W.S. Johnson Speaker:
Roger Y. Tsien

University of California at San Diego

About Roger Tsien:
Roger Y. Tsien, born in 1952, received his A.B. in Chemistry and Physics from Harvard College in 1972. He received his Ph.D. in Physiology in 1977 from the University of Cambridge and remained as a Research Fellow until 1981. He then became an Assistant, Associate, then full Professor at the University of California, Berkeley. In 1989 he moved to the University of California, San Diego, where he is an Investigator of the Howard Hughes Medical Institute and Professor in the Depts. of Pharmacology and of Chemistry & Biochemistry. His honors include First Prize in the Westinghouse Science Talent Search (1968), Searle Scholar Award (1983), Artois-Baillet-Latour Health Prize (1995), Gairdner Foundation International Award (1995), Award for Creative Invention from the American Chemical Society (2002), Heineken Prize in Biochemistry and Biophysics (2002), Wolf Prize in Medicine (shared with Robert Weinberg, 2004), Rosenstiel Award (2006), and E.B. Wilson Medal of the American Society for Cell Biology (shared with M. Chalfie, 2008). He is a member of the National Academy of Sciences and the Royal Society. Dr. Tsien is best known for designing and building molecules that either report or perturb signal transduction inside living cells. These molecules, created by organic synthesis or by engineering naturally fluorescent proteins, have enabled many new insights into signaling via calcium, sodium, pH, cyclic nucleotides, nitric oxide, inositol polyphosphates, membrane and redox potential changes, protein phosphorylation, active export of proteins from the nucleus, and gene transcription. He is now developing new ways to target contrast agents and therapeutic agents to tumor cells based on their expression of extracellular proteases.



Tsien's lecture at the Johnson Symposium:
"Improving In Vivo and Clinical Imaging with Genetically Encoded and Synthetic Molecules"

Fluorescent proteins (FPs) have revolutionized many areas of molecular and cell biology by making many crucial molecules and biochemical events directly visible. Infrared-fluorescent proteins (IFPs) would facilitate fluorescence imaging in intact animals because absorption from tissue hemes drops steeply beyond 600 nm. IFPs would also benefit microscopic imaging by reducing the contribution of cellular autofluorescence, enabling excitation by cheap laser diodes, adding new wavelengths for multicolor labeling, and accepting fluorescence resonance energy transfer from existing FPs. However, published FPs with inbuilt chromophores, i.e. FPs from jellyfish or corals, have not yet exceeded 595/655 nm in excitation/emission maxima respectively. We now show that phytochromes incorporating tetrapyrrole chromophores can be mutated into monomeric IFPs, with typical excitation/emission maxima at 686/713 nm respectively, extinction coefficients >90,000 M 1cm-1, and quantum yield up to 0.08 so far. The IFPs express well in mammalian cells and spontaneously incorporate the chromophore, which can either be supplied exogenously or enzymatically synthesized in situ. Alternatively, the phytochromes can incorporate a different tetrapyrrole, which photogenerates singlet oxygen with a quantum yield of 0.15. Thus a single fusion protein, depending on which chromophore is loaded and what excitation wavelength is applied, should emit either infrared fluorescence or singlet oxygen. The latter enables electron microscopic imaging and chromophore-assisted light inactivation.

Activatable cell penetrating peptides (ACPPs) are polycationic cell penetrating peptides (CPPs) whose cellular uptake is minimized by a polyanionic inhibitory domain and then restored upon proteolysis of the peptide linker connecting the polyanionic and polycationic domains. Local activity of proteases able to cut the linker causes amplified retention in tissues and uptake into cells. We have been optimizing ACPP configurations and translating to nonoptical imaging and therapeutic modalities in murine models of cancer and cardiovascular disease. ACPPs alone are superior to CPPs in vivo because CPPs deposit their cargoes mainly at the site of injection and secondarily in the liver. Tumor uptake of ACPPs is up to 4 fold higher with a matrix metalloproteinase substrate (PLGLAG) as the linker than with a negative control composed of D-amino acids. Conjugation of ACPPs to macromolecular carriers such as dendrimers prolongs pharmacokinetics and increases delivery of payload (Cy5 or Gd-DOTA or both in the same molecule) to tumor for far-red or MR imaging. The dual labeled probe with Cy5 and Gd-DOTA enables whole body MRI scanning followed by fluorescence-guided surgery. Addition of therapeutic cargoes is in progress. Thrombin-cleavable ACPPs accumulate in atherosclerotic plaques and experimental stroke models, so vascular pathologies can also be imaged. The ability of ACPPs to deliver a wide variety of cargoes with enzymatic amplification to protease-expressing tissues in vivo offers significant clinical potential.