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The Skaggs Institute
for Chemical Biology


Scientific Report 2006




Chemical Physiology


B.F. Cravatt, J. Alexander, K. Barglow, M.H. Bracey, E. Carlson, K. Chiang, M. Evans, H. Hoover, W. Li, K. Masuda, A. Mulder, S. Niessen, S. Ortega-Gutierrez, M. McKinney, C. Salisbury, G. Simon, E. Weerapana, 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 the 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. In collaboration with R.C. Stevens, Scripps Research, we solved the first 3-dimensional structure of FAAH. We are using this information to explore the molecular mechanism of action of the enzyme and to design inhibitors of FAAH. 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. Such activity-based probes could be used to record variations in protein function independent of alterations in protein abundance, offering a potentially powerful and complimentary set of tools for proteome analysis. To date, we have generated activity-based probes for more than a dozen enzyme classes, including serine hydrolases, metalloproteases, glutathione-S-transferase, and several oxidoreductases. We are currently using our activity-based probes to explore the roles that enzymes play in variety of physiologic and pathologic processes, especially cancer progression. We are also developing complementary strategies for profiling the complete content of metabolites in cells and tissues (the “metabolome”) to facilitate the assignment of endogenous substrates to enzymes of uncharacterized function.

Publications

Alexander, J.P., Cravatt, B.F. Mechanism of carbamate inactivation of FAAH: implications for the design of covalent inhibitors and in vivo functional probes for enzymes. Chem. Biol. 12:1179, 2005.

Alexander, J.P., Cravatt, B.F. The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases. J. Am. Chem. Soc. 128:9699, 2006.

Drahl, C., Cravatt, B.F., Sorensen, E.J. Protein-reactive natural products. Angew. Chem. Int. Ed. 44:5788, 2005.

Evans, M.J., Cravatt, B.F. Mechanism-based profiling of enzyme families. Chem. Rev. 106:3279, 2006.

Evans, M.J., Saghatelian, A., Sorensen, E.J., Cravatt, B.F. Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling. Nat. Biotechnol. 23:1303, 2005.

Leung, D., Saghatelian, A., Simon, G.M., Cravatt, B.F. Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids. Biochemistry 45:4720, 2006.

McKinney, M.K., Cravatt, B.F. Structure-based design of a FAAH variant that discriminates between the N-acyl ethanolamine and taurine families of signaling lipids. Biochemistry 45:9016, 2006.

Mulder, A.M., Cravatt, B.F. Endocannabinoid metabolism in the absence of fatty acid amide hydrolase (FAAH): discovery of phosphorylcholine derivatives of N-acyl ethanolamines. Biochemistry 45:11267, 2006.

Saghatelian, A., Cravatt, B.F. Assignment of protein function in the postgenomic era [published correction appears in Nat. Chem. Biol. 1:233, 2005]. Nat. Chem. Biol. 1:130, 2005.

Saghatelian, A., McKinney, M.K., Bandell, M., Patapoutian, A., Cravatt, B.F. A FAAH-regulated class of N-acyl taurines that activates TRP ion channels. Biochemistry 45:9007, 2006.

Sieber, S.A., Niessen, S., Hoover, H.S., Cravatt, B.F. Proteomic profiling of metalloprotease activities with cocktails of active-site probes. Nat. Chem. Biol. 2:274, 2006.

Simon, G.M., Cravatt, B.F. Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for α/β-hydrolase 4 in this pathway. J. Biol. Chem. 281:26465, 2006.

 

Benjamin F. Cravatt, Ph.D.
Professor

Cravatt Web Site