Download TETHERING: Fragment-Based Drug Discovery

30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
10.1146/annurev.biophys.33.110502.140409
Annu. Rev. Biophys. Biomol. Struct. 2004. 33:199–223
doi: 10.1146/annurev.biophys.33.110502.140409
c 2004 by Annual Reviews. All rights reserved
Copyright °
TETHERING: Fragment-Based Drug Discovery
Daniel A. Erlanson, James A. Wells,
and Andrew C. Braisted
Sunesis Pharmaceuticals, Inc., 341 Oyster Point Boulevard, South San Francisco,
California 94080; email: [email protected]; [email protected]
Key Words disulfide exchange, small-molecule inhibitors, fragment assembly,
structure-based drug design, molecular recognition
■ Abstract The genomics revolution has provided a deluge of new targets for drug
discovery. To facilitate the drug discovery process, many researchers are turning to
fragment-based approaches to find lead molecules more efficiently. One such method,
Tethering1, allows for the identification of small-molecule fragments that bind to specific regions of a protein target. These fragments can then be elaborated, combined with
other molecules, or combined with one another to provide high-affinity drug leads. In
this review we describe the background and theory behind Tethering and discuss its
use in identifying novel inhibitors for protein targets including interleukin-2 (IL-2),
thymidylate synthase (TS), protein tyrosine phosphatase 1B (PTP-1B), and caspases.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
BACKGROUND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fragment-Based Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Finding Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Designing Fragments for Tethering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
TETHERING AS A FRAGMENT DISCOVERY TOOL . . . . . . . . . . . . . . . . . . . . . . .
IL-2: Probing an Adaptive Binding Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IL-2: Combining Medicinal Chemistry with Tethering . . . . . . . . . . . . . . . . . . . . . .
Thymidylate Synthase: Discovering and Elaborating
a Core Fragment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
TETHERING WITH BREAKAWAY EXTENDERS: DISCOVERY OF
A NEW PHOSPHOTYROSINE MIMETIC FOR PTP-1B . . . . . . . . . . . . . . . . . . . .
TETHERING WITH EXTENDERS: TETHERING AS A FRAGMENT
ASSEMBLY TOOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Discovery of Nonpeptidic Caspase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONCLUSIONS AND FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . .
200
200
200
201
204
204
204
208
209
211
214
215
218
218
1
Tethering is a servicemark of Sunesis Pharmaceuticals, Inc. for its fragment-based drug
discovery.
1056-8700/04/0609-0199$14.00
199
30 Apr 2004
18:12
200
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
INTRODUCTION
Modern molecular biology has overwhelmed drug discovery organizations with
myriad novel drug targets. These targets have not only expanded the familiar
families of G protein–coupled receptors (GPCRs) and enzymes that most drugs
address but have provided new families of enzymes and protein-protein targets.
A major challenge for the drug discovery community is validating this wealth
of targets with small molecules, and ultimately developing drugs for the more
promising targets.
The problem of finding a good small-molecule starting point is one of searching chemical space. The number of possible small (up to 30 nonhydrogen atoms)
drug-like molecules is estimated to be greater than 1060 (8a). Fortunately, many
solutions exist to interrogate a site of interest with small molecules. Structural biology, biochemistry, enzymology, biophysics, and molecular modeling can define
key binding interactions and restrict the chemical search dramatically. Moreover,
the drug discovery process is not purely random. Medicinal and combinatorial
chemistry can improve the binding affinity of a weak starting compound. Nonetheless, given the vastness of chemical space, finding good starting points remains a
significant bottleneck in the drug discovery process.
One way to make chemical diversity space more manageable is to search for
smaller molecules, or even fragments of molecules, and then merge or elaborate
these fragments. Discovering drugs in pieces can reduce the dimensionality of
the search and dramatically improve the chances of finding good starting points
for the drug discovery process against novel drug targets. This review describes a
powerful new technology for doing so.
BACKGROUND
Fragment-Based Drug Discovery
The notion that entities with weak binding affinity can be linked to form higheraffinity molecules was first shown for ligands binding to metal ions, giving rise to
the so-called “chelate effect.” Jencks (38) applied this theory to small molecule–
protein interactions. An early test of this theory (51) showed that free energies
for the binding of fragments of substrates and analogs to beta-hydroxy-betamethylglutaryl coenzyme A (HMG-CoA) reductase were additive and, when corrected for the linking energy, approximated the binding affinity of the parent
molecules. Recent structural work comparing the binding of parent compounds
with that of fragments derived from them in thymidylate synthase neatly shows the
fragments binding in a fashion similar to that of moieties in the parent compound
(69). The concept of additive binding effects has been widely exploited in both
medicinal chemistry and protein engineering.
Additive binding allows the search for drugs in pieces, which offers a tremendous combinatorial advantage over discovery of drugs intact. For example, to
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
201
produce a million-compound library (the size of many pharmaceutical compound
collections), one could link 1000 different fragments asymmetrically in all binary
combinations. Assuming one can screen the fragments separately and then link
them, the diversity of this huge library could be gleaned from a much smaller
collection of compounds, in this case just over 1000. This huge combinatorial
advantage is one of the main drivers for the development of fragment-based drug
discovery methods, but there are additional advantages to fragment-based drug discovery as well. Small fragments can gain access to smaller nooks in the protein that
may not be easily accessible to a larger, nonoptimized scaffold. Small fragments
are synthetically more accessible and often more soluble. Small libraries (of small
fragments) are easier to maintain and fully characterize than massive compound
collections. And “growing” a molecule from a small scaffold can offer a much
simpler chemical pathway than does remodeling a larger and more complicated
scaffold while maintaining a constant molecular weight.
Finding Fragments
NON-TETHERING METHODS Detecting fragments that bind a target can be challenging. Traditional high-throughput screening (HTS) typically relies on inhibition assays. But high concentrations of compound may be needed to find weak
inhibitors. For example, an inhibitor with an IC50 of 1 mM produces less than
10% inhibition when screened at 100 µM, an effect that may fall within the background error range of the screen. Moreover, HTS that employs high compound
concentrations is often not practical, either because many molecules are insoluble
or because it requires prohibitively large amounts of compound. Even more significant is the problem of false positives: Causes other than the desired one-to-one
binding interaction can yield assay inhibition. A variety of chemotypes has been
identified as nonspecifically chemically reactive, and strongly colored compounds
can be problematic in spectrophotometric screens (61). Recently, Shoichet and
colleagues (49) have demonstrated that a number of molecules can act “promiscuously” against a wide range of targets, probably by forming large aggregates.
In response to these pitfalls, many researchers have pursued “positive” selection
procedures (75). The rationale is that, whereas many factors may interfere with
an assay and produce a negative signal that would be misinterpreted as inhibition,
fewer artifacts can produce a positive signal.
The three techniques most frequently used for positively detecting fragments are
nuclear magnetic resonance (NMR), X-ray crystallography, and mass spectrometry (MS). Of these, NMR screening has been most extensively utilized for fragment
discovery. NMR screening can be performed at high compound concentrations and
often provides a good understanding of binding interactions. Structure activity relations by NMR (SAR by NMR), a technique developed by Fesik and coworkers (45,
67), is a powerful and versatile strategy. Other research groups (8, 22, 23, 37) have
developed complementary approaches. X-ray crystallography is unmatched in the
level of detail it provides to deconstruct small molecule–protein interactions, and
30 Apr 2004
18:12
202
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
several groups (7, 44, 53) have used crystallography to discover and develop fragments. Crystallography can also be combined with NMR to yield a hybrid approach
(76). Finally, MS can also provide a positive detection method. Although information available about the site of binding is usually more limited than that obtained
with NMR or crystallography, MS can be extremely high throughput (41, 70).
TETHERING In general, the preceding three methods of fragment discovery rely
on noncovalent interactions between the target protein and small-molecule ligands,
requiring high ligand concentrations for detectable occupancy on the protein. In
contrast, Tethering relies on reversible covalent bond formation between the fragment and the protein of interest. This in effect amplifies the affinity of the fragment
for the target molecule, enabling detection at lower concentrations. The method
also controls the region where selected fragments bind on the protein. Tethering
requires relatively little protein (10–50 mg to screen more than 10,000 fragments)
compared to other fragment-based approaches and is not limited by protein size
or crystallization properties. The only essential requirements for Tethering are a
coarse three-dimensional model of the protein target and the ability to analyze the
protein by MS. Tethering also provides an in situ linking procedure not afforded
by other methods (see below).
The basic method of Tethering is shown in Figure 1 (19). If the target protein
contains a cysteine residue within or near the targeted site, it can be used directly.
Otherwise, site-directed mutagenesis can introduce a cysteine residue. The cysteine
residue should generally be within 5 to 10 Å of the site of interest and should be
relatively surface exposed to facilitate thiol-disulfide exchange (see below). Once
the cysteine is in place, the protein is reacted with a library of disulfide-containing
fragments under partially reducing conditions. In theory, the cysteine should form
a disulfide with each of the fragments, and the exchange should occur rapidly.
Assuming that the reactivity of the disulfide in each fragment is equivalent (see
below), and assuming no noncovalent interaction between the fragments and the
protein, the mixture at equilibrium should consist of the protein disulfide-bonded
to each fragment in equal proportion. However, if one of the fragments has inherent
Figure 1 Tethering fragment-based drug discovery.
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
203
Figure 2 Simplified schematic showing some of the equilibria in Tethering. Fragments
with inherent affinity for the protein (Square B) have a more stable disulfide bond and
predominate at equilibrium.
affinity for the protein of interest, and if it binds to the protein near the introduced
cysteine, then the thiol-disulfide equilibrium will be shifted in favor of the disulfide for this fragment, and this protein-fragment complex will predominate. In
other words, the fragment will be “selected” by the protein. The overall level of
modification can be tuned using a reductant, such as 2-mercaptoethanol (2-ME).
Figure 2 illustrates some of the competing reactions that can occur in a simplified system. Multiple equilibria are possible for a reaction consisting of just two
disulfide-containing fragments, the protein, and a reductant. Even in this simplified case, more than a dozen species will be present. However, if only the protein
species are considered, only four possibilities exist: protein alone, protein + reductant, protein + fragment A, and protein + fragment B. The dominant protein
species can be identified with MS. If each fragment in a given pool has a unique
molecular weight, MS also reveals which fragment binds predominantly to the
protein and, thus, which has the highest intrinsic binding affinity for the protein.
We have demonstrated that a single compound can be selected from a pool of as
many as 100 different fragments (19). Because of resolution limits in the mass
determination of larger proteins and the small size and mass redundancy of the
fragments (generally between 100 and 250 Da), pools of around 10 compounds
are most convenient for screening. To facilitate identification, the pools are constructed so that each fragment differs by at least 5 Da from any other fragment in
the same pool.
Tethering is related to dynamic combinatorial chemistry (DCC), a concept in
which reversible equilibria are exploited to generate receptors or ligands in situ
from fragments (32, 43, 60). The system is generally set up to thermodynamically
select molecules that have a desirable property (for example, high affinity to a
30 Apr 2004
18:12
204
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
receptor) through bond-forming and bond-breaking reactions among a group of
fragments. A variety of bond-forming reactions have been investigated in the case
of DCC, including metal-ligand interactions, oxime formation, imine formation,
boronic acid ester formation, disulfide formation, and others (60). In theory, any
of these chemistries could be used for Tethering. In practice, the thiol-disulfide
exchange is ideal. The cysteine residue is naturally occurring and therefore easy
to introduce into proteins. The disulfide bond can be formed and broken under
extremely mild conditions with no other effects on biological molecules. The
equilibria in a thiol-disulfide exchange system can be tuned to more or less stringent conditions merely by altering the ratio of free thiol to disulfide (26). Finally,
previous research has shown that the stability of intermolecular (56, 68) and intramolecular (14) disulfide bonds can be coupled to the binding energies of the
interacting molecules. A recent example describes using disulfide exchange to
select peptide ligands to the translocase protein Tom20 (55).
Designing Fragments for Tethering
To be useful for drug discovery, fragments should be small (molecular weight
preferably lower than 250 Da), heavily functionalized, and contain no “toxicophores” or other malignant functionalities. Several researchers have written
about the design of compounds for fragment-based discovery methods (23, 36).
Fragments for Tethering must also contain a thiol or disulfide bond. Though
synthetically simple, few disulfide-containing small molecules are commercially
available, so it is necessary to custom build the fragment library. For convenience,
each molecule contains a unique fragment on one side of the disulfide and a
common, small, solubilizing group such as aminoethanethiol on the other side.
A critical assumption unique to Tethering is that the disulfide moiety in each
fragment has a similar reduction potential. In reality, the electronic and steric
context of a disulfide affects its overall stability (31, 42). Because at least a couple
atoms separate the disulfide bond from the variable portion in our fragments, we
observe only small differences in reactivity. Moreover, to minimize the impact of
these differential reactivities, we group molecules so that all fragments in a given
pool have the same connectivity between the variable portion and the disulfide
bond.
TETHERING AS A FRAGMENT DISCOVERY TOOL
IL-2: Probing an Adaptive Binding Site
Protein-protein interfaces are considered one of the most difficult target classes
to inhibit with small molecules, yet they also represent a large and important
class for therapeutic intervention (13, 73). In contrast to enzyme targets, where
substrate analogs can provide starting points and where the active site offers
both a binding pocket to exclude solvent and side chain functionality for binding
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
205
interactions, protein-protein interfaces tend to be large, relatively flat surfaces. Few
well-characterized small-molecule ligands inhibit protein-protein interactions, and
those that have been described are often high molecular weight or exploit an allosteric site.
The cytokine interleukin-2 (IL-2) is a critical component in the immune response (52). Binding of IL-2 to its hetero-trimeric receptor (α, β, and γ chains)
induces T cell proliferation, and IL-2 has been implicated in several immune system disorders. Two antibody therapeutics that target the IL-2α receptor, Zenapax
(Roche) and Simulect (Novartis), have been approved for treating graft-host disease associated with renal transplantation. The efficacy of these drugs validates
the manipulation of the IL-2 signaling pathway in treating diseases of the immune
system and identifies IL-2 as an important therapeutic target. Unlike these protein
therapeutics, small molecules offer the potential for oral bioavailability and more
controlled pharmacokinetics.
IL-2 is also one of the few protein-protein targets for which a well-validated
small-molecule inhibitor has been identified. Hoffman-La Roche identified
a molecule (compound 1, Figure 3, IC50 = 3 µM) that binds specifically and
reversibly to IL-2 (72). We determined the structure of the complex between 1 and
IL-2 to pinpoint the binding site’s location and the specific interactions between
1 and IL-2 (5). The X-ray structure revealed a striking transformation: The surface of unliganded IL-2 is relatively flat and offers no obvious binding pockets,
whereas the structure of the complex reveals a series of significant side chain and
loop movements that create a hydrophobic binding pocket accommodating the
biaryl-alkyne of 1 while the guanidine group forms a bidentate salt bridge with
E62 (Figure 4A). Intriguingly, the region where the small-molecule binding site
is located closely overlaps with the residues that have been implicated as critical
for IL-2Rα binding by alanine-scanning mutagenesis (65, 79). The commonality
between the receptor binding site and the ligand binding site suggests that this
region is a true binding hot spot (17).
The observation that substantial rearrangements occurred in the bound state
established that the binding site on IL-2 was mobile and adapted to 1 upon binding. Thus, targeting these adaptive regions may identify binding sites that are not
obvious from the native protein conformation and that, because of the lability of
the structure, are not amenable to structure-based design.
We applied Tethering to address this binding site with several goals in mind.
First, we wanted to identify fragments that bind to this site to better understand the
kinds of chemical functionality preferred in this region. Second, we were interested
in determining whether these fragments could be used to create new lead molecules
or whether the fragments could be used in conjunction with a medicinal chemistry
program to advance low micromolar hits into high-affinity ligands.
We first prepared a panel of 11 cysteine mutants (Figure 4B) targeting the region
surrounding the binding site of 1 (5). To confirm that the protein was correctly
folded, each mutant was evaluated for its ability to bind IL-2Rβ. Those mutations that were at positions already identified as important for IL-2Rα binding by
30 Apr 2004
18:12
206
AR
¥
WELLS
¥
AR214-BB33-10.sgm
BRAISTED
AR214-BB33-10.tex
ERLANSON
LaTeX2e(2002/01/18)
P1: FHD
Figure 3 IL-2 ligands.
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
207
alanine scanning also affected IL-2Rα binding in our screens. Using Tethering, we
screened all cysteine mutants against a library of approximately 7000 fragments.
The redox conditions used for Tethering were tuned such that hits occurred
at a frequency of 0.1% to 1%. Certain cysteine mutants selected many more hits
than did others; these cysteines mapped to the adaptive binding region, whereas
the hit-poor mutants generally targeted the more fixed regions of IL-2. Many hits
identified in the adaptive region had structural similarities, suggesting specific
SAR. Different cysteine mutants rarely selected the same hits, although similar
fragments with different length linkers were occasionally selected by cysteines
that could access the same regions.
Because the binding of 1 alters the structure of IL-2, we investigated its influence
on the selection of fragments through Tethering. By running the experiments in
either the presence or absence of 1, we determined that some fragments were
competitive with 1 and that others were selected synergistically (35). This approach
has the potential to rapidly identify complementary fragments in the absence of
structural information and thus to overcome a key challenge of fragment assembly:
identifying which fragments can be productively linked.
We initially focused our attention on one of the strongest hits, an indole
glyoxylate-derived fragment (compound 2, Figure 3) that was selected by Y31C
in the absence of 1. A Tethering SAR study of 2 was accomplished by synthesizing analogs containing a common disulfide linker. By pooling the fragments
and allowing them to compete for binding and concomitant disulfide bond formation, Tethering enabled a rapid rank ordering of the fragment binding strength.
Selected compounds were tested as IL-2 inhibitors in an ELISA assay and were
found to inhibit IL2-Rα binding in the 500–600 µM range, suggesting that the
fragments identified through Tethering can be quickly modified to generate weak
but measurable binding activity (see also Thymidylate Synthase, below).
The crystal structure of 2 and IL-2 Y31C was solved, revealing the binding site
for this fragment (Figure 5A). The overlap between the phenyl ring of the fragment
2 and the phenyl ring of 1 is close, suggesting that this adaptive region contains a
consensus aromatic binding pocket. Fewer rearrangements occur upon fragment
2 binding than upon compound 1 binding. Most strikingly, F42 is in the wild-type
conformation compared to the “flipped” conformation observed with the binding
of 1.
The close overlay between 1 and 2 suggested a fragment merging strategy:
Compound 3 (Figure 3), which replaces the phenyl ring of 1 with the indole
glyoxylate 2, was synthesized and found to have an IC50 value of 1.5 µM, a
twofold improvement over 1. The crystal structure of 3 bound to IL-2 was solved,
demonstrating that 3 bound IL-2 as expected (J.D. Oslob, A.C. Braisted, M. Randal
& M.R. Arkin, unpublished data). The orientation of the indole fragment is not
a perfect match with the structure of the covalently bound fragment (Figure 5B),
which may explain why only a modest improvement in activity was observed.
Nonetheless, this result demonstrated the feasibility of merging an established
lead molecule with fragments discovered by Tethering.
30 Apr 2004
18:12
208
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
IL-2: Combining Medicinal Chemistry with Tethering
Our interest in IL-2 as a target led us to pursue 1 with medicinal chemistry as well.
A chemistry effort provided a novel compound series exemplified by compound 4
(Figure 3), but this series struck an activity plateau in the low micromolar range.
We decided to craft new compounds by merging 4 with fragments identified using
Tethering (9).
Because most of the fragments were selected on the adaptive region of IL-2, this
area appeared to have significant potential binding energy. Y31C and L72C were
two of the more hit-rich mutants, and both mutants showed a statistical preference
for small aromatic carboxylic acids. We suspected that hits selected from Y31C
(the mutant that selected 2) might also overlap with the lead compounds, so we
focused on L72C. L72C is located on an α-helix, and analysis of similar structures
from the PDB indicated three possible vectors to orient the fragment. Two of these
vectors would direct the fragment toward solvent, but the third would bury the
fragment into the adaptive region with only minor side chain rearrangements. We
were unable to obtain fragment-conjugated crystal structures, and the adaptivity
of this region makes modeling highly speculative, but the suggestion of a proximal
accessory site indicated how to merge fragments with the soluble lead. On the
basis of the length of the linker and the predicted vector of the disulfide bond, we
designed and tested a series of 20 compounds that attach an aromatic ring with a
predominately acidic functionality off the 4 position of 4 using a two-atom linker.
While the activity of 4 was only 3 µM, 8 of the 20 compounds that merged 4
with fragments from Tethering had nanomolar activity, representing improvements
between 5- and 50-fold (Figure 6). All of the more potent compounds contained a
carboxylic acid, as the results from Tethering had indicated. The most potent compound, 7, inhibits the IL-2:IL2Rα interaction with an IC50 of 60 nM, which makes
it the most potent small-molecule inhibitor yet characterized against a cytokine
target. Subsequent crystallography has revealed the binding mode of 7 (70a). Perhaps most important is the efficiency with which this molecule was identified.
More traditional efforts to improve this series by modifying the core failed to
Figure 6 IC50 values for IL-2 inhibitors designed by appending fragments from Tethering
onto a known binder.
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
209
improve affinity. By incorporating the fragments from Tethering and thereby introducing larger, more directed functionality, we accessed a previously unknown
subsite. Making such large changes to a lead series without any indication of potential benefit is inefficient. Integrating Tethering with medicinal chemistry guides
not only the direction of growth but also what types of chemical functionality to
incorporate.
Thymidylate Synthase: Discovering and Elaborating
a Core Fragment
In addition to identifying novel sites on a protein, Tethering can identify novel
fragments in a known ligand binding site. This strategy can be particularly powerful
when combined with existing information about the active site, as any new fragment
can be merged with elements from known inhibitors to produce new, high-affinity
inhibitors. The first published example of Tethering did just this (19).
The enzyme thymidylate synthase (TS) is an ancient, highly conserved enzyme
present in virtually all organisms. It plays a critical role in the nucleotide synthesis pathway and is especially important in actively dividing cells where DNA
is rapidly synthesized. As a result, it is a prominent cancer target; the widely
prescribed chemotherapeutic agent 5-fluorouracil targets this enzyme. Mechanistically, the enzyme relies on a cysteine located in the active site to facilitate catalysis. Moreover, the catalytic pocket is large, as the enzyme needs to
be able to bind both deoxyuracil monophosphate (dUMP) as well as the cofactor (6R)-5,10-methylenetetrahydrofolic acid (mTHF). The Escherichia coli
version of the enzyme has been extensively studied both crystallographically
and mechanistically (15). These factors made TS an ideal choice for developing
Tethering.
We initially screened TS against a small library of about 1200 disulfidecontaining fragments. Although the TS construct we used contains four cysteines in
addition to the active-site cysteine, examination of the crystal structure and preliminary modification studies demonstrated that only the active-site cysteine is accessible for disulfide-exchange. Of all of the compounds screened, N-arylsulfonamideproline derivatives were most strongly selected, represented by N-tosyl-proline.
Substituents around the phenyl ring showed some variability, but the presence of
the proline ring itself was essential.
To understand this SAR, we determined the crystal structures of N-tosyl-Dproline bound to TS, with and without the disulfide linker, and compared its position with the natural substrates (Figure 7). Significantly, the presence of the
covalent linker minimally affected the overall structure (Figure 8) (19). The crystallographic structure of N-tosyl-D-proline bound to TS shows a hydrogen bond
between a sulfonamide oxygen and Asn177 on TS as well as hydrophobic interactions between the proline ring itself and Trp80; altogether, about 80% of the
small molecule is buried. When we mutated the active-site cysteine to a serine and
introduced a new cysteine residue nearby (L143C), we were also able to select the
30 Apr 2004
18:12
210
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
Figure 9 Inhibition constants (Kis) for N-phenyl-sulfonamide-proline derivatives.
N-tosyl-proline fragment, demonstrating that the same hit can be found from differently placed cysteine residues. X-ray crystallography showed that the fragment
bound similarly to the noncovalent and C146-linked structures, as shown in Figure
8 (19). Thus, the disulfide linkage itself has at most a modest effect on fragment
binding.
Enzymatic assays demonstrated that N-tosyl-D-proline (compound 8, Figure 9)
inhibits TS competitively with respect to dUMP, albeit with a low activity (Ki =
1.1 mM), a level that would almost certainly not have been detected in conventional
HTS. Superimposing the crystallographic structure of fragment 8 with that of TS
bound to its cofactor mTHF (34) (Figure 7) revealed that appending a glutamate
residue at the 4 position of the N-tosyl group would mimic the glutamate residue
of mTHF. Synthesis of this compound 9 (Figure 9) does in fact result in a 30fold increase in activity. The SAR around this glutamate residue indicates that
the α-carboxylate is important for binding (compare compound 9 with compound
11 or compound 12), whereas the γ -carboxylate is much less important (compare
compound 9 with compound 10). In general, the SAR around the glutamate moiety
is similar to that previously observed for mTHF (40) and mTHF mimics (47).
Next, we elaborated the proline side chain to try to further improve the affinity. Compound 13 (Figure 9) revealed that the negative charge is not necessary,
whereas compound 14 showed that some substituent is essential for the inhibitor
to be competitive with respect to dUMP; this observation is consistent with the
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
211
structure shown in Figure 7, which reveals that this side chain is the only portion
of the molecule that extends into the dUMP binding site. These data suggested the
possibility of trying to mimic the phosphate moiety of dUMP directly, which led
to the synthesis of compound 15, which has a Ki of 330 nM.
Introducing an acidic side chain off the proline ring enhanced affinity significantly (compare compound 15 and compound 13). Compound 16, which is
nearly isosteric with compound 15, has a much lower affinity, indicating that the
acid moiety is essential. Compound 17, with a positively charged substituent,
is only partially competitive, suggesting that its side chain does not occupy the
dUMP binding site. To discover the origins of this SAR, we determined the crystal
structure of compound 15 (Figure 10) bound to TS. Compound 15 forms a wellordered complex with TS in which the β-alanine side chain is involved in multiple
hydrogen-bond contacts with residues Tyr209, His207, and Ser167. The first two
of these residues normally interact with the 30 -hydroxyl of the substrate dUMP.
This example demonstrates how Tethering can be used to discover a core fragment that binds to the active site of an enzyme. Structure-based design allowed us
to rapidly improve the affinity of this millimolar hit by more than three orders of
magnitude.
TETHERING WITH BREAKAWAY EXTENDERS: DISCOVERY
OF A NEW PHOSPHOTYROSINE MIMETIC FOR PTP-1B
In the case of TS, we used Tethering within the active site to discover a novel core
fragment that we then elaborated to enhance affinity. This was successful partly
because TS has a large, open active site. However, for many enzymes the active
site is smaller and more easily disrupted. A case in point is the class of protein tyrosine phosphatases, or PTPs. These enzymes, which remove the phosphate group
from phosphotyrosine (pTyr) residues in peptides and proteins, are increasingly
recognized as an important but difficult class of drug targets (39, 78). One of the
primary challenges associated with these enzymes is the fact that the active site has
evolved to recognize the highly charged and therefore nondrug-like pTyr residue
with great specificity. Several research groups have sought to find replacements
for the pTyr residue (3, 11, 45, 54), but few compounds have made the transition
into clinical trials.
PTPs contain a conserved cysteine residue in the active site that is highly reactive
toward electrophiles (4, 28), and may in fact be regulated in vivo through disulfide
bond formation with small endogenous thiols (6). However, the active site is deep
and narrow, with room for little other than the pTyr residue itself. Therefore, we felt
that Tethering from the native active-site cysteine would not be useful for discovering pTyr replacements, as any identified fragments would likely bind outside of the
active site. At the same time, we were reluctant to introduce mutations too close to
the active site for fear of disrupting the fragile and highly tuned catalytic machinery.
30 Apr 2004
18:12
212
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
Figure 11 Tethering with breakaway extenders to identify new fragments within an
active site.
To access the active site for Tethering without disrupting it, we developed Tethering with breakaway extenders, as illustrated in Figure 11 (21). In this approach,
we first introduce a cysteine residue well outside of the active site. We then modify
this residue with a cleavable “breakaway extender,” which, when cleaved, positions a thiol close to the active site. The newly introduced thiol is then accessible
for Tethering. We chose to develop the approach on the antidiabetic target PTP-1B,
which is well characterized structurally, biochemically, and physiologically. We
mutated Arg47 to a Cys (R47C), which places a thiol near the pTyr-1 binding site
(62, 64), but places the mutation well outside the active site.
To validate the concept, we first synthesized prototype extender 18 (Figure 12).
This molecule contains a bromoacetamide moiety designed to react with the R47C
residue as well as an oxalamic acid moiety, a common pTyr mimetic designed to
bind in the pTyr binding site. Earlier experiments demonstrated that simple bromoacetamides react rapidly with the active-site cysteine C215. However, the pTyr
Figure 12 PTP-specific breakaway extenders.
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
213
mimetic part of the prototype extender binds in the active site, sterically blocking
the active-site cysteine from reacting with the bromoacetamide. Binding of the
oxalamic acid moiety in the active site also positions the bromoacetamide to react
with R47C. The design was confirmed by X-ray crystallography, which demonstrated that the prototype extender 18 binds as expected. In fact, the oxalamic acid
moiety superimposes closely on the pTyr moiety of a typical peptide substrate (21).
Having demonstrated that we could protect the active-site cysteine by plugging the pTyr binding pocket and simultaneously modify R47C, we next designed
breakaway extender 19 (Figure 12). Although this extender is roughly the same
length as the prototype extender 18, there are several differences, most notably
the presence of the thioester bond in the middle. Nonetheless, we found that this
breakaway extender cleanly and selectively modified our PTP-1B at R47C, and
we could cleave the thioester efficiently with hydroxylamine.
We then screened the modified protein against our library of approximately
15,000 disulfide-containing fragments. We discovered a number of hits, virtually
all of which are negatively charged. The pyrazine fragment 20 (Figure 13) was of
particular interest: It was by far the strongest hit, and it demonstrated remarkably
sharp SAR. In particular, changes to the cyclohexyl group or removal of the negative charge completely abolished binding. To determine the origin of this SAR, we
first grew crystals of the modified PTP-1B with the cleaved extender and soaked
these with the pyrazine disulfide. X-ray crystallography then revealed the precise
interactions that give rise to the SAR (Figure 13). Particularly noteworthy is the
fact that the cis-cyclohexyl group, which was seen to be critical, sits snugly within
a hydrophobic pocket created by three protein residues. The acid substituent off the
pyrazine is likewise involved in several hydrogen-bond interactions. In addition,
we found that PTP-1B adopts the “open” conformation, in which the “WPD-loop”
that normally packs down against the pTyr is open. Although this conformation has
been observed in a few other pTyr mimetic structures, it is relatively uncommon
and suggests an additional mechanism to gain specificity.
Figure 13 Schematic of selected fragment 20 bound to PTP1B (bb, backbone).
30 Apr 2004
18:12
214
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
Inhibition studies revealed that the pyrazine-cyclohexyl moiety 20 possesses a
relatively weak affinity (Ki = 4.1 mM), but this is actually comparable to pTyr
itself (Km = 4.9 mM) as well as other small pTyr mimetics that have subsequently
been optimized for affinity (3, 11, 54). Therefore, this novel fragment may be a
suitable starting point for a medicinal chemistry effort.
TETHERING WITH EXTENDERS: TETHERING AS A
FRAGMENT ASSEMBLY TOOL
In all of the preceding cases, we used Tethering to identify novel fragments. In the
case of IL-2, we identified an adaptive region of the protein and used some of the
newly identified fragments to enhance a known inhibitor. In the case of TS, we
used structure-based design and medicinal chemistry to optimize the affinity of a
low-affinity hit from Tethering. In the case of Tethering with breakaway extenders
on PTP-1B, we identified a novel pTyr mimetic. But optimizing the binding affinity
of a fragment that binds weakly is often not straightforward, particularly in the
absence of structural information. Ideally, we want to use Tethering not only to
identify fragments but also to incorporate them into a larger molecule to create a
high-affinity inhibitor. To this end, we developed Tethering with extenders (20).
Effectively, this transforms Tethering from a fragment discovery tool to a fragment
assembly tool.
Figure 14 shows a schematic of this approach. As in Tethering with breakaway extenders, we first introduce an extender into the protein of interest; this is
a molecule that (a) has inherent binding affinity for the target protein, (b) reacts
with a specific cysteine (or other residue) in the protein of interest, and (c) contains a (masked) thiol. Fragments identified from an initial Tethering screen are
good candidates for converting into extenders. Whereas breakaway extenders were
cleaved to remove the binding element of the extender, the present technique uses
the binding element as a springboard from which to probe for new fragments: The
Figure 14 Tethering with extenders to identify fragments in the context of a larger
molecule.
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
215
whole assembly is screened against the disulfide library to identify hits that bind
to the protein and also form a disulfide bond to the extender. In effect, this forces
the selection of fragments that bind to the protein of interest in the presence of a
first binding element. Because the linkage length through the disulfide is known, a
synthetic molecule containing binding elements from the extender as well as from
the fragment can be rapidly constructed, in which the disulfide is replaced by a
more stable, drug-like moiety. The resulting molecule should have higher affinity
for the target protein than do either of the component fragments alone.
Discovery of Nonpeptidic Caspase Inhibitors
In developing the approach, we first considered proteins that contain a nucleophilic
active-site cysteine. Such proteins often have known small-molecule inhibitors that
interact with this cysteine and that could serve as starting points for extender design.
Cysteine proteases are a large class of enzymes that contain a catalytic cysteine
residue, have known covalent inhibitors, are pharmaceutically relevant, and are
ideal test cases. The first example to which we applied this approach was the
enzyme caspase-3, a member of the cysteine aspartyl protease family. Caspase-3
is a central executioner in the apoptosis pathway leading to programmed cell death
and has been investigated as a point of therapeutic intervention in diseases ranging
from Alzheimer’s and Parkinson’s to myocardial infarction and sepsis (48, 77).
As do all members of the caspase family, caspase-3 has a linear peptide binding site that neatly accommodates four amino acids residues; tetrapeptides have
been used as starting points for small-molecule inhibitors (71). The most critical
recognition element for all caspases is an aspartyl group immediately preceding the substrate amide bond: The aspartyl residue binds in a highly conserved
pocket in the enzyme. Irreversible inhibitors have been designed by starting with
aspartyl-containing peptides and replacing the C-terminal amide with a ketone
functionalized with a good leaving group alpha to the carbonyl (18, 25). We used
this template to design extender 22, as shown in Figure 15. This extender contains the essential aspartyl group with a reactive arylacyloxymethylketone and a
cleavable thioester. We found that this molecule reacted rapidly and cleanly with
caspase-3, and the thioester was deprotected to give a modified protein that was
then subjected to Tethering. By far the most prominent hit was the salicylic acid
shown at the bottom of Figure 15.
Because we knew the molecular connectivity of the salicylic acid fragment to
the extender, we could readily synthesize an initial “diaphore” containing recognition elements from the extender (in this case, the aspartyl group) and the salicylic
acid fragment identified from Tethering. The disulfide linkage was replaced with
a simple alkane linkage. Instead of the original irreversible arylacyloxymethylketone warhead, we used a reversible aldehyde warhead (46). The resulting molecule
(compound 23, Figure 16) inhibits caspase-3 with a low micromolar affinity. Simple conversion of the flexible 6-amino-hexanoic acid linker to the more rigid paraaminomethyl benzoic acid linker (compound 24) increased the affinity by more
than one order of magnitude, and functionalization of the central aromatic moiety
30 Apr 2004
18:12
216
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
Figure 15 Tethering with extenders on caspase-3.
with nitrogen picked up a hydrogen bond in the active site of the enzyme (compound 25), adding another order of magnitude in increased affinity (12, 20).
We also enhanced the affinity of the initial diaphore by a complementary route.
We noted that the simple alkane-linked diaphore could position an element into
the S2 binding site, and when we explored this possibility, we discovered that
it was possible to enhance affinity 30-fold simply by adding a phenyl group (2)
(compound 27, Figure 16).
Although we were able to rapidly generate two series of potent inhibitors from
a common fragment, other approaches would likely have been ineffective. The
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
P1: FHD
217
Figure 16 Evolution of a hit derived from Tethering with extenders to nanomolar
leads.
simple salicylic acid fragment by itself (i.e., compound 26) showed no inhibition
of the enzyme whatsoever, even at concentrations of several millimolar. Therefore,
this fragment would not have been detected in a conventional screen and may have
been missed even in an NMR screen.
To demonstrate that the approach is general, we made a second extender (extender 28), as shown in Figure 17. This extender has the same features as the first
extender (extender 22), but it contains a phenyl sulfonamide linkage between the
Figure 17 Tethering with extenders on caspase-3 with a different extender.
30 Apr 2004
18:12
218
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
aspartyl group and the thioester and thus is expected to probe a different area of the
caspase. Consistent with these differences, Tethering did not pick up the salicylic
acid fragment, but it did pick up a different fragment, a thiophene sulfone. When
this fragment was converted into a diaphore, the resulting compound 29 inhibited
caspase-3 with a potency in the high nanomolar range (20).
The crystal structures of both extender-modified enzymes complexed to their
respective disulfide hits reveal that the salicylic acid and the thiophene sulfone
bind in the same region of the enzyme, the S4 pocket, even though the linkers take different trajectories to reach this pocket (20) (Figure 18). Also notably, the residue Tyr204 collapses into the S2 pocket in the case of extender 22,
whereas because of the bulk of extender 28 the tyrosine swings back to accommodate the extender. Residues in the S4 binding pocket also readjust slightly to accommodate the different moieties, revealing the adaptability of the protein surface.
Tethering with extenders was also applied to caspase-1, using the same extenders, and a number of different fragments were identified. These were also
converted into diaphores, and in one case a single-digit nanomolar inhibitor was
rapidly identified (T. O’Brien, B.T. Fahr, M. Sopko, J.W. Lam, N.D. Waal, B.
Raimundo, H. Purkey, P. Pham, & M.J. Romanowski, manuscript submitted).
Opportunities
The strength of Tethering with extenders is that it can be applied to any protein for
which a small-molecule inhibitor is known. If the small-molecule inhibitor is covalent, one simply needs to install a masked thiol onto the small-molecule inhibitor
to generate an extender. Otherwise, it is straightforward to add a covalent warhead
to a noncovalent inhibitor for Tethering and to remove it when diaphores are constructed. As described here, this approach is well suited for enzymes that utilize
proteins as their substrates and therefore bind their substrates in extended forms
using fragment-sized subsites. These include proteases (27, 29, 30, 57, 59, 63), kinases (24, 74), and phosphatases (4, 28, 50), all classes of enzymes that are heavily
pursued in the pharmaceutical industry. One of the most exciting uses of Tethering
is to identify a first hit, convert this hit to an extender, and then perform Tethering
with extenders to identify a second hit, such that both fragments are derived from
Tethering. This approach can be generalized to virtually any protein target.
CONCLUSIONS AND FUTURE DIRECTIONS
Fragment-based drug discovery offers great promise for providing new starting
points for drug discovery as well as for facilitating lead optimization. Given the
weak intrinsic affinities of many small fragments for their targets, it is crucial that
any fragment-based method selects for one-to-one binding to a specific site on
the protein. Tethering is well suited for this because the stoichiometry is measured
directly by MS, and the site is localized to the cysteine used for Tethering. The disulfide bond does not compromise the utility of the discovered fragments, because
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
TETHERING
219
the selection is under thermodynamic control and X-ray structures that compare
the disulfide-bonded and free fragments show them to be in close alignment.
At Sunesis we have applied Tethering to over a dozen different drug targets and
have produced a spectrum of useful fragments for each. The chemical information
that Tethering provides can be applied to existing hits to generate higher-affinity
compounds, as was shown for IL-2. Tethering identifies nucleating fragments that
can be grown into free-standing hits, as was shown for TS. Tethering can “mine”
for novel warheads that engage the active sites of enzymes, as shown by the use
of breakaway extenders in PTP-1B. And Tethering with extenders can allow the
assembly of fragments into high-affinity molecules, as was shown for the caspases.
Several new avenues promise to further expand the utility of Tethering. Because
Tethering is a binding assay and does not rely on protein function, it is more versatile than many functional assays. For example, kinases exist in both inactive and
active forms in the cell, and these may have different conformations (33). Though
small molecules that target one form over the other can achieve greater selectivity
(66), functional screens are less able to identify molecules that bind selectively to
inactive forms of the enzyme, a difficulty obviated by Tethering. Finally, Tethering
provides a site-directed basis for discovery so that one can choose the sites to be
explored. This offers the possibility of selectively targeting and even identifying
allosteric (58) sites in proteins that may be missed in a functional screen. Such
allosteric sites may prefer different chemical features that could be advantageous
to the highly charged fragments preferred by substrate sites in phosphatases and
many proteases. In aggregate, we believe fragment-based approaches will prove
to be useful tools in drug discovery and optimization for novel and challenging
protein targets. Tethering is a powerful addition to the arsenal of fragment-based
drug discovery approaches.
ACKNOWLEDGMENTS
We thank all our colleagues at Sunesis Pharmaceuticals for their research and
support in making this work possible, and Monya L. Baker for editorial assistance.
The Annual Review of Biophysics and Biomolecular Structure is online at
http://biophys.annualreviews.org
LITERATURE CITED
1. Deleted in proof
2. Allen DA, Pham P, Choong IC, Fahr B,
Burdett MT, et al. 2003. Identification
of potent and novel small-molecule inhibitors of caspase-3. Bioorg. Med. Chem.
Lett. 13:3651–55
3. Andersen HS, Olsen OH, Iversen LF,
Sorensen AL, Mortensen SB, et al. 2002.
Discovery and SAR of a novel selective and orally bioavailable nonpeptide
classical competitive inhibitor class of
Protein Tyrosine Phosphatase 1B. J. Med.
Chem. 45:4443–59
4. Arabaci G, Xiao-Chuan G, Beebe KD,
30 Apr 2004
18:12
220
5.
6.
7.
8.
8a.
9.
10.
11.
12.
13.
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
Coggeshall KM, Pei D. 1999. αhaloacetophenone derivatives as photoreversible covalent inhibitors of protein tyrosine phosphatases. J. Am. Chem. Soc.
121:5085–86
Arkin MR, Randal M, DeLano WL, Hyde
J, Luong TN, et al. 2003. Binding of
small molecules to an adaptive proteinprotein interface. Proc. Natl. Acad. Sci.
USA 100:1603–8
Barrett WC, DeGnore JP, Konig S, Fales
HM, Keng Y-F, et al. 1999. Regulation of
PTP1B via glutathionylation of the active
site cysteine 215. Biochemistry 38:6699–
705
Blundell TL, Jhoti H, Abell C. 2002.
High-throughput crystallography for lead
discovery in drug design. Nat. Rev. Drug
Discov. 1:45–54
Boehm HJ, Boehringer M, Bur D, Gmuender H, Huber W, et al. 2000. Novel inhibitors of DNA gyrase: 3D structure
based biased needle screening, hit validation by biophysical methods, and 3D
guided optimization. A promising alternative to random screening. J. Med. Chem.
43:2664–74
Bohacek RS, McMartin C, Guida WC.
1996. The art and practice of structurebased drug design: a molecular modeling
perspective. Med. Res. Rev. 16:3–50
Braisted AC, Oslob JD, Delano WL, Hyde
J, McDowell RS, et al. 2003. Discovery
of a potent small molecule IL-2 inhibitor
through fragment assembly. J. Am. Chem.
Soc. 125:3714–15
Deleted in proof
Burke TR Jr, Yao ZJ, Liu DG, Voigt J, Gao
Y. 2001. Phosphoryltyrosyl mimetics in
the design of peptide-based signal transduction inhibitors. Biopolymers 60:32–44
Choong IC, Lew W, Lee D, Pham P, Burdett MT, et al. 2002. Identification of
potent and selective small-molecule inhibitors of caspase-3 through the use of extended tethering and structure-based drug
design. J. Med. Chem. 45:5005–22
Cochran AG. 2001. Protein-protein inter-
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
faces: mimics and inhibitors. Curr. Opin.
Chem. Biol. 5:654–59
Cochran AG, Tong RT, Starovasnik MA,
Park EJ, McDowell RS, et al. 2001. A
minimal peptide scaffold for beta-turn
display: optimizing a strand position in
disulfide-cyclized beta-hairpins. J. Am.
Chem. Soc. 123:625–32
Costi MP, Tondi D, Rinaldi M, Barlocco
D, Pecorari P, et al. 2002. Structure-based
studies on species-specific inhibition of
thymidylate synthase. Biochim. Biophys.
Acta. 1587:206–14
DeLano WL. 2003. PyMOL molecular
graphics system on the World Wide Web.
http://pymol.sourceforge.net/
DeLano WL. 2002. Unraveling hot spots
in binding interfaces: progress and challenges. Curr. Opin. Struct. Biol. 12:14–20
Dolle RE, Hoyer D, Prasad CVC,
Schmidt SJ, Helaszek CT, et al. 1994. P1
aspartate-based peptide α-((2,6-dichlorobenzoyl)oxy)methyl ketones as potent
time-dependent inhibitors of interleukin1b converting enzyme. J. Med. Chem.
37:563–64
Erlanson DA, Braisted AC, Raphael DR,
Randal M, Stroud RM, et al. 2000. Sitedirected ligand discovery. Proc. Natl.
Acad. Sci. USA 97:9367–72
Erlanson DA, Lam JW, Wiesmann C, Luong TN, Simmons RL, et al. 2003. In
situ assembly of enzyme inhibitors using extended tethering. Nat. Biotechnol.
21:308–14
Erlanson DA, McDowell RS, He MM,
Randal M, Simmons RL, et al. 2003. Discovery of a new phosphotyrosine mimetic
for PTP1B using breakaway tethering. J.
Am. Chem. Soc. 125:5602–3
Fejzo J, Lepre C, Xie X. 2003. Application of NMR screening in drug discovery.
Curr. Top. Med. Chem. 3:81–97
Fejzo J, Lepre CA, Peng JW, Bemis GW,
Ajay, et al. 1999. The SHAPES strategy:
an NMR-based approach for lead generation in drug discovery. Chem. Biol. 6:755–
69
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
24. Fry DW, Bridges AJ, Denny WA, Doherty A, Greis KD, et al. 1998. Specific,
irreversible inactivation of the epidermal
growth factor receptor and erbB2, by a
new class of tyrosine kinase inhibitor.
Proc. Natl. Acad. Sci. USA 95:12022–
27
25. Garcia-Calvo M, Peterson EP, Leiting B,
Ruel R, Nicholson D, Thornberry NA.
1998. Inhibition of human caspases by
peptide-based and macromolecular inhibitors. J. Biol. Chem. 273:32608–13
26. Gilbert HF. 1995. Thiol/disulfide exchange equilibria and disulfide bond stability. Methods Enzymol. 251:8–28
27. Graczyk PP. 1999. Caspase inhibitors—
a chemist’s perspective. Restor. Neurol.
Neurosci. 14:1–23
28. Ham SW, Park J, Lee SJ, Yoo JS. 1999.
Selective inactivation of protein tyrosine
phosphatase PTP1B by sulfone analogue
of naphthoquinone. Bioorg. Med. Chem.
Lett. 9:185–86
29. Han MS, Ryu CH, Chung SJ, Kim DH.
2000. A novel strategy for designing irreversible inhibitors of metalloproteases:
acetals as latent electrophiles that interact with catalytic nucleophile at the active
site. Org. Lett. 2:3149–52
30. Hernandez AA, Roush WR. 2002. Recent advances in the synthesis, design and
selection of cysteine protease inhibitors.
Curr. Opin. Chem. Biol. 6:459–65
31. Houk J, Whitesides GM. 1987. Structurereactivity relations for thiol-disulfide interchange. J. Am. Chem. Soc. 109:6825–
36
32. Huc I, Lehn JM. 1997. Virtual combinatorial libraries: dynamic generation of
molecular and supramolecular diversity
by self-assembly. Proc. Natl. Acad. Sci.
USA 94:2106–10
33. Huse M, Kuriyan J. 2002. The conformational plasticity of protein kinases. Cell
109:275–82
34. Hyatt DC, Maley F, Montfort WR. 1997.
Use of strain in a stereospecific catalytic mechanism: crystal structures of Es-
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
P1: FHD
221
cherichia coli thymidylate synthase bound
to FdUMP and methylenetetrahydrofolate. Biochemistry 36:4585–94
Hyde J, Braisted AC, Randal M, Arkin
MR. 2003. Discovery and characterization
of cooperative ligand binding in the adaptive region of interleukin-2. Biochemistry
42:6475–83
Jacoby E, Davies J, Blommers MJ. 2003.
Design of small molecule libraries for
NMR screening and other applications in
drug discovery. Curr. Top. Med. Chem.
3:11–23
Jahnke W, Florsheimer A, Blommers MJ,
Paris CG, Heim J, et al. 2003. Second-site
NMR screening and linker design. Curr.
Top. Med. Chem. 3:69–80
Jencks WP. 1981. On the attribution and
additivity of binding energies. Proc. Natl.
Acad. Sci. USA 78:4046–50
Johnson TO, Ermolieff J, Jirousek MR.
2002. Protein Tyrosine Phosphatase 1B
inhibitors for diabetes. Nat. Rev. Drug.
Discov. 1:696–709
Kamb A, Finer-Moore J, Calvert AH,
Stroud RM. 1992. Structural basis
for recognition of polyglutamyl folates
by thymidylate synthase. Biochemistry
31:9883–90
Kaur S, McGuire L, Tang D, Dollinger
G, Huebner V. 1997. Affinity selection
and mass spectrometry-based strategies to
identify lead compounds in combinatorial libraries. J. Protein Chem. 16:505–
11
Keire DA, Strauss E, Guo W, Noszál B,
Rabenstein DL. 1992. Kinetics and equilibria of thiol/disulfide interchange reactions of selected biological thiols and related molecules with oxidized glutathione.
J. Org. Chem. 57:123–27
Lehn JM, Eliseev A. 2001. Dynamic combinatorial chemistry. Science 291:2331–
32
Lesuisse D, Lange G, Deprez P, Benard
D, Schoot B, et al. 2002. SAR and X-ray.
A new approach combining fragmentbased screening and rational drug design:
30 Apr 2004
18:12
222
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
AR
AR214-BB33-10.tex
ERLANSON
¥
WELLS
¥
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
P1: FHD
BRAISTED
application to the discovery of nanomolar inhibitors of SRC SH2. J. Med. Chem.
45:2379–87
Liu G, Szczepankiewicz BG, Pei Z,
Janowick DA, Xin Z, et al. 2003. Discovery and structure-activity relationship
of oxalylarylaminobenzoic acids as inhibitors of Protein Tyrosine Phosphatase
1B. J. Med. Chem. 46:2093–103
Margolin N, Raybuck SA, Wilson K,
Chen W, Fox T, et al. 1997. Substrate
and inhibitor specificity of interleukin-1βconverting enzyme and related caspases.
J. Biol. Chem. 272:7223–28
Marsham PR, Wardleworth JM, Boyle
FT, Hennequin LF, Kimbell R, et al.
1999. Design and synthesis of potent nonpolyglutamatable quinazoline antifolate
thymidylate synthase inhibitors. J. Med.
Chem. 42:3809–20
McBride CB, McPhail LT, Steeves JD.
1999. Emerging therapeutic targets in
caspase-dependent disease. Expert Opin.
Ther. Targets 3:391–411
McGovern SL, Caselli E, Grigorieff N,
Shoichet BK. 2002. A common mechanism underlying promiscuous inhibitors
from virtual and high-throughput screening. J. Med. Chem. 45:1712–22
Myers JK, Widlanski TS. 1993. Mechanism-based inactivation of prostatic acid
phosphatase. Science 262:1451–53
Nakamura CE, Abeles RH. 1985. Mode
of interaction of beta-hydroxy-betamethylglutaryl coenzyme A reductase
with strong binding inhibitors: compactin
and related compounds. Biochemistry
24:1364–76
Nelson BH, Willerford DM. 1998. Biology of the interleukin-2 receptor. Adv. Immunol. 70:1–81
Nienaber VL, Richardson PL, Klighofer
V, Bouska JJ, Giranda VL, Greer J. 2000.
Discovering novel ligands for macromolecules using X-ray crystallographic
screening. Nat. Biotechnol. 18:1105–8
Niimi T, Orita-Igarashi M, Okazawa M,
Sakashita H, Kikuchi K, et al. 2001. De-
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
sign and synthesis of non-peptidic inhibitors for the syk C-terminal SH2 domain based on structure-based in-silico
screening. J. Med. Chem. 44:4737–40
Obita T, Muto T, Endo T, Kohda D. 2003.
Peptide library approach with a disulfide
tether to refine the Tom20 recognition motif in mitochondrial presequences. J. Mol.
Biol. 328:495–504
O’Shea EK, Rutkowski R, Stafford WF
III, Kim PS. 1989. Preferential heterodimer formation by isolated leucine zippers from fos and jun. Science 245:646–
48
Otto HH, Schirmeister T. 1997. Cysteine
proteases and their inhibitors. Chem. Rev.
97:133–72
Pargellis C, Tong L, Churchill L, Cirillo PF, Gilmore T, et al. 2002. Inhibition
of p38 MAP kinase by utilizing a novel
allosteric binding site. Nat. Struct. Biol.
9:268–72
Powers JC, Asgian JL, Ekici OD, James
KE. 2002. Irreversible inhibitors of serine,
cysteine, and threonine proteases. Chem.
Rev. 102:4639–750
Ramstrom O, Lehn JM. 2002. Drug
discovery by dynamic combinatorial libraries. Nat. Rev. Drug Discov. 1:26–32
Rishton GM. 2003. Nonleadlikeness and
leadlikeness in biochemical screening.
Drug Discov. Today 8:86–96
Salmeen A, Andersen JN, Myers MP,
Tonks NK, Barford D. 2000. Molecular
basis for the dephosphorylation of the activation segment of the insulin receptor
by Protein Tyrosine Phosphatase 1B. Mol.
Cell 6:1401–12
Salto R, Babe LM, Li J, Rose JR, Yu Z,
et al. 1994. In vitro characterization of
nonpeptide irreversible inhibitors of HIV
proteases. J. Biol. Chem. 269:10691–98
Sarmiento M, Puius YA, Vetter SW, Keng
YF, Wu L, et al. 2000. Structural basis of
plasticity in Protein Tyrosine Phosphatase
1B substrate recognition. Biochemistry
39:8171–79
Sauve K, Nachman M, Spence C, Bailon
30 Apr 2004
18:12
AR
AR214-BB33-10.tex
AR214-BB33-10.sgm
LaTeX2e(2002/01/18)
TETHERING
66.
67.
68.
69.
70.
70a.
71.
72.
P, Campbell E, et al. 1991. Localization in
human interleukin 2 of the binding site to
the alpha chain (p55) of the interleukin
2 receptor. Proc. Natl. Acad. Sci. USA
88:4636–40
Schindler T, Bornmann W, Pellicena P,
Miller WT, Clarkson B, Kuriyan J. 2000.
Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science
289:1938–42
Shuker SB, Hajduk PJ, Meadows RP, Fesik SW. 1996. Discovering high-affinity
ligands for proteins: SAR by NMR. Science 274:1531–34
Stanojevic D, Verdine GL. 1995. Deconstruction of GCN4/GCRE into a
monomeric peptide-DNA complex. Nat.
Struct. Biol. 2:450–57
Stout TJ, Sage CR, Stroud RM. 1998.
The additivity of substrate fragments in
enzyme-ligand binding. Structure 6:839–
48
Swayze EE, Jefferson EA, SannesLowery KA, Blyn LB, Risen LM, et al.
2002. SAR by MS: a ligand based technique for drug lead discovery against
structured RNA targets. J. Med. Chem.
45:3816–19
Thanos CD, Randal M, Wells JA. 2003.
Potent small-molecule binding to a dynamic hot spot on IL-2. J. Am. Chem. Soc.
125:15280–81
Thornberry NA, Rano TA, Peterson EP,
Rasper DM, Timkey T, et al. 1997. A
combinatorial approach defines specificities of members of the caspase family and
granzyme B. J. Biol. Chem. 272:17907–
11
Tilley JW, Chen L, Fry DC, Emerson SD,
Powers GD, et al. 1997. Identification of
a small molecule inhibitor of the IL-2/IL-
73.
74.
75.
76.
77.
78.
79.
P1: FHD
223
2Rα receptor interaction which binds to
IL-2. J. Am. Chem. Soc. 119:7589–90
Toogood PL. 2002. Inhibition of proteinprotein association by small molecules:
approaches and progress. J. Med. Chem.
45:1543–58
Tsou HR, Mamuya N, Johnson BD, Reich
MF, Gruber BC, et al. 2001. 6-substituted4-(3-bromophenylamino)quinazolines as
putative irreversible inhibitors of the epidermal growth factor receptor (EGFR)
and human epidermal growth factor receptor (HER-2) tyrosine kinases with enhanced antitumor activity. J. Med. Chem.
44:2719–34
van Dongen M, Weigelt J, Uppenberg J,
Schultz J, Wikstrom M. 2002. Structurebased screening and design in drug discovery. Drug Discov. Today 7:471–78
van Dongen MJ, Uppenberg J, Svensson
S, Lundback T, Akerud T, et al. 2002.
Structure-based screening as applied to
human FABP4: a highly efficient alternative to HTS for hit generation. J. Am.
Chem. Soc. 124:11874–80
Yue T-L, Ohlstein E, Ruffolo R. 1999.
Apoptosis: a potential target for discovering novel therapies for cardiovascular
diseases. Curr. Opin. Chem. Biol. 3:474–
80
Zhang ZY. 2002. Protein tyrosine phosphatases: structure and function, substrate
specificity, and inhibitor development.
Annu. Rev. Pharmacol. Toxicol. 42:209–
34
Zurawski SM, Vega F Jr, Doyle EL,
Huyghe B, Flaherty K, et al. 1993. Definition and spatial location of mouse
interleukin-2 residues that interact with
its heterotrimeric receptor. EMBO J.
12:5113–19
Erlanson.qxd
4/30/2004
7:55 PM
Page 1
TETHERING
C-1
Figure 4 (A) X-ray structure of the complex between IL-2 and 1. Residues established as important for IL-2Rα binding are labeled and colored orange. E62 forms a
critical bidentate salt bridge and the adaptive hydrophobic pocket is created around
residues F42, L72, K35, R38, and K76. (B) Ribbon diagram of IL-2 that shows the
sites chosen for cysteine mutations. Individual mutants were made targeting the binding site of 1. This and all color figures were made using the program PyMOL (16).
Erlanson.qxd
C-2
4/30/2004
ERLANSON
7:55 PM
■
WELLS
Page 2
■
BRAISTED
Figure 5 (A) X-ray structure of the disulfide adduct between IL-2 Y31C and 2. The
side chain of F42 is in a conformation similar to that seen in wild-type IL-2; this conformation would block binding of 1. (B) Overlay of three crystal structures, 1 (yellow), 2 (green) and 3 (blue), demonstrating the overlap between fragment 2 and compound 1 compared with the bound conformation of the compound designed through
fragment merging, 3.
Erlanson.qxd
4/30/2004
7:56 PM
Page 3
TETHERING
C-3
Figure 7 Stereoview showing the crystallographically determined structure of TS bound
to N-tosyl-D-proline without the disulfide linker (compound 8) (blue) superimposed with
the cofactor mTHF (yellow) (34).
Figure 8 Overlay of three structures: N-tosyl-D-proline bound to TS noncovalently
(green), covalently through C146 (red) or covalently through L143C (blue).
Erlanson.qxd
C-4
4/30/2004
ERLANSON
7:56 PM
■
WELLS
Page 4
■
BRAISTED
Figure 10
Superposition of two crystallographically determined structures:
Compound 15 (blue) and mTHF (yellow)/dUMP (yellow) bound to TS (green).
Hydrogen bonds are shown as yellow dashed lines with distances in Ångstroms.
Figure 18 Overlay of caspase-3 modified with either extender 22 (gray) or extender 28 (blue) and their selected disulfide fragments. The thick cylinders are the
extenders and selected fragments, the thin cylinders are protein residues. Reprinted
with permission from Nature Biotechnology (20).