The Skaggs Institute
for Chemical Biology

Scientific Report 2005

Chemical Physiology

B.F. Cravatt, J. Alexander, K. Barglow, M.H. Bracey, K. Chiang, M. Evans, H. Hoover, K. Matsuda, M. McKinney, A. Mulder, S. Niessen, A. Saghatelian, C. Salisbury, S. Sieber, G. Simon, B. Wei

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 the 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 the hydrolase in the regulation of fatty acid amide levels in vivo. We found that transgenic mice that lack FAAH 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 hydrolase. In collaboration with D.L. Boger, the Skaggs Institute, we have identified potent FAAH inhibitors, and using a functional proteomic screen developed by our group, we showed 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 a 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 cancer. Using a combination of pharmacologic and molecular biology approaches, we are examining the role of these enzymes in cancer pathogenesis. Additionally, we are developing chemical probes that target many other enzyme families. To date, we have succeeded in identifying activity-based proteomics probes for more than 10 mechanistically distinct classes of enzymes.

Publications

Jessani, N., Niessen, S., Wei, B.Q., Nicolau, M., Humphrey, M., Ji, Y., Han, W., Noh, D.Y., Yates, J.R. III, Jeffrey, S.S., Cravatt, B.F. A streamlined platform for high-content functional proteomics of primary human specimens. Nat. Methods 2:691, 2005.

Jessani, N., Young, J.A., Diaz, S.L., Patricelli, M.P., Varki, A., Cravatt, B.F. Class assignment of sequence-unrelated members of enzyme superfamilies by activity-based protein profiling. Angew. Chem. Int. Ed. 44:2400, 2005.

McKinney, M.K., Cravatt, B.F. Structure and function of fatty acid amide hydrolase. Annu. Rev. Biochem. 74:411, 2005.

Saghatelian, A., Cravatt, B.F. Global strategies to integrate the proteome and metabolome. Curr. Opin. Chem. Biol. 9:62, 2005.

Saghatelian, A., Trauger, S.A., Want, E.J., Hawkins, E.G., Siuzdak, G., Cravatt, B.F. Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry 43:14332, 2004.

Sieber, S.A., Mondala, T.S., Head, S.R., Cravatt, B.F. Microarray platform for profiling enzyme activities in complex proteomes [published correction appears in J. Am. Chem. Soc. 127:4114, 2005]. J. Am. Chem. Soc. 126:15640, 2004.

Speers, A.E., Cravatt, B.F. A tandem orthogonal proteolysis strategy for high-content chemical proteomics. J. Am. Chem. Soc. 127:10018, 2005.