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Fragment-based lead discovery: a chemical update
Fragment-based lead discovery: a chemical update Daniel A Erlanson Fragment-based lead discovery constructs drug leads from small molecular fragments. In theory, this is a highly efficient method for drug discovery, and the technique has become enormously popular in the past few years. In this review, I describe how a variety of approaches in fragment-based lead discovery — including NMR, X-ray crystallography, mass spectrometry, functional screening, and in silico screening — have produced drug leads. Although the examples show that the technique can reliably generate potent molecules, there is still much work to be done to maintain the efficiency of molecules’ binding affinities as fragments are linked, expanded, and otherwise improved. Addresses Sunesis Pharmaceuticals, Inc., 341 Oyster Point Boulevard, South San Francisco, CA 94080, USA Corresponding author: Erlanson, Daniel A ([email protected])
Current Opinion in Biotechnology 2006, 17:643–652 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Gary Woodnut and Frank S Walsh Available online 3rd November 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.10.007
Introduction Small-molecule drug discovery has always been a struggle of attrition, but in the past few years pressures have mounted to increase efficiency at all stages of the process. A potential solution for lead identification and optimization, fragment-based lead discovery, is becoming increasingly popular [1]. The goal is to build drug leads in pieces, by identifying small molecular fragments and then either linking them or expanding them. The definition of a fragment varies, but usually refers to molecules weighing less than 200–300 Da, with fewer than 15–20 heavy atoms. The concept of fragment-based lead discovery was first proposed a quarter of a century ago [2], but difficulties finding and linking fragments delayed serious pursuit until 1996, when the first practical demonstration was published [3]. Since then, fragment-based lead discovery has become very popular, with numerous demonstrated breakthroughs in finding fragments as well as in linking and expanding them. In fact, the first full book on fragment-based drug discovery has just been published [1]. www.sciencedirect.com
There are also several general reviews [4,5,6–12], as well as more specialized reviews focusing on library design [13], structure-guided fragment screening [14,15], NMR [16,17,18,19,20], X-ray crystallography [21–23], functional screening [24], site-directed ligand discovery and affinity-based ligand discovery [25,26], Tethering1 [27,28], and kinases [29]. Given the wealth of primary and secondary literature on the topic, this review will take a chemocentric focus on specific examples published in the past two years. Previous reviews largely cover earlier work [4,5]. Specifically, this review examines recent fragment-based case studies and analyses how the atom-efficiency of ligands changes as fragments are advanced.
Theoretical advantages of fragment-based lead discovery The basic approach of fragment-based lead discovery is shown in Figure 1. After an initial fragment that binds to a protein of interest is identified, it is either elaborated or combined with other fragments to generate a hit, which is subsequently optimized to produce a lead, and (in the best cases) a drug. Many disparate methods for finding fragments have been developed over the past few years. The strategies to transform a fragment to a lead also range considerably, from small changes to the fragment itself to dramatic substitutions and linkage with other fragments. As suggested in the figure, these distinctions are sometimes more semantic than real, and in theory the same final molecule could be created by linking two fragments, optimizing a single fragment or through some combination. One of the main theoretical advantages of this approach is that there are fewer possible fragments than possible drug-sized molecules. By way of analogy, only two English words consist of one letter: ‘a’ and ‘I’. The number of two-letter words is larger, but still limited; however, the number of six letter words is considerable. Similarly, there are about 107 fragments with up to 12 heavy atoms (excluding 3- and 4-ring containing structures) [30] compared with an estimated 1063 small drug-like molecules with up to 30 heavy atoms [31]. Given that all the laboratories throughout the world are estimated to contain ‘only’ about 108 molecules [32], screening fragments would sample pharmaceutically acceptable chemical diversity space much more efficiently. A second theoretical advantage of fragment-based lead discovery is that the resulting molecules are likely to have a higher ‘ligand efficiency’ than molecules discovered Current Opinion in Biotechnology 2006, 17:643–652
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Figure 1
The basic concept of fragment-based lead discovery. The blue ovoid and red rectangle represent fragments that bind to the target protein. These can be linked or expanded to produce high-affinity ligands.
through conventional methods. An investigation of roughly 150 ligands, many of them drugs, reveals that the free energy of binding increases roughly linearly with increasing ligand size up to about 15 atoms, beyond which there is very little increase; the maximum free-energy contribution per heavy atom is roughly –1.5 kcal/mol [33]. That study, as well as an earlier study [34], inspired the concept of ‘ligand efficiency’, defined as the ratio of the free energy of ligand binding to the number of nonhydrogen atoms [35]. This has been extended to other properties such as polar surface area and molecular weight [36]. Because fragments should have higher ligand efficiencies than typical high-throughput screening (HTS) hits, they should produce ultimately more ‘efficient’ drugs, which should have better pharmaceutical properties (lower molecular weight, better pharmacokinetics, lower toxicity, etc.). Fragments are also less likely to have interfering functionality that would prevent them from binding to a target protein [37]. Below, I consider recent examples of fragments and their derived hits or leads in terms of potency and ligand efficiency. Because the review is limited in scope to small-molecule drug discovery, peptides, dendrimers, and multimeric molecules are not included. Also, where Ki values are not available, IC50 (half maximum inhibition) values are used to calculate ligand efficiency (LE), as has been done previously [35].
Fragment optimization A challenge in providing the current opinion of fragmentbased lead discovery is in limiting the scope of the review. For example, the c-Jun NH2 terminal kinase (JNK) inhibitors recently reported by Zhao et al. [38] began with a HTS hit (Figure 2a) that qualifies as a fragment (MW = 220, 16 heavy atoms). The resulting optimization could easily be called ‘fragment optimization’ or ‘fragment growing’. However, it is also classic medicinal chemistry, albeit with a more ‘lead-like’ than ‘drug-like’ starting point [39]. Such examples are increasingly common in the literature as fragment-like approaches permeate all aspects of medicinal chemistry and fragments become more common in HTS libraries. Capturing all such examples is beyond the scope of this review. HowCurrent Opinion in Biotechnology 2006, 17:643–652
ever, as new techniques are only useful to the extent that they improve on existing techniques, this example serves as a useful benchmark. A contrasting example comes from Plexxikon, which recently identified itself as a major player in fragmentbased lead discovery with its report of a family of phosphodiesterase 4 (PDE4) inhibitors (Figure 2b) [40]. The researchers differentiate their ‘scaffold-based’ approach from other forms of fragment-based lead discovery in several ways, such as starting with larger numbers of larger fragments and including functional screening as well as high-throughput crystallography. Nevertheless, this research is ultimately an elegant example of fragment-based lead discovery, in that the starting fragment (MW = 168, 12 heavy atoms) is likely to have been missed in a standard HTS environment even though it is a highly efficient ligand. Ellman and colleagues reported substrate activity screening, an elegant fragment-based method, to discover protease inhibitors [41]. In this functional assay, fragments are first identified as substrates, and then converted to inhibitors through rapid analoging and the addition of a protease active-site trap. The method was used to identify highly potent cathepsin S inhibitors (Figure 2c). A combination of virtual screening and NMR was used to identify very small fragments that bind to the ATPbinding pocket of the bacterial protein DNA gyrase B (GyrB) [42]. Starting from a weak fragment, the researchers improved potency by iterative computational searches of commercial molecules to achieve biologically active molecules (Figure 2d). A different combination of computational and NMR screening followed by library design helped generate the lactate dehydrogenase inhibitor shown in Figure 2e [43]. In this case, a systems-based approach was used to develop inhibitors of multiple oxidoreductases, although different inhibitors showed selectivity for different enzymes. Anthrax lethal factor, a metalloproteinase, has also yielded to fragment-based ligand discovery. Starting from www.sciencedirect.com
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Figure 2
Examples of fragment-based lead discovery involving fragment optimization. (a) JNK1, (b) PDE4D, (c) cathepsin S, (d) DNA gyrase B, (e) lactate dehydrogenase, (f) anthrax lethal factor, (g) HCV-IRES IIA, (h) IMPDH, (i) PDE4, (j) hNK2 receptor, (k) DPP-IV, (l) DHNA, (m) CDK2, (n,o) thrombin (p) ribonuclease A (q) PTP1B. www.sciencedirect.com
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a library of only about 300 fragments and an NMR-based functional assay [44], researchers used subsequent synthetic elaboration to produce potent compounds with animal efficacy (Figure 2f).
pounds has not been reported, some of them, particularly the CDK2 inhibitor, have impressive ligand efficiencies, suggesting that they will make excellent starting points for lead discovery.
Researchers at Ibis Therapeutics have developed mass spectrometry (MS)-based fragment discovery. They identified a weak binder to the ribosomome IIA subdomain of hepatitis C (HCV-IRES IIA) and subsequently optimized it to become a submicromolar inhibitor (Figure 2g) [45].
Fragment merging and linking
In a nice example of fragment optimization, researchers at UCB Celltech used a combination of modeling and medicinal chemistry to discover inhibitors to the immunology target inosine monophosphate dehydrogenase (IMPDH, Figure 2h) [46,47]. The initial fragment, while only a modest inhibitor, has a very high ligand efficiency, and had been previously discovered by researchers from Roche using a virtual screen [48].
Proteases
Another application to PDE4 inhibitors is shown in Figure 2i. This ‘scaffold-linker-functional group’ approach relies on computational fragment-screening to design combinatorial libraries, and in this case was used to rapidly produce very high potency inhibitors against PDE4 starting from a known inhibitor [49]. Researchers at Menarini Ricerche used fragment-based design to construct a series of hNK2 receptor ligands [50]. Although the compounds could conceivably have been arrived at using combinatorial or traditional medicinal chemistry, the concept of fragment-based lead discovery (Figure 2j) made discovery efforts more efficient. Santhera Pharmaceuticals recently reported a series of dipeptidyl peptidase-IV (DPP-IV) inhibitors derived from both computational and crystallographic screening, followed by medicinal chemistry and fragment-merging with known inhibitors (Figure 2k) [51,52]. Abbott Laboratories, which pioneered the field of fragment-based lead discovery using NMR [3], has also done considerable work with crystallography [53]. Using a crystallographic approach, they discovered 7,8-dihydroneopterin aldolase (DHNA) inhibitors starting from a library of 10 000 fragments (Figure 2l) [54]. Interestingly, although the initial hit was identified by soaking the compounds into pre-formed crystals, structures of the subsequent compounds could only be obtained through co-crystallization. Astex Therapeutics focuses on the use of X-ray crystallography to identify fragments. They have reported a series of crystallographically identified fragments for several targets, including CDK2 (Figure 2m), thrombin (Figure 2n, 2o), ribonuclease A (Figure 2p), and PTP1B (Figure 2q) [55]. Although the elaboration of these comCurrent Opinion in Biotechnology 2006, 17:643–652
There have been considerable recent advances in the area of fragment merging and linking. Select targets and target classes will be covered separately in the following sections.
Proteases are ideally suited for fragment linking strategies. Because they cleave peptides or proteins, proteases usually have extended binding sites, with each aminoacid binding in its own subsite (S). An elegant example of Astex’s ‘Pyramid’ crystallography-based fragment screening applied to the protease thrombin is shown in Figure 3a. Two weak inhibitors were identified as binding in adjacent regions, one in the S1 pocket and the other in the S2–S4 region. These were linked to produce a highaffinity inhibitor, and crystallography shows that this molecule has roughly the same binding mode as the separate fragments [56]. An application of NMR-based screening, this time from Schering-Plough, was used to identify inhibitors of the single-chain hepatitis C virus NS3 protease–NS4A cofactor complex (ns4a–ns3p) [57]. This is a good example of fragment linking guided by the iterative use of NMR structural information (Figure 3b). Researchers at Wyeth were able to optimize an HTS hit against the matrix metalloproteinase MMP-13 (collagenase 3) by using crystallography and structure-based design to introduce a zinc-chelating carboxylic acid moiety (Figure 3c) [58]. Although carboxylic acids are not strong zinc chelators, the rigidity of the linker helps compensate for this weakness and introduces other contacts to the protein. With ‘in situ click chemistry’, proteins catalyse the formation of their own inhibitor from two fragments. In a nice model study using HIV-1 protease, the synthesis of a known nanomolar inhibitor from two fragments was catalyzed much more effectively in the presence of the enzyme than in the presence of bovine serum albumin (Figure 3d) [59]. Another protease that has yielded to fragment-based approaches is caspase-1. Using Sunesis’s Tethering with Extenders [60–62], an S4-binding fragment was linked to an S1-binding fragment to generate a submicromolar inhibitor. Further optimization by making the flexible linker rigid or adding an S2-binding element yielded more potent compounds with improved ligand efficiency (Figure 3e). www.sciencedirect.com
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Figure 3
Examples of fragment-based lead discovery involving fragment merging or linking: proteases, acetychlolinesterase and carbonic anhydrase. (a) Thrombin, (b) ns4a–ns3p, (c) MMP-13, (d) HIV-1 protease, (e) caspase-1, (f–i) acetylcholinesterases, and (j–l) carbonic anhydrases. www.sciencedirect.com
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Figure 4
Other examples of fragment-based lead discovery involving fragment merging or linking. (a) Bcl-2, (b) Bid, (c) p38a, (d) p38a, (e) ketopantoate reductase, (f) 17b-HSD, and (g) 16S rRNA. Current Opinion in Biotechnology 2006, 17:643–652
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Acetylcholinesterase
One of the most productive proteins for fragment linking has been acetylcholinesterase (AChE), owing partly to a deep gorge-like active site with binding pockets near the bottom and the top. For example, a bis-huperzine B ligand binds much more tightly than huperzine B alone (albeit with considerable loss of ligand efficiency), and modeling studies indicate this is due to two-site occupancy (Figure 3f) [63]. A similar approach with heterodimers produced low nanomolar inhibitors (Figure 3g). Xray crystallography of the bound inhibitors demonstrated that the common galanthamine moieties (blue) bind in the same position, while the phthalimide (red) binds near the top of the active site [64]. Another heterodimer approach, this time starting with the very efficient fragment tacrine, produced the picomolar inhibitors shown in Figure 3h [65]. The application of in situ click chemistry to discover AChE inhibitors has produced femtomolar inhibitors [66,67], and crystallography revealed that the protein makes contacts not just with the two fragments but with the triazole linker as well [68]. Subsequent work using a larger set of fragments has produced even more potent and novel compounds (Figure 3i) [69]. Carbonic anhydrase
Several approaches to fragment linking have proven successful with carbonic anhydrase (CA). Click chemistry was used to discover novel inhibitors to bovine CA II in situ (Figure 3j) [70]. In an interesting variation of fragment-based ligand discovery, a surface histidine residue was exploited to bind to a metal-chelating fragment [71]. Crystallography confirmed that at least one of these chelating molecules binds as designed (Figure 3k). Another fragment-discovery method, called ‘encoded self-assembling chemical libraries’, identifies fragments that can bind to the target simultaneously using small molecules attached to oligonucleotides [72]; promising fragments are subsequently linked. When applied to CA, low nanomolar compounds were identified, although their affinity was only modestly better than the sulfonamide fragment alone (blue) (Figure 3l). Other targets
In a striking recent example of fragment-based lead discovery, researchers at Abbott discovered small molecules that inhibit the anti-apoptotic protein Bcl-2 [73,74,75] (Figure 4a). After structure-activity relationships by NMR (SAR by NMR) identified two initial fragments, the carboxylic-acid-containing biphenyl of one fragment was linked to a surrogate of the second hydrophobic fragment, and the resulting molecule was optimized through parallel synthesis and structure-based design. NMR-based rational design was then used to reduce binding to serum albumin and produced a very high affinity molecule, ABT-737, that caused regression of tumors in mice. www.sciencedirect.com
Another example of protein interaction inhibitors discovered using an NMR-based fragment approach is shown in Figure 4b. In this case, fragments of millimolar potency were identified as binding simultaneously to adjacent sites on the proapoptotic member of the Bcl-2 family, Bid, through interligand nuclear Overhauser effects (NOEs), and then linked together to yield a low micromolar inhibitor [76]. Astex discovered two potent p38a MAP kinase inhibitors [77,78]. In both cases (Figure 4c,d), a weak ATP-competitive inhibitor was identified and optimized using crystallography, medicinal chemistry and knowledge of other inhibitors to capitalize on flexible regions of the kinase. The ligand efficiency of these molecules is lower than that of the JNK inhibitor (Figure 2a), but this can possibly be ascribed to the MAP kinase inhibitors binding in an extended mode compared with the JNK inhibitors, which bind in a more classical purine-like mode. As additional ‘extended-mode’ kinase inhibitors are discovered, the effect of binding mode on ligand efficiency will become clearer. In a conceptual reversal of fragment-based lead discovery, researchers at Astex and the University of Cambridge dissected the binding of the cofactor NADPH to the enzyme ketopantoate reductase [79]. Using isothermal titration calorimetry, biochemical inhibition experiments and mutagensis, they were able to determine the enthalpy and entropy contributed by each fragment of the molecule and to identify the ‘hotspots’ in the protein (Figure 4e). Although the overall ligand efficiency of this cofactor is remarkably low, the researchers were able to show that the 20 -phosphate and reduced nicotinamide moieties contribute significantly to the binding affinity. A study of the enzyme type 1 17b-hydroxysteroid dehydrogenase (17b-HSD) provides another example of using distinct binding sites to develop inhibitors (Figure 4f). In this case, a substrate mimic was linked to a cofactor mimic to produce potent inhibitors; however, as in the example above, the ligand efficiency is quite low [80]. Finally, an unusual application of fragment-based drug discovery against a nucleic acid target is shown in Figure 4g [81]. The small-molecule neamine, which binds to two adjacent sites on the A site of the 16S subunit of bacterial ribosomal RNA, was dimerized to produce a more potent analog that showed good antimicrobial activity against a panel of bacteria.
Conclusions Although not exhaustive, the examples presented here span the range from proof-of-concept studies designed to validate new technology, through applications of established technology to discover hits against drug targets, to full-scale medicinal chemistry programs en route to the Current Opinion in Biotechnology 2006, 17:643–652
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clinic. The number and breadth of targets demonstrates that fragment-based lead discovery has left the realm of speculative science to become a standard tool in drug discovery.
4. Erlanson DA, McDowell RS, O’Brien T: Fragment-based drug discovery. J Med Chem 2004, 47:3463-3482. Aside from [1], one of the most complete reviews on the subject.
However, the true power of the approach is far from being fully realized. Starting with fragments that are highly efficient is most likely to achieve reasonably sized final molecules. Of the 11 examples of fragment optimization for which ligand efficiencies are available for both the initial fragment and most potent molecule, only five generated inhibitors that are more efficient than the initial fragments. None of the fragment-linking examples for which data are available have ligand efficiencies greater than both of the initial fragments, and only six of seventeen have greater ligand efficiencies than even one of the fragments.
6.
Fattori D: Molecular recognition: the fragment approach in lead generation. Drug Discov Today 2004, 9:229-238.
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Mitchell T, Cherry M: Fragment-based drug design. Innovations in Pharmaceutical Technology 2005:34-36.
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Jhoti H: A new school for screening. Nat Biotechnol 2005, 23:184-186.
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Zartler ER, Shapiro MJ: Fragonomics: fragment-based drug discovery. Curr Opin Chem Biol 2005, 9:366-370.
Although fragment linking is becoming more common, it continues to be inefficient. Of course, affinity is not the only important parameter in drug discovery, and fragment-based methods can be used to identify fragments with improved stability, solubility or pharmacological properties. In such cases, the ligand efficiency might decline even as the overall quality of the lead improves. Fragment-based lead discovery needs better strategies for linking and expanding fragments without generating unacceptably large molecules. Nonetheless, the field has come remarkably far in the decade since it first became practical [3]. Within the next decade, I predict we will see approved drugs that began their journey to the clinic as fragments.
Disclosure Daniel A Erlanson is an employee of Sunesis Pharmaceuticals, Inc.
Acknowledgements I thank Monya Baker for editorial assistance, and Robert McDowell for useful suggestions on the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Jahnke W, Erlanson DA (Eds): Fragment-Based Approaches in Drug Discovery. Vol 34 in series Methods and Principles in Medicinal Chemistry (Series Eds: R Mannhold, H Kubinyi, G Folkers). Weinheim, Germany: Wiley-VCH; 2006. A comprehensive monograph covering all aspects of fragment-based drug discovery, from theory to library design to practical methodologies and case studies.
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54. Sanders WJ, Nienaber VL, Lerner CG, McCall JO, Merrick SM, Swanson SJ, Harlan JE, Stoll VS, Stamper GF, Betz SF et al.: Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization. J Med Chem 2004, 47:1709-1718. 55. Hartshorn MJ, Murray CW, Cleasby A, Frederickson M, Tickle IJ, Jhoti H: Fragment-based lead discovery using X-ray crystallography. J Med Chem 2005, 48:403-413. 56. Howard N, Abell C, Blakemore W, Chessari G, Congreve M, Howard S, Jhoti H, Murray CW, Seavers LC, van Montfort RL: Application of fragment screening and fragment linking to the discovery of novel thrombin inhibitors. J Med Chem 2006, 49:1346-1355. A beautiful illustration of Astex’s crystallography-centred approach to fragment-based drug discovery. 57. Wyss DF, Arasappan A, Senior MM, Wang YS, Beyer BM, Njoroge FG, McCoy MA: Non-peptidic small-molecule inhibitors of the single-chain hepatitis C virus NS3 protease/ NS4A cofactor complex discovered by structure-based NMR screening. J Med Chem 2004, 47:2486-2498. 58. Wu J, Rush TS III, Hotchandani R, Du X, Geck M, Collins E, Xu ZB, Skotnicki J, Levin JI, Lovering FE: Identification of potent and selective MMP-13 inhibitors. Bioorg Med Chem Lett 2005, 15:4105-4109. 59. Whiting M, Muldoon J, Lin YC, Silverman SM, Lindstrom W, Olson AJ, Kolb HC, Finn MG, Sharpless KB, Elder JH et al.: Inhibitors of HIV-1 protease by using in situ click chemistry. Angew Chem Int Ed Engl 2006, 45:1435-1439. 60. Erlanson DA, Lam JW, Wiesmann C, Luong TN, Simmons RL, DeLano WL, Choong IC, Burdett MT, Flanagan WM, Lee D et al.: In situ assembly of enzyme inhibitors using extended tethering. Nat Biotechnol 2003, 21:308-314. 61. O’Brien T, Fahr BT, Sopko M, Lam JW, Waal ND, Raimundo BC, Purkey H, Pham P, Romanowski MJ: Structural analysis of Current Opinion in Biotechnology 2006, 17:643–652
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caspase-1 inhibitors derived from Tethering. Acta Crystallogr 2005, F61:451-458. 62. Fahr BT, O’Brien T, Pham P, Waal ND, Baskaran S, Raimundo BC, Lam JW, Sopko MM, Purkey HE, Romanowski MJ: Tethering identifies fragment that yields potent inhibitors of human caspase-1. Bioorg Med Chem Lett 2006, 16:559-562. 63. Feng S, Wang Z, He X, Zheng S, Xia Y, Jiang H, Tang X, Bai D: Bis-huperzine B, highly potent and selective acetylcholinesterase inhibitors. J Med Chem 2005, 48:655-657. 64. Greenblatt HM, Guillou C, Guenard D, Argaman A, Botti S, Badet B, Thal C, Silman I, Sussman JL: The complex of a bivalent derivative of galanthamine with torpedo acetylcholinesterase displays drastic deformation of the active-site gorge: implications for structure-based drug design. J Am Chem Soc 2004, 126:15405-15411. 65. Munoz-Ruiz P, Rubio L, Garcia-Palomero E, Dorronsoro I, del Monte-Millan M, Valenzuela R, Usan P, de Austria C, Bartolini M, Andrisano V et al.: Design, synthesis, and biological evaluation of dual binding site acetylcholinesterase inhibitors: new disease-modifying agents for Alzheimer’s disease. J Med Chem 2005, 48:7223-7233. 66. Lewis WG, Green LG, Grynszpan F, Radic Z, Carlier PR, Taylor P, Finn MG, Sharpless KB: Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew Chem Int Ed Engl 2002, 41:1053-1057. 67. Manetsch R, Krasinski A, Radic Z, Raushel J, Taylor P, Sharpless KB, Kolb HC: In situ click chemistry: enzyme inhibitors made to their own specifications. J Am Chem Soc 2004, 126:12809-12818. 68. Bourne Y, Kolb HC, Radic Z, Sharpless KB, Taylor P, Marchot P: Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation. Proc Natl Acad Sci USA 2004, 101:1449-1454.
with ‘two-prong’ inhibitors reveal the molecular basis of high affinity. J Am Chem Soc 2006, 128:3011-3018. 72. Melkko S, Scheuermann J, Dumelin CE, Neri D: Encoded self-assembling chemical libraries. Nat Biotechnol 2004, 22:568-574. 73. Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ et al.: An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 2005, 435:677-681. A tour de force in the application of Abbott’s SAR-by-NMR to a challenging protein–protein interaction. 74. Wendt MD, Shen W, Kunzer A, McClellan WJ, Bruncko M, Oost TK, Ding H, Joseph MK, Zhang H, Nimmer PM et al.: Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo. J Med Chem 2006, 49:1165-1181. 75. Petros AM, Dinges J, Augeri DJ, Baumeister SA, Betebenner DA, Bures MG, Elmore SW, Hajduk PJ, Joseph MK, Landis SK et al.: Discovery of a potent inhibitor of the antiapoptotic protein Bcl-xL from NMR and parallel synthesis. J Med Chem 2006, 49:656-663. 76. Becattini B, Culmsee C, Leone M, Zhai D, Zhang X, Crowell KJ, Rega MF, Landshamer S, Reed JC, Plesnila N et al.: Structureactivity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid. Proc Natl Acad Sci USA 2006. 77. Gill AL, Frederickson M, Cleasby A, Woodhead SJ, Carr MG, Woodhead AJ, Walker MT, Congreve MS, Devine LA, Tisi D et al.: Identification of novel p38a MAP kinase inhibitors using fragment-based lead generation. J Med Chem 2005, 48:414-426. 78. Gill A, Cleasby A, Jhoti H: The discovery of novel protein kinase inhibitors by using fragment-based high-throughput X-ray crystallography. ChemBioChem 2005, 6:506-512.
69. Krasinski A, Radic Z, Manetsch R, Raushel J, Taylor P, Sharpless KB, Kolb HC: In situ selection of lead compounds by click chemistry: target-guided optimization of acetylcholinesterase inhibitors. J Am Chem Soc 2005, 127:6686-6692.
79. Ciulli A, Williams G, Smith AG, Blundell TL, Abell C: Probing hot spots at protein-ligand binding sites: a fragment-based approach using biophysical methods. J Med Chem 2006, 49:4992-5000.
70. Mocharla VP, Colasson B, Lee LV, Roper S, Sharpless KB, Wong CH, Kolb HC: In situ click chemistry: enzyme-generated inhibitors of carbonic anhydrase II. Angew Chem Int Ed Engl 2004, 44:116-120.
80. Poirier D, Boivin RP, Tremblay MR, Berube M, Qiu W, Lin SX: Estradiol-adenosine hybrid compounds designed to inhibit type 1 17b-hydroxysteroid dehydrogenase. J Med Chem 2005, 48:8134-8147.
71. Jude KM, Banerjee AL, Haldar MK, Manokaran S, Roy B, Mallik S, Srivastava DK, Christianson DW: Ultrahigh resolution crystal structures of human carbonic anhydrases I and II complexed
81. Agnelli F, Sucheck SJ, Marby KA, Rabuka D, Yao SL, Sears PS, Liang FS, Wong CH: Dimeric aminoglycosides as antibiotics. Angew Chem Int Ed Engl 2004, 43:1562-1566.
Current Opinion in Biotechnology 2006, 17:643–652
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Current Opinion in Biotechnology 2006, 17:643–652 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Gary Woodnut and Frank S Walsh Available online 3rd November 2006 0958-1669/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.10.007
Introduction Small-molecule drug discovery has always been a struggle of attrition, but in the past few years pressures have mounted to increase efficiency at all stages of the process. A potential solution for lead identification and optimization, fragment-based lead discovery, is becoming increasingly popular [1]. The goal is to build drug leads in pieces, by identifying small molecular fragments and then either linking them or expanding them. The definition of a fragment varies, but usually refers to molecules weighing less than 200–300 Da, with fewer than 15–20 heavy atoms. The concept of fragment-based lead discovery was first proposed a quarter of a century ago [2], but difficulties finding and linking fragments delayed serious pursuit until 1996, when the first practical demonstration was published [3]. Since then, fragment-based lead discovery has become very popular, with numerous demonstrated breakthroughs in finding fragments as well as in linking and expanding them. In fact, the first full book on fragment-based drug discovery has just been published [1]. www.sciencedirect.com
There are also several general reviews [4,5,6–12], as well as more specialized reviews focusing on library design [13], structure-guided fragment screening [14,15], NMR [16,17,18,19,20], X-ray crystallography [21–23], functional screening [24], site-directed ligand discovery and affinity-based ligand discovery [25,26], Tethering1 [27,28], and kinases [29]. Given the wealth of primary and secondary literature on the topic, this review will take a chemocentric focus on specific examples published in the past two years. Previous reviews largely cover earlier work [4,5]. Specifically, this review examines recent fragment-based case studies and analyses how the atom-efficiency of ligands changes as fragments are advanced.
Theoretical advantages of fragment-based lead discovery The basic approach of fragment-based lead discovery is shown in Figure 1. After an initial fragment that binds to a protein of interest is identified, it is either elaborated or combined with other fragments to generate a hit, which is subsequently optimized to produce a lead, and (in the best cases) a drug. Many disparate methods for finding fragments have been developed over the past few years. The strategies to transform a fragment to a lead also range considerably, from small changes to the fragment itself to dramatic substitutions and linkage with other fragments. As suggested in the figure, these distinctions are sometimes more semantic than real, and in theory the same final molecule could be created by linking two fragments, optimizing a single fragment or through some combination. One of the main theoretical advantages of this approach is that there are fewer possible fragments than possible drug-sized molecules. By way of analogy, only two English words consist of one letter: ‘a’ and ‘I’. The number of two-letter words is larger, but still limited; however, the number of six letter words is considerable. Similarly, there are about 107 fragments with up to 12 heavy atoms (excluding 3- and 4-ring containing structures) [30] compared with an estimated 1063 small drug-like molecules with up to 30 heavy atoms [31]. Given that all the laboratories throughout the world are estimated to contain ‘only’ about 108 molecules [32], screening fragments would sample pharmaceutically acceptable chemical diversity space much more efficiently. A second theoretical advantage of fragment-based lead discovery is that the resulting molecules are likely to have a higher ‘ligand efficiency’ than molecules discovered Current Opinion in Biotechnology 2006, 17:643–652
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Figure 1
The basic concept of fragment-based lead discovery. The blue ovoid and red rectangle represent fragments that bind to the target protein. These can be linked or expanded to produce high-affinity ligands.
through conventional methods. An investigation of roughly 150 ligands, many of them drugs, reveals that the free energy of binding increases roughly linearly with increasing ligand size up to about 15 atoms, beyond which there is very little increase; the maximum free-energy contribution per heavy atom is roughly –1.5 kcal/mol [33]. That study, as well as an earlier study [34], inspired the concept of ‘ligand efficiency’, defined as the ratio of the free energy of ligand binding to the number of nonhydrogen atoms [35]. This has been extended to other properties such as polar surface area and molecular weight [36]. Because fragments should have higher ligand efficiencies than typical high-throughput screening (HTS) hits, they should produce ultimately more ‘efficient’ drugs, which should have better pharmaceutical properties (lower molecular weight, better pharmacokinetics, lower toxicity, etc.). Fragments are also less likely to have interfering functionality that would prevent them from binding to a target protein [37]. Below, I consider recent examples of fragments and their derived hits or leads in terms of potency and ligand efficiency. Because the review is limited in scope to small-molecule drug discovery, peptides, dendrimers, and multimeric molecules are not included. Also, where Ki values are not available, IC50 (half maximum inhibition) values are used to calculate ligand efficiency (LE), as has been done previously [35].
Fragment optimization A challenge in providing the current opinion of fragmentbased lead discovery is in limiting the scope of the review. For example, the c-Jun NH2 terminal kinase (JNK) inhibitors recently reported by Zhao et al. [38] began with a HTS hit (Figure 2a) that qualifies as a fragment (MW = 220, 16 heavy atoms). The resulting optimization could easily be called ‘fragment optimization’ or ‘fragment growing’. However, it is also classic medicinal chemistry, albeit with a more ‘lead-like’ than ‘drug-like’ starting point [39]. Such examples are increasingly common in the literature as fragment-like approaches permeate all aspects of medicinal chemistry and fragments become more common in HTS libraries. Capturing all such examples is beyond the scope of this review. HowCurrent Opinion in Biotechnology 2006, 17:643–652
ever, as new techniques are only useful to the extent that they improve on existing techniques, this example serves as a useful benchmark. A contrasting example comes from Plexxikon, which recently identified itself as a major player in fragmentbased lead discovery with its report of a family of phosphodiesterase 4 (PDE4) inhibitors (Figure 2b) [40]. The researchers differentiate their ‘scaffold-based’ approach from other forms of fragment-based lead discovery in several ways, such as starting with larger numbers of larger fragments and including functional screening as well as high-throughput crystallography. Nevertheless, this research is ultimately an elegant example of fragment-based lead discovery, in that the starting fragment (MW = 168, 12 heavy atoms) is likely to have been missed in a standard HTS environment even though it is a highly efficient ligand. Ellman and colleagues reported substrate activity screening, an elegant fragment-based method, to discover protease inhibitors [41]. In this functional assay, fragments are first identified as substrates, and then converted to inhibitors through rapid analoging and the addition of a protease active-site trap. The method was used to identify highly potent cathepsin S inhibitors (Figure 2c). A combination of virtual screening and NMR was used to identify very small fragments that bind to the ATPbinding pocket of the bacterial protein DNA gyrase B (GyrB) [42]. Starting from a weak fragment, the researchers improved potency by iterative computational searches of commercial molecules to achieve biologically active molecules (Figure 2d). A different combination of computational and NMR screening followed by library design helped generate the lactate dehydrogenase inhibitor shown in Figure 2e [43]. In this case, a systems-based approach was used to develop inhibitors of multiple oxidoreductases, although different inhibitors showed selectivity for different enzymes. Anthrax lethal factor, a metalloproteinase, has also yielded to fragment-based ligand discovery. Starting from www.sciencedirect.com
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Figure 2
Examples of fragment-based lead discovery involving fragment optimization. (a) JNK1, (b) PDE4D, (c) cathepsin S, (d) DNA gyrase B, (e) lactate dehydrogenase, (f) anthrax lethal factor, (g) HCV-IRES IIA, (h) IMPDH, (i) PDE4, (j) hNK2 receptor, (k) DPP-IV, (l) DHNA, (m) CDK2, (n,o) thrombin (p) ribonuclease A (q) PTP1B. www.sciencedirect.com
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a library of only about 300 fragments and an NMR-based functional assay [44], researchers used subsequent synthetic elaboration to produce potent compounds with animal efficacy (Figure 2f).
pounds has not been reported, some of them, particularly the CDK2 inhibitor, have impressive ligand efficiencies, suggesting that they will make excellent starting points for lead discovery.
Researchers at Ibis Therapeutics have developed mass spectrometry (MS)-based fragment discovery. They identified a weak binder to the ribosomome IIA subdomain of hepatitis C (HCV-IRES IIA) and subsequently optimized it to become a submicromolar inhibitor (Figure 2g) [45].
Fragment merging and linking
In a nice example of fragment optimization, researchers at UCB Celltech used a combination of modeling and medicinal chemistry to discover inhibitors to the immunology target inosine monophosphate dehydrogenase (IMPDH, Figure 2h) [46,47]. The initial fragment, while only a modest inhibitor, has a very high ligand efficiency, and had been previously discovered by researchers from Roche using a virtual screen [48].
Proteases
Another application to PDE4 inhibitors is shown in Figure 2i. This ‘scaffold-linker-functional group’ approach relies on computational fragment-screening to design combinatorial libraries, and in this case was used to rapidly produce very high potency inhibitors against PDE4 starting from a known inhibitor [49]. Researchers at Menarini Ricerche used fragment-based design to construct a series of hNK2 receptor ligands [50]. Although the compounds could conceivably have been arrived at using combinatorial or traditional medicinal chemistry, the concept of fragment-based lead discovery (Figure 2j) made discovery efforts more efficient. Santhera Pharmaceuticals recently reported a series of dipeptidyl peptidase-IV (DPP-IV) inhibitors derived from both computational and crystallographic screening, followed by medicinal chemistry and fragment-merging with known inhibitors (Figure 2k) [51,52]. Abbott Laboratories, which pioneered the field of fragment-based lead discovery using NMR [3], has also done considerable work with crystallography [53]. Using a crystallographic approach, they discovered 7,8-dihydroneopterin aldolase (DHNA) inhibitors starting from a library of 10 000 fragments (Figure 2l) [54]. Interestingly, although the initial hit was identified by soaking the compounds into pre-formed crystals, structures of the subsequent compounds could only be obtained through co-crystallization. Astex Therapeutics focuses on the use of X-ray crystallography to identify fragments. They have reported a series of crystallographically identified fragments for several targets, including CDK2 (Figure 2m), thrombin (Figure 2n, 2o), ribonuclease A (Figure 2p), and PTP1B (Figure 2q) [55]. Although the elaboration of these comCurrent Opinion in Biotechnology 2006, 17:643–652
There have been considerable recent advances in the area of fragment merging and linking. Select targets and target classes will be covered separately in the following sections.
Proteases are ideally suited for fragment linking strategies. Because they cleave peptides or proteins, proteases usually have extended binding sites, with each aminoacid binding in its own subsite (S). An elegant example of Astex’s ‘Pyramid’ crystallography-based fragment screening applied to the protease thrombin is shown in Figure 3a. Two weak inhibitors were identified as binding in adjacent regions, one in the S1 pocket and the other in the S2–S4 region. These were linked to produce a highaffinity inhibitor, and crystallography shows that this molecule has roughly the same binding mode as the separate fragments [56]. An application of NMR-based screening, this time from Schering-Plough, was used to identify inhibitors of the single-chain hepatitis C virus NS3 protease–NS4A cofactor complex (ns4a–ns3p) [57]. This is a good example of fragment linking guided by the iterative use of NMR structural information (Figure 3b). Researchers at Wyeth were able to optimize an HTS hit against the matrix metalloproteinase MMP-13 (collagenase 3) by using crystallography and structure-based design to introduce a zinc-chelating carboxylic acid moiety (Figure 3c) [58]. Although carboxylic acids are not strong zinc chelators, the rigidity of the linker helps compensate for this weakness and introduces other contacts to the protein. With ‘in situ click chemistry’, proteins catalyse the formation of their own inhibitor from two fragments. In a nice model study using HIV-1 protease, the synthesis of a known nanomolar inhibitor from two fragments was catalyzed much more effectively in the presence of the enzyme than in the presence of bovine serum albumin (Figure 3d) [59]. Another protease that has yielded to fragment-based approaches is caspase-1. Using Sunesis’s Tethering with Extenders [60–62], an S4-binding fragment was linked to an S1-binding fragment to generate a submicromolar inhibitor. Further optimization by making the flexible linker rigid or adding an S2-binding element yielded more potent compounds with improved ligand efficiency (Figure 3e). www.sciencedirect.com
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Figure 3
Examples of fragment-based lead discovery involving fragment merging or linking: proteases, acetychlolinesterase and carbonic anhydrase. (a) Thrombin, (b) ns4a–ns3p, (c) MMP-13, (d) HIV-1 protease, (e) caspase-1, (f–i) acetylcholinesterases, and (j–l) carbonic anhydrases. www.sciencedirect.com
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Figure 4
Other examples of fragment-based lead discovery involving fragment merging or linking. (a) Bcl-2, (b) Bid, (c) p38a, (d) p38a, (e) ketopantoate reductase, (f) 17b-HSD, and (g) 16S rRNA. Current Opinion in Biotechnology 2006, 17:643–652
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Acetylcholinesterase
One of the most productive proteins for fragment linking has been acetylcholinesterase (AChE), owing partly to a deep gorge-like active site with binding pockets near the bottom and the top. For example, a bis-huperzine B ligand binds much more tightly than huperzine B alone (albeit with considerable loss of ligand efficiency), and modeling studies indicate this is due to two-site occupancy (Figure 3f) [63]. A similar approach with heterodimers produced low nanomolar inhibitors (Figure 3g). Xray crystallography of the bound inhibitors demonstrated that the common galanthamine moieties (blue) bind in the same position, while the phthalimide (red) binds near the top of the active site [64]. Another heterodimer approach, this time starting with the very efficient fragment tacrine, produced the picomolar inhibitors shown in Figure 3h [65]. The application of in situ click chemistry to discover AChE inhibitors has produced femtomolar inhibitors [66,67], and crystallography revealed that the protein makes contacts not just with the two fragments but with the triazole linker as well [68]. Subsequent work using a larger set of fragments has produced even more potent and novel compounds (Figure 3i) [69]. Carbonic anhydrase
Several approaches to fragment linking have proven successful with carbonic anhydrase (CA). Click chemistry was used to discover novel inhibitors to bovine CA II in situ (Figure 3j) [70]. In an interesting variation of fragment-based ligand discovery, a surface histidine residue was exploited to bind to a metal-chelating fragment [71]. Crystallography confirmed that at least one of these chelating molecules binds as designed (Figure 3k). Another fragment-discovery method, called ‘encoded self-assembling chemical libraries’, identifies fragments that can bind to the target simultaneously using small molecules attached to oligonucleotides [72]; promising fragments are subsequently linked. When applied to CA, low nanomolar compounds were identified, although their affinity was only modestly better than the sulfonamide fragment alone (blue) (Figure 3l). Other targets
In a striking recent example of fragment-based lead discovery, researchers at Abbott discovered small molecules that inhibit the anti-apoptotic protein Bcl-2 [73,74,75] (Figure 4a). After structure-activity relationships by NMR (SAR by NMR) identified two initial fragments, the carboxylic-acid-containing biphenyl of one fragment was linked to a surrogate of the second hydrophobic fragment, and the resulting molecule was optimized through parallel synthesis and structure-based design. NMR-based rational design was then used to reduce binding to serum albumin and produced a very high affinity molecule, ABT-737, that caused regression of tumors in mice. www.sciencedirect.com
Another example of protein interaction inhibitors discovered using an NMR-based fragment approach is shown in Figure 4b. In this case, fragments of millimolar potency were identified as binding simultaneously to adjacent sites on the proapoptotic member of the Bcl-2 family, Bid, through interligand nuclear Overhauser effects (NOEs), and then linked together to yield a low micromolar inhibitor [76]. Astex discovered two potent p38a MAP kinase inhibitors [77,78]. In both cases (Figure 4c,d), a weak ATP-competitive inhibitor was identified and optimized using crystallography, medicinal chemistry and knowledge of other inhibitors to capitalize on flexible regions of the kinase. The ligand efficiency of these molecules is lower than that of the JNK inhibitor (Figure 2a), but this can possibly be ascribed to the MAP kinase inhibitors binding in an extended mode compared with the JNK inhibitors, which bind in a more classical purine-like mode. As additional ‘extended-mode’ kinase inhibitors are discovered, the effect of binding mode on ligand efficiency will become clearer. In a conceptual reversal of fragment-based lead discovery, researchers at Astex and the University of Cambridge dissected the binding of the cofactor NADPH to the enzyme ketopantoate reductase [79]. Using isothermal titration calorimetry, biochemical inhibition experiments and mutagensis, they were able to determine the enthalpy and entropy contributed by each fragment of the molecule and to identify the ‘hotspots’ in the protein (Figure 4e). Although the overall ligand efficiency of this cofactor is remarkably low, the researchers were able to show that the 20 -phosphate and reduced nicotinamide moieties contribute significantly to the binding affinity. A study of the enzyme type 1 17b-hydroxysteroid dehydrogenase (17b-HSD) provides another example of using distinct binding sites to develop inhibitors (Figure 4f). In this case, a substrate mimic was linked to a cofactor mimic to produce potent inhibitors; however, as in the example above, the ligand efficiency is quite low [80]. Finally, an unusual application of fragment-based drug discovery against a nucleic acid target is shown in Figure 4g [81]. The small-molecule neamine, which binds to two adjacent sites on the A site of the 16S subunit of bacterial ribosomal RNA, was dimerized to produce a more potent analog that showed good antimicrobial activity against a panel of bacteria.
Conclusions Although not exhaustive, the examples presented here span the range from proof-of-concept studies designed to validate new technology, through applications of established technology to discover hits against drug targets, to full-scale medicinal chemistry programs en route to the Current Opinion in Biotechnology 2006, 17:643–652
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clinic. The number and breadth of targets demonstrates that fragment-based lead discovery has left the realm of speculative science to become a standard tool in drug discovery.
4. Erlanson DA, McDowell RS, O’Brien T: Fragment-based drug discovery. J Med Chem 2004, 47:3463-3482. Aside from [1], one of the most complete reviews on the subject.
However, the true power of the approach is far from being fully realized. Starting with fragments that are highly efficient is most likely to achieve reasonably sized final molecules. Of the 11 examples of fragment optimization for which ligand efficiencies are available for both the initial fragment and most potent molecule, only five generated inhibitors that are more efficient than the initial fragments. None of the fragment-linking examples for which data are available have ligand efficiencies greater than both of the initial fragments, and only six of seventeen have greater ligand efficiencies than even one of the fragments.
6.
Fattori D: Molecular recognition: the fragment approach in lead generation. Drug Discov Today 2004, 9:229-238.
7.
Mitchell T, Cherry M: Fragment-based drug design. Innovations in Pharmaceutical Technology 2005:34-36.
8.
Jhoti H: A new school for screening. Nat Biotechnol 2005, 23:184-186.
9.
Zartler ER, Shapiro MJ: Fragonomics: fragment-based drug discovery. Curr Opin Chem Biol 2005, 9:366-370.
Although fragment linking is becoming more common, it continues to be inefficient. Of course, affinity is not the only important parameter in drug discovery, and fragment-based methods can be used to identify fragments with improved stability, solubility or pharmacological properties. In such cases, the ligand efficiency might decline even as the overall quality of the lead improves. Fragment-based lead discovery needs better strategies for linking and expanding fragments without generating unacceptably large molecules. Nonetheless, the field has come remarkably far in the decade since it first became practical [3]. Within the next decade, I predict we will see approved drugs that began their journey to the clinic as fragments.
Disclosure Daniel A Erlanson is an employee of Sunesis Pharmaceuticals, Inc.
Acknowledgements I thank Monya Baker for editorial assistance, and Robert McDowell for useful suggestions on the manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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