Paper Introduction 8.25.2015 Kazuya Matsuo

Associate Professor @ Wayne State University Angew. Chem. Int. Ed. 2015, 54, 9618–9621. Angew. Chem. 2015, 127, 9754–9757. ① HDAC (= Histon Deacetylase) inhibitors for epigenetics Mary Kay H. Pflum Associate Professor @ Wayne State University From her homepage (http://chem.wayne.edu/faculty/pflum/) ② Functional ATP (= Adenosine Triphosphate) analogue for kinase-catalyzed modification From her homepage (http://chem.wayne.edu/faculty/pflum/) Kinase’s co-substrate is ATP

Previous works on kinase-catalyzed labeling kinase substrate (protein) kinase product (protein) Pflum, M. K. H. et al. J. Am. Chem. Soc. 2007, 129, 10-11. Pflum, M. K. H. et al. ACIE 2010, 49, 1627-1630. ATP-biotin Streptavidin (SA) has high affinity with biotin (Kd = 10-15 mol/L) All of these excellent works were done only in vitro because of cell-impermeability of ATP analogue

Basic information on cell membrane permeability outside inside In passive transport Relatively hydrophobic Not high molecular weight (preferably < 500) Positive charge > Negative charge although neutral is the best ※ Active transport (protein-mediated transportation using ATP) is not included H2O permeable Noncharged molecules Peptide ATP ion Macromolecule Impermeable Cell membrane is a big barrier for ATP and its analogue because of their high negative charge Novel ATP analogue for in-cell study ← APB (2) To avoid side reaction, they used not 2º amine but 3º amine Cationic linker was used in APB

Docking simulation of APB and in vitro kinase-catalyzed modification Basic information of kinase • >500 kinases and their thousands of substrates are identified so far • Phosphorylation using ATP • Regulation of metabolic and cell signaling • Disorder of kinases can induce Parkinson’s disease and cancer kinase (ATP) phosphatase Substrate of kinase Product of kinase Docking model of PKA (kinase) with APB Biotin labeling Phosphorylation ※ 50% TFA treatment makes phosphate ester to hydrolyze 1. Biotin group is positioned outside active pocket 2. Catalytic Lys is located around phosphate groups MBP : Substrate protein of PKA PKA reacted with APB in vitro (APB = co-substrate of PKA)

In cellulo kinase-catalyzed modification In vitro (HeLa cells lysate) labeling Fluorescence microscopy analysis ※ HI lysate means heat-denatured lysate including no active kinase After fixation of cells, biotinylated proteins were visualization by SA-Cy5 ※ NHS-biotin show the random labeling of the proteins on cell surface ※ Fixation of cells = Method for imaging and observing cells Fixed cells are not live cells, and the cell membrane is destroyed, so anti-body or fluorescent proteins can easily go into cells. In vitro labeling showed selective biotinylation of kinase substrate using ATP-biotin and APB (this study) Only APB can penetrate cell membrane In cell labeling ※ STSP (staurosporine) : kinase inhibitor In cell biotinylation by endogenous kinase was observed using APB Labeling patterns between lysate (lane 4) and in cell (lane 11) are different In cell labeling results show the real phosphoproteome much better Kinase-catalyzed biotinylation using APB inside cells was achieved, so APB can go through cell membrane. APB is the first cell-permeable ATP analogue.

Conclusion Cell membrane outside inside substrate kinase APB product biotinylation APB is cell-permeable analogue of ATP, which can modify the kinase-substrate inside cells

From Wada-san notebook Questions According to Wada-san’s results and this paper, kinesin does not hydrolyze ATP-Az, but ATP-Az might be applicable to kinase Is ATP recognition in kinesin and kinase completely different ? ATP-Az From Wada-san notebook In both of kinesin and kinase, ① Mg ion activates the electrophilicity of ATP ② Leaving group = ADP Kinesin → Nucleophile = water molecule (activated by kinesin) Kinase → Nucleophile = hydroxyl group (activated by kinase)

I’ll try... ATP-Az Azo-TP caged ATP Structure and activity relationship of γ-phosphate substituted ATP was well-studied Shorter linker of ATP analogue is not preferable in kinase-catalyzed labeling reaction Is ATP-Az with longer linker applicable to kinase or kinesin ? Is this linker length important to show ATPase activity ? ATP-Az Azo-TP Caged ATP before photolysis has no affinity with kinesin and kinase caged ATP Can kinase recognize Azo-TP ? If possible, is Azo-TP photo-controllable for kinase-catalyzed phosphorylation ? I’ll try... Pflum, M. K. H. et al. Bioconjugate Chem. 2012, 23, 2386−2391.