Transcript MATRIX METALLOPROTEINASES: ITS IMPLICATIONS ON THE

RIO BOOTHELLO
DEPARTMENT OF MEDICINAL CHEMISTRY
VIRGINIA COMMONWEALTH UNIVERSITY
EMAIL: [email protected].
Date: 22nd October 2010
THE COMMON PATH
vv
Cardiovascular disorders
Cancer
The Extracellular
Matrix
Skeletal disorders
Matrix
metalloproteinase
CNS disorders
Arthritis
Brinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3, 207-214.
1
EXTRACELLULAR MATRIX
Collagen
Laminin
Plasma membrane
Integrins
Elastin
Rozario, T. Dev. Biol. 2010, 341, 126–140.
2
FUNCTIONS
• Enzymes involved in ECM remodelling
Provides structure
Bone morphogenetic protein 1
Tracks migratory cells
ADAMS
Presents growth factors to receptors
Serine proteases
Senses/transduces mechanical signals
Matrix metalloproteinases
Rozario, T. Dev. Biol. 2010, 341, 126–140.
3
THE TADPOLE ENZYME
1962: Discovered by Jerome Gross and Charles
Lapiere
Anuran tadpole explants
Placed on collagen gel
Collagen degraded
Gross, J. et. al. Proc. Natl. Acad. Sci. 1962, 48, 1014-1022.
4
THE TADPOLE ENZYME
• Amount of collagen degraded
o Area lysed
o Degradation of C14 collagen
• Microscopic studies
Lysed collagen gel
NH2
NH2
COOH
collagenase
cleavage site
COOH
Cleavage of collagen triple helix
Gross, J. et. al. Proc. Natl. Acad. Sci. 1965, 54, 1197-1204.
PDB ID: 1CAG
5
HISTORY
1970
Purification
of human
collagenase
1979
Purification
of TIMP-1
1984
1992
Development
of genomic
clones
Batimastat
Phase I
trial
1993
First
crystal
structure
solved
Brinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3, 207-214.
6
MATRIX METALLOPROTEINASES
• Belong to the metzincin
group of proteases
• Synthesized as inactive
precursors
• Degrade the extracellular
matrix in a concerted manner
PDB ID: 1CK7
McCaw, A. et. al. Nat. Rev. Mol. Cell Biol., 2007, 8, 221-233.
7
STRUCTURE
Catalytic domain
Pro domain
Fibronectin type II domain
Hemopexin domain
Murphy, G. Mol. Aspects Med. 2008, 29, 290–308. PDB ID: 1GXD
8
CLASSIFICATION
Zn2+
MMP -1, -8, -13
Collagenase N
Zn2+
Matrilysins
Membrane
type
N
C
MMP -7, -26
Zn2+
CMT-MMP 1-8
N
Zn2+
Gelatinase
N
N
Signal sequence
Propeptide
C
Catalytic domain
Tail
MMP -2, -9
Transmembrane domain
Hinge region Furin domain
Cysteine switch Hemopexin domain
Cytosolic domain Fibronectin repeat
Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.
9
MECHANISM OF ACTIVATION
PRCGXPD
Cysteine switch peptide
AHEXGHXXGXXH
Catalytic site
His
His
Zn2+
Cys
His
Proenzyme catalytic domain
Hu, J. Nat. Rev. Drug Discovery, 2007, 6, 480-498. PDB ID: 1SLM
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ACTIVATION OF THE PROENZYME
His
His
Zn2+
His His
Stepwise activation
His
His
His
2+ His
Zn
Zn+2
His
Pro
Activation by MT-MMP
SH
Pro
SH
Chemical activation
Intermediate
Proenzyme
Active
form
Ra, H. J.; Parks, W. C. Matrix Biol. 2007, 26, 587–596.
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ROLE PLAYED IN ECM
 Path clearing through the ECM
 ECM proteolysis MMP
generates signaling molecules
MMP
 Degradation of basement membrane
MMP Epithelial cells
 Activation of latent signal
Mesenchymal cells
Degradation
MMPof basement membrane
Proliferation
Cell death
Mesenchymal cell
Cell motility
McCaw, A. et. al. Nat. Rev. Mol. Cell Biol. 2007, 8, 221-233.
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THE CELLULAR MILIEU IN CVS
Myocytes
Collagen IV
Collagen VI
Laminin
Proteoglycans
Fibroblasts
Collagen I
Collagen III
Periostin
Fibronectin
MMPs
Endothelial Cells
Collagen IV
Laminin
Fibronectin
Mast cells/Leukocytes/
Macrophages
Cytokines
Growth factors
MMPs
Vascular Smooth
Muscle cells
Collagen I
Collagen III
Collagen IV
Laminin
Fibronectin
Bowers, S. L. K. et.al. J. Mol. Cell. Cardiol. 2010, 48, 474-482.
13
CONDITIONS INVOLVED
Artery
MMP -3,9,12
Smooth
Elastin
muscle cell
MMP -2,-9 migration degradation Foam cell
Monocyte
Plaque rupture
infiltration
MMP -2, -9
Aneurysms
Myocardial infarction
MMP-2 and plaque
MMP-9
Atherosclerotic
Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.
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ROLE IN THE CVS
GENE
MMP-9
MMP-2
MMP-3
MT1MMP
DELETION
• Reduced LV dilation and
inflammation post-MI
• Reduce LV dilation and
rupture rate post MI
• Reduced LV hypertrophy
OVEREXPRESSION
-
• Severe LV contractile
dysfunction
• Dilated
cardiomyopathy
• Defects in cell proliferation
and cytokine release
• Impaired myocardial scar
maturation
• Decreased connective
tissue malformations
Brew, K. et. al. Biochim. Biophys. Acta 2010, 1803, 55–71.
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TARGETING MMPS
Endogenous inhibitors
Synthetic inhibitors
PDB ID: 1SLM
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ENDOGENOUS INHIBITORS
The tissue inhibitors of metalloproteinases
• Two distinct domains
o N-terminal domain
o C-terminal domain
• Four major types
o TIMP 1- 4
N-Domain
• Broad spectrum inhibitors
• Bind in a 1:1 stoichiometric ratio
Brew, K. et. al. Biochim. Biophys. Acta 2010, 1803, 55-71.
C-Domain
PDB ID: 1BR9
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INHIBITION MECHANISM
Glu67
Cys 1 to Val 4
Primed subsites
Glu 67 to Cys 70 Unprimed subsites
Cys 70
His
His
Zn2+
TIMP
Ser68
S2
Cys
Cys3
Cys 1
Val69
S3
Active site
Zn2+
Val4
His
Thr2
S1’
Brew, K. et. al. Biochim. Biophys. Acta, 2010, 1803, 55-71.
S3’
PDB ID: 1UEA
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TIMP: ROLE IN THE CVS
GENE
TIMP-1
TIMP-2
TIMP-3
TIMP-4
KNOCKOUT
• Greater LV dilation and matrix
loss post-MI
• LV systolic dysfunction postMI
• Aneurysm development
OVEREXPRESSION
• Reduced cardiac
rupture post MI
• Improved LV
systolic function
• Involved in ECM
• Greater LV dilation
• Increased cytokine
processing
• Increased MMP-2 activation
in fibroblasts
• No phenotype
• Decreased
activation of proMMP-2
• N.A
Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.
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TARGETING MMPS
Endogenous inhibitors
Synthetic inhibitors
PDB ID: 1SLM
20
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STRUCTURAL BASIS FOR INHIBITION
S3
S2
S1
Zn2+ S1’ S2’ S3’
S2’
S3
Collagen type peptide inhibitors
P2 P1 ZBG P1’ P2’ P3’
P3
General inhibitors requirements
P3 P2 P1 ZBG
Left side Inhibitors
ZBG P1’ P2’ P3’
Right side Inhibitors
S1
S3’
S2
S1’
ZBG : Zinc binding
group
Dorman, G. et. al. Drugs, 2010, 70, 949-964. PDB ID: 2TCL
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PEPTIDOMIMETIC INHIBITORS
Based on the structure of natural substrate collagen
Isobutyl,
Methyl
Essential
t-butyl
group
for
group
preferred
activity
preferred
Brown, P. D. Medical Oncology, 1997, 14, I- I0.
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THE ZINC BINDING GROUP
• Hydroxamates
• Thiol
• Phosphinates
• Carboxylates
Hu, J. Nat. Rev. Drug Discov. 2007, 6, 480-498.
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MECHANISM OF ZBG
Active enzyme
Hu, J. Nat. Rev. Drug Discovery, 2007, 6, 480-498.
Enzyme-hydroxamate
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BROAD SPECTRUM HYDROXAMATES
• The earliest MMP inhibitors
• Many members of this class entered clinical trials
Ilomastat
MMP-1 = 0.4
MMP-2 = 0.39
MMP-3 = 26
MMP-8 = 0.18
MMP-9 = 0.57
Batimastat
MMP-1= 5
MMP-1=10
Marimastat
MMP-2 = 6
MMP-2 = 4
MMP-3 = 200 MMP-3 = 20
MMP-7 = 20 MMP-8 = 10
MMP-9 = 3
MMP-9 = 1
*All IC50 values are in units of nM
Skiles, J. W. et. al. Curr. Med. Chem. 2004, 11, 2911-2977.
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STRUCTURAL BASIS OF INHIBITION
P2’ substituent
The succinate type backbone was modified
Wide range of
substituent
tolerated
α substituent
Zinc binding group
Improves
Essential for activity
Pharmacokinetic
properties
Hu, J. Nat. Rev. Drug Discov. 2007, 6, 480-498.
P3’ substituent
P1’ substituent
Wide range of
Major
substituent
determinant of
tolerated
activity
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MODIFICATIONS OF THE BACKBONE
Malonic acid type
Glutaric acid type
Sulphonamide type
Sulphone type
Hu, J. Nat. Rev. Drug Discov. 2007, 6, 480-498.
29
SULPHONAMIDE BASED INHIBITOR
Sulphonamide type
Succinic acid type
Developed by Parke-Davis showing μM potency for MMPs
Enzyme
IC50 μM
MMP-1
5.4
MMP-2
0.040
MMP-3
0.038
MMP-7
71
MMP-9
26
MMP-13
0.062
O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, 156-166.
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DEVELOPMENT OF PG-116800
Electron
Electron
Halogen
Halogen
Halogen
withdrawing
donating
atat
at
position
position
position
group
2’
group
3’
4’

4’

Decreased

Decreased
4’
Increased
Decreased
 Increase
selectivity
activity
activity
activity
activity
MMP (IC50 μM)
R
1
2
3
7
9
13
H
5.4
0.04
0.038
71
26
0.062
4’-F
4.2
0.039
0.010
4.8
64
0.043
4’-Br
6.0
0.004
0.007
7.2
7.9
0.008
4’-Cl
6.5
0.011
0.009
7.5
16
0.048
3’-Br
100
0.535
0.290
100
100
0.710
2’-F, 4’-Br
3.6
0.005
0.016
2.1
4.9
0.007
4’-NH2
26
0.036
0.036
31
20
0.105
4’-CF3
4.2
0.013
0.009
7.9
20
0.023
O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, 156-166.
31
DEVELOPING PG-116800
R1
H
Improving the pharmacokinetic profile
MMP IC50 μM
1
2
3
7
9
13
t1/2
(h)
50 0.004
0.010 77
6.7
0.026
15.6
13 0.012
0.012 11
9.5
0.010
25.1
6
0.004
0.007 7.2 7.9
0.008
43.6
7
0.009
0.007 3.1 4.9
0.005
41.8
0.007 15
0.012
3.88
27 0.002
2.3
O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, 156-166.
32
PG-116800
PG-116800
Developed by Parke-Davis
Enzyme
IC50 μM
MMP-1
6
MMP-3
0.007
MMP-7
7.2
MMP-2
0.004
MMP-9
7.9
S1’ pocket
Schematic representation of
crystal structure
Indicated that it could be used in left ventricular failure
O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, 156-166.
33
STUDIES CONDUCTED
Studies in humans
Animal studies
Randomized
trial were conducted for 90 days
• Study end points
Species
Dose
Effects on
Cardiac effects
o LV end diastolic index
MMP
o Pigs
Ejection20mg/kg/day
fraction • MMP-2
• LV dilation
• MMPs
• Results
Rats
5mg/Kg/day • MMP-2
o No Significant changes
• MMP-9
o Musculoskeletal syndrome
Dosing
• Possible causes
Regimen
•
•
LV peak wall stress
LV load
•
•
•
LV dilation
Contractility
Thickness of fibrillar
collagen
MMP
Selectivity
MMP-1
Kaludercic, N. et.al. Cardiovascular Therapeutics, 2008, 26, 24–37.
34
THE S1’ SELECTIVITY POCKET
• The depth of the S1’ tunnel is determined by the
S1’ specificity loop
• Pocket differs for different MMP’s
S1’
MMP-1,
-7
Ct
Shallow pocket
MMP-2, -9
Intermediate loop
pocket
Specificity
Nt
MMP-MMP-8,
1 shallow
MMP- 13 Deep pocket
-13pocket
Deep pocket
PDB ID: 2TCL
PDB ID: 456C
Overlap of the active site of major MMP classes
Devel, L. et. al. Biochimie, 2010. doi:10.1016/j.bioci.2010.07.017.
35
36
α-TETRAHYDROPYRANYL SULFONES
RS-130830
(β-Sulfone)
β- Sulfone
Developed by Roche
2nd Generation
α- Sulfone sulfone
Sulfone
MMP- 1
MMP-2
MMP-3
MMP-9
MMP-13
BA (%)
α
435
0.1
18.1
0.3
0.015
45.8
β
800
0.4
17.5
1
0.6
21.2
α- Sulphone derivative
Developed by Pfizer
*All IC50 values are in units of nM
Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.
37
α-TETRAHYDROPYRANYL SULFONES
R
α- Sulphone
MMP1
MMP2
MMP- MMP-13
9
BA
%
268
0.1
0.4
0.1
5.8
1800
0.3
2.9
0.45
-
435
0.1
0.3
0.15
45.8
400
0.2
0.1
0.3
-
1140
0.1
0.2
0.1
35.9
8000
0.1
1.2
0.8
33
5000
0.6
-
1
42.3
All IC50 values are in units of nM
Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.
38
α-PIPERIDINYL SULPHONES
R1
Selectivity
R2
H
Pharmacokinetics
Piperidine sulphones
MMP1
MMP2
MM99
MMP13
BA
%
10000
1.7
4
1.2
12.4
10000
0.3
1.9
5.5
48.3
10000
0.1
2.3
0.2
36.3
6000
0.2
1.9
0.5
16.6
4000
0.1
0.15
0.1
23
10000
0.1
0.18
0.1
67.9
All IC50 values are in units of nM
Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.
39
STRUCTURAL SELECTIVITY
Arg214
Crystal structure of α-Piperidine sulfone
Leu218
MMP-13, MMP-1 S1’overlap
Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.
40
ANIMAL STUDIES
LV pressure (mmHg)
• Inhibition of post infarction left ventricular dilation
investigated in rat model
50mg/kg
Vehicle
A
α- Tetrahydropyranyl sulfone
MI0.55
B Sham
α- Piperidine sulfone
MI-Veh
0.69
LV volume (mL)
SHAM-Veh
MI-Veh
MI-10mpk
MI-50mpk
A
LV vol.
(mL)
MI-10mpk
A
0.63
B
0.62
MI-50mpk
B
Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.
41
ANIMAL STUDIES
Pharmacokinetic parameter 10mg/kg
Species
Mouse
Rat
Dog
Monkey
BA (%)
51.7
66.6
64
53.8
C
LV pressure (mmHg)
Dose:10mg/kg
Vehicle
LV vol.
(mL)
MISham
0.49
MI-Veh
0.59
C
0.51
LV volume (mL)
SHAM-Veh
MI-Veh
MI-0.01mpk
MI-0.1mpk
MI-1mpk
MI-10mpk
Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.
42
MECHANISM BASED INHIBITORS
kon Rapid
+
Enzyme (E)
koff
Slow
Inhibitor (I)
His
+
E:I
complex
His
His
Zn+2
Substrate (S)
Interaction with Zn
E:S inhibitors
complex
Thiirane
Kcat
kon
koff
His
His
His
Zn+2
+
E
Product (P)
Covalently bound Zn
Ikejiri, M. et. al. J. Biol. Chem. 2005, 280, 33992-34002.
43
THIIRANE TYPE INHIBITORS
R
1
2
3
Enzyme
kon
MMP-2
2.1 x 104
3.5 x 10-4
0.016
MMP-9
4.9 x 103
9.0 x 10-4
0.18
MMP-14
6.9 x 102
6.4 x 10-4
0.9
MMP-2
1.9 x
103
10-3
0.7
MMP-9
-
-
1.0
MMP-14
-
-
4.9
MMP-2
1.2 x 104
1.3 x 10-3
0.11
MMP-9
-
-
0.13
MMP-14
-
-
0.68
M-1.s-1
koff
1.3 x
s-1
Ki
μM
Ikejiri, M. et. al. J. Biol. Chem. 2005, 280, 33992-34002.
A
NO2
B
C
44
TETRACYCLINES
Weak inhibitors of MMPs
• Inhibits smooth muscle cell
proliferation and migration
Doxycycline
• Inhibits MMP-2 and MMP-9
MMP-9 proenzyme
MMP-2 proenzyme
MMP-2 active
Doxycycline μM
0
42
104
208
416
Gelatin zymography of SMC
Doxycycline treated SMC
Untreated SMC
Franco, C. et. al. Am. J. Pathol. 2006, 168, 1697-1709.
45
SUMMARY
MMPs
Inhibitors
Future
strategies
•• Physiological
Inhibitors of MMPs
and
have
from matrix
potent broad
Understanding
thepathological
role
ofevolved
extracellular
functions
spectrumoftoMMP
selective
are
broad
cardiovascular
agents
metalloproteinase
inducer
and other modulators
of MMP
•• Role
Development
of individual
of selective
MMP in inhibitors holds the key to
the progress
progression
of these
isagents
not known
• disease
Developing
newer
strategies
to first understand
the role and then target MMPs
•• Modulators
Understanding
of MMP
the variation
activity in binding site of
needs
MMPstomay
be recognized
be essential
46
ACKNOWLEDGEMENTS
• Dr. Umesh Desai
• The Desai group
• The Department of Medicinal Chemistry
at VCU
47