1. Paper Introduction
Kazuya Matsuo

2. Total Synthesis of Natural Products
Dirk Munich
Total Synthesis of Natural Products
Photochemical Biology (Basically Using Azobenzene derivatives)
The first report of photoneuroscience
Nature Neuroscience  2004, 7,

3. Photoswitchable Inhibitors of Microtubule Dynamics
Optically Control Mitosis and Cell Death
Trauner, D. and Thorn-Seshold O. et al. Cell, 2015, 162,
DOI : /j.cell

4. Target : microtubule composed of tubulin
Tubulin depolymerization inhibitors
paclitaxel, epothilone, docetaxel, discodermolide etc
Mitotic inhibitor
Tubulin polymerization inhibitors
colchicine, combrestatin, vinblastine, halichondrins etc

5. Molecular Design of Photostatins (PSTs)
Key points
Colchicine-based inhibitor
Polymerization inhibitor inspired from stilbene type inhibitor, CA4
Photocontrol
Raipd and spontaneous conversion from cis to trans isomer
Spacio-temporal control using photoirradiation (reversible photoswitch)
Prodrug strategy
Active drug is released after some enzymatic reaction occurs in cell or in vivo
Improvement of water-solubility

6. The toxicity by PST-1 derivatives depends on photo-irradiation condition★
Crystal violet assay = Measurement of viable cells
EC50 390nm is up to 100 times greater than under the dark condition ★ ★
nm : Most toxic
(> 250 times more toxic than dark condition)
MTT assay = Measurement of cytotoxicity

7. PSTs induce mitotic arrest and cell death in a light-dependent manner★
PARP : poly(ADP-ribose) polymerase
= repair of DNA damage induced by cellular stress
PST induced apoptosis type cell death (G2/M arrest) depending on blue light illumination

8. PSTs bind tubulin and visualize tubulin polymerization
colchicine-competitive inhibitory assay
Cis-PST-1 binds to the same domain in tubulin with colchicine
Immunofluorescence imaging of endogenous tubulin in MDA-MB-231
Dark condition
☞ trans-PST-1 had no effects on MT structure
Blue light illumination
☞ cis-PST-1 caused dose-dependent MT depolymerization

9. The cell liability by PST depends on light-irradiation
※EB3-mCherry is expressed in MDA-MB-231 cells
EB3 is the end-binding protein of microtubule
Assembly at plus tips of polymerizing MTs (called comet)
Disassembly in phases of MT shrinkage
Analyses of EB3 comet
Total number (5B)
Lifetime (5C)
Speed (5D)
Distance traveled (5E)
Trans-cis photoisomerizations of PST-1 inside living cells has achieved optical switching of MT polymerization dynamics.

10. Photo-control of MT structure and function in vivo
C. elegans (about 1000 cells , 1mm length)
The first multicellular organism to have its whole genome sequenced
C. elegans embryos
※Synchronicity of several blastomeres
= Cell division occurs at the same timing
Blue light (cis PST) = Inhibit the cell division
Green light (trans PST) = Normal cell division
Mouse cremaster muscle tissue
Blue light (cis PST) = destruction of microtubule network
Dark condition (trans PST) = no effects

11. Conclusion
They have achieved...
1. Perfect photo-controllable microtubule inhibitor, PST was developed
(cis-PSTs are two orders of magnitude more potent than the trans-PSTs)
2. PST works well in cellulo, ex vivo and in vivo
3. PST control the dynamics of microtuble in living organisms