Reactive Oxygen Species(ROS)- Development of
Innovative Drugs andAnalytical Methods Literature Seminar 2014.9.6
D1 Yohei Seki

Contents
1. Introduction
2. Probe for the redox status

3. Recent progress of antioxidants
4. New strategy for therapeutic agents
5. Future outlook and summary
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Contents
1. Introduction
2. Probe for the redox status

3. Recent progress of antioxidants
4. New strategy for therapeutic agents
5. Future outlook and summary3

1-1. What is a Reactive Oxygen Species (ROS)

Free radicalsandnon-radical ROS
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1-2. History of free radicals
1. Triphenylmethyl radical was discovered in 1900.

2. “Free radical theory” was articulated of aging in 1957 speculating
endogenous oxygen radicals were generated in cells and resulted
in a pattern of cumulative damage.
3. Superoxide dismutase was discovered in 1969.

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1-2. History of free radicals
4. Superoxide was produced from leukocytes in 1973.
5. Endothelium-derived relaxing factor (EDRF) was NO in 1987.

The nobel prize in physiology or medicine in 1998
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1-3. The sources and cellular resposes to ROS

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1-4. Mitochondrial ROS production8Contents
1. Introduction
2. Probing for the redox status

3. Recent progress of antioxidants
4. New strategy for therapeutic agents
5. Future outlook and summary9

2-1. Spin trapping and labelling using ESR
Spin trapping

Spin labelling
Thiol-selective

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2-1. Spin trapping and labelling using ESR

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2-2. Redox-sensitive probe
N-oxylradical moiety

122-2. Redox-sensitive probe13

2-2. Redox-sensitive probe

Parkinson’s disease model14

2-2. Redox-sensitive probe
N-oxylradical-enhanced MRI

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2-2. Redox-sensitive probe

A: Total antioxidant capacity
B: ROS level162-2. Redox-sensitive probe
1. Fast reduction and 2. low r1 of N-oxylradical were problematic.
Modification of N-oxylradical
Signal intensity formula of SE method

r1 and SBM formular1↑, T1↓, and tR↓17

2-2. Redox-sensitive probe
Modification of N-oxylradicals for long in vivo lifetime
Inductive effects on rates of reduction of ascorbate

Six-membered N-oxylradical > Five-membered N-oxylradical
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2-2. Redox-sensitive probe
Modification of N-oxylradicals for long in vivo lifetime
Reduction profiles of nitroxides with ascorbate

Sterically shielded pyrrolidine N-oxylradical 3 was slowest.
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2-2. Redox-sensitive probe
Synthesis of sterically shielded pyrrolidine N-oxylradical 320

2-2. Redox-sensitive probe
N-oxylradical-enhanced MRITEEPONE was found to have high resistivity against bio-reduction.
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2-2. Redox-sensitive probe
Modification of N-oxylradicals for long in vivo lifetime and high r1

Spirocyclohexyl N-oxylradicals and PEG chains conjugated to dendrimer
scaffold were effective.
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2-2. Redox-sensitive probe
N-oxylradical-enhanced MRI

Spirocyclohexyl N-oxylradicals and PEG chains conjugated to dendrimer
scaffold enhanced MRI in mice for over 1 h.
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Contents
1. Introduction
2. Probing for the redox status

3. Recent progress of antioxidants
4. New strategy for therapeutic agents
5. Future outlook and summary24

3-1. What is an antioxidant
Native antioxidants
Mechanism 1Mechanism 2

Sole drug which has antioxidant activity as its main effect25

3-1. What is an antioxidant
Proposed radical scavenging pathway of uric acid

UA can be equivalent to p-aminophenol or hydroquinone.
263-1. What is an antioxidant
Radical scavenging activity of uric acid analogs

5-hydroxyindolinones (2a) showed sufficient activity with highsolubility and low cytotoxity.
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3-2. Recent therapeutic antioxidant
Characteristics of H2
H2 is a colorless, odorless, tasteless andcombustible diatomic gas.
H2 is expected as a promising energy carrier in futureenergy system.
H2 potentials for antioxidants
H2 reduces the・OH that is produced by radiolysis or photolysisof water.
H2 behaves as an inert gas in the absence of catalysts or at bodytemperature.

H2 can be dissolved in water up to 0.8 mM under atmospheric pressure.
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3-2. Recent therapeutic antioxidant
H2 has rapid diffusion.

Formation of highly ROS
Fenton reaction
Weise reaction

・OH is the strongest
free radical.

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3-2. Recent therapeutic antioxidant
H2 dissolved in medium reduced・OH in cultured cell.

H2 decreased

・OH levels.

H2 protected
mitochondria.
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3-2. Recent therapeutic antioxidant

a-c: H2 decreased
signals of・DMPO-OH.
d-e: H2 showed
selective reduction ofSpin-trappingin cultured cellcellular・OH
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3-2. Recent therapeutic antioxidant
H2 dissolved in solution reduced・OH in cell-free systems.
H2 reduced・OH and ONOOsomewhat.

323-2. Recent therapeutic antioxidant
Inhalation of H2 protected against ischemia-reperfusion injury.

H2 suppressed not only the initial brain
injury but also its progressive damage.

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Contents
1. Introduction
2. Probing for the redox status

3. Recent progress of antioxidants
4. New strategy for therapeutic agents
5. Future outlook and summary34

4-1. NO releaser
NO is a colorless gas that is unstable underphysiological conditions (t1/2 = 0.1-5 s).

NO releasers, which can store and release NO, have beendeveloped for biological research on the roles of NO or
as candidates of chemotherapeutic agents.

UV light

Transition metal

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4-2. Blue-light-controllable NO releaser
Design a visible-light-controllable NO releaser
NO release from BNN

Photoinduced electron transfer36

4-2. Blue-light-controllable NO releaser
Synthesis of NOBL-1 (1)374-2. Blue-light-controllable NO releaser
Spin-trappingin cell-free systems

Detection of NO generation from NOBL-1 in cell-free systems

Temporally precise control of NO releasefrom NOBL-1 could be achieved.

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4-2. Blue-light-controllable NO releaser
Fluorescence imaging of NO release from NOBL-1 in cultured cell.

Changes in tension of rat aorta ex vivo39

4-2. Blue-light-controllable NO releaser
Proposed mechanism of photodecomposition of NOBL-1

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Contents
1. Introduction
2. Probing for the redox status

3. Recent progress of antioxidants
4. New strategy for therapeutic agents
5. Future outlook and summary41

5. Application to ROS-dependent chemical reaction
Redox active catalyst mediated by ROS
Selective-activation

Non-toxicity

Water solubility

Cell permeability

Today’s seminar: N-oxylradical moiety

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5. Application to ROS-dependent chemical reaction
Ros-dependent chemical reaction and changing the functionof protein

Living cell
ROS locally increased
Serious human diseases
Cancer, diabetes, etc.

Changing thefunction of protein
Is it possible to apply this system to controling the ROS ? 43

5. Summary
Section 2
Probing for the redox status using N-oxylradicals

・Various kinds of N-oxylradicals for probing have been developed.
・Slow reduction and high r1 of N-oxylradical were important.
Section 3
Developments of small molecules for antioxidants

・Drug which has antioxidant activity as its main effect is onlyedarabone.
・H2 has potential as recent therapeutic antioxidant.

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5. Summary
Section 4
Visible-light-controllable NO releaser

・NOBL-1 which bears NO releasing moiety tethered to antenna
moiety released NO on blue-light irradiation.

・NOBL-1 can be a useful tool for understanding NO action in tissues
and may also have potential for phototherapy.

Section 5ROS-dependent chemical reaction
Chemical biology tool
Medicinal application
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Appendix 2-2. Redox-sensitive probe
SE method

r1 and SBM formula46

2-2. Redox-sensitive probe47

3-2. Recent therapeutic antioxidant
Diffusion of H2 in cell48

5. Strategy for detection of ROS and related protein
First step: ROS-dependent activation of catalyst

Second step: Trptophan-selective conjugation

Challenge:
Development of reagent activated ROS-dependently
and conjugating selectively to tryptophan

2-2. Redox-sensitive probe50