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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. 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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).