D analogs, appropriately,
include osteoporosis by balancing bone-mineral mobilization,
secondary hyperparathyroidism to reduce PTH-gene transcription
and blocking chief cell hyperplasia, autoimmune diseases such
as psoriasis and asthma, organ-transplant rejection, benign
prostate hyperplasia, involuntary bladder control, blood pressure
control by suppressing renin biosynthesis, type 1 diabetes
and insulin secretion by affecting pancreatic β-cell
function, anti-inflammatory events via cyclooxygenase-2 inhibition,
and cancer via the established antiproliferative and prodifferentiating
effects on a variety of cell lines, such as breast, prostate
and colon.
The chemist must strive for selective molecular modifications
of vitamin D to balance the potential function as a nuclear
receptor agonist, antagonist or reverse agonist, and at the
same time maintain tissue specificity and sufficient metabolic
stability with a constant look out for hypercalcemia and hyperphosphatemia.
While the discovery of agonist or even “superagonist”
activity is the more popular goal in molecular design, the
prospect of finding ligands that selectively stabilize an
antagonistic conformation of the VDR LBD within the VDR-RXR-DVRE
construct, to actually prevent induction of transactivation,
is also of potential therapeutic value. The adaptability of
the VDR to accept ligands that are deprived of many structural
elements common to 1,25(OH)2D3 and demand
drastically changed space requirements, however, underlines
the difficulty to employ modern drug design technologies.
The degree of affinity of a vitamin-D analog to the VDR appears
to be of lesser importance than its alignment with specific
contact sites in the LBD to produce different VDR conformations
with modified transcriptional consequences. It is not surprising,
therefore, that some 3000 vitamin D related compounds have
already been synthesized, mostly by ‘trial, error and
lessons from the past’, with the goal to minimize or
eliminate hypercalcemic side effects while maintaining sustained
plasma levels, the desired transactivation potencies and cell
specificities. Inspired more by the chemist’s intuition
and less by in-silico technologies, therefore, this effort
has provided invaluable insights into the molecular anatomy
of vitamin D analogs that circumscribe the binding affinity
and determine its function as agonist or antagonist, the disease-modifying
potential and, eventually, the inherent clinical value.
The authors of this volume provide us with a panoramic view
of the current research in the vitamin D arena. They expand
our view of the VDR on a molecular level and analyze the functionalities
involved in ligand recognition of the VDR and the requirements
for the differential action of vitamin-D analogs to function
as agonistic or antagonistic VDR ligands. They rely on sophisticated
techniques such as x-ray crystallography, molecular dynamics
simulations and total alanine scanning mutational analysis
to explore the three-dimensionality and the inner lining of
the LBD. Based on the malleable ligand-VDR LBD interaction,
the possible non-genomic functions of the VDR are explored.
Although still nebulous and fragmented, our knowledge of the
role of the activated VDR in ligand-receptor signaling, especially
the signaling of monocytic differentiation is gaining lucidity
and the immunoregulatory activities of VDR agonists, contrapositive
to their anti-neoplastic activities, are scrutinized. The
authors expose the intriguing chemical avenues toward new
compounds whose directions are outlined by these studies.
The resulting guidelines, combined with the chemist’s
ingenuity, will engender novel vitamin-D analogs with minimal
side effects and maximum therapeutic potential. Some of them
will surely emerge from the chrysalis of R&D and enter
the pantheon of drugs of clinical prominence.
This Editorial would not be complete without expatiating on
the commercial aspects of vitamin-D related drug candidates
and their ranking within an R&D program. Due to the broad
spectrum of established and prognosticated clinical applications
of vitamin-D analogs, the emphasis of drug discovery has shifted
mainly to industrial laboratories. Unlike conventional drug
research, which commences at rudimentary levels, the vitamin-D
platform proffers simultaneously not only the framework of
a new chemical entity but also the disease targets thus leading
to an accelerated conclusion of the drug discovery phase.
An abbreviated discovery phase has a pronounced effect on
all resources. The diagram shown above illustrates the requirement
of a drug candidate to pass from a value of 0 at the initiation
of research to unity at the successful completion of phase
III clinical trial. The interplay of the essential goals along
this path and the risk factors are represented by a triangle
wherein the hypotenuse is indicative of the required milestones,
the vertical leg marks the activities and estimated costs
and the time coordinate is on the horizontal leg. Each ongoing
event on the vertical coordinate (cost data taken from BIO.IT
World 2004, vol. 3, # 1, p. 18) is continuously monitored
by the success index si wherein the subscript “i”
refers to the particular event. A value of unity signifies
a successful conclusion; a value of null represents failure.
The intrinsic value of a drug candidate is the product of
all si values. Thus, a value of si =
1 empowers further advancement but as soon as a value of si
= 0 is encountered the value of the drug candidate reverts
to zero. Such an event is the more deleterious the later it
occurs. Although oversimplified, the display emphasizes the
high-risk gamble of R&D and our predilection of the vitamin-D
platform with the drastically reduced discovery efforts and
the concomitant benefit in the form of si = 1.
In the light of the gargantuan estimated cost for a single,
new chemical entity to reach the market, ranging from 1.7
(C&EN 2003, vol. 81, #50, p.8) to 3 billon Dollars (BIO.IT
World, 2005, vol. 4, #11, p. 26), an achievement of si
= 1 is of monumental significance.
Hubert Maehr, Ph.D.
Guest Editor
[Back to top]
Ligand Binding Domain of Vitamin D Receptors
Natacha Rochel and Dino Moras
The vitamin D receptor, a member of the nuclear receptor
subgroup NR1I, is regulated by 1α,25(OH)2D3
to control calcium metabolism, cell proliferation and differentiation
and immunomodulation. The therapeutic applications of vitamin
D metabolites are wide. To develop efficient therapy, the
elucidation of the structure-function relationships of VDR
and its ligands are essential. In this review we will focus
on the current structural understanding of the interactions
of ligands in the ligand binding pocket of the VDR. These
structures revealed the mutual adaptability of the ligands
and the protein. In silico modeling has further revealed a
possible new pocket in the VDR LBD responsible of the non-genomic
action mediated by VDR. With the availability of all these
structural information on VDR LBD, new ligands that are more
selective, such as non-steroidal ligands, could be designed
by taking into account the flexibility of some VDR regions.
Tissue selectivity may also be achieved by developing ligands
that specifically activate the non-genomic pathway.
[Back to top]
Detailed Molecular Understanding of Agonistic
and Antagonistic Vitamin D Receptor Ligands
Carsten Carlberg and Ferdinand Molnár
The vitamin D receptor (VDR) is an endocrine member of the
nuclear receptor superfamily and binds the biologically most
active vitamin D metabolite, 1α,25-dihydroxyvitamin
D3 (1α
,25(OH)2D3). The VDR ligand-binding
domain is a molecular switch, since its ligand-triggered interactions
with corepressor and coactivator proteins are the central
molecular events of nuclear 1α
,25(OH)2D3 signaling. 1α
,25(OH)2D3 analogues have been
developed with the goal to improve the biological profile
of the natural hormone for a therapeutic application either
in hyperproliferative diseases, such as psoriasis and different
types of cancer, or in bone disorders, such as osteoporosis.
Most of the analogues described to date are agonists, with
a few having been identified as antagonists. Only the two
side chain analogue Gemini and some of its derivatives act
under restricted conditions as inverse agonists. In this review
we discuss the molecular mechanisms of these different type
of analogues based on crystal structure data, molecular dynamics
simulations and biochemical assays.
[Back to top]
Ligand Recognition by Vitamin D Receptor: Total
Alanine Scanning Mutational Analysis of the Residues Lining
the Ligand Binding Pocket of Vitamin D Receptor
Sachiko Yamada and Keiko Yamamoto
We performed total alanine scanning mutational analysis (ASMA)
of the residues lining the ligand binding pocket (LBP) of
the human vitamin D receptor (hVDR) to investigate allosteric
effects of ligands in the function of nuclear receptors (NRs).
This was accomplished for the first time in the NR superfamily.
The effects of ligand structure were also examined in this
system (termed 2D-ASMA) using 8 representative VDR ligands.
The results clearly revealed the role and importance of all
amino acid residues lining the LBP and the relationships between
ligand binding and transcriptional potency. 2D-ASMA indicated
ligand-specific ligand-protein interactions, which are essential
in determining the transactivation potency of the ligand.
Taking the results as a whole, we suggest a ligand-mediated
allosteric network, which allows transmission of information
from ligands to the interfaces of the VDR in association with
protein cofactors and was shown to be linked to a part of
the network identified by statistical coupling analysis (SCA).
[Back to top]
Vitamin D Receptor Signaling of Monocytic Differentiation
in Human Leukemia Cells: Role of MAPK Pathways in Transcription
Factor Activation
G.P. Studzinski, E. Garay, R. Patel, J. Zhang and X. Wang
Among the many important physiological functions of the activated
vitamin D receptor (VDR) is the signaling of monocytic differentiation,
first demonstrated by conversion of malignant myeloid leukemia
cells to nonproliferating cells with mature monocyte/macrophage
appearance. However, the understanding of how 1, 25-dihydroxyvitamin
D3 (1,25D) signals monocytic differentiation is still developing.
Recent advances summarized here include the role of the principal
“mitogen-activated protein kinase” (MAPK) pathways,
their potential downstream target the CCAAT/enhancer binding
protein β
(C/EBP β),
cell cycle related proteins, and cyclin-dependent kinase 5
(Cdk5) in 1,25D-induced differentiation.
The precise steps by which activated VDR signals differentiation
are incompletely understood in any of the cell types known
to respond to 1,25D. We have focused our studies on HL60 cells,
a widely available cell line derived from a patient with promyeloblastic
leukemia, with the goal of achieving as clear a picture as
possible with the currently available tools. In this model,
outlined in Fig. 1, a plausible sequence of events is presented,
with the caveats that these are not the only pathways activated
by liganded VDR, and that several other pathways, also operative,
remain to be convincingly demonstrated. The details of the
scheme will be discussed in the sections below.
[Back to top]
Recent Results on A-Ring Modification of 1α,25
Dihydroxyvitamin D3: Design and Synthesis of VDR-Agonists
and Antagonists with High Biological Activity
Nozomi Saito, Shinobu Honzawa, and Atsushi Kittaka
The structure-activity relationships of 1α,25-dihydroxyvitamin
D3 on simultaneous modification at both C2α
and CD-ring side chain, including 20-epimerization, double
side chain (gemini), and vitamin D receptor (VDR) antagonists
TEI-9647 and TEI-9648 lactone rings, and also on simultaneous
modifications at both C2 and C10 positions, i.e.,
C2 modified active 19-norvitamin D3, have been
studied in our laboratory to find new seeds of B-seco-steroidal
medicine for treating bone diseases, psoriasis, secondary
hyperparathyroidism, and certain kinds of cancers. We developed
an efficient and systematic route to the 2α-substituted
1α,25-dihydroxyvitamin
D3 analogs, i.e., VDR-agonists (20-epi-2-4,
double side chain 13a-c, 19-nor
15a-c) and antagonists (36a-c, 37a-c).
The A-ring precursors (11a-o) for these analogs
were synthesized from D-glucose as a chiral template. In the
19-nor series, we used radical coupling reaction for preparing
the A-ring parts from (–)-quinic acid, and the resulting
2-substituted A-ring moiety was coupled with 25-hydroxy Grundmann’s
ketone utilizing Julia olefination to connect between the
C5 and C6 positions. We also synthesized the highly potent
VDR-antagonists by introducing the 2α-functional
group to the TEI-9647 and TEI-9648 skeletons.
[Back to top]
C-20 Cyclopropyl Vitamin D3 Analogs
Milan R. Uskokovic, Percy Manchand, Stanislaw Marczak,
Hubert Maehr, Pawel Jankowski, Luciano Adorini and G. Satyanarayana
Reddy
The formal C-20 methylation of 1,25-dihydroxy vitamin D3
(calcitriol) and bridging of two methyl groups produces spiro[cyclopropane-1,
20’-calcitriol], colloquially referred to as C-20 cyclopropylcalcitriol,
which is much more active in MLR for suppression of interferon-γ
release than calcitriol, and hypercalcemia in mice is elicited
at a ten-fold lower dose when compared to calcitriol. Introduction
of the Δ16,17-double bond, modification
of the side chain by 23-unsaturation and replacement of the
methyl groups at C-26 and C-27 with trifluoromethyl moieties
create a highly active series of vitamin D analogs. As previously
observed in the calcitriol series, the presence of the C-16
double bond in the cyclopropyl analogs also arrests metabolic
side-chain oxidation in the at the C-24 oxo level in UMR 106
cells. The enhanced biological activity is ascribed, at least
in part, to the improved resistance toward metabolic degradation.
[Back to top]
Vitamin D Receptor Agonists, Cancer and the Immune
System: An Intricate Relationship
Luciano Adorini, Kenn C. Daniel and Giuseppe Penna
Vitamin D receptor (VDR) agonists can inhibit cell growth,
promote apoptosis, and induce differentiation of many cell
types, in addition to inhibiting metastasis and angiogenesis,
all desirable properties for a drug to control cancer. However,
from an immunological perspective, the immunomodulatory properties
of VDR agonists are apparently just opposite to the main aims
of cancer immunotherapy: boosting the immune response and
breaking tumor-related tolerance. While it may be possible
to identify VDR agonists with enhanced anti-proliferative/pro-differentiative
and reduced immunomodulatory activities as anti-cancer agents,
a complementary approach could rely on identifying clinical
indications where their systemic immunomodulatory properties
could be minimized. Superficial bladder cancer, where treatments
are usually administered by vesical instillation, may represent
such an indication. We have observed a strong synergism in
vitro between calcitriol and doxorubicin or epirubicin
in the inhibition of bladder cancer cell proliferation. Thus,
calcitriol and doxorubicin or epirubicin in combination may
have clinical value in the management of superficial bladder
cancer.
[Back to top]
Vitamin D Receptor Agonists: Opportunities and
Challenges in Drug Discovery
Tadakatsu Takahashi and Kazumi Morikawa
1α,25-dihydroxyvitamin
D3 (1,25(OH)2D3) is an important
hormone that regulates metabolism of calcium and phosphorus
in small intestine, kidney, and bone, and its physiological
action is expressed as ligand-dependent transcription activity
mediated by vitamin D receptor (VDR). The VDR is found in
various organs and cells including small intestine, kidney,
and bone. In addition to the regulation of calcium metabolism,
1,25(OH)2D3 is involved in various biological
reactions such as differentiation induction, antiproliferative
effect, immunomodulatory effect, and regulation of cytokine
and parathyroid hormone secretion. Thus, 1,25(OH)2D3
is expected to become a therapeutic drug for various related
diseases. At present, a number of vitamin D derivatives are
clinically applied to psoriasis, secondary hyperparathyroidism
and osteoporosis but hypercalcemia and hypercalciuria are
major concerns. Therefore, the current focus is directed toward
new vitamin D derivatives with weak calcemic effects and a
wide therapeutic window. In this summary, recent developments
of new vitamin D derivatives for application in clinical treatment
are described.
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