Design and Development of Superoxide Dismutase Mimetics as

Design and Development of
Superoxide Dismutase
Mimetics as Therapeutics
Traditional
Approach
Block negative effects
of enzymes
A
B
MetaPhore’s
Approach
Amplify positive
effects of enzymes
C
D
SYNZYMES: A CASE STUDY
Mimics of Superoxide Dismutase : mSOD
Superoxide Shunts
HOO.
H+
SOD
Catalyst
Normal Hydrogen Peroxide
Detoxification
O2-.
Neutrophil-Driven
Immune Responses
O2 + H2O2
Catalase or
Glutathione Peroxidase
O2 + H2O
(Myeloperoxidase)
+
Cl-
OCl- - bacteriocide
Reactive Oxygen Metabolites
HOO
Lipids,
DNA,
Catecholamines,
Steroids, etc.
.
H+
Arginine
-.
O2
SOD
Catalyst
FeIII
NO.
NOS
OONO-
Storage
Proteins
O2 + H2O2
Chain Autoxidation Products
Fe
H+
II
[OONOH]
.
OH
(Hydroxyl Radical)
Cellular
Damage
Peroxynitrite
Cytotoxic
NO2.
Biochemical Impact of Excess
Superoxide Anions
DNA
Damage
Degrades Synovial
Fluid & Collagen
Depolymerize
Hyaluronic
Acid
–
O2
Cytokine
Release
ONOO–
Generation
Lipid
Peroxidation
Stimulates Release of
Inflammatory Mediators
Superoxide is eliminated under physiological conditions
by a set of superoxide dismutase enzymes that convert
it to hydrogen peroxide which is then converted to water
and oxygen by catalase.
Mangenese superoxide dismutase is localized in
mitochondria while Cu/Zn superoxide dismutases are in
the cytoplasm and extracellular.
Bovine Cu/Zn SOD (Orgatein®) was used as a
pharmaceutical in Europe for many years for the
treatment of osteoarthritis.
In animal models, SOD enzymes have been shown
efficacious in models of ischemia-reperfusion injury,
inflammation, radiation damage, etc.
DISADVANTAGES OF PROTEIN SOD
THERAPY
Lack of Oral Activity
Lack of Access to Intracellular Space where
Superoxide is Produced
Immunogenicity
Short Half-Lives in Serum
Project Goal: SOD mimics
To develop a revolutionary advance
in the small molecule approach to
pharmacological intervention.
Ribbon diagram of crystal structure of human superoxide dismutase
dimer; active sites are within blue ribbons
Enzyme Mimetic
confidential
TECHNOLOGY: mSODs
10 years of research
>$50 Million
SOD
•
•
•
88kDA
kcat = 108-109 M-1s-1
superoxide hydrogen peroxide
mSOD
•ca. 500Da
•kcat = 107-109 M-1s-1
•superoxide hydrogen peroxide
N
H
Cl
N
N
Mn
N
Cl
N
H
H
M40403
H
D. P. Riley and R. H. Weiss, J. Am. Chem. Soc. 116:387-388, 1994
Stopped-Flow UV
Assay for SOD
Mimetics
D. P. Riley and R. H. Weiss, J. Am. Che
Soc. 116:387-388, 1994
Traditional Approach
to Synthesis of
Pentaazacrowns
Gave Poor Yields
Cyclic Peptide Approach to
Cyclopentaazacrowns
Aston et al. Tetrahedron Lett. 35:3687, 1994
SOD Mimetics Used to Develop Models
of Mechanisms
Changes in Chirality of
Substituents and
Introduction of Cyclic
Constraints Modified
Complex Geometry
D. P. Riley, Susan L. Henke, Patrick J.
Lennon and Karl Aston, Inorg. Chem.
38:1908-1917, 1999
Stereochemical Effects
H
H
N
N Cl N
Mn
Cl
N
H
N
H
H
H
all-Rkcat (pH=7.4)= 1.2 x 10+8 M-1 s-1
H
N
N Cl N
Mn
Cl
H
N
N
H
H
R,R,S,Skcat (pH=7.4): Inactive
R,R,S,S,-isomer is rigidly planar; i.e., will not fold, thus no catalytic activity
Ligands Used to Test Predictive Ability
of Model
Mn(II): E = 32.2 kcal
Mn(III): 31.0 kcal
The folded six-coordinate Mn(II) structure is very close to that of the sixcoordinate Mn(III) indicating a favorable geometry for facile electron transfer
& hence fast SOD catalysis. (Science, 286, 304 (1999)).
MECHANISMS FOR
CONVERSION OF
SUPER-OXIDE TO
HYDROGEN PEROXIDE
VIA INNER SPHERE OR
OUTER SHERE
OXIDATION OF MN(II)
D. P. Riley, Susan L. Henke, Patrick J. Lennon
and Karl Aston, Inorg. Chem. 38:1908-1917, 1999
H
N
H
Cl N
Mn
HN
Cl
N
H
SC-68328
CH3
CH3
NH
6000-fold stability
increase, while
retaining catalytic
activity
N
HN
Cl
N
Mn
N
H
Cl
N
H
M40403
H
Synthesis of M40403
NH2
1. TrtCl, CH2Cl2
N
2. Glyoxal,
MeOH
NH2
H2/Pd on C
N
CH3OH
NHTrt TrtHN
96%
NH
HN
NHTrt TrtHN
100%
conc. HCl
TrtCl
96%
OHC
N
NH
CHO
HN
•4HCl
+
NH2
+ MnCl2
H2N
M40400
H
N
N Cl N
Mn
Cl
Pd/C, 150 psig H2
ropanol
H2
H
H
N
N
H
1 hr, >98% yield
N
N Cl N
Mn
Cl
N
M40402
Intermediate
M40403
H
N
H
99.3% purity
in 1 pot reaction->93% Chem Yield with
No Chromatography or
Crystallization needed
H
Cl
N
H
N
Mn
Cl
H
H
N
Cl
N
N
N
H
H
N
Cl
S
M40403
Mn
N
Cl
N
N
N
S
M40401
*Riley, et.al, Inorg. Chem., 2001, 40(8), 1779.
H
H
H
H
N
N
Mn
Cl
S
H
N
M40484
N
S
H
Chemical History of SODm
SAR studies utilizing the [Mn([15]aneN5)Cl2] lead reveals that
extremely high chemical stability is achieved with added substituents
to carbon centers of the macrocyclic ligand (Inorg.Chem.,35, 5213,
1996)
SAR studies reveal that catalytic activity can be dramatically affected
with the stereochemistry, number, and position of such substituents
(JACS, 119, 995 (1997)).
Mechanistic understanding developed that led to a molecular
modeling paradigm (MM) allowing prediction of catalytic activity
(Inorg. Chem., 38, 1908 (1999); and ibid., 40(8), 1779 (2000).)
Bis-cyclohexylpyridine class of SOD mimic affords high activity and
high stability with excellent in vivo efficacy (Science, 286, 304
(1999)).
Metabolism of M40403
H
H
3
H
N
N Cl N
Mn
Cl
H
N
N
H
Liver Oxidases
H
H
3
N
N Cl N
Mn
Cl
H
N
N
H
M40403
M40414
kcat (pH=7.4) = 1.6 x 10+7
kcat (pH=7.4) = 1.35 x 10+7
log P = -0.3
log P = -0.9
H
H
N
N Cl N
Mn
Cl
N
H
N
isomer
Native Enzyme
M40403
One of two
subunits.
–
Inflammation
O2
Cardiovascular/CNS
Ischemia-reperfusion injury
Septic shock
Organ transplantation
Stroke
Pain
IBD/Crohn’s
Oncology
Osteoarthritis
Rheumatoid arthritis Pain
Psoriasis
Inhibition of tumor growth
Reduction of side effects associated with
chemotherapy and radiation therapy
Superoxide –
central role
SOD mimetics
effective in multiple
models
Rheumatoid arthritis
Blocks TNF
release
Asthma
Many diseases
severe/
chronic Pain
Persist
j
acu /
mod
PAIN
Traditional NSAIDs provide unsatisfactory relief in
models of severe chronic pain (neuropathic pain, visceral
pain, cancer pain)
Opportunities exist beyond opioids and NSAIDs for the
treatment of acute and severe pain
Roles of Superoxide Anions (O2 •) in Pain
Damage nerve endings
Direct algesic
action
Sensitize primary
afferent neurons
–
O2
Increase spinal release of
excitatory amino acids
(NMDA)
Deactivate NE, a
potent endogenous
analgesic
Superoxide anions
9
Decomposed
Superoxide anions
Vehicle
8
7
SOD-PEG
6
5
4
3
2
1
0
0
50
100
150
200
Latency changes (sec.)
Latency changes (sec.)
10
Superoxide anions
10
M40403(6mpk)
M40403(3mpk)
8
M40403(1mpk)
6
4
2
0
0
50
Time (min) post superoxide anion
100
150
M40404 which lacks SODm activity does not block this response.
Drugs given i.v. 15min before challenge
200
% inhibition of hyperalgesia
Rapid onset
Long duration
0
0
25
25
50
50
75
75
100
0
10
20
30
40
50
60
100
vehicle
1 mg/kg
3 mg/kg
6 mg/kg
0
Time (min) post drug
drug administered iv 3 hr after carrageenan
1
2
3
4
5
6
Time (hr) post drug
7
M40403 Restores Analgesia
in Morphine-tolerant Mice
120
80
Compound given 5 min
prior to morphine
60
40
0.03
0.01
Tol
0
mg/kg, i.v
1
20
Naive
% Analgesia
100
Mechanism: SOD-mimics in Ischemia
– Reperfusion-Mediated Injury
Ischemia
Reperfusion
-
O2
Lipid
peroxidation &
cellular damage
TNF
Neutrophil
activation/
sequestration
Primary & Secondary
Organ Damage
O-2 concentration
0
Control
I (30min)
I+R (60s)
+SC-67066 (0.65mol/kg)
+SC-67066(1.25mol/kg)
+SC-67066(2.5mol/kg)
+SC-65512(2.5mol/kg)
10
20
.
HO2
.
H2O2
Initiation
HO
.
OH
HO2
O2
O
Propagation
O
OH
O
OH
HO
NHR
HO
NHR
R=H, Norepinephrine
R=Me, Epinephrine
RHN
.
"Quinone"
OONOStoichiometric
.
oxidation
O
.
A Single Reaction with HO2 Initiates a
-
"Chain Autoxidation" Affording Multiple
Conversions to "Adrenochromes"!
OH
O
+
N
Polymerize
R
"Adrenochromes"
Crosslinked Proteins
Cytotoxic
Melanin
Salvemini, MacArthur, Riley, PNAS, 2000, 97(17), 9753
MetaPhore Pharmaceuticals
PAC Conformational Template
vs. Type I -Turn
Pentaazacrown complexed with Mn(II, red ball) showing
overlap of i, i+1, and i+2 side-chain orientations (yellow)
Arg
Asp
Tyr
T r
Arg
Arg
Tyr
Trp
MAC
TENDAMISTAT
T ND M ST T
MIMICRY OF CHIRAL MAC WITH WRY SEQUENCE
OF TENDAMISTAT THAT INHIBITS -AMYLASE
Trp
Entropy and Solvation
1. Lafont, V., A.A. Armstrong, H. Ohtaka, Y. Kiso, L. Mario
Amzel, and E. Freire, Compensating enthalpic and entropic
changes hinder binding affinity optimization. Chem Biol Drug Des,
2007. 69(6): p. 413-22.
2. Young, T., R. Abel, B. Kim, B.J. Berne, and R.A. Friesner,
Motifs for molecular recognition exploiting hydrophobic enclosure in
protein-ligand binding. Proc Natl Acad Sci U S A, 2007. 104(3): p.
808-13.
Enzyme Mimetic
Structured Waters