key: cord-328483-sj8i9ss2 authors: Jaegle, Mike; Wong, Ee Lin; Tauber, Carolin; Nawrotzky, Eric; Arkona, Christoph; Rademann, Jörg title: Protein‐Templated Fragment Ligations—From Molecular Recognition to Drug Discovery date: 2017-05-31 journal: Angew Chem Int Ed Engl DOI: 10.1002/anie.201610372 sha: doc_id: 328483 cord_uid: sj8i9ss2 Protein‐templated fragment ligation is a novel concept to support drug discovery and can help to improve the efficacy of protein ligands. Protein‐templated fragment ligations are chemical reactions between small molecules (“fragments”) utilizing a protein's surface as a reaction vessel to catalyze the formation of a protein ligand with increased binding affinity. The approach exploits the molecular recognition of reactive small‐molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations. In this way, chemical synthesis and bioassay are integrated in one single step. This Review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein‐templated ligation products. The chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed. Protein-binding,b ioactive molecules,t he starting points to future drugs,are in most cases identified and optimized in two clearly separated steps:first small molecules are synthesized chemically or isolated from natural sources,t hen their biochemical properties are investigated in bioassays (Figure 1A) . Both steps can be repeated iteratively during the optimization process.A ccordingly,t he classical process for lead discovery is characterized by the strict separation of the generation of chemical libraries and their screening for biological activities in aprotein-based or cellular assay. Over the last two decades,h owever,a na lternative concept of drug discovery has emerged that aims at the integration of chemical synthesis and bioassays. [1] [2] [3] This approach exploits the molecular recognition of reactive small-molecule fragments by proteins both for assembly of the chemical ligand and for the identification of bioactive fragment combinations and is,t herefore,d enominated as "protein-templated fragment ligation". Protein-templated fragment ligation is an attractive alternative to classical drug discovery for several reasons:T he combination of chemical synthesis and bioassay in one step considerably confines the effort for chemical synthesis to the most active fragment combinations and, thus,i sh ighly efficient, saving time,money,energy,and chemical resources. Only small libraries of af ew hundred to at housand reactive fragments are required to cover the relevant chemical space and to test huge numbers of potential fragment combinations or fragment ligation products.M oreover,f ragment ligations enable the detection of low-affinity fragments for as patially resolved protein binding site,since the binding of the reactive primary fragment or protein probe amplifies the binding of asecondary fragment at ap recisely defined location. In this Review we will present the current state and the future potential of protein-templated fragment ligations in drug discovery.For this purpose we propose acomprehensive definition of protein-templated fragment ligations: Chemical reactions between two or more small molecules ("fragments") that utilize the proteinssurface as acatalyst to accelerate the formation of protein ligands with increased binding affinity are defined as protein-templated fragment ligations. This definition encompasses both reversible and irreversible ligation reactions.W ec onsider the combined coverage of both reaction types af itting approach, as all templated ligations share the same biophysical principles,pose the same challenges for the detection of products,a nd contribute excellent examples to the drug discovery process.M oreover, the definition creates ac lear distinction between templated fragment ligations and other catalytic transformations exerted by proteins similar to the turn-over of enzyme substrates.R emarkably,p rotein-templated fragment ligation reactions following this definition are the established mode of action of several clinically admitted drugs,which suggests that the reactions can indeed proceed efficiently under physiological conditions.F or example,t he anti-Parkinson drug carbidopa binds to the enzyme DOPAd ecarboxylase and reacts with the cofactor pyridoxal phosphate to form ahydrazone as the active inhibitor ( Figure 2A ). [4] Likewise,t he anticonvulsive drug vigabatrin reacts in ap rotein-templated mode with the cofactor of GABAt ransaminase to form aM ichael acceptor intermediate,w hich leads to the irreversible inhibition of the enzyme ( Figure 2B ). [5] Other examples Protein-templated fragment ligation is anovel concept to support drug discovery and can help to improve the efficacy of protein ligands. Protein-templated fragment ligations are chemical reactions between small molecules ("fragments") utilizing aproteinss urface as areaction vessel to catalyzethe formation of aprotein ligand with increased binding affinity.T he approache xploits the molecular recognition of reactive small-molecule fragments by proteins both for ligand assembly and for the identification of bioactive fragment combinations.Inthis way, chemical synthesis and bioassayare integrated in one single step.T his Review discusses the biophysical basis of reversible and irreversible fragment ligations and gives an overview of the available methods to detect protein-templated ligation products.T he chemical scope and recent applications as well as future potential of the concept in drug discovery are reviewed. of protein-templated fragment ligations in admitted drugs are found for selegiline [6] (reacts with cofactor FADi nthe depression target monoamine oxidase) and for isoniazid (reacts with NAD + in mycobacterium tuberculosis catalase). As fragment ligations are driven by thermodynamic interactions between the fragments and the protein, Section 2 considers the biophysical basis of protein-templated fragment ligations to highlight the conceptual potential of the method. Special emphasis will then be given to the detection of products of fragment ligation reactions,which is amajor and general challenge in fragment ligation assays (Section 3). In Section 4wewill give an overview of the chemical reactions used so far in templated ligations and also discuss possible future extensions of this reaction set. Reversible reactions, which have been studied in the context of dynamic covalent chemistry [7] [8] [9] [10] [11] [12] [13] [14] and irreversible reactions,also denominated as target-guided synthesis (TGS), [15] are treated together,a sb oth reaction categories deliver examples of templated fragment ligations and in many cases it is difficult to categorize one reaction unambiguously.R epresentative recent applications of templated ligations in fragment-based drug discovery are reported in Section 5, thus demonstrating how far the method has developed to date.F inally,i n Section 6w ew ill discuss the current state of protein-templated fragment ligations,considering the strengths of this method and the requirements for it to succeed. Thec omplementarity of the method with classical ligand screening and fragment-based methods will be considered, thereby leading to an outlook on the relevance and further development of proteintemplated fragment ligations for future drug discovery. In protein-templated fragment ligations the protein serves as acatalyst for the assembly of protein ligands from protein- Mike Jaegle, Ee Lin Wong, Carolin Tauber,and Eric Nawrotzkya re PhD students in the group, coming from academic backgrounds in chemistry, pharmacy,a nd molecular biology from universities in Freiburg (M.J.), Johor Bahru, Taipei, and Seoul (E.L.W.), Berlin (C.T.), and Leipzig (E.N.). They all work on projects involving protein-templated reactions on various protein targets including proteases, phosphatases, and protein-protein interactions. Christoph Arkona studied biochemistry at the Leibniz University in Hannover and joined the group as asenior scientist after finishing aP hD in plant biochemistry and many years of drug development in the biotech industry. The photo shows from left to right:Mike Jaegle, Eric Nawrotzky,Jçrg Rademann,C arolin Tauber,C hristoph Arkona, and Ee Lin Wong. binding small-molecule fragments.T he molecule fragments are chemically reactive and are linked covalently to yield fragment combinations with improved binding affinities and biological activities.T he process requires ac hemically reactive,d ynamic system that is able to adapt on the molecular level by the formation and-in the case of reversible reactions-recleavage of covalent chemical bonds and by evolving into at hermodynamically more favored state,t hus furnishing optimized protein ligands.S uch adaptive systems can be considered as examples of molecular learning, where the term "learning" is applied here to describe an adaptive, chemically evolving and self-optimizing system. Theadditivity of free binding energies is the driving force of protein-templated fragment ligations ( Figure 3 ). [16, 17] Tw o fragments bind to the protein independently and without any overlapping of their free energies of binding, DG 1 and DG 2 . [18] Thel inking of two fragments by ar eversible or irreversible chemical reaction forms aligation product with afree binding energy DG lig = DG 1 + DG 2 + X,w here X represents the deviation from the addition of the binding energies.Provided that the covalent linking of the two fragments is additive (X = 0), the obtained fragment combination product will display abinding affinity that is the product of the binding affinities of its fragments: K D = e ÀDG/RT = K D1 K D2 . [19, 20] Fore xample,t wo fragments with K D values of 1mm will result in af ragment ligation product of 1 mm.Infact, fragment combinations can also be strongly superadditive,asaresult of the additional binding energy of the linker or entropic gain, which results in an even stronger enhancement of binding affinities. [19] Likewise,t he linking of two fragments can reduce the binding energy of the ligation product below additivity if the linker contributes unfavorably to the binding. Thea nalytical detection of proteinbinding fragments constitutes am ajor challenge and has been as ignificant limitation to the development and exploitation of fragment-based methods in drug discovery.T he main reason for this detection problem is the low affinity of protein-binding fragments requiring high fragment concentrations to generate and possibly saturate the detection signal. In some assays,s uch as fluorescence anisotropy or saturation transfer difference (STD) NMR, high protein concentrations can be used instead to saturate the binding of the small-molecule ligand. Compared to other fragment-based methods without ligation, the detection problem, however,i ss ignificantly reduced in protein-templated fragment ligations due to the higher affinity of the fragment ligation product formed. As aresult, fragment ligation assays can, in principle,detect even low-affinity fragments that would not be identified in other fragment assays,for example,because of concentration limits. In general, fragments have to be present at concentrations that exceed their dissociation constants (K D values) by afactor of at least 10 for as trong, saturated detection signal to be measured. In contrast, in the case of afragment ligation assay,c oncentrations of the starting fragment below the K D value are typically used to effect only partial inhibition, which can be saturated by the formation of the stronger binding ligation product. Although the detection of fragment ligation products is thereby strongly facilitated compared to single fragments,i t remains ac hallenge for several reasons.A st he ligation products possess ah igher binding affinity than the starting fragments to the target protein, their formation is autoinhibitory and the amount of ligation product is limited strictly by the concentration of the protein template.A s ar esult, fragment ligation products have to be detected against astrong background of excess nonreacted fragments. Other challenges can arise from the reactivity of fragments in the fragment ligation assays.Although some reactive fragments such as those used in dipolar cycloaddition reactions are truly bioorthogonal, several fragment ligations rely on electrophiles that might also react with protein nucleophiles as reaction partners.C ontrol experiments that distinguish the effect of one fragment from the effect of af ragment combination are routinely conducted to avoid interference of such reactions with the assay read-out. In addition, it is generally recommended in hit validation to use independent secondary assays to detect false-positive hit fragments. Several classical analytical methods have been widely applied and adapted to the detection of templated fragment ligations ( Figure 4 ). Most studies in the field have used liquid chromatography,usually in combination with mass spectrometry (LC-MS). [13, [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] In some examples,s pecific methods of NMR spectroscopy were developed. [31] [32] [33] [34] [35] [36] In addition, fragment ligations have been studied by X-ray crystallography. [37] [38] [39] [40] [41] [42] Finally,asapowerful complement, the detection of fragment ligation products by various bioassays has been developed over recent years. [1, 10, [43] [44] [45] [46] [47] [48] Thed etection of fragment ligation products by LC-MS has become substantially easier over the last two decades because of the rapid development of this method in terms of chromatographic separation and the sensitivity of mass detectors. [13, [25] [26] [27] [28] Not only detection but also quantification and structure elucidation of ligation products is facilitated by using extracted-ion or single-ion chromatography and MS-MS techniques.S ome limitations remain, however.A s chromatographic separation takes some time,L C-MS detection is best suited for irreversible or quasi-irreversible ligation reactions. [15, 49] In the case of reversibly formed ligation products,t heir chemical fixation by ac hemical reaction or ap Hs hift can be an option. This strategy was followed, for example,i nt he seminal report by Huc and Lehn on "virtual chemical libraries", in which the unstable imine ligation products were converted into stable amines by chemical reduction during the ligation reaction. [21] Asecond limitation of LC-MS detection can arise from the buffer systems used in fragment ligation reactions.Many buffer ions used in ligation assays interfere with the detection of ligation products in the mass detector,w hereas buffer salts such as ammonium formiate or acetate that are highly compatible with LC-MS may interfere with the ligation reaction by adding reactive nucleophiles in strong excess.Inrecent years,the detection of protein-binding fragments by native protein MS has developed into am ature technology and it is very likely that this method will also be useful for the detection of fragment ligation products. [50] [51] [52] [53] NMR spectroscopy has the big advantage over LC that it can monitor ligation reactions directly in the assay solution. As standard NMR spectroscopy requires high (mm)c oncentrations of fragment ligation products in volumes of around 0.5 mL for the chemical analysis,l arge amounts of proteins are required. [54] Therefore,t he method is only applicable to proteins that are available in large quantities and only af ew experiments with low throughput can be conducted. The throughput can be enhanced if mixtures of reactive fragments are employed that undergo reversible,c ovalent ligation reactions. [33, [55] [56] [57] These mixtures are denominated as dynamic combinatorial libraries (DCL), or dynamic covalent libraries, and can in principle form all possible combinations of the available fragment building blocks,that is,inthe case of n and m complementary reactive building blocks,anumber of n m possible products are obtained. Thep rotein template shifts the equilibrium in favor of the best binding and, thus,m ost stabilized ligation product, which is then detected by one of the classical analytical methods. Several NMR methods have been adapted specifically for the detection of fragment ligation products.R amstrçm and co-workers have investigated 1 HSTD NMR spectroscopy for the detection of hemithioacetals formed by ap rotein-templated reaction in aqueous medium ( Figure 5 ). [58] 1 HSTD NMR spectroscopy is am ethod that exploits the selective transfer of proton magnetization from the protein to reversibly bound ligands and was used here to identify binding fragments from aD CL. Foraproof of concept study, bgalactosidase was selected as the target protein that catalyzes the hydrolysis of O-b-galactosides and contains no cysteine residues in the active site.Amixture of five thiols and two aldehydes was employed, thereby resulting in the potential formation of ten hemithioacetals.Itwas demonstrated that 1b-mercapto-d-galactose binds more strongly to the target enzyme than do the four other thiols and strongly enhanced the signals of both aldehydes in the STD spectrum-presumably through the formation of hemothioacetals. Surprisingly,t he binding of the two sugars could not be suppressed by the addition of substrate,t hus suggesting additional allosteric binding sites for these fragments.S ignificant inhibition of substrate hydrolysis could be confirmed for one thiol-aldehyde combination, which also suggests the formation of hemithioacetals as active inhibitors. Another application of STD NMR spectroscopy was published recently by the Hirsch research group. [36] Recently, 11 BNMR spectroscopy was applied by Claridge and coworkers for the detection of fragment ligation products (see Figure 10 in Section 5). [59] In addition to ligand-based NMR spectroscopy,t here have been intensive applications of protein-based NMR methods for the detection of protein-binding fragments. [60, 61] Protein NMR spectroscopy uses in many cases proteins that have been produced with 13 Cand 15 Nisotopes to enhance the NMR signals.F ragment binding can then be observed, for example,in2DHSQC experiments from perturbations of the chemical shifts.T he method was introduced as "structureactivity relationships (SAR) by NMR spectroscopy" and has the big advantage of furnishing information on the binding site of fragments if the signals in the NMR spectra are assigned to the protein structure. [62] Protein-based NMR spectroscopy should also be av aluable method for the investigation of fragment ligation reactions, although we could not find applications of it so far. X-ray crystallography has found broad application in fragment-based drug discovery,a nd its attractiveness comes from the detailed structural information it reveals of the fragment-protein complex. [63] This information can be extremely helpful in the design of fragment combination products.S everal studies have so far used protein crystallography for the detection of fragment ligation products. [37] [38] [39] [40] [41] [42] Ther apid and parallel detection of potent fragment ligation products formed in proteintemplated reactions was finally realized by the introduction of bioactivity-based assays.The initial strategy was denominated as dynamic ligation screening (DLS), as reversible ligation reactions were first investigated. DLS increased the sensitivity of ligand detection considerably,a nd the site-directed discovery of low affinity fragments with millimolar K D values was realized ( Figure 6 ). [10, 43] Bioactivity-based detection methods require only minimal amounts of protein;u sually low nanomolar concentrations of the protein are sufficient. They can be conducted using standard assay equipment such as microtiter plates and automated pipetting devices used for the handling of fragment libraries.I ne nzyme assays,t he sensitivity is further enhanced by the catalytic activity of the Figure 5 . Application of 1 HSTD NMR spectroscopy for detecting the proteintemplated formation of hemithioacetals from adynamic combinatoriall ibrary and enzymatic selection of the best inhibitor. [58] protein targets resulting in the rapid turnover of many of the fluorogenic or chromogenic substrate molecules.F luorescence resonance energy transfer (FRET) has also been used for the detection of fragment combinations. [44] To reach the highest sensitivity,t he primary reactive fragment has to be added to the enzyme assay at ac oncentration that results in 10-20 %i nhibition, thereby leaving am easurement window of 80-90 %f or detection of the enhanced inhibition after addition of the secondary fragment. Va rious bioassay formats have so far been adapted to dynamic ligation screening ( Figure 7) . As already described, fluorogenic substrates can be used in competition assays, competing with the fragment ligation product for the enzymesb inding site.I nt his assay the best fragment combination results in the strongest inhibition of the enzymatic reaction ( Figure 7A) . Alternatively,the substrate itself can be constructed with ar eactive group that allows for fragment ligations in which the ligated fragment can increase the affinity of the substrate to the target protein. These substrate-enhancement assays ( Figure 7B )e nable the site-directed detection of proteinbinding fragments in secondary binding sites that lead to accelerated turnover of the substrate. [45] Competitive binders can also be detected in this assay format. In the special case that the reactive,fluorogenic molecule is completely inactive without ligation of af ragment, it has been denominated as ap resubstrate and is especially sensitive to the detection of chemically unstable,t ransient ligation products such as hemiacetals and hemithioacetals. [46] Them ethod has been used successfully for the identification of secondary-site binding fragments of serine proteases [46] and of protein tyrosine phosphatases (PTPs), [45] where it was capable of detecting specific secondary-site binders as astarting point for the construction of PTP-specific inhibitors. Thep rinciple of bioactivity-based detection of fragment ligation products has also been extended from enzymatic assays to protein binding assays,thus enabling the identification of ligands of non-enzymatic protein-binding sites.P rotein-binding assays are principally conducted either in homogeneous solution or heterogeneously after attachment of one binding partner to as olid phase or surface.A homogeneous protein-binding assay for protein-templated ligation reactions has been demonstrated by using fluorescence polarization (FP), also known as fluorescence anisotropy,for detection ( Figure 7C ,D). [47] Forthis assay,areactive protein-binding peptide fragment was labeled with af luoro- Angewandte Chemie Reviews phore.T he FP ligand (10 nm)w as dissolved in ab uffer containing the target protein at ac oncentration leading to about 50 %l igand binding.T hen al ibrary of fragments was screened for those modulating the FP.T he assay enabled the identification both of competing ( Figure 7C )a nd of enhancing fragments ( Figure 7D )i nas ingle experiment, and the templating effect exerted by the protein was quantified for the best enhancing fragment combination. Reductive amination of the labeled fragment with the best enhancing fragment resulted in ap icomolar inhibitor.S imilar to the enzyme inhibition assays,h omogeneous protein-binding assays such as FP assays can be conducted in high-throughput equipment, in small assay volumes,a nd at low (nm)p rotein concentrations,p rovided aligand with sufficient affinity is available as astarting point. Label-free,h omogeneous protein-binding assays will be an attractive complement for the detection of bioactive fragment ligation products.F or example,thermal shift assays are used increasingly for the detection of protein-ligand complexes. [65, 66] Them ethod monitors the defolding ("melting") of proteins at increasing temperatures by using fluorophores that show increased fluorescence when they come into contact with the hydrophobic core of unfolding proteins. Fragment binding can be observed by as ignificant enhancement of the proteinsmelting temperature because of the free binding energy of the ligand. Even more thermodynamic information about the binding of fragments is revealed by isothermal titration calorimetry and future studies will most likely use this technique-although it requires al arge quantity of protein and can be conducted only at low throughput. [67] [68] [69] [70] Heterogeneous protein-binding assays have been applied to the investigation of protein-fragment binding. These methods are labelfree with respect to the fragments;h owever,c ovalent immobilization or labeling with affinity probes such as biotin are required. [71] [72] [73] [74] Recently,t he bioactivity-based detection of fragment ligation products has been further extended from protein assays to even more complex cellular experiments. [75, 76] Ohkanda and co-workers have reported the intracellular generation of the inhibitor 3 of the 14-3-3 protein by intracellular oxime ligation (Figure 8 ). The1 4-3-3 protein is involved in protein-protein interactions (PPIs), which are especially difficult to inhibit, as interactions of large and dynamic interaction surfaces have to be disrupted by molecules that are still able to penetrate cells through cellular uptake.T he authors realized that the reactive aldehyde derivative of fusicoccin A( Fc, 1)b inds in ah ydrophobic cavity adjacent to the binding site of peptide 2,which contains ah ydroxylamine functionality.T hey were able to demonstrate the templated formation of ah eterobivalent ligation product, the potent oxime inhibitor 3,i nvitro in the presence of 14-3-3 protein. Next, the authors investigated the intracellular formation of 3 in stably transformed HEK293 (Flag 14-3-3 z cell line). HPLC analysis showed the formation of 3 in cells treated with 1 and 2,w hich led to the highest cytotoxic effect. In contrast, chemically synthesized 3 was inactive in the cells,p ossibly because of the large molecular size that impedes their cell-penetrating properties (Figure 8 ). [77] Thus,t his study demonstrates that even large molecules that potentially modulate PPIs can be generated through intracellular protein-templated fragment ligation. In summary,the considerable progress made in detection methods over the last few years,e specially in bioactivitybased methods,b ut also in the classical analytical methods and biophysical detection strategies,h as facilitated the identification of bioactive fragment ligation products in protein-binding,enzymatic,and cellular assays.This development will enable the discovery of further examples for templated fragment ligation reactions in the future and thereby contribute to the successful application of fragment ligation assays. Numerous reaction types have so far been employed for templated fragment ligations.Here,wewill give an overview of these reaction types,focusing on the recent additions to this repertoire and giving an outlook on further extensions that can reasonably be expected. From ap ractical viewpoint, the degree of reversibility of the ligation reaction is an important criterium, as it affects the detection and isolation of the ligation products.T hus,r eaction types used for templated fragment ligations are categorized here according to their reversibility ( Table 1) . Additions of heteronucleophiles to aliphatic or aromatic aldehydes/ketones typically represent reversible ligation reactions that equilibrate rapidly in aqueous solutions as aresult of low activation barriers.There are,however, marked differences with respect to reversibility and kinetics depending on the reacting nucleophile. Hemiacetals and hemithioacetals are rapidly formed in water from aldehydes,alcohols,and thiols through templated fragment ligations,although they cannot be isolated as stable ligation products. [46, 58, 78, 79] Theformation of both hemiacetals and hemithioacetals as ligation products is impeded by competition of the heteronucleophiles with ah igh molar excess of water (55 m), which furnishes hydrates as alternative reaction products.Nevertheless,itcould be demonstrated by using NMR spectroscopy [58] and bioactivity-based assays [46] that aprotein template is able to shift the ligation equilibrium and, thus,t oenable the identification of hemiacetals and hemithioacetals as ligation products. [46] In contrast to the hemiacetals," full" acetals or dithioacetals (obtained by the addition of two alcohol or thiol nucleophiles to one carbonyl group) have not been reported as templated ligation products and cannot be expected to be formed because of the high activation barrier to the carbocation intermediates in this reaction. [80] [81] [82] Many templated ligation reactions involve nitrogen nucleophiles that react with carbonyl electrophiles.T he addition of primary amines to aldehydes furnishes hemiaminals as intermediates,w hich react further to form imines. [126, 127] Imine formation is also ap rocess that equilibrates rapidly and requires nonprotonated amines for reactivity. [10, 43, 44, 46, 47, 83, 84] Thus,t he process depends on the pK a value of the amine nucleophile and the pH value of the reaction buffer.I mines are considerably more stable than hemiacetals,e specially those formed from aromatic amines, and can even be isolated in some cases. One consequence of the increased stability of imines is that the equilibration of dynamic combinatorial libraries may take significantly longer than the equilibration of hemiacetals or hemithioacetals.T he stability of amine-aldehyde ligation products can be further enhanced if the aldehyde carries acidic a-hydrogen atoms,w hich allow the formation of enamines.A nother possibility is the chemical fixation of imines by an irreversible reaction, for example,r eductive amination to yield secondary amines. [86, 99] Thestability of the ligation products of nitrogen nucleophiles with aldehydes is further elevated in the case of hydroxylamines,h ydrazines, and acyl hydrazides. [14, 77, [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] 100] Theformed products,oximes and (acyl) hydrazones,a re stable at physiological pH values and can be isolated by standard procedures,s uch as column chromatography.A ccordingly,t he ligation reaction is equilibrated very slowly or aniline has to be added as acatalyst for the formation of the hydrazine or oxime. [86, 87, 89, 99] Alternatively,the equilibration of acyl hydrazones can be accelerated by the addition of acid. [98] Further ligations of heteronucleophiles with aldehydes are awaiting further investigation. Forexample,thiols can be added to imines to furnish N,S-acetals in al igation equilibrium that can possibly be shifted by interaction with aprotein template. [43, 46, 112] Bioactivity data obtained from the investigation of ap resubstrate have suggested that the trimeric complexes of aldehyde,a mine,a nd thiol have as tronger affinity to the protein target, as expressed by ar educed K M value and increased turnover of the enzyme substrates. [46] Likewise,t he observed superadditive binding and inhibition of peptide aldehydes and amines quantified by the FP assay can be interpreted by the formation of such thioaminal products. [43, 47] Mannich bases,aspecial form of stable aminals, can also be expected to be suitable for protein-templated reactions,atopic which is currently under investigation in our group. Further templated fragment ligations involve the addition of heteronucleophiles to Celectrophiles other than aldehydes.T hio-Michael additions have been reported in several cases,a nd the products can usually be isolated, even though the reaction can be reversible if the product favors retroaddition through b-elimination. [46, 117] Alkylations are usually truly irreversible ligations;i nm ost cases thiolates are employed as nucleophiles for highest reactivity,and aliphatic halogenides,e poxides,o rs ulfonates have been used as electrophiles. [118] [119] [120] These studies suggest that alternative Celectrophiles should be useful in protein-templated reactions.For example,nucleophilic substitutions at electron-poor aryl moieties could be an interesting extension of the reaction repertoire. Boric acid and boronic acids have been used as alternative electrophiles.T he use of diols as bisnucleophiles furnishes boronate esters as ligation products with limited stability. [59, 125] Other popular examples of reversible reactions that furnish stable products that can be isolated are disulfide formation and disulfide exchange reactions through the use of thiolates as the reactive nucleophiles under slightly basic and nonreducing conditions. [101] [102] [103] [104] [105] [106] [107] Asimilar exchange reaction of thiolates has been reported for the thioester exchange reaction. [33, 55, 82, [108] [109] [110] C À Cb ond-forming reactions are of special interest in templated ligations,a st hey can extend the choice and diversity of the accessible ligations products considerably.F ew examples have been reported to date. Ramstrçm and co-workers have described ar eversible templated Henry-aldol addition of nitroalkanes to aldehydes,i n which the formed, best-bound secondary alcohol was preferably acylated by the lipase as the protein target in the assay. [56] Although no further reports on templated aldol reactions have yet been published, this reaction type should be highly suitable for fragment ligation chemistry considering the detailed literature on aldol reactions in water and under mild reaction conditions. [56, 111, 116, 128] Likewise,a dditions of cyanide anions and isocyanides have been reported in water, which suggests that Passerini and Ugi reactions might be highly suitable for protein-templated reactions.T he same applies to the CÀCb ond formations by alkene and alkyne metathesis reactions,which have so far been demonstrated as ligation reactions in water. [112] [113] [114] [115] Classical examples of irreversible templated reactions have been reported for dipolar cycloaddition reactions, including azide-alkyne ligations that furnish 1,4-disubstituted a) Hemiacetals, b) hemithioacetals, c) acetals. d) thioacetals, e) N,S-acetals, f) imines, g) hydrazones, g')acylhydrazones, h) oximes, i) boronates, j) disulfides, k) thioesters, l) alkene metathesis, m) alkyne metathesis, n) nitroaldols o) alkylation, p) amidation between sulfonylazides and thioacids, q) 1,3-dipolarcycloaddition,r)addition, s) ring opening, t) amidation between amines and active esters. ,2,3-triazoles,a nd sulfonylazide-thioacid ligations that provide sulfonylamides through ac yclic intermediate. [24, 25, 76, [118] [119] [120] [121] [122] [123] [124] It is clear that numerous other ligations based on cycloaddition reactions may also succeed. Besides cycloaddition reactions,h owever, irreversible ligations have not yet been investigated much and future extensions are necessary to provide ligation products that cover alarger part of the biologically relevant chemical space.I ng eneral, chemoselective reactions in water are required for asuccessful ligation. Ideally,the reaction should provide alinker between the reacting fragments that supports binding or at least does not interfere with it. Fore xample,t he structural analysis of the world drug index (a collection of molecules with reported bioactivity) revealed that amide linkages are privileged linkers in bioactive compounds. [129] Thus,aprotein-templated amidation reaction can be considered an especially useful extension of the fragment ligation repertoire.V ery recently the first background-free protein-templated amidation reactions were discovered. [48] Other reaction types that could considerably extend the opportunities of fragment ligations are reactions that are able to connect fragments without al inker remaining in the product, such as cross-coupling reactions or reactions forming heterocycles as the connection between two fragments instead of ac lassical linker moiety. Having defined fragment ligation reactions,e xplained their biophysical background, useful detection strategies,a nd the underlying chemical transformations,w ea re now going to focus on the application of the concept for the discovery and optimization of protein ligands.W ew ill start with reversible ligation reactions and then proceed to the irreversible ones. Many of the first applications of reversible,t emplated fragment ligations were reported for dynamic combinatorial libraries (DCLs). As ar ecent representative example of this line of research, we highlight the studies by Hirsch and co-workers. [36, 98] Theauthors employed endothiapepsin as atarget and model enzyme for other aspartic acid proteases.N ine hydrazides (4-12)and one bisaldehyde were used to form adynamic library derived from two fragment hits [36] that bind to adjacent binding pockets of endothiapepsin ( Figure 9 ). [98] To ease analysis, two sublibraries (DCL-1 and DCL-2) were formed that consisted of four and five hydrazides.R eversed-phase HPLC and LC-MS were employed to analyze the templated formation of the bisacylhydrazone ligation products in the presence of endothiapepsin. Out of the potential 78 different bisacylhydrazones and 12 monoacylhydrazones,o nly 6p ossible fragment combinations showed significant amplification of the HPLC signal in the presence of the endothiapepsin template.T wo of the found combinations, 13 and 16,w ere synthesized and tested in af luorescence-based biochemical assay.B oth were reported to be active,and the best inhibitor, 13,showed a240fold increase in potencyc ompared to the starting fragments, thus illustrating as uccessful example of at emplated threefragment ligation reaction. Thea uthors demonstrated in this study that, unlike other fragment optimization methods such as fragment growing or merging, templated fragment ligation is especially sensitive for discovering combined fragments with superadditivity,w here their ligand efficiencies were not only maintained but improved. In their approach, the challenge lay in preserving the binding mode of the fragments and finding aw ell-fitting linker that provides additional interactions to the target. Despite the positive results,t he study also demonstrates the limitations of DCL, namely the tedious analysis of complex combinatorial mixtures and the limitation to few dynamic reactions that deliver non-druglike products such as acylhydrazones. Figure 9 . Protein-templated fragmentligations using adynamic combinatoriall ibrary. [98] A) Isophthalaldehyde 3 and nine hydrazides, 4-12 form up to 81 bisacylhydrazones. Bi)The model protein endothiapepsin is equilibrated with the DCL for the formation of potential inhibitors. Bii) HPLC analysis shows that the formation of the bisacylhydrazone 13 was amplified in the presence of the enzyme. Biii)Binding modes of 19 (purple) and 13 (cyan) were determined by Xray crystallography (PDB IDs:4 KUP and 5HCT respectively). C) The best compound 13 exhibits a240-fold improvement in potency and higher ligand efficiencyc ompared to the starting fragment 19. Thes ame limitations are displayed in the study by Claridge and co-workers who applied 11 BNMR spectroscopy for the detection of serine protease inhibitors from aDCL. [59] Foraproof-of-concept study, a-chymotrypsin (aCT) was recruited as amodel enzyme,a nd boronic acids were ligated with added sugar molecules to form improved enzyme inhibitors.T he formation of ternary complexes of enzyme, boronic acid, and sugar diol was monitored by 11 BNMR and 1 H-WaterLOGSY (water-ligand observed by gradient spectroscopy). These findings encouraged the groups of Schofield and Claridge to apply native mass spectrometry to detect hits from aD CL. Reversibly formed boronate esters were identified as inhibitors of prolyl hydroxylase domain isoform 2( PHD2), an enzyme of the 2-oxoglutarate (2-OG) oxygenase family (Figure 10 ). [130] PHD2 is an Fe II -containing 2-OG oxygenase which helps to regulate human hypoxic response, thus making it an excellent drug target for the treatment of anemia and ischemia-related diseases. In the experiments,aheterocyclic iron-binding boronic acid fragment was incubated with several pools of diol ligands and subsequently analyzed by native MS.T he best-binding fragment combinations were identified by the shift in protein mass and converted into stable,boron-free inhibitors by using Suzuki cross-coupling reactions,w hich resulted in inhibitors of the enzyme being active down to the nanomolar range.The results of the fragment ligation assays were further validated in another study by using a 1 HNMR based method. [131] Thetethering approach is aclassical example of reversible templated fragment ligations using disulfide exchange reactions. [132] This method exploits the reversible disulfide exchange reaction between an atural or an engineered cysteine residue on the protein surface and ad isulfidecontaining small-molecule fragment in solution. Further developments by the Erlanson group using so-called "extenders" made this approach even more applicable for fragment combination reactions. [133] In 2008 they reported an inhibitor screening that targeted the aurora kinase ( Figure 11 ). [134] The first step was to introduce ac ysteine residue near the ATP binding site.N ext, an extender was synthesized containing two disulfide-containing residues and ad iaminopyridine group,w hich is known to bind to the purine binding site. One disulfide residue was able to react and exchange with the introduced cysteine residue,t he other one was able to bind as econdary disulfide fragment. If this secondary fragment was able to interact with the adaptive region of the protein adjacent to the extender,athermodynamically stabilized disulfide bond was formed. This stabilized complex was subsequently detected by MS measurements through modification of the proteinsm ass.I nt heir experiments,alibrary containing roughly 4500 disulfide-containing fragments was screened in pools of 10 compounds,w hich resulted in the identification of several fragment combinations.Afew modifications of the corresponding fragments led to the formation of astable inhibitor with affinity in the micromolar range.Asimilar approach has been successfully applied to another kinase,3 -phosphoinositide-dependent protein kinase-1 (PDK1), by attaching the extender through an irreversible alkylation reaction. [135] Dynamic ligation screening was introduced in 2008 to overcome the detection limits of combinatorial methods such as DCL. Thef irst bioactivity-based detection of dynamically formed fragment ligation products has been demonstrated for Figure 10 . A) Schematic representation of the generation of boronate acid/ester leads using protein-directed dynamic combinatorial libraries containing diols and boronic acids. [130] B) The identification of potent boronate ester conjugates by using ab oronic acid "scaffold ligand" leads to the discovery of first and second generation stable analoguest hat inhibit PHD2 in the nanomolar range. the SARS coronavirus main protease (SARS-CoV M pro , Figure 12 ). [43] SARS-CoV M pro is av irus-encoded cysteine protease essential for the replication of the virus inside the infected host cells.I nhibitors targeting M pro are,t herefore, relevant as potential antivirals. [136, 137] Since proteases possess defined binding pockets for the side chains of peptides,itwas possible to develop ap eptide aldehyde inhibitor that positions an electrophilic aldehyde precisely at the active site of the protease.Dynamic ligation screening of afragment library of 234 diverse nucleophiles revealed several fragment hits that targeted the S1' pocket of the enzyme with K I values in the millimolar range.T he best fragment hit was converted into the corresponding aldehyde fragment and assayed again for enhanced inhibition against an amine library,t hereby providing asecondary hit fragment for the adjacent S1 pocket of the enzymesb inding site.O nly two iterations of dynamic ligation screening starting from apeptide inhibitor resulted in the selection of two millimolar-active small-molecule fragments,a nd the covalent combination of these fragments by reductive amination revealed an entirely nonpeptidic inhibitor of SARS-CoV M pro with a K I value of 2.9 mm. Further examples of bioactivity-based dynamic ligation assays are summarized in Table 2 . Caspase 3isaprotein that plays an essential role in the execution phase of cell apoptosis and, therefore,ap otential drug target in traumatic brain injury and amyotrophic lateral sclerosis as well as Alzheimers and Parkinsonsdisease.T he protein was tested with ananomolar fluorophore-labeled peptidyl ketoaldehyde inhibitor and atotal of 7397 fragments,including 4019 nucleophilic and primary amines in 384-well microtiter plates,byapplying the FP binding assay described above.Weobserved no change for most of the fragments tested. 78 fragments led to lower FP signals (negative cooperativity) and 176 fragments evoked asignificantly stronger FP signal (positive cooperativity) than the control. 21 fragments were confirmed as competitive inhibitors of caspase-3 with K I < 10 mm.T he best cooperatively binding fragment B was linked covalently to the starting ligand A by reductive amination, thereby resulting in apotent inhibitor of caspase-3 with a K I value of 80 pm. Dynamic ligation assays were further implemented for secondary-site screening of four closely related protein tyrosine phosphatases (PTPs): human PTP1B,P TPN7, PTPN12 (= SHP2), and mycobacterial MPTPA. [45] In general, the development of specific PTP inhibitors is considered to be amajor challenge that raises doubts on the druggability of this physiologically relevant class of enzymes.B yu sing 4-formyl- Figure 11 . Atethering approach using ad ynamice xtender has been applied to screen for fragments binding to Aurora kinase. [134] Angewandte Chemie Reviews phenyl phosphate as an electrophilic PTP substrate,s pecific secondary-site binding fragments could be identified for each of the PTPs from al ibrary of only 110 primary amines.T he specific secondary site binders were detected using a"dynamic substrate enhancement" assay (see Figure 7B ): active fragments form al igation product with the PTP substrate which results in ahigher stability of the enzyme-substrate complex and alowered K M value.This leads to an amplified release of phosphate ions,w hich was determined in aM alachite green assay.R eplacement of the phenyl phosphate substrate by anoncleavable phosphotyrosine mimetic and covalent linking to the selected MPTPA-specific amine fragment furnished inhibitors of protein tyrosine phosphatase A( MPTPA) from Mycobacterium tuberculosis,w ith no activity for the other three PTPs.T his result suggests that second-site targeting of PTPs enables the development of selective PTP inhibitors. Dynamic ligation screening has been further extended toward aspartic proteases,t his time by employing af luorescence resonance energy transfer (FRET) assay. [44] Ap eptide aldehyde was used as adirecting probe to identify the activesite binding fragments of the aspartic protease b-secretase (BACE-1). This enzyme is known to be the main culprit in the aggregation of amyloid-b-peptides,w hich is am ajor pathological hallmark of AlzheimersDisease (AD). Thus,considerable efforts have been made to discover BACE-1 inhibitors for potential therapeutic treatment of AD.I nstead of reversible hemithioacetal formation, the aldehyde hydrate was formed to bind the catalytic aspartic acid dyad through formation of ah ydrogen bond. Dynamic ligation of the peptide aldehyde with an amine nucleophile reversibly yielded an imine product. Thep eptide aldehyde,w hich serves as ac hemically reactive inhibitor (CRI), revealed 3-(3-aminophenyl)-2H-chromen-2-one to be ac ompetitive BACE1 inhibitor.T he identified 3-(aminophenyl)coumarin fragment was used as astarting point for hit optimization and al ow micromolar (K I = 3.7 mm)B ACE1 inhibitor was developed. Another application of bioactivity-based detection has been reported recently for the aspartic protease endothiapepsin. [64] In the most recent application, reversible protein-templated fragment ligation has been extended toward the discovery of irreversible inhibitors of enteroviral proteases ( Figure 13 ). [85] Epoxyaldehyde 23,amodified partial structure of the known inhibitor of cysteine proteases E-64 and aweak irreversible inhibitor of the 3C protease of Coxsackie 3B virus,w as tested with al ibrary of 850 primary amine fragments.5 -Aminopyrazolone 24 was discovered as af ragment hit that led to asuperadditive inhibition of the protease and covalent modification of the enzyme by addition of both fragments.I terative optimization of ligation products of 23 and 24 finally led to sub-micromolar,b road-spectrum inhibitors of enteroviral proteases,w ith 300-to 500-fold acceleration of the protein deactivation rate and no significant crossreactivity with nonviral proteases. Early studies in the area of irreversible protein-templated fragment ligations were conducted by the Sharpless group and denominated as kinetic target-guided synthesis.T hey used dipolar cycloadditions of azides and alkynes for in situ click reactions templated by the protein acetylcholinesterase (AChE), which is at arget in the treatment of Alzheimers disease. [30] In af ollow-up study from 2005, the method was again applied to the same target, with the goal of identifying and synthesizing af ragment combination that occupied both the active center and the peripheral binding site ( Figure 14 ). [123] As astarting point, the established AChE inhibitor tacrine was used, which binds to the active center of the enzyme. Tacrine was modified by introducing an azide functionality.A compound collection of 23 terminal acetylenes was composed of peripheral site binding fragments and tested to detect the formation of the 1,2,3-triazole by dipolar cycloaddition. The first LC-MS results confirmed the proteinstemplating effect during the ligation reaction of the bound fragments.O nly in the presence of the enzyme were 1,5-disubstituted (syn) triazoles formed, with only little or no background reaction. Having confirmed the reaction with single fragment pairs, the authors turned their attention to reactions containing mixtures of up to 10 acetylene compounds.Indeed the protein template induced the formation of highly potent fragment combinations with dissociation constants in the low picomolar or even femtomolar range. Azide-alkyne ligations have also been applied to other protein targets. [76, [121] [122] [123] [124] [125] 138] Fore xample,t he formation of aknown nanomolar inhibitor of HIV protease containing an anti-substituted 1,2,3-triazole was accelerated in the presence Figure 12 . Developmento fafragment-based, nonpeptidic SARS-CoV main protease inhibitor starting from ap eptide aldehyde. [43] Dynamic ligation screening of alibrary of nucleophiles yielded the amine 20, which binds to the S1' subpocket of the protease. Amine 20 was converted into the electrophile 21,w hich furnished asecondary hit fragmenti nthe next iteration. Reductivea mination of fragment 21 and the best amine hit from the secondary screening yielded inhibitor 22. of the enzyme. [138] In this case,t he reaction was considerably slower than that reported for the pico-/femtomolar AChE inhibitors,a nd this time ac lear background reaction that furnished the syn-triazole was observed. Protein-templated azide-alkyne "click" reactions have also been successfully applied to the Abl tyrosine kinase. [121] TheF ukase research group has demonstrated the templated synthesis of functional mimetics of the Grb2-SH2 domain which inhibited the growth of cancer cells in vitro. [76] To obtain the best inhibitor through the templated reaction of azide and alkyne fragments it is crucial to exclude even tiny amounts of copper ions from the reaction. Theg roups of Miyata and Finn reported the in situ synthesis of an inhibitor of the histone deacetylase 8(HDAC-8) from an alkyne fragment bearing ah ydroxamate and an azide fragment. [122] Surprisingly,t he formed ligation product was not the best inhibitor by far and contained a1 ,4disubstituted anti-triazole instead of the more active syntriazol. Ad etailed scrutiny of these results revealed that the reaction contained traces of Cu I ions,w hich were bound by the metal-binding site of HDAC-8 and were sufficient to catalyze the reaction to the less-favored cycloaddition product. In addition to azide-alkyne ligations,other dipolar cycloadditions have also been investigated. Reactions of thioacids with sulfonylazide fragments,which provide acylsulfonamides via ac yclic intermediate,c onstitute especially successful examples and have been denominated as sulfo-click reactions. In 2011, the Manetsch group demonstrated protein-templated, irreversible ligation reactions with ap articularly challenging target:the PPI domain Bcl-X L . [120] Theinteraction of Bcl-X L with the BH3 peptide is ac rucial step in the regulation of apoptosis (programmed cell death). Inhibitors of this interaction might be potent anticancer agents.I n ascreening for new modulators,the Bcl-X L protein was used as atemplate for the protein-templated sulfo-click reaction of as ulfonylazide and at hioacid fragment ( Figure 15 ). It could be shown that formation of the micromolar Bcl-X L inhibitor proceeded in aprotein-templated reaction;less inhibitor was Reviews 7372 www.angewandte.org formed without the protein or on blocking the binding site by the BH3 peptide,while addition of an inactive mutated BH3 peptide had no effect. Protein-templated, irreversible fragment ligations were recently established for the formation of amide bonds (amidation reactions), one of the most relevant fragment linkages in bioactive compounds and clinically admitted drugs ( Figure 16 ). [47] Ac ollection of active esters 28-40 covering ab road range of chemical reactivity was incubated with 4aminomethylbenzamidine (41)a nd protein factor Xa, adrug target from the blood coagulation cascade,t od iscover conditions for protein-templated amidation reactions.T wo active ester fragments,phenyl ester 39 and trifluoroethyl ester 40, displayed aclear protein-templated amidation of 41 in the substrate competition assay and in LC-MS,a nd afforded the nanomolar inhibitor 42 from two weakly binding fragments with millimolar affinities.Extracted-ion chromatography with aQ TOFd etector was used to quantify the progress of the protein-templated formation of inhibitor 42.Interestingly,the reaction of the trifluoroethyl ester 40 proceeded without detectable background reaction and was autoinhibited at as aturation concentration of 10 nm of the free inhibitor 42. Theinhibitor displayed aremarkable superadditive enhancement of its free binding energy (the K I value was 29 nm instead of 3 mm for the additive case), which was proven to result from the relatively decreased entropy of binding because of fragment linking.T he protein-inhibitor complex was crystallized and the obtained high-resolution structure allowed the authors to rationalize the templated amidation reaction in steric and mechanistic detail. Figure 13 . Discovery of irreversible inhibitors of Coxsackie virus B3 3C protease by using reversible protein-templated fragment ligations. [85] Nucleophilic amine fragment 24 binds to the S1 pocket of the enzyme (A) to reversibly form af ragment ligation product with the biselectrophilic warhead 23 (B). Next the epoxide is opened by attack of the cysteine side chain in the active site, as detected by LC-MS (C). The ligation product displays asuperadditive inhibitory effect in the FRET assay and is optimized to potent broadband inhibitors of enteroviral and rhinoviral 3C proteases. Figure 14 . DevelopmentofAChE inhibitors by protein-templated fragment ligation reactions. [123] The AChE inhibitor tacrine was modified with an azide functionality and acts as an anchoring molecule in the active site of the protein. Mixtures of various acetylenes were added and the templated reaction furnished two highly potent syn-1,2,3triazoles. Protein-templated fragment ligations have been established as an alternative and complementary route to access bioactive protein ligands over recent years.S ignificant progress has been realized on several fronts.I mproved and specialized analytical and bioanalytical methods have contributed both to ab road implementation and ap rofound understanding of the method. Thechemical scope of ligation Figure 15 . Templated formation of sulfonamide inhibitor SZ7TA2 of the protein Bcl-X L from fragments sulfonylazide SZ7 and thioacid TA2. [119] A) Reference compound obtained by chemical synthesis. B) Templatedr eaction:fragments incubated in the presence of target protein;B cl-X L serves as atemplate for the formation of SZ7TA2. C) Backgroundr eaction:f ragments incubated without Bcl-X L lead only to small amounts of the product. D) Blocking of the Bcl-X L binding site by the Bim-BH3 peptide suppresses the product formation.E )Mutated Bim-BH3 has only alow affinity towards Bcl-X L ,t hereby leading to no inhibition of the templated fragmentl igation reaction. Figure 16 . Protein-templated amidation of active esters 39/40 with 4-aminomethylbenzamidine (41)f urnished the nanomolar inhibitor of the protein factor Xa. [47] The protein-templated, background-free reaction was demonstrated in ab ioactivity-based assay and the autoinhibitory kinetics of inhibitorf ormation was proven by QTOF-MS. The crystal structure of the protein-inhibitor complex enabled the pathway of the templated reaction to be modeled. Reviews 7374 www.angewandte.org reactions has been continually extended, and further extensions to reactions that deliver templated fragment ligation products can be expected. After reviewing the biophysical background, the underlying chemistry,and actual applications of this method, we will now consider its strengths and limitations and finally give an outlook on the future possibilities and developments of this technique as part of the drug discovery process. Theb iggest advantage of protein-templated fragment ligations compared to other fragment-based methods is that it enables the site-directed, spatially resolved identification of fragments that bind to ap recisely defined protein pocket. Other fragment-based methods,f or example X-ray crystallography and some of the NMR-based methods,a lso deliver structural information on ligand binding,h owever, they cannot be used to screen specifically for one binding site. Site-directed fragment detection is realized by the structure and by the defined binding of the reactive starting fragment. Thes econd major advantage of protein-templated fragment ligation assays is their sensitivity caused by the (super-) additive binding enhancement of ligated fragments.A s ar esult, fragment ligation assays can identify fragments that are not detectable with other fragment-based discovery methods or only at considerably higher fragment or protein concentrations.Thus,the method may provide protein ligands with alternative structures and with improved ligand efficiencies.A sathird advantage,p rotein-templated fragment ligations have practical and economic benefits,mainly arising from the integration of the synthesis of fragment combinations and the detection of bioactivity in one step.Asaresult, the effort and the resources required for the chemical synthesis of bioactive ligands are reduced considerably,a s only bioactive fragment combinations need to be resynthesized for further structural and functional validation. Whereas classical high-throughput screening uses large libraries of drug-like molecules to cover the chemical space,f ragment ligation screening needs only small libraries of reactive fragments to sample the chemical space of all potential fragment combinations. One general requirement of fragment ligation assays is the need for areactive starting fragment. This could be problematic if no suitable ligand of atarget protein is known. In such cases,c omplementary methods such as classical screening or structure-based design are indispensable to provide starting points for fragment ligation. Another major requirement of protein-templated ligation assays is the availability of ligation reactions that cover the relevant chemical space and are compatible with the conditions of the protein assay.Although, as shown in Sections 4 and 5, the number of chemical reactions that have been adapted to fragment ligation screening is constantly growing, continuous research efforts will be needed to make more of the privileged drug-like fragment linkages accessible in ligation assays.I np articular, C À Cb ond-forming reactions, formations of heterocycles,a nd direct connections between cyclic fragments,w hich are often constructed through crosscoupling reactions,need further investigation. Thec ritical evaluation shows that protein-templated fragment ligations can currently be considered as ab roadly applicable method that can, and should, contribute to the modern drug discovery process by complementing the other established and successful methods when its specific virtues are required:s patially resolved and site-directed screening and high sensitivity for the identification of novel fragments as well as the further chemical development of known protein ligands. Future research will show if the specific advantages of the method will be able to provide clinical candidates with improved properties.T he ultimate significance of proteintemplated fragment ligations will depend on to what extent they will contribute to fulfill the promise of fragment-based drug discovery:F ind better drugs with high potency, specificity,a nd fewer off-target related side effects.M ost likely, protein-templated fragment ligations will contribute to this goal in close collaboration with other fragment-based and classical lead discovery approaches.B eyond the practical success of the method in the drug discovery process,proteintemplated ligation reactions are also potent and attractive research and training tools that teach us how partial structures of am olecule can interact additively to create high-affinity protein ligands.T hrough this,t he method helps us to understand how molecular recognition makes the molecules of life work and how molecular evolution can proceed. Howt ocite: Angew.C hem. Int Rademann in Fragment-based Drug Discovery Lessons and Outlook Proc.N atl. Acad. Sci Proc. Natl. Acad. Sci Proc.N atl. Acad. Sci Proc.N atl. Acad. Sci Proc.N atl. Acad. Sci Expert Opin. Drug Discovery Angew.C hem. Int Angew.C hem. Int Drug Discovery Today Dynamic Combinatorial Chemistry Drug Discovery Today Ramstrçm in Dynamic Combinatorial Chemistry in Drug Discovery,B ioorganic Chemistry,a nd Materials Science Proc.Natl. Acad. Sci Proc.N atl. Acad. Sci Revised manuscript received Version of record online Theauthors declare no conflict of interest.