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The Skaggs Institute
for Chemical Biology
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.
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