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, andin silicoscreening — 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 (erlanson@sunesis.com)
Current Opinion in Biotechnology2006,17:643–652
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.
DOI10.1016/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 optimiza-tion, fragment-based lead discovery, is becoming increas-ingly 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 weigh-ing 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 frag-ment-based drug discovery has just been published [1].
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. Pre-vious reviews largely cover earlier work [4,5]. Specifi-cally, 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 inFigure 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 Eng-lish 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 107fragments with up to 12 heavy atoms (excluding 3- and 4-ring containing structures) [30] com-pared with an estimated 1063small 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
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 non-hydrogen atoms [35]. This has been extended to other properties such as polar surface area and molecular weight [36]. Because fragments should have higher ligand effi-ciencies than typical high-throughput screening (HTS) hits, they should produce ultimately more ‘efficient’ drugs, which should have better pharmaceutical proper-ties (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 Kivalues are not available, IC50(half maximum
inhibi-tion) values are used to calculate ligand efficiency (LE), as has been done previously [35].
Fragment optimization
A challenge in providing the current opinion of fragment-based 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 ‘frag-ment growing’. However, it is also classic medicinal chemistry, albeit with a more ‘lead-like’ than ‘drug-like’ starting point [39]. Such examples are increasingly com-mon in the literature as fragment-like approaches perme-ate all aspects of medicinal chemistry and fragments become more common in HTS libraries. Capturing all such examples is beyond the scope of this review.
How-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 fragment-based lead discovery with its report of a family of phos-phodiesterase 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 frag-ment-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 screen-ing, an elegant fragment-based method, to discover pro-tease 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 iden-tify 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 ATP-binding pocket of the bacterial protein DNA gyrase B (GyrB) [42]. Starting from a weak fragment, the research-ers 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 inFigure 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 644 Pharmaceutical biotechnology
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.
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)hNK2receptor,(k)DPP-IV,(l)DHNA,(m)CDK2,(n,o)thrombin
a library of only about 300 fragments and an NMR-based functional assay [44], researchers used subsequent syn-thetic elaboration to produce potent compounds with animal efficacy (Figure 2f).
Researchers at Ibis Therapeutics have developed mass spectrometry (MS)-based fragment discovery. They iden-tified 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]. In a nice example of fragment optimization, researchers at UCB Celltech used a combination of modeling and medicinal chemistry to discover inhibitors to the immu-nology 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].
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 start-ing from a known inhibitor [49].
Researchers at Menarini Ricerche used fragment-based design to construct a series of hNK2receptor 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 frag-ment-based lead discovery using NMR [3], has also done considerable work with crystallography [53]. Using a crystallographic approach, they discovered 7,8-dihydro-neopterin 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 crystal-lography to identify fragments. They have reported a series of crystallographically identified fragments for sev-eral targets, including CDK2 (Figure 2m), thrombin (Figure 2n, 2o), ribonuclease A (Figure 2p), and PTP1B (Figure 2q) [55]. Although the elaboration of these
com-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.
Fragment merging and linking
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 sec-tions.
Proteases
Proteases are ideally suited for fragment linking strate-gies. Because they cleave peptides or proteins, proteases usually have extended binding sites, with each amino-acid binding in its own subsite (S). An elegant example of Astex’s ‘Pyramid’ crystallography-based fragment screen-ing 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 high-affinity 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 cofac-tor 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 (collage-nase 3) by using crystallography and structure-based design to introduce a zinc-chelating carboxylic acid moi-ety (Figure 3c) [58]. Although carboxylic acids are not strong zinc chelators, the rigidity of the linker helps compensate for this weakness and introduces other con-tacts to the protein.
With ‘in situ click chemistry’, proteins catalyse the for-mation 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 cat-alyzed 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).
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.
648 Pharmaceutical biotechnology
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.
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 occu-pancy (Figure 3f) [63]. A similar approach with hetero-dimers produced low nanomolar inhibitors (Figure 3g). X-ray 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 frag-ment 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 IIin situ (Figure 3j) [70]. In an interesting variation of frag-ment-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 dis-covery, 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 car-boxylic-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.
Another example of protein interaction inhibitors discov-ered 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 micro-molar inhibitor [76].
Astex discovered two potent p38aMAP kinase inhibitors [77,78]. In both cases (Figure 4c,d), a weak ATP-compe-titive inhibitor was identified and optimized using crystal-lography, 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 dehy-drogenase (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 antimi-crobial 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 estab-lished technology to discover hits against drug targets, to full-scale medicinal chemistry programs en route to the
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.
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.
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 frag-ment-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 Pharma-ceuticals, Inc.
Acknowledgements
I thank Monya Baker for editorial assistance, and Robert McDowell for useful suggestions on the manuscript.
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