First test of SlideShare...

1. ACE Inhibitors Block MMP-9 Activity
Daisuke Yamamoto and Shinji Takai
Biomedical Computation Center and
Department of Pharmacology
Osaka Medical College, Japan

2. Tonight’s Menu
• Background of our research -
Two Zn2+ metalloproteases (ACE & MMP-9) and
inhibitors, relating to ECM remodeling and MI.
• Molecular structures of ACE inhibitors on the
MMP-9 active site and inhibitory activities of ACE
inhibitors to MMP-9. We have used lisinopril,
imidapril and captopril as typical ACE inhibitors.
• Pharmacological implications and importance for
development of new MMP-9 inhibitors.

3. Background-1
• ACE (angiotensin converting enzyme) is a Zn metalloprotease
converting angiotensin-I to angiotensin-II. ACE inhibitors were
designed to target ACE active site since 1970’s, and have
been used as anti-hypertension drug clinically.
• Recently, it has reported that some ACE inhibitors prevented
myocardial infarction and remodeling of ECM, but it was not
clear that all of this effect would be due to angiotensin-II.
• In addition, few reports said that ACE inhibitors inhibit matrix
metalloproteinase 9 (MMP-9) weakly on in vitro assay system.

4. Background-2
ACE
converts
angiotensin-­‐I
to
angiotensin-­‐II
by
hydrolysis
of
the
pepEde
bond
between
Phe8
and
His9.
S1
S2’
Phe8
Lyz511
Pro7
angiotensin-I
His9
Leu10
Val379
S2
Zn2+
Val380
His383
Glu162
ACE
acEve
site
His387
Glu411
S1’
Asp377
Molecular structure of ACE active site in the C domain has a Zn catalytic
center chelating to His, Asp residues. S1’(substrate C-terminus binding
site 1) has (-) charged residues, and S2’ has (+) charged residues,
recognizing [-His-Leu-COO-] of angiotensin-I C-terminus.

5. Background-3 Binding mode between ACE and ACE inhibitor
A typical ACE inhibitor, lisinopril, was stabilized in ACE active site by a
chelate bond to the Zn active center, hydrogen bonds to peptide
binding site and specific interactions with S1, S1’, and S2’ site of ACE.
(R.Natesh, et al. Nature v.142 p.551-554 2003, PDB code; 1O86)

6. Background-4 Lisinopril binding to the ACE active site
Nitrogen
Oxygen
Sulfur
Zinc
X-ray crystal structure of ACE – lisinopril complex
(R.Natesh, et al. Nature v.142 p.551-554 2003, PDB code; 1O86)

7. Background-5
• Matrix metalloproteinase (MMP)-9 is a Zn dependent
endopeptidase, and is one of key effectors of ECM
remodeling. MMP-9 has a different substrate specificity
from ACE. (- Pro – X – X ^ [Hydropohobic] – [Ser/Thr] -)
• It has been reported that levels of MMP-9 increase after
MI in human and animals, and cardiac dysfunction and
mortality after MI are suppressed by MMP-9 inhibitors
and in MMP-9 null mice.
• In addition, few reports said that ACE inhibitors inhibit
matrix metalloproteinase 9 (MMP-9) weakly on in vitro
assay system.

8. Background-6 Binding mode between MMP and MMP-9 inhibitor
An-1, one of MMP-9
carboxylate inhibitors, was
stabilized in MMP-9 active site
by a chelate bond to the Zn
active center, hydrogen bonds
to peptide binding site and
hydrophobic interactions with
S1 and S1’ site of MMP-9.
(A. Tocowitcz, et al.
J. Mol. Biol. v.371 p.989-1006 2007,
PDB code; 2OW0)

9. Background-7 An-1 binding to the MMP-9 active site
Nitrogen
Oxygen
Sulfur
Zinc

10. Background-8 Structure of proMMP-9
AcEve
domain
FnII
(fibronecEn
type
II)
domains
Precursor
domain
The
precursor
of
human
MMP9
includes
the
precursor
domain,
the
acEve
domain
and
three
FnII
(fibronecEn
type
II)
domains.
The
precursor
domain
is
inserted
into
the
acEve-­‐
site
cleY,
blocking
access
to
the
catalyEc
zinc.
(PA.
Elkins,
et
al.
Acta
Cryst..
D58,
1182-­‐1192
(2002)
PDB
code;
1L6J)

11. Background-9 Activation of proMMP-9
AcEve
domain
Precursor
chain
AcEve
center
The
precursor
chain
is
inserted
into
the
acEve-­‐site
cleY,
blocking
access
to
the
catalyEc
zinc
by
Cys99,
and
its
direcEon
is
reverse
to
the
substrate
binding.
The
acEvaEon
by
APMA
in
MMP-­‐9
assay
system
cleaves
between
Ala93
and
Met94,
and
Phe110
is
shiYed
to
the
area
occupied
by
Phe107
(shown
in
some
X-­‐ray
structures).
MMP-­‐9
is
reported
to
be
also
acEvated
by
chymase
in
the
inflammaEon,
cleaving
between
Phe110
and
Glu111.

12. Investigations in our research are …
1. Can ACE inhibitors inhibit MMP-9 directly?
2. How do ACE inhibitors bind to MMP-9 active
site?
3. What kind of interaction is important for the
binding to MMP-9?

13. Inhibitory Activity of Lisonopril -1
In
this
slide,
ACE
and
MMP-­‐9
acEviEes
1,
3
and
7
days
aYer
myocardial
infarcEon
in
the
infarcted
leY
ventricles
of
hamsters
are
shown.
ACE
acEvity
was
significantly
increased
3
and
7
days
aYer,
but
not
1
day
aYer
myocardial
infarcEon.
On
the
other
hand,
MMP-­‐9
acEvity
was
significantly
increased
1
day
aYer
myocardial
infarcEon.
Three
and
7
days
aYerwards
it
declined
progressively
back
towards
its
pre-­‐infarcEon
level.
(S.
Takai,
et
al.
Eur.
J.
Pharmacol.
v.568,
p.231-­‐233,
2007)

14. Inhibitory Activity of Lisonopril -2
Lisinopril
significantly
inhibited
both
ACE
and
MMP-­‐9
acEviEes
1
day
aYer
myocardial
infarcEon,
but
candesartan
cilexeEl
(an
ARB;
angiotensin-­‐II
receptor
blocker)
did
not.
It
was
supported
that
lisinopril
might
directly
inhibit
MMP-­‐9
acEvity
without
angiotensin-­‐II.
(S.
Takai,
et
al.
Eur.
J.
Pharmacol.
v.568,
p.231-­‐233,
2007)

15. Inhibitory Activity of Lisinopril & Imidapril
• A
ACE
and
MMP-­‐9
acEvity
in
the
normal
(N),
placebo
(P)-­‐,
lisinopril
(L)-­‐
and
imidapril
(I)-­‐treated
groups
1
day
aYer
MI
are
shown.
(⁎P<0.05
and
⁎⁎P<0.01
vs.
placebo.
†P<0.05
vs.
lisinopril.)
Both
of
lisinopril
and
imidapril
significantly
suppressed
the
ACE
acEvity
compared
with
placebo,
but
there
was
no
significant
difference.
MMP-­‐9
acEvity
was
also
significantly
suppressed
by
lisinopril
and
by
imidapril,
and
it
was
significantly
lower
in
the
group
given
imidapril
than
lisinopril.
(D.
Yamamoto,
et
al.
J.
Mol.
Cell.
Cardiol.
v.43,
p.670-­‐676,
2007)

16. Inhibitory Activity of Captopril
To
confirm
the
inhibitory
effects
of
captopril
on
ACE
and
MMP-­‐9
acEviEes,
human
plasma
samples
from
6
paEents
who
had
on
acute
myocardial
infarcEon
were
treated
with
NH2–Ph–Hg–Ac
(APMA),
and
then
were
incubated
with
captopril
for
1
h
at
37°C.
Captopril
inhibited
ACE
acEvity
in
a
dose-­‐dependent
manner
(IC50;
45
nmol/l)
(A),
and
it
also
dose
dependently
inhibited
MMP-­‐9
acEvity
at
the
same
dose
level
(IC50;
87
nmol/l)
(B).
(D.
Yamamoto,
et
al.
Eur.
J.
Pharmacol.
V.588,
p.277-­‐279,
2008)

17. Investigations in our research are …
1. Can ACE inhibitors inhibit MMP-9 directly?
2. How do ACE inhibitors bind to MMP-9 active
site?
3. What kind of interaction is important for the
binding to MMP-9?

18. Molecular Modeling - MMP9 + Lisinopril
Modeling
Methods;
We
constructed
molecular
models
of
MMP-­‐9
–
ACE
inhibitor
complex
by
structural
merging
of
MMP-­‐9
–
MMP-­‐9
inhibitor
complex
(1GKC
chain
B)
and
ACE
–
ACE
inhibitor
complex
(1O86).
Based
on
X-­‐ray
structures
of
previous
studies,
two
interacEon
modes
for
the
MMP-­‐9
-­‐
ACE
inhibitor
complex
can
be
envisaged,
in
which
phenyl
ethyl
group
of
ACE
inhibitor
is
either
in
the
MMP-­‐9
S1
or
S1’
site.
(called
as
“mode
A”
and
“mode
B”
in
this
presentaEon)
Molecular
models
were
opEmized
structurally
by
energy
minimizaEon
and
100
picoseconds
molecular
dynamics
simulaEons,
using
MMFF94x
force
field
and
water
molecules
randomly-­‐distributed
in
a
5Å
shell
around
each
model.
All
operaEons
of
the
modeling
were
performed
using
a
package
for
molecular
structure
analyses,
MOE
(Molecular
OperaEng
Environment,
Chemical
CompuEng
Group
Inc.).
(D.
Yamamoto,
et
al.
Biochem.
Biophys.
Res.
Commun.
v.354,
p.981-­‐984,
and
J.
Mol.
Cel
Cardiol.
v.43,
p.670-­‐676,
2007)

19. Lisinopril binding to the MMP-9 active site
InteracEon
mode
A
of
lisinopril
to
MMP-­‐9
acEve
site.
This
model
was
constructed
under
two
condiEons
;
the
central
carboxy
group
was
chelate-­‐binding
to
the
catalyEc
zinc,
and
phenyl
ethyl
group
closed
to
S1
site
by
hydrophobic
interacEon.
>NH
and
>C=O
groups
of
lisinopril
were
interacted
to
“pepEde
binding
site”
of
MMP-­‐9.
The
lysinyl
–NH3+
group
was
interacted
to
the
entrance
of
S1’
site.

20. Lisinopril binding to the MMP-9 active site
Nitrogen
Oxygen
Sulfur
Zinc
Lisinopril
is
shown
by
green.
The
lysinyl
moiety
did
not
enter
to
S1’
site
(deep
“P1’
hole”),
because
of
the
charge
repulsion
with
Arg424.
From
this
effect,
molecular
distorEon
might
be
shown
in
the
lysinyl
moiety.
Phenyl
ethyl
group
was
in
the
wide
S1
site
without
Phe110
residue.
This
S1
structure
was
prepared
considering
the
acEvaEon
of
MMP-­‐9
by
chymase.

21. Lisinopril binding to the MMP-9 active site
InteracEon
mode
B
of
lisinopril
to
MMP-­‐9
acEve
site.
This
model
was
constructed
under
the
condiEon
where
the
phenyl
ethyl
group
closed
to
S1’
site
by
hydrophobic
interacEon.
>NH
and
terminal
carboxyl
groups
of
lisinopril
were
interacted
to
Ala189
mainchain
and
His190
side
chain
by
hydrogen
bonds.
The
lysinyl
–NH3+
group
was
exposed
to
the
water
phase.

22. Lisinopril binding to the MMP-9 active site
Nitrogen
Oxygen
Sulfur
Zinc
The
phenyl
ethyl
group
was
Eghtly-­‐binding
to
the
S1’
site,
but
molecular
distorEon
was
shown
in
MMP-­‐9
acEve
site,
especially
His405
and
His411.
Linsinopril
in
mode
B
seems
to
be
not
easy
to
be
formed
comparing
with
in
mode
A.
However,
this
interacEon
mode
would
not
be
effected
by
the
status
of
S1
site
relaEng
to
the
MMP-­‐9
acEvaEon.

23. Imidaprilat binding to the MMP-9 active site
We
also
constructed
the
complex
of
imidaprilat,
and
the
interacEon
mode
A
is
shown.
Imdaiprilat
is
an
acEve
form
of
imidapril,
and
do
not
have
the
lysinyl
group
of
lisinopril.
In
this
interacEon
mode,
imidaprilat
was
fixed
on
the
MMP-­‐9
acEve
site
by
similar
interacEons
shown
in
the
lisinopril
complex.

24. Imidaprilat binding to the MMP-9 active site
Nitrogen
Oxygen
Sulfur
Zinc
Imidaprilat
is
shown
by
orange.
By
the
lack
of
lysinyl
group,
imidaprilat
was
easy
to
access
to
the
acEve
site
without
the
molecular
distorEon
shown
in
lisinopril.

25. Imidaprilat binding to the MMP-9 active site
InteracEon
mode
B
of
imidaprilat
to
MMP-­‐9
acEve
site.
In
this
interacEon
mode,
imidaprilat
was
fixed
on
the
MMP-­‐9
acEve
site
by
same
interacEons
shown
in
the
lisinopril
complex.

26. Imidaprilat binding to the MMP-9 active site
Nitrogen
Oxygen
Sulfur
Zinc
In
this
interacEon
mode,
significant
difference
was
not
shown
comparing
with
the
mode
B
of
the
lisinopril
complex.

27. Summary-1 mode A or mode B?
• Imidapril had more stronger inhibitory activity to
MMP-9 than lisinopril.
• In the interaction mode A, imidaprilat was easy
to access to the MMP-9 active site comparing
with lisinopril. Significant difference was not
shown between lisinopril and imidaprilat in the
mode B.
• In the mode B, the phenyl ethyl group on S1’
site might cause the molecular distortion of
MMP-9.
We think that the molecular structure of mode A is
easy to be formed comparing with it of mode B.

28. Captopril binding to the MMP9 active site
Nitrogen
Oxygen
Sulfur
Zinc
We
also
constructed
molecular
models
for
captopril.
Randomly-­‐docking
iteraEons
were
used
for
the
modeling,
because
of
the
lack
of
big
hydrophobic
moeity,
such
as
phenyl
ethyl
group.
Two
typical
conformaEons
of
captopril
are
shown.
In
these
binding
modes,
the
binding
of
captopril
would
not
be
effected
by
the
status
of
S1
site.

29. Captopril binding to the MMP9 active site
• AN
Chelate
bond
Chelate
bond
Captopril
–SH
-­‐
-­‐
-­‐
-­‐
-­‐
Zn(2+)
Captopril
–SH
-­‐
-­‐
-­‐
-­‐
-­‐
Zn(2+)
Hydrogen
bonds
Hydrogen
bonds
Captopril
>C=O
-­‐
-­‐
-­‐
-­‐
-­‐
Leu188
N
Captopril
>C=O
-­‐
-­‐
-­‐
-­‐
-­‐
Ala191
N
Ala189
N
Captopril
–COO(-­‐)
-­‐
-­‐
-­‐
His190
ND1
Hydrophobic
interacEon
Hydrophobic
interacEon
Capto.
Pyrrolidine
-­‐
-­‐
-­‐
-­‐
Leu187
Capto.
Pyrrolidine
-­‐
-­‐
-­‐
-­‐
Leu187
Leu188
Tyr179

30. Summary-2 Status of the S1 site
• Captopril had the inhibitory activity to MMP-9 as
same as to ACE, in human plasma on acute MI.
Lisinopril and imidapril had very weak activities to
MMP-9 in human plasma (data not shown), but
these two ACE inhibitors inhibited the MMP-9 in LV
1 day after MI remarkably.
• The binding of captopril would be not effected by
the status of S1 site, and the bindings of lisinopril
and imidaprilat would be effected by the S1 site
status in the interaction mode A.
We think that the Phe110 residue would remain in the
S1 site of plasma MMP-9, but it would be deleted in
LV MMP-9, because of the activation by chymase-like
enzymes.

31. Summary-2 Status of the S1 site
APMA
treatment
of
assay
system
ProMMP-­‐9
An-­‐1
complex
Chymase
at
inflammaEon
areas
The
deleEon
of
Phe110
causes
the
extension
of
S1
site,
and
the
phenyl
ethyl
group
in
the
mode
A
interacts
to
Imidaprilat
complex
this
wide
S1
site.
Captopril
would
able
to
bind
to
MMP-­‐9
(mode
A)
in
both
statuses
of
the
S1
site,
because
of
its
small
size.

32. Investigations in our research are …
1. Can ACE inhibitors inhibit MMP-9 directly?
2. How do ACE inhibitors bind to MMP-9 active
site?
3. What kind of interaction is important for the
binding to MMP-9?

33. Summary-3 Pharmacological Implications
• Chelate binding to catalytic zinc and hydrogen bonds
with “peptide binding site” are very important for MMP-9
inhibition. These interactions are also shown in the
MMP-9 – MMP-9 inhibitor complex and the inactive
proMMP-9.
• Lysinyl group and terminal carboxyl group (simulated
groups of His9 and C-terminus of angiotensin-I) are not
essential for MMP-9 inhibition.
• The wide S1 site of MMP-9 might have to be considered
clinically in the MMP-9 inhibition at inflammation areas.
We think that these findings will be applicable to the new
development of effective MMP-9 inhibitors based on ACE
inhibitors.
(D. Yamamoto, et al, Curr. Med. Chem. v.16, p.1349-1354, 2009)

34. Acknowledgements
• Dr. Merry L. Lindsey and Prof. Thomas K. Borg, thank
you very much for the invitation to this symposium. And
Dr. Merry L. Lindsey introduced our article in “Editorial”
of JMCC (2007).
• Dr. Mizuo Miyazaki, professor emeritus of Dep.
Pharmacology in our college, has constructed the base
related to our research, especially pharmacological
functions of A-II, ACE and chymase.
We are very grateful to these persons.

35. New Tricks for Old Dogs…
Dr. Snoopy A.C.E.i. said;
“My patent had been expired already,
but I can still exert myself!!!”