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: · inhibitors · molecular probes · natural products · structure– activity relationships

Angewandte Chemie

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Researchers in the post- 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 - 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 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 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 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 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 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 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 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 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 ; it features the 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 or xenical, tetrahydrolipstatin was inhibition. In this section, we highlight representative natural approved by the Food and Drug Administration in March products that modify key catalytic residues in enzyme active 1999 and has been successful in helping obese patients lose sites. weight.[26b,27] Obesity affects approximately one third of the U.S. population,[26b] and many problems are thought to stem from this chronic condition, including diabetes mellitus 2.1. Lipstatin type II. This disease may be prevented or brought under control with an Orlistat regimen.[28] Lipstatin was isolated from Streptomyces toxytricini on the Tetrahydrolipstatin is also an inhibitor of the thioesterase basis of its potent, selective, and irreversible inhibition of domain of fatty acid synthase (FAS), an enzyme associated pancreatic lipase (Scheme 1).[14] The structure of lipstatin was with tumor cell proliferation.[29] Administration of orlistat to determined by a combination of spectroscopic and chemical prostate tumor cells causes apoptosis; the drug also halts methods,[15] and was confirmed by chemical synthesis.[16] growth of xenograft tumors in mice. Interestingly, it was an

Carmen Drahl obtained her BA in chemistry Benjamin Cravatt studied biological sciences at Drew University in 2002. She received her (BS) and history (BA) at Stanford Univer- MA at Princeton University in the laboratory sity. He then conducted research with D. of G. McLendon. Currently, she is conduct- Boger and R. Lerner and received his PhD ing graduate studies at Princeton under the from The Scripps Research Institute (TSRI) direction of E. J. Sorensen. Her research in 1996. He joined the faculty at TSRI in interests include the synthesis and biological 1997 as a member of the Skaggs Institute evaluation of reactive natural products. She for Chemical Biology and the departments is the recipient of a Barry M. Goldwater Fel- of Cell Biology and Chemistry. His research lowship, as well as predoctoral fellowships group is developing and applying new tech- from Eli Lilly and the National Science nologies to elucidate the roles of enzymes in Foundation. the physiological and pathological processes of the nervous system and in cancer.

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Scheme 1. The lipstatin family of natural products; all members feature a four-membered b-lactone ring.

ABPP screen of prostate cancer cell proteomes with a ate hydrolyzes very slowly to a hydroxy acid that still bears fluorophosphonate–rhodamine probe that allowed research- the formylleucine side chain, thus almost completely restoring ers to characterize Orlistat as a FAS thioesterase inhibitor and enzyme activity within 24 hours.[22a, 30a] a potential antitumor therapeutic.[29] This type of reactivity is similar to that of the b-lactam The chemical reactivity of the b-lactone group of tetrahy- antibiotics, which target peptidases involved in bacterial cell- drolipstatin and its analogues is the basis for their biological wall biosynthesis (Scheme 3). The active-site serine nucleo- effects. The catalytic active-site serine nucleophile of pancre- phile in the proteins targeted by penicillin is believed to atic lipase attacks the electrophilic b-lactone at the carbonyl attack the carbonyl carbon of the lactam ring to furnish a ring- carbon C1 to form a b-hydroxy serine ester (Scheme 2, opened penicilloyl-enzyme.[31] Clavulanic acid has a more route a).[5, 30] Theoretically, attack could also occur at C3 to complicated proposed mechanism, but it shares with lipstatin form a stable serine ether (Scheme 2, route b). Modified the principal features of inhibition through ring opening and tetrahydrolipstatins (as obtained after enzyme attack and recovery of enzyme activity through the slow subsequent following adduct hydrolysis) were synthesized and then hydrolysis of an enamine.[32] compared with the enzyme reaction results. Products with The simplicity of the reactive motif in the lipstatin family inverted stereochemistry at C3, which would result from is as instructive as the scaffold that frames it. Many nucleophilic attack at this atom, were observed in trace physiological nucleophiles could conceivably react with and amounts at most. These data support the carbonyl carbon open the lactone ring. The long alkyl chains of these natural atom as the site of attack.[30] The carbonyl carbon atom may products presumably direct them toward enzymes with lipid be activated by a hydrogen-bond donor in the active site of substrates. The reactivation period for pancreatic lipase by the lipase, or the relief of ring strain alone may be sufficient to hydrolysis of the acyl-enzyme intermediate is interesting, drive the ring-opening process. In the absence of a water- particularly in light of a study that demonstrated a threefold insoluble substrate and at pH 7.0, the acyl-enzyme intermedi- increase in the reactivation rate constant (kr) in the presence

of lipid–water interfaces (kr increased from 2000 to Erik J. Sorensen received his BA degree from 6000 minÀ1).[33] Enzymes found in different environments Syracuse University. Under the guidance of might therefore have different turnover rates. Part of the K. C. Nicolaou at the University of Califor- noted selectivity of lipstatin may come not only from selective nia, San Diego, he obtained his PhD in targeting of the small molecule to pancreatic lipase, but also 1995 and co-authored the book Classics in Total Synthesis. He subsequently worked as from the selected lack of turnover of the lipase–lipstatin a postdoctoral fellow with S. Danishefsky. In complex. Variation of the scaffold around the reactive b- 1997, he began his independent career at lactone motif should allow lipstatin-like chemical probes to be The Scripps Research Institute and became redirected to target other lipase enzymes or even other associate professor in 2001. In 2003, he enzyme families altogether. moved to Princeton University, where he is the Arthur Allan Patchett Professor in organic chemistry.

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Scheme 2. Two possible mechanisms for the covalent modification of pancreatic lipase by lipstatin. The reaction occurs exclusively at the b-lactone carbonyl group.

Scheme 3. Principal features of b-lactam antibiotic reactivity.

2.2. Lactacystin of the highly conserved N-terminal threonine residue to inhibit the -like and -like proteasome Lactacystin (Scheme 4) was isolated by Omura and co- activities in irreversible fashion.[38b] The proteasome is an workers from a Streptomyces strain in a screen for natural abundant ATP-dependent cellular that degrades products that induce neurite outgrowth.[34] Its interesting damaged proteins.[39] With few exceptions, proteasomes act on structure was elucidated by X-ray crystallography and NMR proteins that are tagged for destruction through the covalent spectroscopy,[35] and confirmed by the total syntheses carried attachment of the small protein ubiquitin.[39] The proteasome out by several research groups.[36] Lactacystin is biosynthe- consists of a barrel-like core, in which controlled sized from three units: leucine, isobutyrate (and/or valine), takes place. The core is capped at each end by a regulatory and .[37] protein.[39] The N-terminal threonine residue modified by Further investigations showed that lactacystin does not lactacystin serves as the active-site nucleophile of the protea- mimic nerve growth factor;[38] instead, it reacts specifically some.[38b,40] The proteasome lacks a catalytic triad, but with the proteasome, binding covalently to the hydroxy group requires a basic group to accept a proton from threonine in

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of the N-acetylcysteine leaving group. The resulting omuralide is more cell- permeable than the parent com- pound,[40, 49] and it inhibits proteasome activities 15 to 20-fold faster than lactacystin.[38] Carbon atom C4 of this intermediate lactone is electrophilic and is presumed to undergo attack by the hydroxy group of the catalytic threonine residue[44b] to yield an ester- linked omuralide–proteasome adduct (Scheme 5). The “masking” of the reactive b-lactone in its open form is important, as it may help lactacystin avoid spurious targets. Sev- eral research groups have shown that Scheme 4. Lactacystin and related proteasome inhibitors. Velcade is a synthetic inhibitor lactacystin labels all active proteasome clinically approved for the treatment of multiple myeloma. b subunits.[39b,50] Interestingly, it was the transition state. This is most likely the job of the a-amino group of Thr1, which is ideally situated to accept a proton in the crystal structure of the proteasome in complex with lactacystin.[41] Alterna- tively, the conserved Lys33 residue may fulfill this role, since mutation of this residue abolishes pro- teasome activity.[42] Lactacystin inhibits neither the serine proteases trypsin and chymotrypsin, nor the cysteine proteases papain, the calpains, or cathe- psin B;[38b] it does, however, covalently inhibit the Scheme 5. Lactacystin is lactonized to its cell-permeable, protein-reactive derivative, omuralide, which then covalently inactivates the proteasome. A.[43] As a fairly selective proteasome inhibitor, lactacystin has proven to be a valuable probe to study the cellular role of the ubiquitin– demonstrated that replacement of the catalytic N-terminal proteasome pathway.[44] threonine group with serine produced a fivefold increase in Different families of proteasome inhibitors have been the rate of proteasome inactivation by omuralide. This developed over the years to better understand the ubiquitin– replacement also increased the rate of hydrolysis of the proteasome system. The earliest synthetic inhibitors, the C- acyl-enzyme intermediate.[51] This result may explain why terminal peptide aldehydes, showed too little selectivity for omuralide and lactacystin inhibit very few serine proteases, as the proteasome over serine and cysteine proteases.[40] Certain serine–omuralide adducts may undergo rapid hydrolysis to tripeptides modified at the C terminus by a vinyl sulfone or relieve inhibition.[44a] It is instructive to note how the activity glyoxal act as mechanism-based inhibitors of the proteaso- and selectivity of lactacystin are exquisitely controlled by me.[39b,45] Boronic acid peptides (in which the aldehyde intrinsic chemical reactivity. pharmacophore has been replaced by a boronate group) are The structure of the proteasome from Saccharomyces more potent and selective for the proteasome. The flagship cerevisiae co-crystallized with omuralide[41] was determined boronate compound, PS-341,[40, 46] was recently clinically by X-ray analysis, and confirmed the covalent linkage approved for the treatment of multiple myeloma (velcade between the threonine side chain hydroxy group and omur- (bortezomib), Scheme 4). This validates the proteasome as a alide. The N-terminal amino group is not modified by the therapeutic target for at least this type of cancer and natural product. Omuralide makes four hydrogen-bond con- potentially other forms as well.[47] Unlike lactacystin, how- tacts with the main chain of the proteasome catalytic subunit. ever, Velcade reversibly inhibits the chymotrypsin-like activ- The C9 isopropyl moiety of omuralide projects into a ity of the proteasome.[40] The most clinically advanced hydrophobic pocket of the proteasome to lend additional lactacystin analogue is PS-519 (Scheme 4), a cell-permeable binding affinity. Corey and co-workers have shown that variant that features an n-propyl substitution at C7. This replacement of the omuralide C9 isopropyl group with a compound is more potent than the natural product and has phenyl group abolishes all activity.[52] Interestingly, salino- entered phase II clinical trials for acute stroke.[40,48] sporamide A (Scheme 4), a natural product 35-fold more The reactivity of lactacystin is brought about by its latent potent than omuralide, possesses a cyclohexene moiety in b-lactone group, termed clasto-lactacystin b-lactone or omur- place of the isopropyl group, yet it shares the bicyclic ring alide (Scheme 4).[49] Derivatives that cannot form the b- structure of omuralide.[53] Feling and co-workers suggested lactone are inactive. Omuralide is formed in the extracellular that this may be indicative of the different ways by which fluid from cyclization (lactonization) of lactacystin with loss omuralide and salinosporamide A interact with the protea-

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some.[53] Corey and co-workers recently reported the first trophy, Alzheimers disease, cancer, and cataract formation enantiospecific total synthesis of salinosporamide A[54] with a all have a putative link to dysfunction.[63] In procedure that yields sufficient material to carry out biolog- addition, because cysteine proteases are a critical component ical analyses of the efficacy of salinosporamide A as a of the Plasmodium life cycle, they are regarded as viable potential anticancer agent. Moreover, congeners of salino- targets for potential antimalarial drugs.[64] In an effort to sporamide A were recently prepared for the possible treat- improve the cell permeability of leads, the agmatine group ment of proteasome-mediated disease.[55] It will be interesting was replaced with neutral moieties, such as isoamylamine in to learn whether salinosporamide A has improved upon the the case of E-64c (Scheme 6).[65] In 1986, the ethyl ester of E- delicately balanced chemistry of lactacystin. 64c was removed from phase III clinical trials in Japan for muscular dystrophy indications because its efficacy did not meet expectations, and the drug covalently modified proteins 2.3. E-64 of other mechanistic classes.[63d,66] However, by virtue of its inhibition of calpain, this same E-64 derivative has found use In 1978, the Hanada group pursued a specific inhibitor of in eye drops for the prevention and treatment of cataracts.[67] cysteine proteases to expedite investigation of protease E-64 is still frequently used to facilitate discovery and biological functions. They succeeded in isolating a highly classification of newly isolated papain-class enzymes.[68] potent and irreversible inhibitor, E-64, from the mold Molecular probes for ABPP inspired by the E-64 motif Aspergillus japonicus.[56] The structure of E-64 was deter- have proven to be of great value for tracking the activities of mined principally through classical chemical reactivity test- both and cysteine proteases in whole proteomes, ing, IR spectroscopy, and 1H NMR spectroscopy, and was and in the design and screening of inhibitors for these subsequently confirmed by a chemical synthesis of its enzymes.[68a, b] For example, a screen of plant extracts revealed enantiomer.[57] The structure contains agmatine, the arginine cysteine protease activity for three proteins that had not been decarboxylation product 1-amino-4-guanidinobutane previously characterized.[68a] In a comparative screen of (Scheme 6). To identify an E-64 pharmacophore, a compre- mature and senescent leaf extracts, ABPP demonstrated that a change in mRNA transcript levels does not always correlate with protease activity.[68a] In addition, a cell-permeable E-64 probe revealed that increased cathepsin activity is associated with the angiogenic vasculature and invasive fronts of carcinomas.[69] Based on these results, administration of a broad- spectrum cathepsin inhibitor was performed, which impaired tumor growth and invasiveness. The reaction of the E-64 epoxide with papain was elucidated by 13C NMR spectroscopy.[70] Attack by the catalytic cysteine nucleophile occurs at the epoxide C2 [70,71] atom. Backside attack in SN2 fashion inverts the configuration at C2 of the product thioether (Scheme 7). The key contacts between cysteine pro- Scheme 6. E-64 and other l-trans-(S,S)-epoxysuccinic acid derivatives. teases and E-64 that are responsible for situating the inhibitors epoxide group for regioselective nucleo- hensive library of derivatives and fragments was synthesized. philic attack have been extensively characterized in a series of Both the epoxide and the carboxyl group derived from l- crystal structures.[57, 71,72] In the structure of the E-64–papain trans-(S,S)-epoxysuccinic acid were found to be indispensable complex, the N-terminal carboxyl group of E-64 is positioned for inhibition of papain.[58] Notably, other peptidyl epoxide in the , while the carbonyl group adjacent to the natural products that act as covalent inhibitors of the epoxide group interacts with the histidine catalytic base.[71] proteasome have also been identified, including epoxomi- The mechanistic characterization of the interaction between cin[59] and eponemicin[60] (Scheme 6). E-64 and cysteine proteases of the papain class is an excellent In addition to papain, E-64 is a covalent inhibitor of example of the power of convergent research efforts in several other cysteine proteases, including B, H, chemical synthesis, classical biochemistry, and structural and L, stem bromelain, and ficin.[61] The revelation that the biology. high affinity, active-site-directed reversible inhibitor leupep- tin decreased the rate of E-64 binding cemented the idea that E-64 is an active-site-directed inhibitor of cysteine pro- 3. Natural Products that Target “Non-Nucleophilic” teases.[61] These proteases exert their catalytic activity with Residues in Enzyme Active Sites the assistance of an active-site ion pair between cysteine and histidine residues.[62] In papain, these residues are Cys25 and Several natural products inhibit enzymes through the His159.[62] covalent modification of active-site residues that are not Aberrant cysteine protease activity leads to many disease essential for catalysis. Although some of these residues states. Inflammatory and traumatic processes, muscular dys- appear to perform supportive catalytic roles, they do not

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Scheme 7. The active-site cysteine residue of papain attacks the electrophilic epoxide of E-64 in an SN2 fashion. participate as nucleophiles directly; other residues seem 1).[6b,84] Methionine are metalloproteases altogether superfluous for catalysis. In either case, however, responsible for co-translational removal of the N-terminal covalent modification of these residues results in enzyme methionine residue in specific protein targets.[6b,85] These inactivation. Herein, we highlight selected examples of enzymes were originally thought to use cobalt as the active- natural products that target “non-nucleophilic” residues in site metal center, but more recent studies indicate that enzyme active sites. manganese is likely the physiologically relevant .[86] In the enzyme active site, the metal ions are coordinated by Asp251, Asp262, His331, Glu364, Glu459, and a water 3.1. Fumagillin molecule.[87] The nucleophilicity of the water molecule is enhanced by coordination to the manganese ion; it attacks the A 1949 report demonstrated that concentrates of Asper- scissile amide bond of the substrate.[88] MetAP-mediated gillus fumigatus cultures possessed antibiotic activity against removal of methionine is necessary for post-translational .[73] Tarbell and co-workers character- modifications such as myristoylation.[89] A single amino acid ized the chemical behavior of the active component, fuma- residue in MetAP-2, Ala362, confers sensitivity to ovalicin, gillin, including the acid sensitivity of its characteristic and a Thr362Ala mutation of the human MetAP-1 gene spiroepoxide unit.[74] The constitution and stereochemistry inserted into yeast results in ovalicin-sensitive yeast colo- of fumagillin were confirmed by X-ray crystallography nies.[90] (Scheme 8).[75] In 1972, Corey and Snider described their The connection between inhibition of MetAP-2 and the elegant studies that culminated in the first chemical synthesis medicinal utility of fumagillin appears complex and remains of this natural product.[76] Subsequent work has produced a unclear.[91] It has been speculated that a connection exists at 26-step synthesis of (À)-fumagillol,[77] the saponification the level of protein myristoylation.[92] Without removal of N- product of fumagillin, and a 13-step racemic synthesis of terminal methionine residues, proteins cannot be myristoy- fumagillol.[78] Fumagillin and its close relatives ovalicin[79] and lated in vivo, which in turn, could disrupt their localization FR65814 (Scheme 8)[80] possess a rare sesquiterpene carbon and function. Therefore, the hypothesis is that fumagillin skeleton that has been shown by labeling experiments to indirectly causes cell-cycle arrest by interfering with myris- originate biosynthetically from b-trans-bergamotene, which toylation, which leads to aberrant subcellular localization of arises from farnesyl pyrophosphate.[81] Other natural products proteins that are not yet identified.[92] Despite a mechanism of not necessarily in the fumagillin family, but which contain a action that remains enigmatic, fumagillin is firmly established spiroepoxide moiety include curvularol[82] and FR901464.[83] as a potent antiparasitic agent.[93] During the 1950s, fumagillin Fumagillin and other members of its family target was used for treatment of amoebiasis in humans, caused by methionine 2 (MetAP-2), but not the closely Endamoeba histolytica, and is still used today for Nosema related enzyme methionine aminopeptidase 1 (MetAP- disease in honeybees, caused by the protozoan Nosema

Scheme 8. Fumagillin and other protein-reactive spiroepoxides. TNP-470 and CKD-731 are synthetic compounds.

Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org 5795 Reviews B. F. Cravatt, E. J. Sorensen, and C. Drahl

apis.[93a,b] More recently, fumagillin has shown efficacy against microsporidial keratoconjunctivitis of the eye, caused by a single-celled .[94] Fumagillin and its semisynthetic derivative TNP-470 (Scheme 8) are the only treatments for microsporidiosis in HIV-positive patients[95] and block the in vitro growth of Plasmodium falciparum and Leishmania donavani, the organisms that cause malaria and leishmaniasis, respectively.[96] The broad anti-infective activity of fumagillin does not come without a price; it is toxic to mammals which indicates its low selectivity for parasitic over mammalian MetAP-2.[97] The research group of Folkman stimulated a renaissance of interest in fumagillin with their discovery that this natural product inhibits tumor-induced angiogenesis.[98] TNP-470 is also a promising small-molecule inhibitor of angiogenesis and has progressed into clinical trials as a potential anticancer drug.[99] By using a homology modeling approach from the crystal structure of MetAP-2, Han and co-workers designed Figure 1. X-ray crystal structure of human MetAP-2 complexed with fumagillin. The covalent bond between C11 of the natural product CKD-731 (Scheme 8), an analogue that is 1000-fold more e [100] (yellow) and N 2 of the active site His231 (cyan) is shown; the rest of potent against endothelial cell proliferation than TNP-470. the molecular surface of MetAP-2 is colored blue. New data suggest that MetAP-2 may not be the biolog- ically relevant target of fumagillin, and that MetAP-2 function is not necessary for the growth of endothelial state analogues and crystallographic analysis yielded further cells.[101] However, this study could not completely rule out insight into the catalytic mechanism of E. coli MetAP,[102–104] a functional role for MetAP-2, because knockdown of the suggesting that His79 of E. coli MetAP (equivalent to His231 peptidase by siRNA was incomplete. The authors raised the of human MetAP-2) actually stabilizes the tetrahedral intriguing possibility that another MetAP family member intermediate that results when the peptide is attacked by an may exist in humans that could also act as a target for activated hydroxide through direct interaction with the fumagillin.[101] nitrogen atom of the scissile peptide bond. Thus, the histidine The key reactive motif of fumagillin is its spiroepoxide residue modified by fumagillin appears to play an important structure.[6] Removal of the spiroepoxide unit results in a supportive role in catalysis, consistent with the dramatic 1000-fold decrease in MetAP-2 inhibition.[84] Fumagillin also reduction in enzyme activity observed for a His231Asn possesses a side chain epoxide group; this epoxide was MetAP-2 mutant.[84] More generally, fumagillin targeting of demonstrated to be dispensable for MetAP-2 inhibitory MetAP-2, which uses a metal-activated water molecule as the activity.[84] In the X-ray crystal structure of human MetAP-2 nucleophile for peptide-bond hydrolysis, underscores the complexed with fumagillin, a covalent bond between the remarkable ability of natural products to selectively label imidazole nitrogen (Ne2) of a histidine residue (His231) and the active sites of enzymes that do not themselves engage in the methylene of the spiroepoxide is evident (Figure 1).[87] It covalent catalysis. has been speculated that His231 acts as a general base during catalysis, as it serves as a nucleophile to open the spiroepoxide ring.[84] It was thought that His231, along with the binuclear 3.2. Wortmannin metal center, could activate a water molecule for attack at the scissile amide bond, after which another histidine in the active The antifungal antibiotic wortmannin (Scheme 10) was site, His339, could protonate the leaving group.[84] However, isolated from Penicillium wortmanii and its complex structure the active site geometry in E. coli MetAP-2 does not was elucidated by chemical degradation and NMR spectro- corroborate this proposal.[102] Scheme 9 depicts one model scopic methods.[105] The hallmark of wortmannin is its reactive for fumagillin-mediated labeling of MetAP-2.[103] Transition- furan ring, which is fused between C4 and C6 of a steroid

Scheme 9. In one model for MetAP-2 modification by fumagillin, a histidine group in the MetAP-2 substrate is able to open a protonated epoxide. Fumagillin binding is enhanced at low pH.[103]

5796 www.angewandte.org  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809 Angewandte Enzyme Inhibitors Chemie framework.[106] This feature is also found in viridin, deme- mycin (mTOR), DNA-dependent protein kinase (DNA- thoxyviridin, the marine-derived pp60v-src protein tyrosine PK),[114] myosin light chain kinase (MLCK), the gentamicin kinase inhibitors halenaquinone and halenaquinol, and hibis- resistance enzyme AAC(6’)-APH(2’’), and a membrane- cone C (gmelofuran), which is found in the heartwood of bound PI 4-kinase.[115] Gmelina arborea and the Jamaican Blue Mahoe PI 3-kinases are involved in a large number of fundamen- (Scheme 10).[107] tal cellular processes, including apoptosis, proliferation, cell motility, and adhesion, and have been implicated in the malignant transforma- tion of cells. Increased levels of PI 3- kinase products have been observed in colorectal tumors and in breast can- cers.[116] It was also reported that dephosphorylation of PI 3-kinase prod- ucts suppresses tumor formation.[117] As inhibitors of PI 3-kinase in vertebrates, wortmannin and its structural relatives are considered to have potential as therapeutic agents for the treatment of human neoplasms and other disease states such as diabetes, inflammation, platelet aggregation, atherosclerosis, and osteoporosis. A semisynthetic wort- mannin library yielded ten compounds that were advanced to pharmacokinetic and toxicity studies.[118] PX-866 (Scheme 10), a diallylamino wortman- nin derivative lacking the furan ring, Scheme 10. The wortmannin family of steroidal natural products. PX-866 was synthesized from demonstrated good pharmacokinetics wortmannin. and toxicity and exhibited prolonged inhibition of PI 3-kinase in vivo. This compound also augmented the antitu- Wortmannin[108] and its structural relative viridin[109] are mor effects of the established chemotherapeutic agent biosynthesized from the triterpenoid alcohol lanosterol[110] cisplatin.[119] It was speculated that loss of the furan ring was and are potent cell-permeable inhibitors of the lipid kinase redeemed by improved chemical stability or a better fit in the phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme is a ATP-binding pocket.[118] central component of two major receptor-mediated signal nucleophiles react efficiently with the doubly transduction pathways:[111] the G-protein-coupled receptor activated and highly electrophilic strained furan ring of pathway and the receptor tyrosine kinase pathway. PI 3- wortmannin by attacking C20 to give vinylogous carbamates kinase phosphorylates at position 3 of the inositol ring of through an addition–elimination mechanism.[120] Wipf and co- phosphatidylinositol (PtdIns), leading to the production of workers recently synthesized a library of 94 C20-substituted the second messengers PtdIns-3-phosphate, PtdIns-3,4- semisynthetic wortmannin derivatives by reaction of the bisphosphate, and PtdIns-3,4,5-triphosphate. The precise natural product with a wide variety of amine and thiol nature of the PI 3-kinase catalytic mechanism is unknown.[112] nucleophiles.[118] Wymann and co-workers discovered that this In porcine PI 3-kinase, contact with the a- and b-phosphate chemistry also occurs in the active site of PI 3-kinase.[121] The groups of ATP is mediated by the conserved Lys833 and e-amino group of Lys802 of PI 3-kinase (Lys833 in porcine Ser806 side chains, respectively. An early mechanistic model PI 3-kinase)[122] attacks C20 of wortmannin which results in involved deprotonation of the lipid substrate hydroxy group furan ring-opening to form a vinylogous carbamate by Asp946 to generate a nucleophile that attacks the g- (Scheme 11). This covalent bond-forming event irreversibly phosphate of ATP. Although mutations of this amino acid inhibits the enzyme. The acid lability of the vinylogous abolish activity, the crystal structure of PI 3-kinase indicates carbamate group complicated efforts to isolate wortmannin- that this particular aspartate is not positioned properly for a labeled peptides. Fortunately, this group could be reduced role as catalytic base. Another residue, His948, has been with sodium cyanoborohydride to give a considerably more offered as an alternative base, but there is no experimental stable amine–wortmannin adduct. ATP, adenine, and FSBA evidence to support this claim.[112b] Alternatively, PI 3-kinase (5’-para-fluorosulfonylbenzoyladenosine) each at 1 mm inter- may not possess a base catalyst. Instead, it may operate fered with the alkylation of PI 3-kinase by wortmannin when through a dissociative transition state.[113] added before the inhibitor, although substances containing In addition to its well-studied lipid kinase target, wort- nucleophilic amino acid side chain groups had no effect at the mannin has been demonstrated to inhibit other serine/ same concentration. FSBA is a derivative of adenine that threonine kinases, including the mammalian target of rapa- reacts covalently with nucleophilic amino acids and has been

Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org 5797 Reviews B. F. Cravatt, E. J. Sorensen, and C. Drahl

3.3. Aspirin

In a letter to the Right Honorable George, Earl of Macclesfield and President of the Royal Society, the Reverend Edward Stone wrote on April 25, 1763: “There is a bark of an English tree, which I have found by experience to be a powerful astringent, and very efficacious in curing aguish and intermitting disorders.”[125] Stone was not the first to observe the fever and pain-reducing properties of the extracts of the willow tree. In the year 200 B.C., the Greek physician Hippocrates prescribed willow bark Scheme 11. Lysine 802 in PI 3-kinase opens the doubly activated furan ring of wortmannin by an and leaves for pain relief. Willow leaves were addition–elimination mechanism to yield a vinylogous carbamate. described by the Roman Pliny the Elder in his Natural History. Today, a modified version of used to map nucleotide binding sites.[121] The PI 3-kinase salicylic acid, the active constituent in willow bark, named substrate PtdIns-4,5-bisphosphate also effectively competes acetylsalicylic acid or aspirin (Scheme 12),[126] is one of the with wortmannin binding. These findings led Wymann and co- most widely used remedies in the world. workers to conclude that the wortmannin target site in PI 3- kinase is in a nucleotide binding site in proximity to the substrate binding site. These postulates were confirmed by the X-ray crystal structure of a complex between wortmannin and porcine PI 3-kinase.[122] The role of Lys802, as mentioned above, may be the stabilization of the a-phosphate of ATP in the binding pocket, as has been speculated for Lys72 in protein kinase A (PKA).[121] Lysine 72 anchors the nonlabile phosphate Scheme 12. Aspirin and salicylic acid, the natural product from which the drug was first synthetically derived. groups of ATP and neutralizes their charges by a salt bridge. Replacement of Lys72 with Ala in yeast PKA leads [123] to an 800-fold decrease in the Vmax value. Whereas Lys72 Salicylic acid and structurally related secondary metabo- of PKA and perhaps Lys802 of PI3-kinase appear important lites, the salicylates, are major defensive compounds that are for enzyme activity, they probably participate in a primarily present in nearly all higher ,[127] and deter the feeding of structural role rather than through direct involvement in a variety of herbivores.[127] The biosynthesis of salicylic acid catalysis. The protein microenvironment may be responsible (Scheme 13) may be traced to the phenylpropanoid path- for an enhanced nucleophilicity of the targeted lysine; way,[127] which begins with the formation of trans-cinnamic alternatively, the positioning of wortmannin in kinase active acid through the phenylalanine ammonia -catalyzed sites may preclude productive interactions with catalytic elimination of ammonia from phenylalanine.[127a] From this residues. Regardless, the potency of wortmannin should point, two pathways are possible, and different plant species encourage consideration of cofactor binding sites as targets may use either or both.[127b] The first involves the removal of a for small-molecule probes. This may expand the menu of two-carbon unit from cinnamic acid, leading to benzoic acid, proteins amenable to functional proteomic methods like followed by ortho-hydroxylation to salicylic acid. The second ABPP. Indeed, quite recently, a rhod- amine-tagged wortmannin analogue was synthesized and used in proteo- mic studies to identify mammalian polo-like kinase as an additional target of this natural product.[124] This kinase is an important protein for mitosis that is known to be over- expressed in various human cancers. The wortmannin-based probe suc- cessfully detected kinase activity changes due to treatment with drugs and in different stages of the cell cycle. Importantly, these results sug- gest that wortmannin is a good lead for the development of polo-like kinase inhibitors for cancer therapy. Scheme 13. Two biosynthetic pathways for salicylic acid in plants.

5798 www.angewandte.org  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809 Angewandte Enzyme Inhibitors Chemie reverses the order of the two steps, resulting first in ortho- It is recognized that aspirin inhibits the cyclooxygenase coumaric acid. The decarboxylation step is thought to activity of the COX enzymes, but not their peroxidase proceed in a fashion similar to the b-oxidation events of activity.[137] Aspirin appears to exert its inhibitory effects by fatty acid breakdown.[127] This was supported by studies in disrupting substrate (arachidonic acid) binding to the cyclo- which the decarboxylation step in cell-free plant extracts was oxygenase active site, as the serine residue acetylated by found to be ATP and CoA-dependent.[128] aspirin (Ser530 in COX-1, Ser516 in COX-2) is not critical for In 1860, Hermann Kolbe of Marburg University in catalysis.[133b] Germany carried out a laboratory synthesis of salicylic acid Studies of active-site COX residues have provided insight and its sodium salt from phenol, carbon dioxide, and sodium. into the mechanism of COX enzymes, as well as that of aspirin Kolbes student, Friedrich von Heyden, established a factory acetylation. The mechanism of acetyl transfer involves several for the bulk production of salicylates in Radebeul, near amino acids in the COX active site, including conserved Dresden. The unpleasant side effect of salicylate, gastric tyrosine and arginine residues (Scheme 14).[138] In 1990, irritation, revealed itself quite rapidly as the drug became spectral and biochemical studies revealed that Tyr385 is widely available. Felix Hoffmann, a chemist at Bayer, essential for COX-1 cyclooxygenase activity.[139] Structurally, ameliorated the problem in 1897 by synthesizing a more this tyrosine is properly positioned to perform the rate- easily tolerated derivative, acetylsalicylic acid.[129] Heinrich limiting step of cyclooxygenation: abstraction of the 13-pro-S Dreser, the director of research at Bayer, coined the term hydrogen atom from arachidonate.[139,140] A Tyr385Phe “aspirin” from a combination of Spirsäure (salicylic acid) with mutant of COX-2 displays virtually no serine acetylation by an initial “a” for acetyl, and a patent was granted in 1899. aspirin, and a Tyr348Phe mutant displays reduced activity For nearly a century, the molecular mechanism of aspirin and aspirin acetylation.[141] The latter result confirms the remained a mystery. However, in 1971, research by Vane importance of Tyr348, which forms a hydrogen bond to indicated that aspirin inhibits prostaglandin biosynthesis.[130] Tyr385 and helps to position the substrate. Arginine 120 of This seminal finding explained both the beneficial properties COX-1 is an important residue for both substrate recognition and side effects of aspirin, and culminated in the awarding of a and imparting sensitivity to inhibitors that contain a free share of the 1982 Nobel Prize in Physiology or Medicine to carboxylic acid.[142] Analysis of a crystal structure of COX-1 Vane.[131] The prostaglandins are paracrine hormones that act suggests that the guanidino moiety interacts with the arach- on cells proximal to their place of synthesis. A trauma to cells idonate carboxylate group.[143] Furthermore, reversible inhib- results in an increase in local biosynthesis of prostaglandins, itors with a free carboxylic acid, such as flurbiprofen, were which cause elevated body temperature, inflammation, and ineffective against COX-1 enzymes with a mutation at pain.[132] Aspirin was found to acetylate and irreversibly Arg120.[142,143] The corresponding COX-2 Arg-to-Gln inactivate prostaglandin endoperoxide (PGG/H) synthase, mutant was later found to have a 73% reduction of also termed cyclooxygenase (COX).[133] In addition, aspirin acetylation of Ser516 in COX-2.[141] acetylates human hemoglobin and human serum albumin.[134] Based on the above experimental data, researchers have The COX enzyme has two known isoforms, the constit- described a best possible mechanism for serine acetylation by utive COX-1 and the inducible COX-2.[135] COX enzymes aspirin (Scheme 14). The amino acid numbering scheme catalyze the first two steps in the metabolism of arachidonic presented in the following paragraph corresponds to COX-1. acid to prostaglandins. The cyclooxygenase activity incorpo- Initial interaction of the carboxylate group of aspirin with rates two oxygen atoms into arachidonic acid to produce Arg120 stabilizes its location in the substrate-binding pocket. prostaglandin G2 (PGG2). PGG2 is then subjected to the The hydroxy groups of Tyr385 and Tyr348 then act as an peroxidase section of these enzymes, which catalyzes a extended hydrogen-bonding network. This directs the acetyl reduction of the hydroperoxy group of PGG2 to the hydroxy group of aspirin toward Ser530, and increases the reactivity of [133b,136] endoperoxide, PGH2. aspirin by stabilizing the developing negative charge of the

Scheme 14. An intricate network of hydrogen bonds and other interactions position aspirin to acetylate Ser530 of cyclooxygenase-1.

Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org 5799 Reviews B. F. Cravatt, E. J. Sorensen, and C. Drahl

tetrahedral addition intermediate. The possibility of transient ture.[148] Although the peptidic nature of the toxins was tyrosine acetylation was excluded by testing the acetylation of confirmed in 1959,[149] their amino acid compositions were not a Ser530Ala mutant, for which no O-acetyltyrosine was determined until many years later.[150] The first complete detected.[141] These data are all corroborated by the X-ray structure was determined by NMR spectroscopy and MS in crystal structure of COX-1 inactivated by bromoaspirin 1984,[151] and was soon followed by characterization of other (bromoacetylsalicylic acid). Bromoaspirin was used so that family members.[152] The toxins are cyclic heptapeptides with its location could be identified definitively in a relatively low- variable l-amino acid residues at two positions labeled X and resolution (3.4 Š) crystal structure from the presence of the Y. They contain an Adda residue, an unusual aromatic b- heavy bromine atom. The phenolic oxygen atom of Tyr385 is amino acid featuring an unsaturated alkyl chain (Scheme 15). within hydrogen-bonding distance to the acetyl group.[144] An unambiguous determination of the configuration of Adda In summary, an elaborate scheme of hydrogen bonding was published in 1988, confirming the structure as and ionic interactions serves to position aspirin in the COX (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl- active site, increasing its effective concentration and enhan- (4E,6E)-decadienoic acid.[153] Various names were present in cing its reactivity, which leads to the selective acetylation of the literature for Adda-containing cyclic peptide natural Ser530. The X-ray structure also lends insight into the products, but ultimately the name microcystin was chosen inhibitory effect of aspirin acetylation. Ser530 lies in a (Scheme 15).[154] highly conserved region[133b] along a hydrophobic channel that The microcystin family comprises more than sixty mem- leads from the surface of the enzyme to the catalytic tyrosine. bers;[155] the vast majority differ in the nature of two variable The aspirin acetyl group extends outward into the channel, amino acids, or in the methylation states of the dehydroala- perhaps in a manner sufficient to prevent arachidonic acid nine and/or aspartic acid groups. In naming new microcystins, from reaching the active site.[133b,144] two letters are appended to abbreviate amino acids at the Finally, it is important to note that salicylic acid also variable positions. Biosynthetic studies on microcystins were possesses anti-inflammatory effects, but does not itself particularly focused on the identification of the origin of the covalently modify COX enzymes. Discussion concerning the carbon atoms in the Adda unit and the methylaspartic acid precise mechanism of action of salicylate is ongoing.[145] Thus, residue. Methylaspartic acid originates from acetyl-CoA and the conversion of this natural product to its semisynthetic pyruvic acid via a methylsuccinic acid intermediate, and the derivative aspirin had the fortuitous effects of conferring Adda unit derives from acetate; certain methyl groups are mechanistic irreversibility and anointing principal protein methionine-derived.[156] Typically, microcystins are biosynthe- targets for therapeutic intervention. sized non-ribosomally.[157] Analyses of microcystin synthetase have yielded genes for polyketide synthases.[158] Researchers also sought to understand the molecular 3.4. Microcystin nature of the toxicity of microcystins, and not long after the structures of these natural products were revealed, it was in the blue-green algae blooms of con- determined that they are potent inhibitors of serine/threonine taminated drinking water have been the cause of many animal phosphatases 1 and 2A,[7,147b,159] and other less-thoroughly deaths. Beginning in 1878,[146] it became evident that blooms characterized phosphatase family members.[160] Reversible of the genera Oscillatoria, Anabaena, Nostoc, and Microcystis protein phosphorylation is a critical mechanism for the in freshwater and marine environments were to blame for regulation of cellular processes.[161] More than 98% of protein related incidents of acute toxicity.[147] The active components, phosphorylation in eukaryotic cells occurs on serine and a family of potent hepatotoxins, were isolated and a vigorous threonine residues.[160] Serine/threonine phosphatases 1 and research effort initiated toward determining their struc- 2A (PP1 and PP2A) have been implicated in the regulation of

Scheme 15. Two prototypical microcystin family members; positions X and Y may represent any of several different amino acids. Ndha =N-methyl dehydroalanine.

5800 www.angewandte.org  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809 Angewandte Enzyme Inhibitors Chemie such diverse events as glycogen metabolism, synaptic plasti- to phosphatases was not observed when the proteins were city, cell-cycle progression, embryogenesis, apoptosis and denatured with sodium dodecyl sulfate prior to the addition of smooth-muscle contraction.[162] The structures, activities, and the natural product, indicating that the interaction depends inhibition properties of PP1 and PP2A have been reviewed on an intact active site.[7] extensively.[163] Significantly, these enzymes do not possess a Microcystins bind to PP1 with subnanomolar affinity;[170] catalytic nucleophile; instead, they activate a water molecule in the crystal structure of microcystin-LR bound to PP1, the for catalysis with protein-bound metal ion cofactors.[163] Adda side chain makes contacts with a conserved hydro- Indiscriminate inhibition of the many essential functions phobic groove, and the carboxyl group of glutamate and the performed by PP1 and PP2A may contribute to the severe adjacent carbonyl oxygen atom coordinate the metal-binding toxicity of the microcystins; between one and two micrograms site indirectly through water molecules.[171] The leucine side constitutes a lethal intraperitoneal dose in mice.[147b] Although chain of the toxin is situated in a loop of the C-terminal the broad toxicity of microcystins undermines their potential domain; in addition, the carboxyl group of methylaspartate as therapeutic agents, research groups have synthesized hydrogen bonds to Arg96, a residue crucial for interactions variants and truncated versions of the toxin family to better with phosphate groups on substrates. Hence, access to the understand the properties that confer toxicity and specific- active site is completely blocked. ity.[164] So far, only one total synthesis of a microcystin family The extensive interactions between microcystins and member has been completed.[165] These natural products have phosphatase active sites suggest that covalent reactivity facilitated the functional characterization of phosphatases contributes only partly to the mode of action of these natural and phosphatase–protein complexes in proteomes.[159b,166] A products. Microcystins immobilized to a Sepharose bead microcystin-LR–rhodamine conjugate reacted with and through an aminoethanethiol adduct retained inhibitory detected numerous serine/threonine phosphatases in a crude activity over PP1 and PP2A.[159b] Microcystins-LR, YR, and cell lysate sample, and could record changes in phosphatase RR were converted synthetically to their glutathione and activity levels.[166] These probes expanded the list of enzyme cysteine conjugates; both of these thiols reacted with the families amenable to functional proteomic analysis. dehydroalanine group, yet the 1,4-addition products retained À1 Several bioactive natural products, such as leptomy- a measure of in vivo toxicity (LD50 = 38 mgkg for micro- [167] [168] [169] À1 cin B, isoavenaciolide, and pironetin have been cystin-LR; LD50 = 630 mgkg for its glutathione conju- shown to act as carbon-based electrophiles, similar to the gate).[171] Notably, such adducts may not be resistant toward a,b-unsaturated carbonyl element of the methyl-dehydroala- retro-Michael reaction in vivo, and reversible adduct forma- nine residue of microcystin. In 1995, two research groups tion may explain the observed biological activity.[172] These simultaneously published work confirming the conjugate results imply that the dehydroalanine residue probably does addition chemistry of the dehydroalanine moiety of micro- not play a role in early enzyme–inhibitor interactions. cystin with PP1 at Cys273 near the enzyme C terminus.[7,159a] Mechanistic studies by Craig and co-workers confirmed a In PP2A, the analogous Cys266 is the targeted residue.[159a] two-step mechanism for irreversible inhibition by micro- The amino acid sequence Ser-Ala-Pro-Asn-Tyr-Cys, which cystins (Scheme 16).[173] In the first step, the rapid noncova- includes the cysteine residue modified by microcystin, is lent binding and inhibition occurs with all the contacts highly conserved in Ser/Thr phosphatases.[7] The covalent detailed above. The slower covalent modification of Cys273 linkage between the various microcystins and PP1 is stable to occurs over the course of hours.[173] The tendency for the heat and acid. However, covalent binding of microcystin-YR conjugate addition reaction is likely enhanced by the high

Scheme 16. Microcystin inactivates PP1 prior to the slow covalent attachment to Cys273. However, the network of noncovalent interactions is essential for a successful conjugate addition.

Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org 5801 Reviews B. F. Cravatt, E. J. Sorensen, and C. Drahl

effective concentration of microcystin in the well-folded compounds constitute part of a family of structurally similar binding pocket, as evidenced by the control reaction with natural products, including callystatin A, ratjadone, kazusa- denatured protein discussed above. It is also not out of the mycin, anguinomycin, leptofuranin and leptolstatin question that the nucleophilicity of the target cysteine may be (Scheme 17).[177] Structures of the leptomycins were deter- enhanced in its local protein environment. Regardless, it is mined by NMR spectroscopy, MS, and IR spectroscopy.[178] important to note that mutagenesis studies have found that They feature an unsaturated fatty acid with a terminal d- Cys273 is not required for catalysis; in fact, the activity of a lactone ring. Leptomycin B has a polyketide origin; it may be Cys273Ser mutant is indistinguishable from that of the wild- traced back to four acetate units, seven propionate units, and type enzyme.[174] Thus, microcystins provide a salient example one butyrate unit.[179] Leptomycin B interferes with DNA of how an intricate architecture of noncovalent interactions synthesis stages of the cell cycle,[180] presumably a result of its with enzymes can facilitate covalent modification on non- inhibitory effect on nuclear transport.[181] A nanomolar bind- catalytic active-site residues. Notably, similar strategies have ing partner for leptomycin B was discovered and named been employed to convert tight-binding reversible inhibitors maintenance region 1 (CRM1).[182] CRM1, also into covalent inactivators of the EGF receptor[175a–c] and p90 known as exportin 1, is an evolutionarily conserved receptor ribosomal protein S6 kinase[175d] by the introduction of for the leucine-rich nuclear export signal (NES)[183] of electrophilic groups in proximity to cysteine residues in the proteins and is an essential mediator of NES-dependent active sites of these proteins. nuclear export of proteins and ribonucleoprotein complexes in eukaryotic cells.[182,184] In conjunction with CRM1, the protein Rev allows 4. Targeting Nonenzymatic Proteins partially processed HIV mRNAs to be transported outside the nucleus for translation, enabling the HIV to hijack Although most protein-reactive natural products target the cellular protein synthesis machinery.[182, 185] Agents like the active sites of enzymes, there are exceptions. Here, we leptomycin B that block the interaction of Rev with nuclear highlight one such natural product, leptomycin B, which transport proteins have the potential to inhibit HIV-1 covalently modifies a receptor protein involved in nuclear replication.[186] Leptomycin B has also been used to accumu- transport. late the tumor suppressor p53 in the nucleus.[187] This subcellular localization leads to p53 activation, cell-cycle arrest, and apoptosis. Hence, leptomycin could represent a 4.1. Leptomycin B novel therapeutic approach for HIV and cancer treat- ment.[187,188] A Streptomyces strain isolated from soil samples yielded Leptomycin B binds to CRM1 and covalently modifies two novel antifungal antibiotics. The active components were Cys529 in a central conserved region of the protein, which purified, isolated, and termed leptomycins A and B.[176] These inhibits recognition of the NES; ratjadone was recently found

Scheme 17. The leptomycin family of natural products.

5802 www.angewandte.org  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809 Angewandte Enzyme Inhibitors Chemie to share this mechanism (Scheme 18).[167,181,186a,189] Research- an enzyme. Despite this, leptomycin B labels Cys529 with ers found that a single mutation, Cys529Ser, in Schizosac- exquisite specificity, suggesting that this residue resides in a charomyces pombe was sufficient to greatly reduce sensitivity structured small-molecule binding site. One intriguing possi- to leptomycin B.[167,190] To determine which portion of the bility is that the central conserved region of CRM1 contains a hydrophobic pocket that is capable of interacting with both leucine-rich NESs and the distinct architecture of leptomy- cin B in a manner similar to enzyme active sites that bind substrates and inhibitors that often share little structural homology.[167]

5. Summary and Outlook

In this review, we have attempted to highlight the diversity of mechanisms by which natural products engage and covalently modify proteins and protein active sites. The specific compounds discussed are meant to serve as repre- sentatives of the different classes of protein-reactive natural products discovered so far, rather than as a comprehensive list. Notably, the reactivity of these natural products is not restricted to a specific type of amino acid, but rather can occur on a range of active-site residues, including serine (lipstatin, aspirin), threonine (lactacystin), lysine (wortmannin), cys- Scheme 18. The a,b-unsaturated lactone of leptomycin reacts by teine (E-64, microcystin), and histidine (fumagillin). More- conjugate addition with Cys529 of exportin. over, these residues do not need to serve a catalytic function in the target enzyme class (for example, Cys273 in PP1) or, for natural product serves as the pharmacophore, Kobayashi and that matter, to be part of an enzyme active site (as is the case colleagues performed extensive studies on a series of for Cys529 in CRM1). This underscores the creative ability of analogues of callystatin. The a,b-unsaturated lactone was nature to exploit “spurious” nucleophiles in small-molecule found to be the crucial reactive element.[191] Other research binding sites for the selective modification of proteins. groups reduced the lactone moiety to a saturated analogue[192] Although all the natural products discussed herein inactivate or formed a Michael adduct with nitromethane;[185a] both of their cognate protein targets by modifying amino acid these events greatly decreased inhibition.[192] Furthermore, N- residues, other mechanisms of enzyme inhibition are possible. acetylcysteine methyl ester and leptomycin B reacted to give For example, gabaculine (Scheme 19), a rare amino acid a stable 1,4-addition product, as determined by NMR spectroscopy.[167] All these results lend credence to the idea that leptomycin binds covalently through an attack on its electrophilic a,b-unsaturated lactone function by the thiol group of CRM1 Cys529 (Scheme 18). The role of Cys529 in CRM1 is not yet clear. This residue does not seem to be necessary for CRM1 to function. The Scheme 19. The natural product gabaculine is a conformationally Cys529Ser mutant mediates nuclear export just as effectively constrained GABA analogue. GABA =g-aminobutyric acid. as wild-type CRM1, and it is not temperature-sensitive.[167] Moreover, wild-type CRM1 has a isolated from the mold Streptomyces toyocaensis,[196] inacti- threonine in place of cysteine at the analogous position.[167] vates several related aminotransferases by covalently reacting It is difficult to speculate further about the nature of the with the enzymes pyridoxal phosphate cofactor CRM1–leptomycin B interaction, as a structure of the com- (Table 1).[8c,197] plex has yet to be determined.[188, 193] This information is What lessons might be learned from protein-reactive crucial to the development of new drug candidates for the natural products that could assist in the design of chemical inhibition of nuclear translocation. Leptomycin B itself has a probes for the burgeoning field of activity-based protein high toxicity profile, as demonstrated in a phase I clinical trial, profiling (ABPP)?[9] ABPP is a functional proteomic method and was not recommended for further development.[194] that aims to develop active-site-directed probes that label Several syntheses of leptomycin B and its relatives have many enzymes in parallel, thereby facilitating their collective been carried out in an effort to understand the function of this functional characterization in samples of high biological family of compounds.[191a,195] The premise that some of these complexity. The application of ABPP methods has so far reagents might selectively shut down transport of specific enabled the discovery of enzyme activities that are up- mRNA molecules is enticing and warrants further research. regulated in diseases like cancer,[69,198] obesity,[199] and In summary, CRM1 is a particularly notable target for a malaria;[200] it has also enabled the design of selective protein-reactive natural product, because this protein is not inhibitors for these enzymes.[201]

Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org 5803 Reviews B. F. Cravatt, E. J. Sorensen, and C. Drahl

Table 1: Representative protein-reactive natural products and their Their discovery and characterization should continue to offer targets. fascinating insight into the seemingly endless array of small- Natural Target Labeled Catalytic molecule–protein interactions that occur in nature. The product residue nucleophile? lessons learned from these studies should in turn guide lipstatin pancreatic lipase Ser152 yes chemical biologists in their efforts to achieve a paramount lactacystin proteasome Thr1 yes objective of post-genomic science—the design of selective b subunits small-molecule probes for the functional analysis of every E-64 papain Cys25 yes protein. (Cys proteases) fumagillin MetAP-2 His-231 no We thank K. Barglow and Dr. E. C. Taylor for critical reading wortmannin PI 3-kinase Lys802 no of this manuscript, and Dr. M. H. Bracey for assistance with aspirin COX-1 Ser530 no COX-2 Ser516 no the generation of figures. This research was supported by the microcystin PP1 Cys273 no NIH (CA087660), NIGMS (GM065483), a Bristol Myers- PP2A Cys266 no Squibb Unrestricted Grant in Synthetic Organic Chemistry, the leptomycin B CRM1 Cys529 no Skaggs Institute for Chemical Biology, and Princeton Univer- [a] gabaculine aminotransferases PLP cofactor no sity. C.D. gratefully acknowledges predoctoral fellowships [a] PLP =pyridoxal phosphate. from Eli Lilly and the National Science Foundation.

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