A Small Molecule Approach for Studying Signaling Pathways Related to Cell Motility and Cancer Scott Lokey, Chemistry and Biochemistry The laboratory of Scott Lokey uses a small molecule approach, called chemical genetics, to study signaling pathways related to cell cycle checkpoints and the actin cytoskeleton. In one study, Lokey and his co-workers are developing screens of natural compounds that can be used to examine how cells detect their own DNA damage. Studies such as these might lead to development of a new class of chemotherapeutic agents.

Proteomics

Chematic diagram representing the principle of 2d DIGE. Differential protein levels in cell extracts (experiment vs. control) are visualized by conjugating total cytoplasmic protein with either of two Cy dyes with different fluorescent emission spectra but identical masses and electrophoretic mobility. The labeled extracts are mixed and separated by 2-d electrophoresis. An imager scans the gel at the two Cydye emission wavelengths and image analysis software enables spots of different intensity to be identified. Robotic equipment excises spots of interest, performs tryptic digestion in situ, and MALDI-TOF mass spectrometry is used to identify proteins based on masses of tryptic fragments.

The advent of new technologies has brought about a sea change in our ability to study processes at genome- and proteome-wide scales. Proteomics can be broadly defined as the attempt to profile changes in expression levels, activity, and post-translational status of the entire complement of proteins in a cell. There are several methods for approaching this goal. Among these, a recently developed method, called 2D DIGE, combines 2-dimensional electrophoresis with a differential labeling technique analogous to that used in transcriptional profiling arrays ("gene chips"). The UCSC Department of Chemistry and Biochemistry has recently set up a 2D DIGE system, and our group is currently using the technique to decipher biological pathways related to cell motility and cancer. In addition, we are developing methods to enhance the sensitivity of 2D DIGE, for example, by selectively targeting phosphorylated proteins or proteins located in specific subcellular locations. Finally, we are interested in using proteomics methods to identify the protein targets of biologically active small molecules.

Diversity-oriented Synthesis, Natural Products, and High-throughput Screening

A primary interest in my lab is the identification of bioactive compounds related to cell division and cancer. These compounds can be used as tools to dissect complex cellular pathways involved in cellular proliferation, possibly leading to new therapeutics. Our strategy is to combine chemical synthesis, natural product purification, and modern biological screening methods to discover compounds with interesting and specific modes of action. For example, one area of interest in our lab is the DNA-damage checkpoint, in which a complex signaling pathway detects DNA lesions (e.g., from ionizing and UV radiation) and signals to the cell cycle machinery to pause for repair. We are developing a screen for compounds that specifically block the DNA-damage checkpoint. These compounds can be useful as probes for studying DNA damage detection and may lead to a new class of chemotherapeutic agents.

As input into our screens we have chosen both natural product and synthetic libraries. In collaboration with the laboratory of Phil Crews here at UCSC, we have begun to generate a library of thousands of semi-pure marine sponge fractions formatted for high-throughput screening. We are setting up a Center for Chemical Diversity to perform preparative HPLC and mass spectrometry, combined with robotic fraction collection, on large numbers of crude sponge and fungal extracts to create a highly diverse compound collection for input into biological assays performed here, at UC San Francisco, and with our industrial collaborators. We have also purchased a microcapillary NMR probe capable of obtaining 1D and 2D spectra on 10 µg of material, which will allow us to identify active structures without the need for repurification.

We also use natural products as inspiration in the development of synthetic libraries based on cyclic peptide templates. We are developing synthetic methods to introduce backbone modifications that increase geometric diversity, improve cell permeability, and allow access to an enormous number of structurally complex compounds based on peptide scaffolds. Work on the synthesis of N-methylated cyclic peptides has led to studies on the conformational basis of membrane permeability in peptide and non-peptide model systems. Among our synthetic research interests are 1. the solid phase synthesis of thiazoles and oxazoles; 2. site-selective incorporation of N-methyl amides; and use of the Pd-catalyzed intramolecular Heck reaction to create bridged cyclic peptides, and total synthesis of the natural product phalloidin.

Nanobiotechnology

Biomolecules often self-assemble in cells to yield dynamic complexes with highly ordered architectures on the nanometer scale. Thus, cell biology provides an entry point into the study and manipulation of nanometer-sized objects. We are currently investigating the use of actin filaments and microtubules as nanomolecular protein "wires". For example, one project area involves the nucleation of microtubules with taxol-labeled gold nanoparticles. Crosslinking the intra-tubule particles with bifunctional molecules will allow us to generate linear nanoparticle arrays with potential applications in nanofabrication and molecular computation. Challenges in this area include surface analysis using techniques such as atomic force microscopy, confocal fluorescence and electron microscopy.