Download Protein-Reactive Natural Products

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
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Chemie
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Enzyme Inhibitors
Researchers in the post-genome era are confronted with the daunting
task of assigning structure and function to tens of thousands of
encoded proteins. To realize this goal, new technologies are emerging
for the analysis of protein function on a global scale, such as activitybased protein profiling (ABPP), which aims to develop active sitedirected chemical probes for enzyme analysis in whole proteomes. For
the pursuit of such chemical proteomic technologies, it is helpful to
derive inspiration from protein-reactive natural products. Natural
products use a remarkably diverse set of mechanisms to covalently
modify enzymes from distinct mechanistic classes, thus providing a
wellspring of chemical concepts that can be exploited for the design of
active-site-directed proteomic probes. Herein, we highlight several
examples of protein-reactive natural products and illustrate how their
mechanisms of action have influenced and continue to shape the
progression of chemical proteomic technologies like ABPP.
1. Introduction
The sequencing of the human genome has transformed
the way in which scientists think about biology. However,
there is a vast gulf between the wealth of gene sequence
information and our knowledge of gene function. Researchers
are now confronting the task of understanding the cellular
and molecular functions of thousands of predicted gene
products. The genome gives rise to the proteome, and it is the
combinatorial interactions among proteins that make living
organisms so complex at the molecular level. Elucidating the
three-dimensional structures and cellular functions of all
proteins encoded by prokaryotic and eukaryotic genomes and
deciphering the architectures of protein–protein interaction
networks of cells and tissues are among the great challenges
and opportunities facing new generations of life scientists. To
reach this understanding, the development of new technologies and concepts to expedite global analyses of protein
function is required.[1] A growing number of research
laboratories are exploring biology with chemistry-based
strategies that are capable of yielding insight into the role of
individual proteins in complex biological systems. The field of
chemical synthesis has often played a major role in this
process, perhaps most visibly through facilitating the identification of protein targets of bioactive natural products.[2]
For centuries, natural products have been used for
medicinal purposes.[3] Life forms that lack immune systems,
in particular, biosynthesize natural products of unparalleled
structural diversity, some of which modulate biological
function with exquisite specificity. It is tantalizing to consider
the wealth of secondary metabolites that remains undiscovered. Microorganisms, for example, are a fertile and diverse
source for new chemical entities, and researchers are actively
developing ways to exploit their metabolic diversity.[4] A
provocative subset of biologically active natural products is
endowed with electrophilic functional groups that covalently
modify nucleophilic residues in specific protein targets.
Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809
From the Contents
1. Introduction
5789
2. Natural Products that Target
Catalytic Nucleophiles in
Enzyme Active Sites
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3. Natural Products that Target
Non-Nucleophilic Residues in
Enzyme Active Sites
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4. Targeting Nonenzymatic
Proteins
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5. Summary and Outlook
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Lipstatin,[5] fumagillin,[6] and microcystin[7] embody the
chemistry of the carbonyl group, the epoxide, and the
electron-deficient alkene, respectively, and are prominent
examples of protein-reactive natural products. These and
related secondary metabolites are important because they
have yielded insight into the cellular functions of key
enzymes. Most natural products that covalently modify
proteins possess structural features that render them chemically reactive, whereas others bear latent reactivity; by posing
as innocuous substrates, they are activated only by catalytic
turnover in their enzyme targets.[8]
Protein-reactive natural products are highly attractive as
molecular probes for protein activity profiling experiments,
because they provide information about enzyme active sites
in complex proteomes. Moreover, the diversity of mechanisms employed by reactive natural products to target enzyme
active sites can serve as a valuable guide for the de novo
design of affinity agents for the proteomic profiling of specific
classes of enzymes. Such activity-based protein profiling
(ABPP) endeavors[9] provide a more direct readout of
enzyme activity in proteomes, as opposed to inferring this
critical parameter from mRNA or protein levels, neither of
which reflect the myriad post-translational mechanisms that
regulate enzyme function in vivo.[10]
[*] Prof. B. F. Cravatt
The Skaggs Institute for Chemical Biology and
The Departments of Chemistry and Cell Biology
The Scripps Research Institute
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: (+ 1) 858-784-8023
E-mail: [email protected]
C. Drahl, Prof. E. J. Sorensen
Department of Chemistry
Princeton University
Princeton, NJ 08544 (USA)
Fax: (+ 1) 609-258-1980
E-mail: [email protected]
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 molecules in many ways, and the number of natural products that
covalently modify proteins is likely very large. In coming to
grips with this subject, we have chosen to address selected
reactive natural products for which the protein targets are
well-characterized, and we have placed particular emphasis
on natural products that exert their activities in eukaryotic
systems. For a more detailed discussion of classical examples
of protein-reactive natural products that target prokaryotic
enzymes, such as the b-lactam family of antibiotics, which
inhibit peptidases involved in cell wall biosynthesis, the
reader is referred to some authoritative review articles.[11]
Furthermore, natural products such as DNA-alkylating
agents that covalently modify non-protein biomolecules
have been extensively reviewed elsewhere, and are not
discussed herein.[12] Finally, throughout this review, we
attempt to emphasize themes that may be useful in conceiving
novel chemical proteomics probes; as will become apparent,
the target of natural product action need not be a catalytic
nucleophile, or for that matter even an enzyme. Strategies
other than the general electrophile–nucleophile interaction
offer valuable lessons that may be harnessed by chemical
biologists to further expand the proteome space amenable to
analysis by ABPP.[13] From the examples below, we hope it is
evident that bioactive natural products provide key tools and
concepts that can be exploited for the characterization of
protein function on a global scale.
2. Natural Products that Target Catalytic Nucleophiles in Enzyme Active Sites
Many natural products have cleverly exploited the
catalytic mechanisms of enzymes to elicit selective, covalent
inhibition. In this section, we highlight representative natural
products that modify key catalytic residues in enzyme active
sites.
2.1. Lipstatin
Lipstatin was isolated from Streptomyces toxytricini on the
basis of its potent, selective, and irreversible inhibition of
pancreatic lipase (Scheme 1).[14] The structure of lipstatin was
determined by a combination of spectroscopic and chemical
methods,[15] and was confirmed by chemical synthesis.[16]
Lipstatin features a b-lactone ring with two linear carbon
chains of six and thirteen atoms, respectively. The longer of
the two is doubly unsaturated and contains an N-formylleucine ester side chain. Structural analysis revealed a striking
similarity between lipstatin and esterastin, a reported inhibitor of esterase which features a different amino acid side
chain, N-acetylasparagine.[17] Other members of this b-lactone
enzyme inhibitor family include the panclicins,[18] the ebelactones,[19] and valilactone (Scheme 1).[20] The research group of
Bacher later found that lipstatin is derived biosynthetically
from 3-hydroxytetradeca-5,8-dienoic acid,[21] which forms
from a condensation of two mycolic acid[22] components of
14 and 8 carbon atoms, both of which originate from fatty acid
catabolism. Protons at the C2 and C3 positions, and one
proton at C4 are derived from water.[21c] This is in contrast
with the initial assumption that lipstatin had a polyketide
origin; early [13C]acetate feeding experiments were inconclusive.[21d] A total synthesis of lipstatin from (S)-N-formylleucine and dimethyl-(S)-( )-malate was recently described,[23] and there is also a comprehensive body of synthetic
work on tetrahydrolipstatin,[16, 24] which is obtained by the
catalytic hydrogenation of lipstatin.[15]
Pancreatic lipase is the target of lipstatin and its derivatives. This lipase possesses an active-site charge-relay system
similar to serine proteases; it features the catalytic triad of
His 263, Asp 176, and Ser 152.[25] Pancreatic lipase is responsible 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
results in the passage of fats through the stool. Tetrahydrolipstatin limits the absorption of dietary fat by blocking the
activity of pancreatic lipase, as well as carboxylester lipase,
human milk lipase, and gastric lipase.[22a] Now available under
the trade names orlistat or xenical, tetrahydrolipstatin was
approved by the Food and Drug Administration in March
1999 and has been successful in helping obese patients lose
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
type II. This disease may be prevented or brought under
control with an Orlistat regimen.[28]
Tetrahydrolipstatin is also an inhibitor of the thioesterase
domain of fatty acid synthase (FAS), an enzyme associated
with tumor cell proliferation.[29] Administration of orlistat to
prostate tumor cells causes apoptosis; the drug also halts
growth of xenograft tumors in mice. Interestingly, it was an
Carmen Drahl obtained her BA in chemistry
at Drew University in 2002. She received her
MA at Princeton University in the laboratory
of G. McLendon. Currently, she is conducting graduate studies at Princeton under the
direction of E. J. Sorensen. Her research
interests include the synthesis and biological
evaluation of reactive natural products. She
is the recipient of a Barry M. Goldwater Fellowship, as well as predoctoral fellowships
from Eli Lilly and the National Science
Foundation.
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Benjamin Cravatt studied biological sciences
(BS) and history (BA) at Stanford University. He then conducted research with D.
Boger and R. Lerner and received his PhD
from The Scripps Research Institute (TSRI)
in 1996. He joined the faculty at TSRI in
1997 as a member of the Skaggs Institute
for Chemical Biology and the departments
of Cell Biology and Chemistry. His research
group is developing and applying new technologies to elucidate the roles of enzymes in
the physiological and pathological processes
of the nervous system and in cancer.
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Enzyme Inhibitors
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
fluorophosphonate–rhodamine probe that allowed researchers to characterize Orlistat as a FAS thioesterase inhibitor and
a potential antitumor therapeutic.[29]
The chemical reactivity of the b-lactone group of tetrahydrolipstatin and its analogues is the basis for their biological
effects. The catalytic active-site serine nucleophile of pancreatic lipase attacks the electrophilic b-lactone at the carbonyl
carbon C1 to form a b-hydroxy serine ester (Scheme 2,
route a).[5, 30] Theoretically, attack could also occur at C3 to
form a stable serine ether (Scheme 2, route b). Modified
tetrahydrolipstatins (as obtained after enzyme attack and
following adduct hydrolysis) were synthesized and then
compared with the enzyme reaction results. Products with
inverted stereochemistry at C3, which would result from
nucleophilic attack at this atom, were observed in trace
amounts at most. These data support the carbonyl carbon
atom as the site of attack.[30] The carbonyl carbon atom may
be activated by a hydrogen-bond donor in the active site of
the lipase, or the relief of ring strain alone may be sufficient to
drive the ring-opening process. In the absence of a waterinsoluble substrate and at pH 7.0, the acyl-enzyme intermediErik J. Sorensen received his BA degree from
Syracuse University. Under the guidance of
K. C. Nicolaou at the University of California, San Diego, he obtained his PhD in
1995 and co-authored the book Classics in
Total Synthesis. He subsequently worked as
a postdoctoral fellow with S. Danishefsky. In
1997, he began his independent career at
The Scripps Research Institute and became
associate professor in 2001. In 2003, he
moved to Princeton University, where he is
the Arthur Allan Patchett Professor in
organic chemistry.
Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809
ate hydrolyzes very slowly to a hydroxy acid that still bears
the formylleucine side chain, thus almost completely restoring
enzyme activity within 24 hours.[22a, 30a]
This type of reactivity is similar to that of the b-lactam
antibiotics, which target peptidases involved in bacterial cellwall biosynthesis (Scheme 3). The active-site serine nucleophile in the proteins targeted by penicillin is believed to
attack the carbonyl carbon of the lactam ring to furnish a ringopened penicilloyl-enzyme.[31] Clavulanic acid has a more
complicated proposed mechanism, but it shares with lipstatin
the principal features of inhibition through ring opening and
recovery of enzyme activity through the slow subsequent
hydrolysis of an enamine.[32]
The simplicity of the reactive motif in the lipstatin family
is as instructive as the scaffold that frames it. Many
physiological nucleophiles could conceivably react with and
open the lactone ring. The long alkyl chains of these natural
products presumably direct them toward enzymes with lipid
substrates. The reactivation period for pancreatic lipase by
hydrolysis of the acyl-enzyme intermediate is interesting,
particularly in light of a study that demonstrated a threefold
increase in the reactivation rate constant (kr) in the presence
of lipid–water interfaces (kr increased from 2000 to
6000 min 1).[33] Enzymes found in different environments
might therefore have different turnover rates. Part of the
noted selectivity of lipstatin may come not only from selective
targeting of the small molecule to pancreatic lipase, but also
from the selected lack of turnover of the lipase–lipstatin
complex. Variation of the scaffold around the reactive blactone motif should allow lipstatin-like chemical probes to be
redirected to target other lipase enzymes or even other
enzyme families altogether.
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B. F. Cravatt, E. J. Sorensen, and C. Drahl
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
Lactacystin (Scheme 4) was isolated by Omura and coworkers from a Streptomyces strain in a screen for natural
products that induce neurite outgrowth.[34] Its interesting
structure was elucidated by X-ray crystallography and NMR
spectroscopy,[35] and confirmed by the total syntheses carried
out by several research groups.[36] Lactacystin is biosynthesized from three units: leucine, isobutyrate (and/or valine),
and cysteine.[37]
Further investigations showed that lactacystin does not
mimic nerve growth factor;[38] instead, it reacts specifically
with the proteasome, binding covalently to the hydroxy group
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of the highly conserved N-terminal threonine residue to
inhibit the chymotrypsin-like and trypsin-like proteasome
activities in irreversible fashion.[38b] The proteasome is an
abundant ATP-dependent cellular protease that degrades
damaged proteins.[39] With few exceptions, proteasomes act on
proteins that are tagged for destruction through the covalent
attachment of the small protein ubiquitin.[39] The proteasome
consists of a barrel-like core, in which controlled proteolysis
takes place. The core is capped at each end by a regulatory
protein.[39] The N-terminal threonine residue modified by
lactacystin serves as the active-site nucleophile of the proteasome.[38b, 40] The proteasome lacks a catalytic triad, but
requires a basic group to accept a proton from threonine in
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Enzyme Inhibitors
Scheme 4. Lactacystin and related proteasome inhibitors. Velcade is a synthetic inhibitor
clinically approved for the treatment of multiple myeloma.
of the N-acetylcysteine leaving group.
The resulting omuralide is more cellpermeable than the parent compound,[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 esterlinked omuralide–proteasome adduct
(Scheme 5). The “masking” of the
reactive b-lactone in its open thioester
form is important, as it may help
lactacystin avoid spurious targets. Several research groups have shown that
lactacystin labels all active proteasome
b subunits.[39b, 50] Interestingly, it was
the transition state. This is most likely the job of the
a-amino group of Thr 1, which is ideally situated to
accept a proton in the crystal structure of the
proteasome in complex with lactacystin.[41] Alternatively, the conserved Lys 33 residue may fulfill this
role, since mutation of this residue abolishes proteasome activity.[42] Lactacystin inhibits neither the
serine proteases trypsin and chymotrypsin, nor the
cysteine proteases papain, the calpains, or catheScheme 5. Lactacystin is lactonized to its cell-permeable, protein-reactive derivative,
psin B;[38b] it does, however, covalently inhibit the
omuralide, which then covalently inactivates the proteasome.
[43]
serine protease cathepsin A. 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
the rate of proteasome inactivation by omuralide. This
Different families of proteasome inhibitors have been
replacement also increased the rate of hydrolysis of the
developed over the years to better understand the ubiquitin–
acyl-enzyme intermediate.[51] This result may explain why
proteasome system. The earliest synthetic inhibitors, the Cterminal 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
relieve inhibition.[44a] It is instructive to note how the activity
tripeptides modified at the C terminus by a vinyl sulfone or
glyoxal act as mechanism-based inhibitors of the proteasoand selectivity of lactacystin are exquisitely controlled by
me.[39b, 45] Boronic acid peptides (in which the aldehyde
intrinsic chemical reactivity.
The structure of the proteasome from Saccharomyces
pharmacophore has been replaced by a boronate group) are
cerevisiae co-crystallized with omuralide[41] was determined
more potent and selective for the proteasome. The flagship
boronate compound, PS-341,[40, 46] was recently clinically
by X-ray analysis, and confirmed the covalent linkage
between the threonine side chain hydroxy group and omurapproved for the treatment of multiple myeloma (velcade
alide. The N-terminal amino group is not modified by the
(bortezomib), Scheme 4). This validates the proteasome as a
natural product. Omuralide makes four hydrogen-bond contherapeutic target for at least this type of cancer and
tacts with the main chain of the proteasome catalytic subunit.
potentially other forms as well.[47] Unlike lactacystin, howThe C9 isopropyl moiety of omuralide projects into a
ever, Velcade reversibly inhibits the chymotrypsin-like activhydrophobic pocket of the proteasome to lend additional
ity of the proteasome.[40] The most clinically advanced
binding affinity. Corey and co-workers have shown that
lactacystin analogue is PS-519 (Scheme 4), a cell-permeable
replacement of the omuralide C9 isopropyl group with a
variant that features an n-propyl substitution at C7. This
phenyl group abolishes all activity.[52] Interestingly, salinocompound is more potent than the natural product and has
entered phase II clinical trials for acute stroke.[40, 48]
sporamide A (Scheme 4), a natural product 35-fold more
potent than omuralide, possesses a cyclohexene moiety in
The reactivity of lactacystin is brought about by its latent
place of the isopropyl group, yet it shares the bicyclic ring
b-lactone group, termed clasto-lactacystin b-lactone or omurstructure of omuralide.[53] Feling and co-workers suggested
alide (Scheme 4).[49] Derivatives that cannot form the blactone 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 proteaAngew. Chem. Int. Ed. 2005, 44, 5788 – 5809
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B. F. Cravatt, E. J. Sorensen, and C. Drahl
some.[53] Corey and co-workers recently reported the first
enantiospecific total synthesis of salinosporamide A[54] with a
procedure that yields sufficient material to carry out biological analyses of the efficacy of salinosporamide A as a
potential anticancer agent. Moreover, congeners of salinosporamide A were recently prepared for the possible treatment of proteasome-mediated disease.[55] It will be interesting
to learn whether salinosporamide A has improved upon the
delicately balanced chemistry of lactacystin.
2.3. E-64
In 1978, the Hanada group pursued a specific inhibitor of
cysteine proteases to expedite investigation of protease
biological functions. They succeeded in isolating a highly
potent and irreversible inhibitor, E-64, from the mold
Aspergillus japonicus.[56] The structure of E-64 was determined principally through classical chemical reactivity testing, IR spectroscopy, and 1H NMR spectroscopy, and was
subsequently confirmed by a chemical synthesis of its
enantiomer.[57] The structure contains agmatine, the arginine
decarboxylation
product
1-amino-4-guanidinobutane
(Scheme 6). To identify an E-64 pharmacophore, a compre-
Scheme 6. E-64 and other l-trans-(S,S)-epoxysuccinic acid derivatives.
hensive library of derivatives and fragments was synthesized.
Both the epoxide and the carboxyl group derived from ltrans-(S,S)-epoxysuccinic acid were found to be indispensable
for inhibition of papain.[58] Notably, other peptidyl epoxide
natural products that act as covalent inhibitors of the
proteasome have also been identified, including epoxomicin[59] and eponemicin[60] (Scheme 6).
In addition to papain, E-64 is a covalent inhibitor of
several other cysteine proteases, including cathepsins B, H,
and L, stem bromelain, and ficin.[61] The revelation that the
high affinity, active-site-directed reversible inhibitor leupeptin decreased the rate of E-64 binding cemented the idea that
E-64 is an active-site-directed inhibitor of cysteine proteases.[61] These proteases exert their catalytic activity with
the assistance of an active-site ion pair between cysteine and
histidine residues.[62] In papain, these residues are Cys 25 and
His 159.[62]
Aberrant cysteine protease activity leads to many disease
states. Inflammatory and traumatic processes, muscular dys-
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trophy, AlzheimerFs disease, cancer, and cataract formation
all have a putative link to cysteine protease dysfunction.[63] In
addition, because cysteine proteases are a critical component
of the Plasmodium life cycle, they are regarded as viable
targets for potential antimalarial drugs.[64] In an effort to
improve the cell permeability of leads, the agmatine group
was replaced with neutral moieties, such as isoamylamine in
the case of E-64c (Scheme 6).[65] In 1986, the ethyl ester of E64c 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
of other mechanistic classes.[63d, 66] However, by virtue of its
inhibition of calpain, this same E-64 derivative has found use
in eye drops for the prevention and treatment of cataracts.[67]
E-64 is still frequently used to facilitate discovery and
classification of newly isolated papain-class enzymes.[68]
Molecular probes for ABPP inspired by the E-64 motif
have proven to be of great value for tracking the activities of
both animal and plant cysteine proteases in whole proteomes,
and in the design and screening of inhibitors for these
enzymes.[68a, b] For example, a screen of plant extracts revealed
cysteine protease activity for three proteins that had not been
previously characterized.[68a] In a comparative screen of
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 broadspectrum 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
atom.[70, 71] Backside attack in SN2 fashion inverts the
configuration at C2 of the product thioether
(Scheme 7). The key contacts between cysteine proteases and E-64 that are responsible for situating the
inhibitorFs epoxide group for regioselective nucleophilic attack have been extensively characterized in a series of
crystal structures.[57, 71, 72] In the structure of the E-64–papain
complex, the N-terminal carboxyl group of E-64 is positioned
in the oxyanion hole, while the carbonyl group adjacent to the
epoxide group interacts with the histidine catalytic base.[71]
The mechanistic characterization of the interaction between
E-64 and cysteine proteases of the papain class is an excellent
example of the power of convergent research efforts in
chemical synthesis, classical biochemistry, and structural
biology.
3. Natural Products that Target “Non-Nucleophilic”
Residues in Enzyme Active Sites
Several natural products inhibit enzymes through the
covalent modification of active-site residues that are not
essential for catalysis. Although some of these residues
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
altogether superfluous for catalysis. In either case, however,
covalent modification of these residues results in enzyme
inactivation. Herein, we highlight selected examples of
natural products that target “non-nucleophilic” residues in
enzyme active sites.
3.1. Fumagillin
A 1949 report demonstrated that concentrates of Aspergillus fumigatus cultures possessed antibiotic activity against
Staphylococcus aureus.[73] Tarbell and co-workers characterized the chemical behavior of the active component, fumagillin, including the acid sensitivity of its characteristic
spiroepoxide unit.[74] The constitution and stereochemistry
of fumagillin were confirmed by X-ray crystallography
(Scheme 8).[75] In 1972, Corey and Snider described their
elegant studies that culminated in the first chemical synthesis
of this natural product.[76] Subsequent work has produced a
26-step synthesis of ( )-fumagillol,[77] the saponification
product of fumagillin, and a 13-step racemic synthesis of
fumagillol.[78] Fumagillin and its close relatives ovalicin[79] and
FR65814 (Scheme 8)[80] possess a rare sesquiterpene carbon
skeleton that has been shown by labeling experiments to
originate biosynthetically from b-trans-bergamotene, which
arises from farnesyl pyrophosphate.[81] Other natural products
not necessarily in the fumagillin family, but which contain a
spiroepoxide moiety include curvularol[82] and FR901464.[83]
Fumagillin and other members of its family target
methionine aminopeptidase 2 (MetAP-2), but not the closely
related enzyme methionine aminopeptidase 1 (MetAP-
1).[6b, 84] Methionine aminopeptidases are metalloproteases
responsible for co-translational removal of the N-terminal
methionine residue in specific protein targets.[6b, 85] These
enzymes were originally thought to use cobalt as the activesite metal center, but more recent studies indicate that
manganese is likely the physiologically relevant cofactor.[86] In
the enzyme active site, the metal ions are coordinated by
Asp 251, Asp 262, His 331, Glu 364, Glu 459, and a water
molecule.[87] The nucleophilicity of the water molecule is
enhanced by coordination to the manganese ion; it attacks the
scissile amide bond of the substrate.[88] MetAP-mediated
removal of methionine is necessary for post-translational
modifications such as myristoylation.[89] A single amino acid
residue in MetAP-2, Ala 362, confers sensitivity to ovalicin,
and a Thr 362 Ala mutation of the human MetAP-1 gene
inserted into yeast results in ovalicin-sensitive yeast colonies.[90]
The connection between inhibition of MetAP-2 and the
medicinal utility of fumagillin appears complex and remains
unclear.[91] It has been speculated that a connection exists at
the level of protein myristoylation.[92] Without removal of Nterminal methionine residues, proteins cannot be myristoylated in vivo, which in turn, could disrupt their localization
and function. Therefore, the hypothesis is that fumagillin
indirectly causes cell-cycle arrest by interfering with myristoylation, which leads to aberrant subcellular localization of
proteins that are not yet identified.[92] Despite a mechanism of
action that remains enigmatic, fumagillin is firmly established
as a potent antiparasitic agent.[93] During the 1950s, fumagillin
was used for treatment of amoebiasis in humans, caused by
Endamoeba histolytica, and is still used today for Nosema
disease in honeybees, caused by the protozoan Nosema
Scheme 8. Fumagillin and other protein-reactive spiroepoxides. TNP-470 and CKD-731 are synthetic compounds.
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apis.[93a, b] More recently, fumagillin has shown efficacy against
microsporidial keratoconjunctivitis of the eye, caused by a
single-celled fungus.[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
CKD-731 (Scheme 8), an analogue that is 1000-fold more
potent against endothelial cell proliferation than TNP-470.[100]
New data suggest that MetAP-2 may not be the biologically relevant target of fumagillin, and that MetAP-2
function is not necessary for the growth of endothelial
cells.[101] However, this study could not completely rule out
a functional role for MetAP-2, because knockdown of the
peptidase by siRNA was incomplete. The authors raised the
intriguing possibility that another MetAP family member
may exist in humans that could also act as a target for
fumagillin.[101]
The key reactive motif of fumagillin is its spiroepoxide
structure.[6] Removal of the spiroepoxide unit results in a
1000-fold decrease in MetAP-2 inhibition.[84] Fumagillin also
possesses a side chain epoxide group; this epoxide was
demonstrated to be dispensable for MetAP-2 inhibitory
activity.[84] In the X-ray crystal structure of human MetAP-2
complexed with fumagillin, a covalent bond between the
imidazole nitrogen (Ne2) of a histidine residue (His 231) and
the methylene of the spiroepoxide is evident (Figure 1).[87] It
has been speculated that His 231 acts as a general base during
catalysis, as it serves as a nucleophile to open the spiroepoxide
ring.[84] It was thought that His 231, along with the binuclear
metal center, could activate a water molecule for attack at the
scissile amide bond, after which another histidine in the active
site, His 339, could protonate the leaving group.[84] However,
the active site geometry in E. coli MetAP-2 does not
corroborate this proposal.[102] Scheme 9 depicts one model
for fumagillin-mediated labeling of MetAP-2.[103] Transition-
Figure 1. X-ray crystal structure of human MetAP-2 complexed with
fumagillin. The covalent bond between C11 of the natural product
(yellow) and Ne2 of the active site His 231 (cyan) is shown; the rest of
the molecular surface of MetAP-2 is colored blue.
state analogues and crystallographic analysis yielded further
insight into the catalytic mechanism of E. coli MetAP,[102–104]
suggesting that His 79 of E. coli MetAP (equivalent to His 231
of human MetAP-2) actually stabilizes the tetrahedral
intermediate that results when the peptide is attacked by an
activated hydroxide through direct interaction with the
nitrogen atom of the scissile peptide bond. Thus, the histidine
residue modified by fumagillin appears to play an important
supportive role in catalysis, consistent with the dramatic
reduction in enzyme activity observed for a His 231 Asn
MetAP-2 mutant.[84] More generally, fumagillin targeting of
MetAP-2, which uses a metal-activated water molecule as the
nucleophile for peptide-bond hydrolysis, underscores the
remarkable ability of natural products to selectively label
the active sites of enzymes that do not themselves engage in
covalent catalysis.
3.2. Wortmannin
The antifungal antibiotic wortmannin (Scheme 10) was
isolated from Penicillium wortmanii and its complex structure
was elucidated by chemical degradation and NMR spectroscopic methods.[105] The hallmark of wortmannin is its reactive
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 binding site is able to open a
protonated epoxide. Fumagillin binding is enhanced at low pH.[103]
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framework.[106] This feature is also found in viridin, demethoxyviridin, the marine-derived pp60v-src protein tyrosine
kinase inhibitors halenaquinone and halenaquinol, and hibiscone C (gmelofuran), which is found in the heartwood of
Gmelina arborea and the Jamaican Blue Mahoe
(Scheme 10).[107]
mycin (mTOR), DNA-dependent protein kinase (DNAPK),[114] myosin light chain kinase (MLCK), the gentamicin
resistance enzyme AAC(6’)-APH(2’’), and a membranebound PI 4-kinase.[115]
PI 3-kinases are involved in a large number of fundamental cellular processes, including apoptosis, proliferation, cell
motility, and adhesion, and have been
implicated in the malignant transformation of cells. Increased levels of PI 3kinase products have been observed in
colorectal tumors and in breast cancers.[116] It was also reported that
dephosphorylation of PI 3-kinase products 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 wortmannin library yielded ten compounds
that were advanced to pharmacokinetic
and
toxicity
studies.[118]
PX-866
(Scheme 10), a diallylamino wortmannin 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 antitumor effects of the established chemotherapeutic agent
Wortmannin[108] and its structural relative viridin[109] are
cisplatin.[119] It was speculated that loss of the furan ring was
biosynthesized from the triterpenoid alcohol lanosterol[110]
redeemed by improved chemical stability or a better fit in the
and are potent cell-permeable inhibitors of the lipid kinase
ATP-binding pocket.[118]
phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme is a
central component of two major receptor-mediated signal
Amine nucleophiles react efficiently with the doubly
transduction pathways:[111] the G-protein-coupled receptor
activated and highly electrophilic strained furan ring of
wortmannin by attacking C20 to give vinylogous carbamates
pathway and the receptor tyrosine kinase pathway. PI 3through an addition–elimination mechanism.[120] Wipf and cokinase phosphorylates at position 3 of the inositol ring of
phosphatidylinositol (PtdIns), leading to the production of
workers recently synthesized a library of 94 C20-substituted
the second messengers PtdIns-3-phosphate, PtdIns-3,4semisynthetic 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
chemistry also occurs in the active site of PI 3-kinase.[121] The
In porcine PI 3-kinase, contact with the a- and b-phosphate
groups of ATP is mediated by the conserved Lys 833 and
e-amino group of Lys 802 of PI 3-kinase (Lys 833 in porcine
Ser 806 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 Asp 946 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 wortmanninthat this particular aspartate is not positioned properly for a
labeled peptides. Fortunately, this group could be reduced
role as catalytic base. Another residue, His 948, 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 interfered with the alkylation of PI 3-kinase by wortmannin when
may not possess a base catalyst. Instead, it may operate
added before the inhibitor, although substances containing
through a dissociative transition state.[113]
nucleophilic amino acid side chain groups had no effect at the
In addition to its well-studied lipid kinase target, wortsame concentration. FSBA is a derivative of adenine that
mannin has been demonstrated to inhibit other serine/
reacts covalently with nucleophilic amino acids and has been
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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
and leaves for pain relief. Willow leaves were
Scheme 11. Lysine 802 in PI 3-kinase opens the doubly activated furan ring of wortmannin by an
addition–elimination mechanism to yield a vinylogous carbamate.
described by the Roman Pliny the Elder in his
Natural History. Today, a modified version of
salicylic acid, the active constituent in willow bark, named
used to map nucleotide binding sites.[121] The PI 3-kinase
acetylsalicylic acid or aspirin (Scheme 12),[126] is one of the
substrate PtdIns-4,5-bisphosphate also effectively competes
with wortmannin binding. These findings led Wymann and comost widely used remedies in the world.
workers to conclude that the wortmannin target site in PI 3kinase 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 Lys 802, as mentioned above, may be the
stabilization of the a-phosphate of ATP in the binding pocket,
as has been speculated for Lys 72 in protein kinase A
Scheme 12. Aspirin and salicylic acid, the natural product from which
(PKA).[121] Lysine 72 anchors the nonlabile phosphate
the drug was first synthetically derived.
groups of ATP and neutralizes their charges by a salt
bridge. Replacement of Lys 72 with Ala in yeast PKA leads
to an 800-fold decrease in the Vmax value.[123] Whereas Lys 72
Salicylic acid and structurally related secondary metabolites, the salicylates, are major defensive compounds that are
of PKA and perhaps Lys 802 of PI 3-kinase appear important
present in nearly all higher plants,[127] and deter the feeding of
for enzyme activity, they probably participate in a primarily
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 pathfor 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 lyase-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 rhodamine-tagged wortmannin analogue
was synthesized and used in proteomic 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 overexpressed in various human cancers.
The wortmannin-based probe successfully detected kinase activity
changes due to treatment with drugs
and in different stages of the cell
cycle. Importantly, these results suggest 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.
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reverses the order of the two steps, resulting first in orthocoumaric acid. The decarboxylation step is thought to
proceed in a fashion similar to the b-oxidation events of
fatty acid breakdown.[127] This was supported by studies in
which the decarboxylation step in cell-free plant extracts was
found to be ATP and CoA-dependent.[128]
In 1860, Hermann Kolbe of Marburg University in
Germany carried out a laboratory synthesis of salicylic acid
and its sodium salt from phenol, carbon dioxide, and sodium.
KolbeFs student, Friedrich von Heyden, established a factory
for the bulk production of salicylates in Radebeul, near
Dresden. The unpleasant side effect of salicylate, gastric
irritation, revealed itself quite rapidly as the drug became
widely available. Felix Hoffmann, a chemist at Bayer,
ameliorated the problem in 1897 by synthesizing a more
easily tolerated derivative, acetylsalicylic acid.[129] Heinrich
Dreser, the director of research at Bayer, coined the term
“aspirin” from a combination of SpirsJure (salicylic acid) with
an initial “a” for acetyl, and a patent was granted in 1899.
For nearly a century, the molecular mechanism of aspirin
remained a mystery. However, in 1971, research by Vane
indicated that aspirin inhibits prostaglandin biosynthesis.[130]
This seminal finding explained both the beneficial properties
and side effects of aspirin, and culminated in the awarding of a
share of the 1982 Nobel Prize in Physiology or Medicine to
Vane.[131] The prostaglandins are paracrine hormones that act
on cells proximal to their place of synthesis. A trauma to cells
results in an increase in local biosynthesis of prostaglandins,
which cause elevated body temperature, inflammation, and
pain.[132] Aspirin was found to acetylate and irreversibly
inactivate prostaglandin endoperoxide (PGG/H) synthase,
also termed cyclooxygenase (COX).[133] In addition, aspirin
acetylates human hemoglobin and human serum albumin.[134]
The COX enzyme has two known isoforms, the constitutive COX-1 and the inducible COX-2.[135] COX enzymes
catalyze the first two steps in the metabolism of arachidonic
acid to prostaglandins. The cyclooxygenase activity incorporates two oxygen atoms into arachidonic acid to produce
prostaglandin G2 (PGG2). PGG2 is then subjected to the
peroxidase section of these enzymes, which catalyzes a
reduction of the hydroperoxy group of PGG2 to the hydroxy
endoperoxide, PGH2.[133b, 136]
It is recognized that aspirin inhibits the cyclooxygenase
activity of the COX enzymes, but not their peroxidase
activity.[137] Aspirin appears to exert its inhibitory effects by
disrupting substrate (arachidonic acid) binding to the cyclooxygenase active site, as the serine residue acetylated by
aspirin (Ser 530 in COX-1, Ser 516 in COX-2) is not critical for
catalysis.[133b]
Studies of active-site COX residues have provided insight
into the mechanism of COX enzymes, as well as that of aspirin
acetylation. The mechanism of acetyl transfer involves several
amino acids in the COX active site, including conserved
tyrosine and arginine residues (Scheme 14).[138] In 1990,
spectral and biochemical studies revealed that Tyr 385 is
essential for COX-1 cyclooxygenase activity.[139] Structurally,
this tyrosine is properly positioned to perform the ratelimiting step of cyclooxygenation: abstraction of the 13-pro-S
hydrogen atom from arachidonate.[139, 140] A Tyr 385 Phe
mutant of COX-2 displays virtually no serine acetylation by
aspirin, and a Tyr 348 Phe mutant displays reduced activity
and aspirin acetylation.[141] The latter result confirms the
importance of Tyr 348, which forms a hydrogen bond to
Tyr 385 and helps to position the substrate. Arginine 120 of
COX-1 is an important residue for both substrate recognition
and imparting sensitivity to inhibitors that contain a free
carboxylic acid.[142] Analysis of a crystal structure of COX-1
suggests that the guanidino moiety interacts with the arachidonate carboxylate group.[143] Furthermore, reversible inhibitors with a free carboxylic acid, such as flurbiprofen, were
ineffective against COX-1 enzymes with a mutation at
Arg 120.[142, 143] The corresponding COX-2 Arg-to-Gln
mutant was later found to have a 73 % reduction of
acetylation of Ser 516 in COX-2.[141]
Based on the above experimental data, researchers have
described a best possible mechanism for serine acetylation by
aspirin (Scheme 14). The amino acid numbering scheme
presented in the following paragraph corresponds to COX-1.
Initial interaction of the carboxylate group of aspirin with
Arg 120 stabilizes its location in the substrate-binding pocket.
The hydroxy groups of Tyr 385 and Tyr 348 then act as an
extended hydrogen-bonding network. This directs the acetyl
group of aspirin toward Ser 530, and increases the reactivity of
aspirin by stabilizing the developing negative charge of the
Scheme 14. An intricate network of hydrogen bonds and other interactions position aspirin to acetylate Ser 530 of cyclooxygenase-1.
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tetrahedral addition intermediate. The possibility of transient
tyrosine acetylation was excluded by testing the acetylation of
a Ser 530 Ala mutant, for which no O-acetyltyrosine was
detected.[141] These data are all corroborated by the X-ray
crystal structure of COX-1 inactivated by bromoaspirin
(bromoacetylsalicylic acid). Bromoaspirin was used so that
its location could be identified definitively in a relatively lowresolution (3.4 L) crystal structure from the presence of the
heavy bromine atom. The phenolic oxygen atom of Tyr 385 is
within hydrogen-bonding distance to the acetyl group.[144]
In summary, an elaborate scheme of hydrogen bonding
and ionic interactions serves to position aspirin in the COX
active site, increasing its effective concentration and enhancing its reactivity, which leads to the selective acetylation of
Ser 530. The X-ray structure also lends insight into the
inhibitory effect of aspirin acetylation. Ser 530 lies in a
highly conserved region[133b] along a hydrophobic channel that
leads from the surface of the enzyme to the catalytic tyrosine.
The aspirin acetyl group extends outward into the channel,
perhaps in a manner sufficient to prevent arachidonic acid
from reaching the active site.[133b, 144]
Finally, it is important to note that salicylic acid also
possesses anti-inflammatory effects, but does not itself
covalently modify COX enzymes. Discussion concerning the
precise mechanism of action of salicylate is ongoing.[145] Thus,
the conversion of this natural product to its semisynthetic
derivative aspirin had the fortuitous effects of conferring
mechanistic irreversibility and anointing principal protein
targets for therapeutic intervention.
3.4. Microcystin
Cyanobacteria in the blue-green algae blooms of contaminated drinking water have been the cause of many animal
deaths. Beginning in 1878,[146] it became evident that blooms
of the genera Oscillatoria, Anabaena, Nostoc, and Microcystis
in freshwater and marine environments were to blame for
related incidents of acute toxicity.[147] The active components,
a family of potent hepatotoxins, were isolated and a vigorous
research effort initiated toward determining their struc-
ture.[148] Although the peptidic nature of the toxins was
confirmed in 1959,[149] their amino acid compositions were not
determined until many years later.[150] The first complete
structure was determined by NMR spectroscopy and MS in
1984,[151] and was soon followed by characterization of other
family members.[152] The toxins are cyclic heptapeptides with
variable l-amino acid residues at two positions labeled X and
Y. They contain an Adda residue, an unusual aromatic bamino acid featuring an unsaturated alkyl chain (Scheme 15).
An unambiguous determination of the configuration of Adda
was published in 1988, confirming the structure as
(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl(4E,6E)-decadienoic acid.[153] Various names were present in
the literature for Adda-containing cyclic peptide natural
products, but ultimately the name microcystin was chosen
(Scheme 15).[154]
The microcystin family comprises more than sixty members;[155] the vast majority differ in the nature of two variable
amino acids, or in the methylation states of the dehydroalanine and/or aspartic acid groups. In naming new microcystins,
two letters are appended to abbreviate amino acids at the
variable positions. Biosynthetic studies on microcystins were
particularly focused on the identification of the origin of the
carbon atoms in the Adda unit and the methylaspartic acid
residue. Methylaspartic acid originates from acetyl-CoA and
pyruvic acid via a methylsuccinic acid intermediate, and the
Adda unit derives from acetate; certain methyl groups are
methionine-derived.[156] Typically, microcystins are biosynthesized non-ribosomally.[157] Analyses of microcystin synthetase
genes have yielded genes for polyketide synthases.[158]
Researchers also sought to understand the molecular
nature of the toxicity of microcystins, and not long after the
structures of these natural products were revealed, it was
determined that they are potent inhibitors of serine/threonine
phosphatases 1 and 2A,[7, 147b, 159] and other less-thoroughly
characterized phosphatase family members.[160] Reversible
protein phosphorylation is a critical mechanism for the
regulation of cellular processes.[161] More than 98 % of protein
phosphorylation in eukaryotic cells occurs on serine and
threonine residues.[160] Serine/threonine phosphatases 1 and
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.
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such diverse events as glycogen metabolism, synaptic plasticity, cell-cycle progression, embryogenesis, apoptosis and
smooth-muscle contraction.[162] The structures, activities, and
inhibition properties of PP1 and PP2A have been reviewed
extensively.[163] Significantly, these enzymes do not possess a
catalytic nucleophile; instead, they activate a water molecule
for catalysis with protein-bound metal ion cofactors.[163]
Indiscriminate inhibition of the many essential functions
performed by PP1 and PP2A may contribute to the severe
toxicity of the microcystins; between one and two micrograms
constitutes a lethal intraperitoneal dose in mice.[147b] Although
the broad toxicity of microcystins undermines their potential
as therapeutic agents, research groups have synthesized
variants and truncated versions of the toxin family to better
understand the properties that confer toxicity and specificity.[164] So far, only one total synthesis of a microcystin family
member has been completed.[165] These natural products have
facilitated the functional characterization of phosphatases
and phosphatase–protein complexes in proteomes.[159b, 166] A
microcystin-LR–rhodamine conjugate reacted with and
detected numerous serine/threonine phosphatases in a crude
cell lysate sample, and could record changes in phosphatase
activity levels.[166] These probes expanded the list of enzyme
families amenable to functional proteomic analysis.
Several bioactive natural products, such as leptomycin B,[167] isoavenaciolide,[168] and pironetin[169] have been
shown to act as carbon-based electrophiles, similar to the
a,b-unsaturated carbonyl element of the methyl-dehydroalanine residue of microcystin. In 1995, two research groups
simultaneously published work confirming the conjugate
addition chemistry of the dehydroalanine moiety of microcystin with PP1 at Cys 273 near the enzyme C terminus.[7, 159a]
In PP2A, the analogous Cys 266 is the targeted residue.[159a]
The amino acid sequence Ser-Ala-Pro-Asn-Tyr-Cys, which
includes the cysteine residue modified by microcystin, is
highly conserved in Ser/Thr phosphatases.[7] The covalent
linkage between the various microcystins and PP1 is stable to
heat and acid. However, covalent binding of microcystin-YR
to phosphatases was not observed when the proteins were
denatured with sodium dodecyl sulfate prior to the addition of
the natural product, indicating that the interaction depends
on an intact active site.[7]
Microcystins bind to PP1 with subnanomolar affinity;[170]
in the crystal structure of microcystin-LR bound to PP1, the
Adda side chain makes contacts with a conserved hydrophobic groove, and the carboxyl group of glutamate and the
adjacent carbonyl oxygen atom coordinate the metal-binding
site indirectly through water molecules.[171] The leucine side
chain of the toxin is situated in a loop of the C-terminal
domain; in addition, the carboxyl group of methylaspartate
hydrogen bonds to Arg 96, a residue crucial for interactions
with phosphate groups on substrates. Hence, access to the
active site is completely blocked.
The extensive interactions between microcystins and
phosphatase active sites suggest that covalent reactivity
contributes only partly to the mode of action of these natural
products. Microcystins immobilized to a Sepharose bead
through an aminoethanethiol adduct retained inhibitory
activity over PP1 and PP2A.[159b] Microcystins-LR, YR, and
RR were converted synthetically to their glutathione and
cysteine conjugates; both of these thiols reacted with the
dehydroalanine group, yet the 1,4-addition products retained
a measure of in vivo toxicity (LD50 = 38 mg kg 1 for microcystin-LR; LD50 = 630 mg kg 1 for its glutathione conjugate).[171] Notably, such adducts may not be resistant toward
retro-Michael reaction in vivo, and reversible adduct formation may explain the observed biological activity.[172] These
results imply that the dehydroalanine residue probably does
not play a role in early enzyme–inhibitor interactions.
Mechanistic studies by Craig and co-workers confirmed a
two-step mechanism for irreversible inhibition by microcystins (Scheme 16).[173] In the first step, the rapid noncovalent binding and inhibition occurs with all the contacts
detailed above. The slower covalent modification of Cys 273
occurs over the course of hours.[173] The tendency for the
conjugate addition reaction is likely enhanced by the high
Scheme 16. Microcystin inactivates PP1 prior to the slow covalent attachment to Cys 273. However, the network of noncovalent interactions is
essential for a successful conjugate addition.
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effective concentration of microcystin in the well-folded
binding pocket, as evidenced by the control reaction with
denatured protein discussed above. It is also not out of the
question that the nucleophilicity of the target cysteine may be
enhanced in its local protein environment. Regardless, it is
important to note that mutagenesis studies have found that
Cys 273 is not required for catalysis; in fact, the activity of a
Cys 273 Ser mutant is indistinguishable from that of the wildtype enzyme.[174] Thus, microcystins provide a salient example
of how an intricate architecture of noncovalent interactions
with enzymes can facilitate covalent modification on noncatalytic active-site residues. Notably, similar strategies have
been employed to convert tight-binding reversible inhibitors
into covalent inactivators of the EGF receptor[175a–c] and p90
ribosomal protein S6 kinase[175d] by the introduction of
electrophilic groups in proximity to cysteine residues in the
active sites of these proteins.
4. Targeting Nonenzymatic Proteins
Although most protein-reactive natural products target
the active sites of enzymes, there are exceptions. Here, we
highlight one such natural product, leptomycin B, which
covalently modifies a receptor protein involved in nuclear
transport.
4.1. Leptomycin B
A Streptomyces strain isolated from soil samples yielded
two novel antifungal antibiotics. The active components were
purified, isolated, and termed leptomycins A and B.[176] These
compounds constitute part of a family of structurally similar
natural products, including callystatin A, ratjadone, kazusamycin, anguinomycin, leptofuranin and leptolstatin
(Scheme 17).[177] Structures of the leptomycins were determined by NMR spectroscopy, MS, and IR spectroscopy.[178]
They feature an unsaturated fatty acid with a terminal dlactone ring. Leptomycin B has a polyketide origin; it may be
traced back to four acetate units, seven propionate units, and
one butyrate unit.[179] Leptomycin B interferes with DNA
synthesis stages of the cell cycle,[180] presumably a result of its
inhibitory effect on nuclear transport.[181] A nanomolar binding partner for leptomycin B was discovered and named
chromosome maintenance region 1 (CRM1).[182] CRM1, also
known as exportin 1, is an evolutionarily conserved receptor
for the leucine-rich nuclear export signal (NES)[183] of
proteins and is an essential mediator of NES-dependent
nuclear export of proteins and ribonucleoprotein complexes
in eukaryotic cells.[182, 184]
In conjunction with CRM1, the protein Rev allows
partially processed HIV mRNAs to be transported outside
the nucleus for translation, enabling the HIV virus to hijack
the cellular protein synthesis machinery.[182, 185] Agents like
leptomycin B that block the interaction of Rev with nuclear
transport proteins have the potential to inhibit HIV-1
replication.[186] Leptomycin B has also been used to accumulate 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
novel therapeutic approach for HIV and cancer treatment.[187, 188]
Leptomycin B binds to CRM1 and covalently modifies
Cys 529 in a central conserved region of the protein, which
inhibits recognition of the NES; ratjadone was recently found
Scheme 17. The leptomycin family of natural products.
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Chemie
Enzyme Inhibitors
to share this mechanism (Scheme 18).[167, 181, 186a, 189] Researchers found that a single mutation, Cys 529 Ser, in Schizosaccharomyces pombe was sufficient to greatly reduce sensitivity
to leptomycin B.[167, 190] To determine which portion of the
an enzyme. Despite this, leptomycin B labels Cys 529 with
exquisite specificity, suggesting that this residue resides in a
structured small-molecule binding site. One intriguing possibility 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 leptomycin 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
Scheme 18. The a,b-unsaturated lactone of leptomycin reacts by
conjugate addition with Cys 529 of exportin.
natural product serves as the pharmacophore, Kobayashi and
colleagues performed extensive studies on a series of
analogues of callystatin. The a,b-unsaturated lactone was
found to be the crucial reactive element.[191] Other research
groups reduced the lactone moiety to a saturated analogue[192]
or formed a Michael adduct with nitromethane;[185a] both of
these events greatly decreased inhibition.[192] Furthermore, Nacetylcysteine methyl ester and leptomycin B reacted to give
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 Cys 529 (Scheme 18).
The role of Cys 529 in CRM1 is not yet clear. This residue
does not seem to be necessary for CRM1 to function. The
Cys 529 Ser mutant mediates nuclear export just as effectively
as wild-type CRM1, and it is not temperature-sensitive.[167]
Moreover, wild-type Saccharomyces cerevisiae CRM1 has a
threonine in place of cysteine at the analogous position.[167]
It is difficult to speculate further about the nature of the
CRM1–leptomycin B interaction, as a structure of the complex has yet to be determined.[188, 193] This information is
crucial to the development of new drug candidates for the
inhibition of nuclear translocation. Leptomycin B itself has a
high toxicity profile, as demonstrated in a phase I clinical trial,
and was not recommended for further development.[194]
Several syntheses of leptomycin B and its relatives have
been carried out in an effort to understand the function of this
family of compounds.[191a, 195] The premise that some of these
reagents might selectively shut down transport of specific
mRNA molecules is enticing and warrants further research.
In summary, CRM1 is a particularly notable target for a
protein-reactive natural product, because this protein is not
Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809
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 representatives 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), cysteine (E-64, microcystin), and histidine (fumagillin). Moreover, these residues do not need to serve a catalytic function
in the target enzyme class (for example, Cys 273 in PP1) or, for
that matter, to be part of an enzyme active site (as is the case
for Cys 529 in CRM1). This underscores the creative ability of
nature to exploit “spurious” nucleophiles in small-molecule
binding sites for the selective modification of proteins.
Although all the natural products discussed herein inactivate
their cognate protein targets by modifying amino acid
residues, other mechanisms of enzyme inhibition are possible.
For example, gabaculine (Scheme 19), a rare amino acid
Scheme 19. The natural product gabaculine is a conformationally
constrained GABA analogue. GABA = g-aminobutyric acid.
isolated from the mold Streptomyces toyocaensis,[196] inactivates several related aminotransferases by covalently reacting
with the enzymeFs pyridoxal phosphate cofactor
(Table 1).[8c, 197]
What lessons might be learned from protein-reactive
natural products that could assist in the design of chemical
probes for the burgeoning field of activity-based protein
profiling (ABPP)?[9] ABPP is a functional proteomic method
that aims to develop active-site-directed probes that label
many enzymes in parallel, thereby facilitating their collective
functional characterization in samples of high biological
complexity. The application of ABPP methods has so far
enabled the discovery of enzyme activities that are upregulated in diseases like cancer,[69, 198] obesity,[199] and
malaria;[200] it has also enabled the design of selective
inhibitors for these enzymes.[201]
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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B. F. Cravatt, E. J. Sorensen, and C. Drahl
Table 1: Representative protein-reactive natural products and their
targets.
Natural
product
Target
Labeled
residue
Catalytic
nucleophile?
lipstatin
lactacystin
pancreatic lipase
proteasome
b subunits
papain
(Cys proteases)
MetAP-2
PI 3-kinase
COX-1
COX-2
PP1
PP2A
CRM1
aminotransferases
Ser 152
Thr 1
yes
yes
Cys 25
yes
His -231
Lys 802
Ser 530
Ser 516
Cys 273
Cys 266
Cys 529
PLP cofactor[a]
no
no
no
no
no
no
no
no
E-64
fumagillin
wortmannin
aspirin
microcystin
leptomycin B
gabaculine
[a] PLP = pyridoxal phosphate.
Their discovery and characterization should continue to offer
fascinating insight into the seemingly endless array of smallmolecule–protein interactions that occur in nature. The
lessons learned from these studies should in turn guide
chemical biologists in their efforts to achieve a paramount
objective of post-genomic science—the design of selective
small-molecule probes for the functional analysis of every
protein.
We thank K. Barglow and Dr. E. C. Taylor for critical reading
of this manuscript, and Dr. M. H. Bracey for assistance with
the generation of figures. This research was supported by the
NIH (CA087660), NIGMS (GM065483), a Bristol MyersSquibb Unrestricted Grant in Synthetic Organic Chemistry, the
Skaggs Institute for Chemical Biology, and Princeton University. C.D. gratefully acknowledges predoctoral fellowships
from Eli Lilly and the National Science Foundation.
Received: March 10, 2005
The profound influence of protein-reactive natural products on the maturation of ABPP can be discerned at several
levels. First, and perhaps most clear, many ABPP probes are
themselves derivatives of natural products, including biotin/
fluorophore-tagged reagents that target cysteine proteases
(based on E-64),[68a, b, 201b] phosphatases (based on microcystin),[166] and kinases (based on wortmannin).[124] A second
class of ABPP probes also borrows a strategy of natural
products by modifying conserved catalytic nucleophiles in
enzyme active sites. These probes, which include fluorophosphonate and electrophilic ketone reagents for serine[202]
and cysteine[203] hydrolases, respectively, typically exhibit a
broad target spectrum within a given family of enzymes. Even
ABPP probes that incorporate photoreactive (rather than
electrophilic) groups could be considered as followers of the
precedents set by natural products, as they too exploit classselective functional groups to bind conserved elements of
enzyme active sites.[204] Finally, the principles derived from
protein-reactive natural products have also inspired the
emergence of a third approach for ABPP that explores the
proteome reactivity of structurally diverse libraries of electrophilic agents.[199, 205] In this combinatorial, or “nondirected”
approach for ABPP, probe libraries have been designed to
contain carbon electrophiles, based in large part on the rich
diversity of natural products that have exploited this versatile
reactive group, to label enzyme active sites (for example,
fumagillin, wortmannin, and microcystin). To date, nondirected ABPP efforts have identified active-site proteomics
probes for more than 10 mechanistically distinct enzyme
classes.[9, 199] Remarkably, most of these enzymes lack cognate
affinity agents, underscoring the value of nondirected ABPP
for the discovery of novel molecular probes of enzyme
function.
Looking forward, we anticipate that our understanding of
the full spectrum of mechanisms employed by natural
products to target protein active sites is still far from
complete. Given the central role that natural products have
played in the elucidation of the biochemical and cellbiological activities of proteins, we strongly advocate the
sustained pursuit of these special pharmacological agents.
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