The Skaggs Institute
for Chemical Biology

Scientific Report 2007

Chemical Physiology

B.F. Cravatt, K.T. Barglow, J.L. Blankman, M.H. Bracey, E.E Carlson, M. Dix, H. Hoover, W.W. Li, J.Z. Long, B.R. Martin, K. Masuda, M.K. McKinney, S. Niessen, C.M. Salisbury, G.M. Simon, J. Thomas, E. Weerapana, B. Wei, A.T. Wright

We are interested in understanding complex physiology and behavior at the level of chemistry and molecules. At the center of cross talk between different physiologic processes are endogenous compounds that serve as a molecular mode for intersystem communication. However, many of these molecular messages remain unknown, and even in the instances in which the participating molecules have been defined, the mechanisms by which these compounds function are for the most part still a mystery.

We are investigating a family of chemical messengers termed the fatty acid amides, which affect many physiologic functions, including sleep and pain. In particular, one member of this family, oleamide, accumulates selectively in the cerebrospinal fluid of tired animals. This finding suggests that oleamide may function as a molecular indicator of an organism's need for sleep. Another fatty acid amide, anandamide, may be an endogenous ligand for the cannabinoid receptor in the brain.

The in vivo levels of chemical messengers such as the fatty acid amides must be tightly regulated to maintain proper control over the influence of the messengers on brain and body physiology. We are characterizing a mechanism by which the level of fatty acid amides can be regulated in vivo. Fatty acid amide hydrolase (FAAH) degrades fatty acid amides to inactive metabolites. Thus, FAAH effectively terminates the signaling messages conveyed by fatty acid amides, possibly ensuring that these molecules do not generate physiologic responses in excess of their intended purpose.

We are using transgenic and synthetic chemistry techniques to study the role of FAAH in regulating fatty acid amide levels in vivo. We have created transgenic mice that lack FAAH and have found that these animals have highly elevated levels of fatty acid amide in the brain that correlate with reduced pain behavior, suggesting that FAAH may be a new therapeutic target for the treatment of pain and related neural disorders. In collaboration with R.C. Stevens, Scripps Research, we solved the first 3-dimensional structure of FAAH. We are using this information to design potent and selective inhibitors of the enzyme. In studies with D.L. Boger, the Skaggs Institute, we have identified potent FAAH inhibitors and using a functional proteomic screen developed by us, have shown that these inhibitors are highly selective for this enzyme. We are also interested in proteins responsible for the biosynthesis of fatty acid amides.

A second major focus in the laboratory is the design and use of chemical probes for the global analysis of protein function. The evolving field of proteomics, defined as the simultaneous analysis of the complete protein content of given cell or tissue, encompasses considerable conceptual and technical challenges. We hope to enhance the quality of information obtained from proteomics experiments by using chemical probes that indicate the collective catalytic activities of entire classes of enzymes. Using activity-based probes that target the serine and metallo hydrolases, we have identified several enzymes with altered activities in human cancers. Using a combination of pharmacologic and molecular biology approaches, we are now testing the role that these enzymes play in cancer pathogenesis. Additionally, we are developing chemical probes that target many other enzyme families and the endogenous substrates of the enzymes.

Publications

Barglow, K.T., Cravatt, B.F. Activity-based protein profiling for the functional annotation of enzymes. Nat. Methods 4:822, 2007.

Carlson, E.E., Cravatt, B.F. Chemoselective probes for metabolite enrichment and profiling. Nat. Methods 4:429, 2007.

Evans, M.J., Morris, G.M., Wu, J., Olson, A.J., Sorensen, E.J., Cravatt, B.F. Mechanistic and structural requirements for active site labeling of phosphoglycerate mutase by spiroepoxides. Mol. Biosyst. 3:495, 2007.

Hanson, S.R., Hsu, T.L., Weerapana, E., Kishikawa, K., Simon, G.M., Cravatt, B.F., Wong, C.-H. Tailored glycoproteomics and glycan site mapping using saccharide-selective bioorthogonal probes. J. Am. Chem. Soc. 129:7266, 2007.

Li, W., Blankman, J.L., Cravatt, B.F. A functional proteomic strategy to discover inhibitors for uncharacterized hydrolases. J. Am. Chem. Soc. 129:9594, 2007.

Macpherson, L.J., Dubin, A.E., Evans, M.J., Marr, F., Schultz, P.G., Cravatt, B.F., Patapoutian, A. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 445:541, 2007.

Salisbury, C.M., Cravatt, B.F. Activity-based probes for proteomic profiling of histone deacetylase complexes. Proc. Natl. Acad. Sci. U. S. A. 104:1171, 2007.

Wan, L.E., Saghatelian, A., Chong, L.W., Zhang, C.L., Cravatt, B.F., Evans, R.M. Maternal PPARγ protects nursing neonates by suppressing the production of inflammatory milk. Genes Dev. 21:1895, 2007.

Wright, A.T., Cravatt, B.F. Chemical proteomic probes for profiling cytochrome P450 activities and drug interactions in vivo. Chem. Biol. 14:1043, 2007.