Vitamin D current outlook, design and synthesis of active analogs

UNIVERSIDAD DE SANTIAGO DE COMPOSTELA
Departamento de Química Orgánica
Santiago de Compostela E-15782, España
Antonio Mouriño: antonio.mourino@usc.es

Vitamin D: Current Outlook, Design
and Synthesis of Active Analogs
Development of Active vitamin D Analogues

Birmingham. DECIDE. Nov 14, 2013
miércoles 27 de noviembre de 2013

Alimentos
Food

20%

80%
VIT-D3
PRE-D3

7-Deshidrocolesterol
7-Dehydrocholesterol

25-D3
1,25-D3

Forma Activa
Active form 1,25D


Vitamin D Metabolism

7-Dehydrocholesterol

Previtamin D3

Kidney

Cisoid form

Liver

Transoid form

1,25D
(calcitriol)

Vitamin D3

.

ICA
BCM

BIOLOGICAL
RESPONSES

HO

H
H

HO

OH

1α,25-(OH)2-D3

.


.

ICA
BCM

BIOLOGICAL
RESPONSES

DIFFERENTIATION
PROLIFERATION

HO

H

Miyaura et al
Biochem. Biophys. Res.
Commun.1981, 102, 937

H

HO

OH

1α,25-(OH)2-D3

.


.

ICA
BCM
DIFFERENTIATION
PROLIFERATION
CANCER
HO

H

Miyaura et al
Biochem. Biophys. Res.
Commun.1981, 102, 937

H

HO

OH

1α,25-(OH)2-D3
PSORIASIS

IMMUNOLOGY
(TRANSPLANTATION)


BONE DISEASES
(OSTEOPOROSIS)

.

DT-71


OH
H
H

HO


OH

HO

H
H

HO


OH

Mode of action of 1α,25-(OH)2-D3

Cytoplasm

Nucleus


Mode of action of 1,25D

Cytoplam

Nucleus



Cytoplam

Nucleus
RXR



Cytoplam

Nucleus
RXR

coA

Target gene

m-RNA


Cytoplam

Nucleus
RXR

coA

Target gene
Proteins
m-RNA

Vitamin D Nuclear Receptor (VDR)


Receptor Nuclear de la Vitamina D (VDR)

DNA



DNA

Shaffer PL, Gewirth DT.
EMBO J. 2002, 21, 2242-2252



1α,25-(OH)2-D3



1α,25-(OH)2-D3

Moras D. y col.
Mol. Cell 2000, 5, 173-179


Ligand Binding Domain

H12
H2
H11
H3

H5

H6
H8

H4
H9

H1


Ligand Binding Domain

Ser278

2.91
2.83

2.81
2.83
2.86
2.82

Ser237


COLLABORATIONS
Prof Roger Bouillon and Mieke Verstuyf
Leuven University

Transactivation, Calcemic Effects,
Cell Differentiation-Proliferation

Prof Alberto Muñóz
Institute of Biomedical Investigations
CSIC-Madrid

Transactivation,
Cell Diffentiation-Proliferation
(colon cancer cell lines)

Prof. Román Pérez Fernández
Faculty of Medicine-USC
Santiago Compostela

Transactivation, Calcemic Effects,
Cell Differentiation-Proliferation

Prof. Dino Moras and Natacha Rochel
IGBMC-Illkirch-France

Structural Studies


Superposition of Diyne (Docking Conformation)
with 1,25D (Crystallographic Structure)


Drop containing crystals of Moras mutant VDR LBD complexed to 4c
Prof. Dino Moras (GBMC-Illkirch-Francia)

HO

Diyne-LBD(VDR)Complex

HO

H

OH
VDR

Crystallographic Structue of the Diyne
superimposed with 1,25D

N Rochel, S Hourai, X Pérez, A Rumbo, A Mouriño
Arch. Biochem. Biophys. 2007, 460, 172-176


OH

H

12
H

HO


OH

1,25D with a Me Group at C-12


Design, Syntheses and Biological Assays of
1,25D Analogs with Side Chains at C-12
1,25D Analogs with Side Chains at C-12.
Vitamin Analogues with Side Chains atC-12
J. Med. Chem. 2006, 49, 1509-1516

OH

OH

n

Exploration of side
chains at C-12

n = 1, 2, 3

?
H

HO


OH

12

H

Docking

HO

OH

(104%)

(113%)

(112%)

J. Med. Chem. 2012, 55, 8642

in silico LBD(VDR)

100

104

113

112

Affinity for hVDR

100

17

22

13

Transactivation (10-9 M)

100

40

36

57

Ca Effects

100

0

18

1


Analogs of 1,25D with Side Chains at C-18


.


comparably to “1,25”
DS-A y DS-B Induce cell differentiation in colon
cancer cell lines (SW-480-ADH) similarly to 1,25D
A
48 h
Vehicle

1α,25-D3

DS-A

DS-B

le 5-D 3
ic ,2
eh 1α
V
DS-A DS-B

B
Time (h)

48

8

1.0

1.6

48

8

48

8

48

E-cadherin

6.3 1.7 4.6 0.8 1.5

DS-A and DS-B activate VDR transactivation in
human colon cancer cells (SW480-ADH).
VDRE activation (fold increase)

30
25
20
15
10
5
0
10- 9 10- 8 10- 7 10- 9 10- 8 10- 7 10- 9 10- 8 10- 7

1α,25-D3


DS-A

DS-B

DS-A and DS-B inhibit cell proliferation (50% vs 1,25)
Cell proliferation (% inhibition)

50

40

30

20

10

0
1α,25-D3 DS-A


DS-B

Structures of Superagonists MC1288 y KH1060

Superagonists: Potent inducers of cell
differentiation

Superimposition of the crystallographic
structures of 1,25D and superagonists
KH1060 y MC1288
Moras D et al.
PNAS 2001, 98, 5491-5496


Rational Design of a New Superagonist

New Superagonist?


54

A

B

Isomer A induces transactivation 12 times better than
1,25D at 10-10 M, The isomer B behaves as 1,25D.

A

1,25


B

58

DESIGN OF A NEW SUPERAGONIST

+

ISOMER A


=

2-Me-1,25D
BINDS 4 TIMES
BETTER TO VDR
THAN 1,25D

A-Me
NEW
SUPERAGONIST?

29

Interaction Energies with the

A-Me

B-Me

TRANSACTIVATION ACTIVITY
A-Me is 13 times more potent than 1,25D and 4
times more potent than A at 10-10 M

1,25D


A

A-Me

65

Biological Activity Similar than 1,25D
• Proliferación celular en HL-60


• Diferenciación celular en HL-60

66

Ser237
Superimposition
of the New
Metabolite with
1,25D
W2

Ser278
W1

Tyr147

Arg274

Tyr143
Gln277


W3

Prof. Román Pérez-Fernández


Vitamin D current outlook, design and synthesis of active analogs

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Vitamin D current outlook, design and synthesis of active analogs