Paper Introduction 7.21.2015 Kazuya Matsuo

Total Synthesis of Natural Products Dirk Trauner @Ludwig-Maximilians-University Munich Total Synthesis of Natural Products Photochemical Biology (Basically Using Azobenzene derivatives) The first report of photoneuroscience Nature Neuroscience  2004, 7, 1381 - 1386.

Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death Trauner, D. and Thorn-Seshold O. et al. Cell, 2015, 162, 403-411. DOI : 10.1016/j.cell.2015.06.049

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

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

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 ★ ★ 380-390 nm : Most toxic (> 250 times more toxic than dark condition) MTT assay = Measurement of cytotoxicity

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

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

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.

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

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