Reviews B. F. Cravatt, E. J. Sorensen, and C. Drahl DOI: 10.1002/anie.200500900 Natural Products Chemistry Protein-Reactive Natural Products Carmen Drahl, Benjamin F. Cravatt,* and Erik J. Sorensen* Keywords: enzymes · inhibitors · molecular probes · natural products · structure– activity relationships Angewandte Chemie 5788 www.angewandte.org 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809 Angewandte Enzyme Inhibitors Chemie Researchers in the post-genome era are confronted with the daunting From the Contents task of assigning structure and function to tens of thousands of encoded proteins. To realize this goal, new technologies are emerging 1. Introduction 5789 for the analysis of protein function on a global scale, such as activity- 2. Natural Products that Target based protein profiling (ABPP), which aims to develop active site- Catalytic Nucleophiles in directed chemical probes for enzyme analysis in whole proteomes. For Enzyme Active Sites 5790 the pursuit of such chemical proteomic technologies, it is helpful to derive inspiration from protein-reactive natural products. Natural 3. Natural Products that Target Non-Nucleophilic Residues in products use a remarkably diverse set of mechanisms to covalently Enzyme Active Sites 5794 modify enzymes from distinct mechanistic classes, thus providing a wellspring of chemical concepts that can be exploited for the design of 4. Targeting Nonenzymatic active-site-directed proteomic probes. Herein, we highlight several Proteins 5802 examples of protein-reactive natural products and illustrate how their 5. Summary and Outlook 5803 mechanisms of action have influenced and continue to shape the progression of chemical proteomic technologies like ABPP. 1. Introduction Lipstatin,[5] fumagillin,[6] and microcystin[7] embody the chemistry of the carbonyl group, the epoxide, and the The sequencing of the human genome has transformed electron-deficient alkene, respectively, and are prominent the way in which scientists think about biology. However, examples of protein-reactive natural products. These and there is a vast gulf between the wealth of gene sequence related secondary metabolites are important because they information and our knowledge of gene function. Researchers have yielded insight into the cellular functions of key are now confronting the task of understanding the cellular enzymes. Most natural products that covalently modify and molecular functions of thousands of predicted gene proteins possess structural features that render them chemi- products. The genome gives rise to the proteome, and it is the cally reactive, whereas others bear latent reactivity; by posing combinatorial interactions among proteins that make living as innocuous substrates, they are activated only by catalytic organisms so complex at the molecular level. Elucidating the turnover in their enzyme targets.[8] three-dimensional structures and cellular functions of all Protein-reactive natural products are highly attractive as proteins encoded by prokaryotic and eukaryotic genomes and molecular probes for protein activity profiling experiments, deciphering the architectures of protein–protein interaction because they provide information about enzyme active sites networks of cells and tissues are among the great challenges in complex proteomes. Moreover, the diversity of mecha- and opportunities facing new generations of life scientists. To nisms employed by reactive natural products to target enzyme reach this understanding, the development of new technolo- active sites can serve as a valuable guide for the de novo gies and concepts to expedite global analyses of protein design of affinity agents for the proteomic profiling of specific function is required.[1] A growing number of research classes of enzymes. Such activity-based protein profiling laboratories are exploring biology with chemistry-based (ABPP) endeavors[9] provide a more direct readout of strategies that are capable of yielding insight into the role of enzyme activity in proteomes, as opposed to inferring this individual proteins in complex biological systems. The field of critical parameter from mRNA or protein levels, neither of chemical synthesis has often played a major role in this which reflect the myriad post-translational mechanisms that process, perhaps most visibly through facilitating the identi- regulate enzyme function in vivo.[10] fication of protein targets of bioactive natural products.[2] For centuries, natural products have been used for [3] [*] Prof. B. F. Cravatt medicinal purposes. Life forms that lack immune systems, The Skaggs Institute for Chemical Biology and in particular, biosynthesize natural products of unparalleled The Departments of Chemistry and Cell Biology structural diversity, some of which modulate biological The Scripps Research Institute function with exquisite specificity. It is tantalizing to consider 10550 North Torrey Pines Road, La Jolla, CA 92037 (USA) the wealth of secondary metabolites that remains undiscov- Fax : (+1)858-784-8023 ered. Microorganisms, for example, are a fertile and diverse E-mail: [email protected] source for new chemical entities, and researchers are actively C. Drahl, Prof. E. J. Sorensen Department of Chemistry developing ways to exploit their metabolic diversity.[4] A Princeton University provocative subset of biologically active natural products is Princeton, NJ 08544 (USA) endowed with electrophilic functional groups that covalently Fax : (+1)609-258-1980 modify nucleophilic residues in specific protein targets. E-mail: [email protected] Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 5789 Reviews B. F. Cravatt, E. J. Sorensen, and C. Drahl Nature engages protein targets with reactive small mol- Lipstatin features a b-lactone ring with two linear carbon ecules in many ways, and the number of natural products that chains of six and thirteen atoms, respectively. The longer of covalently modify proteins is likely very large. In coming to the two is doubly unsaturated and contains an N-formylleu- grips with this subject, we have chosen to address selected cine ester side chain. Structural analysis revealed a striking reactive natural products for which the protein targets are similarity between lipstatin and esterastin, a reported inhib- well-characterized, and we have placed particular emphasis itor of esterase which features a different amino acid side on natural products that exert their activities in eukaryotic chain, N-acetylasparagine.[17] Other members of this b-lactone systems. For a more detailed discussion of classical examples enzyme inhibitor family include the panclicins,[18] the ebelac- of protein-reactive natural products that target prokaryotic tones,[19] and valilactone (Scheme 1).[20] The research group of enzymes, such as the b-lactam family of antibiotics, which Bacher later found that lipstatin is derived biosynthetically inhibit peptidases involved in cell wall biosynthesis, the from 3-hydroxytetradeca-5,8-dienoic acid,[21] which forms reader is referred to some authoritative review articles.[11] from a condensation of two mycolic acid[22] components of Furthermore, natural products such as DNA-alkylating 14 and 8 carbon atoms, both of which originate from fatty acid agents that covalently modify non-protein biomolecules catabolism. Protons at the C2 and C3 positions, and one have been extensively reviewed elsewhere, and are not proton at C4 are derived from water.[21c] This is in contrast discussed herein.[12] Finally, throughout this review, we with the initial assumption that lipstatin had a polyketide attempt to emphasize themes that may be useful in conceiving origin; early [13C]acetate feeding experiments were incon- novel chemical proteomics probes; as will become apparent, clusive.[21d] A total synthesis of lipstatin from (S)-N-formyl- the target of natural product action need not be a catalytic leucine and dimethyl-(S)-(À)-malate was recently de- nucleophile, or for that matter even an enzyme. Strategies scribed,[23] and there is also a comprehensive body of synthetic other than the general electrophile–nucleophile interaction work on tetrahydrolipstatin,[16,24] which is obtained by the offer valuable lessons that may be harnessed by chemical catalytic hydrogenation of lipstatin.[15] biologists to further expand the proteome space amenable to Pancreatic lipase is the target of lipstatin and its deriva- analysis by ABPP.[13] From the examples below, we hope it is tives. This lipase possesses an active-site charge-relay system evident that bioactive natural products provide key tools and similar to serine proteases; it features the catalytic triad of concepts that can be exploited for the characterization of His263, Asp176, and Ser152.[25] Pancreatic lipase is respon- protein function on a global scale. sible for the hydrolysis of dietary triacylglycerols to fatty acids and monoacylglycerols.[26] This catabolic process is critical for proper fat absorption; inhibition of pancreatic lipase activity 2. Natural Products that Target Catalytic Nucleo- results in the passage of fats through the stool. Tetrahydro- philes in Enzyme Active Sites lipstatin limits the absorption of dietary fat by blocking the activity of pancreatic lipase, as well as carboxylester lipase, Many natural products have cleverly exploited the human milk lipase, and gastric lipase.[22a] Now available under catalytic mechanisms of enzymes to elicit selective, covalent the trade names orlistat