Chemical Biology Probes from Advanced DNA-encoded Libraries

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Chemical Biology Probes from Advanced DNA-encoded Libraries Hazem Salamon, Mateja Klika Škopić, Kathrin Jung, Olivia Bugain, and Andreas Brunschweiger* Faculty of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-Hahn-Straße 6, D-44227 Dortmund, Germany ABSTRACT: The identification of bioactive compounds is a crucial step toward development of probes for chemical biology studies. Screening of DNA-encoded small molecule libraries (DELs) has emerged as a validated technology to interrogate vast chemical space. DELs consist of chimeric molecules composed of a low-molecular weight compound that is conjugated to a DNA identifier tag. They are screened as pooled libraries using selection to identify “hits.” Screening of DELs has identified numerous bioactive compounds. Some of these molecules were instrumental in gaining a deeper understanding of biological systems. One of the main challenges in the field is the development of synthesis methodology for DELs. technological advances have been made in the field.13 For example, encoding strategies have been developed that allowed for synthesis of compound libraries that are larger than discrete screening libraries by orders of magnitude. Technology to read out that many bar code sequences is nowadays affordable. Progress in chemistry methodology has widened access to compound classes beyond peptides. Thus, vast chemical space can be interrogated. Screening of DELs has enabled identification of several bioactive compounds. Among them are several starting points for the development of small molecule ligands for challenging targets, a number of chemical biology probes that gave insight into biological systems, and even clinical candidates. However, expanding the chemical space covered by DELs is likely to be crucial for successful screening campaigns. This defines an important challenge in the field: the development of chemical methodology for the synthesis of DELs.

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he discipline of chemical biology seeks to provide answers to biological questions using tools designed by chemists. For example, one focus of research in chemical biology is the development of chemically synthesized organic compounds that modulate target protein function.1 Such compounds are in wide use as “probes” to elucidate biological processes. They can make valuable contributions in systems biology studies to investigate the role of selected proteins in complex signaling or metabolic pathways.2 In biomedical projects, probes can provide crucial information as to whether modulation of target protein function might eventually prove useful as a therapeutic strategy. Publication of the sequence of the human genome more than a decade ago disclosed all protein-coding genes and thereby provided chemists with a plethora of target proteins for development of tool compounds.3 Still, the number of proteins for which no small molecule probes are available far exceeds that for which tool compounds exist.4 One important source of bioactive small molecules is the screening of small molecule libraries in biochemical assays.5 Screening libraries typically contain from tens of thousands of small molecules in academic screening units to millions of compounds in pharma companies. Approximately 60% of the screening campaigns in the drug industry were reported to yield hits.5 Thus, one burning question is whether the high failure rate in screening campaigns is attributable to the (incomplete) coverage of chemical space by the screening libraries. An alternative technology to conventional HTS to assess the ligandability6 of target proteins is the screening of DNAencoded compound libraries (DELs).7−10 Due to the amplifiable DNA-tag, DELs can be screened as pools by affinity-based selection, obviating the need for HTS infrastructure. The concept of encoding chemically synthesized compounds with DNA was proposed for the first time three decades ago.11 In the early times, DNA was used to encode peptide libraries on polymer beads.12 Since then, important © XXXX American Chemical Society

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ENTRIES TO DNA-ENCODED LIBRARIES DELs are composed of chimeric compounds that consist of a small molecule covalently linked to a single- (ss) or doublestranded (ds)DNA (Figure 1a). The DNA sequence contains terminal PCR-primer regions and internal coding regions unambiguously identifying the small molecule. An often-used format for the synthesis of DELs is the DNA-recorded combinatorial split-and-pool approach (Figure 1b). Library synthesis is initiated with the conjugation of building blocks to short, synthetic ss- or dsDNA oligonucleotides. The DNA contains a linker moiety for conjugation chemistry, most often with a primary amino group which allows for amide coupling. The linker also serves as spacer between the small molecule and its DNA bar code identifier. Each building block is then Received: November 27, 2015 Accepted: January 13, 2016

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DOI: 10.1021/acschembio.5b00981 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 1. (a) A DNA-encoded compound; (b) DNA-recorded mix-and-split combinatorial synthesis; (c) DNA-templated chemistry leading to DNA-encoded libraries of macrocycles; (d) assembly of the “yoctoreactor;” (e) assembly of DNA-small molecule fragment conjugates.

encoded by a specific DNA sequence. The products of the first cycle of synthesis and encoding are pooled and split for the next cycle. This iterative combinatorial procedure has in one example been repeated four times, giving rise to a library numbering four billion small molecules, an impressive library size that is impossible to achieve without encoding.14 For library synthesis, a relatively narrow range of reactions are available to the chemist (see section below). Methods to encode the building blocks include Klenow fill-in of two partially complementary DNA strands,15 T4 DNA ligation,16 and chemical ligation of short alkyne- and azide-modified ssDNA by Cu(I)-catalyzed cycloaddition.17,18 A refined format of DNA-encoded chemistry is the DNA-templated chemistry (Figure 1c).19 Here, the codons of a template ssDNA-reactant conjugate recruit anticodon sequences by Watson−Crick basepairing that carry a reactant via a cleavable linker moiety. Hybridization of the template DNA with the anticodon DNA forces their conjugated reactants into proximity to facilitate a chemical reaction. For this approach, a template DNA has to be synthesized for each final compound, and also all building blocks need to be coupled to anticodon DNAs via cleavable linkers. DNA-templated chemistry has been used to build up a ca. 13 000-membered library of macrocyclic structures.20,21 A

strategy allowing for use of a universal template DNA has been described recently.22 The capability of partially complementary ssDNA strands for assembly of defined structures has been exploited for the synthesis of DELs.23,24 One example for DNA assembly facilitated synthesis of DELs is the “yoctoreactor” approach (Figure 1d).24 The yoctoreactor is a three- or four-way junction assembled by partially complementary DNA-reactant conjugates that encode their reactant. The junction forces the DNAconjugated reactants into proximity to synthesize the desired compounds. After each reaction, the coding DNAs are ligated, and one of the reactants is cleaved from its coding DNA.24 For fragment screening, small molecule fragments are attached to complementary oligonucleotides. In ESAC (Encoded Self-Assembling Chemical libraries), a set of DNAs that are conjugated with fragments at the 3′-end are hybridized with a second set of DNAs that are conjugated at the 5′-end to present a matrix of fragments in combinatorial fashion (Figure 1e).25 In a different approach, PNA-fragment conjugates were assembled on a DNA-template strand.26 The technology is particularly attractive to identify protein ligands that bind to two distinct binding sites of proteins, though choosing the right linker chemistry for the selected fragments can be challenging. Introducing the linker as part of the selection process by DNAB


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Figure 2. Chemical space covered by the majority of published DNA-encoded libraries. (a) Peptoid libaries; (b) libraries based on diaminosubstituted scaffolds; (c) libraries furnished by nucleophilic aromatic substitution; (d) libraries synthesized by cross-coupling reactions, e.g. biaryl libraries; (e) cyclohexene-based libraries synthesized by Diels−Alder cycloaddition; (f) benzimidazole libraries; (g) macrocycle libraries synthesized by DNA-templated chemistry. PG: protective group. The encoding procedure has been omitted for clarity.

compromised by harsh reaction conditions, most notably oxidizing agents, and acidic pH. Thus, the tool box of reactions at disposal for chemists is restricted. Amine-displaying scaffolds or building blocks to which reactants can be conjugated by carbonyl reactions are heavily exploited for library synthesis. Initial accounts describing DNA-encoded chemistry made use of Fmoc-protected natural and unnatural amino acids as building blocks yielding libraries with peptoid character (Figure 2a).15,32,33 This synthesis strategy was subsequently expanded by orthogonally protected diamine-containing scaffold structures (Figure 2b).34,35 Nucleophilic aromatic substitution of reactive heteroaromatic halides has extensively been used to synthesize DELs. A seminal report on DNA-encoded chemistry described the stepwise substitution of cyanuric chloride yielding large triaminotriazine libraries (Figure 2c).16 Also, other heteroaromatic halides that yield heterocyclic amidine or guanidine products upon substitution with N-nucleophiles were useful building blocks for library synthesis.36,37 More

encoded dynamic combinatorial chemistry is an approach to address this problem.27,28

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SYNTHESIS METHODOLOGY FOR AND CHEMICAL SPACE COVERED BY DELS Ideally, a screening library covers chemical space as comprehensively as possible. The composition of a library again depends on the availability of chemistry methodology. DNA poses several challenges to develop methodology for library synthesis. First, DNA is insoluble in many organic solvents. Strategies to overcome this problem are performing the initial step(s) of a DEL synthesis on solid phase-bound, base- and phosphate-protected DNA;29 forming complexes of DNA with positively charged surfactants that allow one to dissolve the DNA in organic solvents;30 or immobilizing DNA on ion-exchange resin, which then allows the addition of reagents and reactants dissolved in organic solvents to the resin-bound DNA.31 Second, the integrity of DNA can be C


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Figure 3. Screening technologies. (a) Selection against immobilized target proteins; (b) interaction determination using unpurified proteins; (c) binder trap selection; (d) ExoI protection assay. POI, protein of interest; PE, primer extension.

cases.37,40−42 Early examples of very large DELs generated by four cycles of synthesis and encoding yielded hits with a molecular weight in the range of 550−700 Da (e.g., 13, Figure 4).14,16,48 This is beyond the molecular weight range of the majority of orally available drugs absorbed by passive diffusion.49 More recent small molecule DELs were designed to yield hits with a lower molecular weight. These benefited from progress in the development of DNA-compatible synthesis methodology and were synthesized through three17,24 or even only two34,39,41 cycles of synthesis and encoding. Incorporation of well-known target protein binding motifs into a library is a highly successful strategy to identify protein binders with improved binding properties. Examples for such binding motifs are the aryl sulfonamides that bind to carbonic anhydrases, hinge binding motifs that target kinases, and key amino acid residues of protein−protein interactions.40,50,16,46 Quality and Analysis of DELs. The quality of DELs, i.e. an even representation of all compounds in a library, is an important parameter for successful screens. Failed reactions reduce the actual number of compounds in a library, and a library synthesis step with wildly different product yields may make the interpretation of screening results more difficult (see below). Some strategies can be implemented to improve library quality. Prior to the synthesis of DELs, the reactivity of all building blocks that are to be incorporated into a library is evaluated.8,29 During DEL synthesis, excess reagents are removed by simple ethanol precipitation of the DNA or ion exchange chromatography.38,51 Ion-pair reverse phase chromatography of pooled intermediates also removes nonreacted DNA tags but carries the risk of losing library members.16 Library quality can be improved post synthesis by conjugation of an affinity handle to unreacted library constituents and subsequent removal of these compounds by affinity purification.52 For the analysis of DELs, methods are needed to follow the encoding steps and to analyze the preparative organic steps. The introduction of the DNA barcodes is followed by polyacrylamide or agarose gel electrophoresis.8,15 Distinct DNA-small molecule conjugates that are used to initiate library synthesis can be characterized by ion pair RP-HPLC and massspectrometric methods such as ESI-MS15 or MALDI-MS.20−22 Analysis of small pools of DNA-small molecule conjugates has been done by mass spectrometry following S1 Nuclease digest

recently, the spectrum of methodology has been expanded by palladium-catalyzed cross-coupling reactions leading to combinatorial biaryl libraries (Figure 2d).17,37,38 Further reactions that can be used for library synthesis are nucleophilic substitution of reactive aliphatic halides, e.g. α-haloacetylamides leading to secondary amines;32,39 Cu(I)-catalyzed alkyne−azide cycloaddition;23 Horner−Wadsworth−Emmons reaction with reactive phosphonium ylids; and reduction of aromatic nitro groups.23,37 Also, a range of protective groups for amines and carboxylic acids has been shown to be compatible with DNA.37 In contrast to appendage reactions, the synthesis of DELs by reactions that yield (hetero)cyclic scaffold structures and thereby add an element of scaffold diversity has been shown in only a few cases. The Diels−Alder cycloaddition was successfully employed to synthesize a library of substituted cyclohexenes (Figure 2e).40 More recently, hits from a benzimidazole-based DEL were reported (Figure 2f).41,42 For a few further heterocycles, the applicability to synthesize DELs has been demonstrated.37 The synthesis of a 2.4-millionmember dihydropyridine library by the yoctoreactor technology was mentioned but without disclosure of any technical details of the synthesis.24 DNA-templated chemistry provided an entry to macrocyclic peptides. Because of their size and increased complexity as compared to small molecules, macrocycles were proposed as a promising approach to address challenging targets such as protein−protein interactions.43−45 DNAencoded macrocycles were synthesized by consecutive amide couplings and a final ring closure step either by Horner− Wadsworth−Emmons reaction (Figure 2g) or by Cu(I) catalyzed alkyne−azide cycloaddition.21,22,46 Topological diversity of the macrocycle libraries was achieved through incorporation of different cyclic amino acids and linker structures for the ring closing step into the peptide chain. As DNA-encoded small molecules are synthesized in modular fashion by robust DNA-compatible conjugation reactions, DELs are characterized by exceptional appendage diversity, i.e., very large numbers of building blocks are appended to rather few central scaffolding structures. The topological diversity of DELs is defined either by suitably functionalized scaffold structures14,35,38,40,47 or by incorporation of diverse bifunctionalized building blocks into the library.36,48 Introduction of topological diversity by scaffold-generating synthesis methodology, e.g., heterocyclic chemistry or cycloaddition reactions, has so far been demonstrated in only a few D


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Figure 4. Hits from DELs.

to cut the DNA to single nucleotides.21 The conversion rates during later steps of library synthesis involving large pools of DNA-small molecule conjugates are assessed by ion-pair RPHPLC.16 Characterization of regio- or stereoisomers during DEL synthesis evades mass-spectrometric methods. Here, synthesis and conjugation of authentic samples to DNA is a method to ascertain formation of certain isomers.20,40,47 Otherwise, a pragmatic strategy to deal with compounds that potentially occur as isomeric mixtures in the library is to defer their characterization to the hit evaluation phase, i.e., once they show up as hits in a screen, to synthesize, isolate, and test the individual isomers in biological assays.16,53

Proteins can be immobilized in nondirected fashion on reactive surfaces such as CNBr-activated sepharose56 or by directed immobilization using a tag. Protein tags that are routinely used are the His-tag,8 biotintag,34 and Flag-tag.36 The amounts of protein required for selection experiments are minuscule, typically single- to doubledigit micrograms, i.e., in the low nanomole to picomole range.8,33 Usually, a pooled DEL is incubated at low nanomolar concentrations with the immobilized target protein. The protein is present in the assay in vast excess as compared to the amount of individual library members. Thus, the surface with the immobilized protein serves as an affinity matrix. The protein binders are enriched by repeated washing steps versus nonbinding compounds. Additives to the selection buffer such as certain salts or cofactors were exploited to select a binder for a desired protein conformation.35,42 Counter- or parallel screens with several target proteins can be performed to select compounds with a desired target binding profile to aid

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SCREENING TECHNOLOGIES FOR DELS DNA-encoded libraries allow for screening by selection (Figure 3a).54 The selection assay requires the immobilization of a recombinantly expressed, purified target protein55 on the surface of a resin8,15 or magnetic beads (Figure 3a).8,34 E


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ACS Chemical Biology prioritization of hits.57,58 Elution of the binders is either followed by further rounds of selection or by PCRamplification. The DNA bar tags of the amplicon are then read by DNA sequencing.8,15,40,59 Hits are identified by counting the sequences relative to control selection experiments and the nonselected library. In selection experiments, binders are enriched according to their residence time on the target protein. Several screening campaigns showed a correlation for the enrichment of the DNA bar tags and the KD values of the final protein binders.33,34,39 However, enrichment of codes also depends on library quality and the amount of protein immobilized on the solid support.59 Still, many proteins resist isolation, lose their native conformation upon immobilization, or lose critical interaction partners during the isolation process. For those proteins, alternative selection techniques are needed. For a technology called “interaction determination using unpurified proteins,” (IDUP) proteins were tagged with a short ssDNA bar code using SNAP tag technology (Figure 3b). These formed a hairpin upon binding of a small molecule ligand-ssDNA conjugate, primer extension followed by PCR, then amplified the codes of the protein and of the ligand which were read by sequencing.60 The selection experiment was performed in cell lysates where all native interaction partners of the target proteins are present. DNA-tagged proteins are also employed in a selection assay called binder trap (Figure 3c). These constructs are incubated with DELs in solution. Rapid emulsification of the complex mixture traps the protein ligand pairs into droplets where the DNA sequences from the small molecule ligand and from the protein are ligated into PCRamplifiable strands that can be sequenced.24 A selection assay design that relied on protection of ssDNA-small molecule ligands from Exonuclease digest upon binding to their target proteins and subsequent photo-cross-linking allowed for enrichment of these ligands from a DEL (Figure 3d).61 In this assay, design target proteins neither need to be isolated nor need to be tagged. The selection assay is an elegant technique to identify bioactive small molecules. However, one needs to keep in mind that a hit from a DEL is a protein binder. It does not necessarily perturb the functional activity of a target protein. Thus, hits from DELs require follow-up experiments to investigate their mechanism of action.

can be capitalized upon for rational design of selective PI3Kα inhibitors. A relatively small, i.e., only 13 000-membered, library of macrocycles (Figure 2g) showed a high hit rate, returning several micromolar inhibitors for the kinases Scr, Pim1, MK2, Akt3, and p38α MK2.65 The Scr-inhibiting macrocycles showed a high degree of selectivity within the Src family and were therefore more closely studied. A crystal structure of an optimized compound (3, Figure 4) revealed a unique binding mode, with the macrocycle binding both to the ATP-binding site and the substrate peptide binding site.66 Screening of an ethylenediamide- and an N-substituted carboxamide-based DEL in which members were designed to meet the rule-offive criteria49 yielded a new class of inhibitors of tankyrase 1 (TNKS1), a member of the family of poly(ADP-ribose)polymerases (PARPs).34,39 Among the hits, 4 (Figure 4) revealed N1-phenyl-substituted dihydrouracil as a novel PARPbinding fragment for development of potent and selective inhibitors in the family of poly(ADP-ribose)polymerases (PARPs). Evidence from animal knockout studies and population studies suggested mitochondrial branched chain aminotransferase (BCATm) as a target to treat metabolic diseases. This enzyme was an elusive target for small molecule inhibitors. A large screening effort involving DELs numbering in total 14 billion compounds led to the identification of compound 5 (Figure 4).38 A crystal structure of compound 5 binding to its target can be exploited for rational drug design. Triaminotriazine libraries yielded inhibitors for the Znmetalloproteases ADAMTS-4 and -5 with nanomolar potency and good selectivity versus a panel of zinc-metalloprotases.14,67 Neither series of compounds displayed any metal ion-binding motif, and the ADAMTS-5 inhibitor competed effectively with the enzyme substrate in a biochemical assay. Compound 6 (Figure 4), a potent, nanomolar, pan-SIRT1−3 inhibitor allowed for analysis of its binding mode for subsequent compound optimization.36 Screening of DELs against soluble epoxide hydrolase (sEH), an enzyme hydrolyzing endogenous lipid epoxides, delivered several starting points for drug development.17,68 The sEH inhibitor 7 (Figure 4) is the first reported example of a clinical candidate that traces its origin to a DEL screen.69 With the increasing occurrence of therapy-resistant pathogens, there is a need to develop anti-infective compounds with novel modes of action.70 The enoyl-ACP reductase InhA likely is a target of the antitubercular drug isoniazid. In an effort to develop InhA inhibitors with a different mode of action, the diacylated aminoproline 8 (Figure 4) was optimized from a DEL screening hit. It inhibited InhA with an IC50 of 4 nM in vitro and displayed a MIC90 of 0.5 μM. The HCV protein NS4B is required for viral replication and thus constitutes a promising target for antiviral treatment. A DEL screen delivered compound 9, which displays a pharmacophore distinct from prior identified NS4B inhibitors and gave a partially different resistance profile in vitro.57 Several screening efforts were directed at interfaces of protein−protein interactions with the aim to disrupt communication of proteins. They resulted in small molecule ligands for challenging target proteins such as IL-2 (10),33 BclxL (11),56 and TNFα (12),47 with low nanomolar to micromolar affinity which may serve as starting points for the development of more potent inhibitors of these disease-relevant targets. The interaction of LFA lymphocyte function associated antigen 1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1) was inhibited by the triaminotriazine 13 (Figure 4)

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HITS FROM DELS Screening of DNA-encoded libraries has delivered bioactive low molecular weight ligands for representatives of several protein families across the human proteome and for target proteins from pathogenic organisms, underpinning the versatility of the screening technology. Targeting kinases is a highly successful approach to treat diseases.62 Unsurprisingly, many screening efforts have been directed to this enzyme family. Screening of triaminotriazinebased DELs identified nanomolar ATP-competitive inhibitors of Aurora A kinase and p38 MAP kinase two kinases.16 A more recent DEL delivered the starting point for development of a selective and brain-penetrant GSK-3β-inhibitor (1, Figure 4).63 A library of substituted biphenyls yielded 2 (Figure 4), an equipotent single-digit nanomolar inhibitor of wild-type and H1047R-mutated phosphoinositide 3-kinase α (PI3Kα), a mutation frequently observed in cancer.64 X-ray structures of the compound cocrystallized with its target revealed a binding mode different from ATP-competitive “type 1” inhibitors that F


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Figure 5. Chemical biology probes from DELs.

“biology,” but feedback from the use of them has also improved our understanding of the requirements for the “chemical” probes.74,75 In the following paragraphs, we highlight a number of hits from DELs that were successfully used in chemical biology studies to cast light on functions of their target proteins in phenotypes. Protein Arginine Deiminase 4-Inhibitor. Protein arginine deiminases 1−4 (PADs 1−4) catalyze the hydrolytic deamination of basic arginine residues, also called protein citrullination.76 PAD expression is associated with several diseases.77 It is key to the formation of neutrophil extracellular traps (NETs) which excessively occur in diseases caused by dysregulation of the immune system, e.g., sepsis.77 The regulation of PADs is not well understood; their enzymatic activity is increased by orders of magnitude upon calcium binding and probably affected by protein binding partners.78 Screening of a DNA-encoded library head-to-head against the calcium-bound and calcium-free conformation of PAD4 and subsequent compound optimization identified the indolylsubstituted benzimidazole inhibitor GSK484 (16, Figure 5), a selective, reversible PAD4 inhibitor with nanomolar potency against both conformations of the enzyme.42 Crystallization of the inhibitor-bound protein showed an induced-fit binding mode likely unique to PAD4. This binding mode explained the isoform selectivity of GSK484, which was moreover highly selective when profiled against a panel of proteins. The ease with which 16 could be fluorescently labeled and also converted into a functional pull-down probe for chemoproteomics again illuminates one advantageous feature of probes identified from DELs: a position for compound labeling is known a priori. GSK484 was then successfully employed in in vivo studies. It validated the crucial role of PAD4 catalytic activity in the formation of neutrophil extracellular traps (NETs), demonstrated that PAD4 is a druggable target for small molecule inhibitors, and established that the compound qualifies as a useful chemical biology probe for PAD4 in disease models. Wip1 Phosphatase Inhibitor. Protein phosphatases catalyze protein dephosphorylation and thereby terminate the intracellular signaling of kinases. Several phosphatases might constitute drug targets.79 However, development of small

with nanomolar potency.48 A drug discovery program aimed at the identification of novel inhibitors of inhibitor of apoptosis proteins (XIAP, cIAPs) nicely illustrated how an initial hit (14, Figure 4) from a library of DNA-programmed macrocycles with micromolar in vitro activity was improved through a strategy combining synthesis of focused DELs and structure-guided compound optimization.46 However, as most of the screening activities took place in pharmaceutical research departments, the published compounds likely represent only the tip of an iceberg.

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EXPLOITING THE LINKER MOIETY FOR CHEMICAL BIOLOGY PROBES A feature that distinguishes hits from DELs from screening hits discovered by other methods is the ready availability of a position for compound modification. The DNA-tag has been replaced with moieties to improve the compounds’ physicochemical properties,36 by dyes to facilitate biochemical assays and to visualize the target protein in its cellular environment,33,47,56,48 with a photo reactive linker moiety to label the compounds’ binding site,71 and with a chemically reactive group to obtain a pulldown probe for proteomic studies.71 An intriguing application of small molecule DEL hits is their use as homing agents for drugs.72 Screening a DNAencoded dual pharmacophore fragment library identified a combination of acetazolamide binding to the catalytic site and a bulky lipophilic ligand that bound to a secondary site of the tumor-marker protein carbonic anhydrase IX (CAIX).50,73 Connecting these two fragments with a previously in vivo validated Asp2 linker72 yielded a CAIX-ligand with approximately 40-fold improved affinity for the target protein. The compound was successfully labeled (15, Figure 5).50 The improved protein binding properties of the compound then translated into an increased residence time of 15 in a mouse renal cell carcinoma model which compared well with targeting of CAIX by antibodies.

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CHEMICAL BIOLOGY STUDIES WITH PROBES FROM DELS Accumulating experience with the use of small molecule probes in chemical biology studies has not only helped to understand G


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vitro, and high selectivity versus CNOT6, a closely related enzyme that cleaves 3′-5′-oligoadenylates.41 In studies in HeLa cells, 18 confirmed a regulatory role for PDE12 in the 2−5Amediated IFN response. Inhibition of PDE12 catalytic activity by 18 reduced infection of both HeLa and human small-airway epithelial cells by different virus strains indicating that PDE12 could be a viable host target to treat virus infections. Notably, 18 is a rare example of a small molecule that competes with an oligoribonucleotide for binding to a protein target, and it demonstrated that DNA-encoded libraries can be screened on RNA-binding proteins. RIP3 Kinase Inhibitor. Genetic studies revealed receptorinteracting protein kinase 3 (RIP3) kinase to be a key player in necroptosis, an alternative form of programmed cell death.88 The kinase was proposed as a target in inflammatory diseases, but mice with a mutated inactive RIP3 kinase died prematurely from Casp8-mediated apoptosis, raising concerns about its suitability as a target for therapeutic intervention.89 Conventional HTS and a screen of a DEL identified three different RIP3 inhibitors.89 The DEL-derived compound GSK840 (19, Figure 5) inhibited human RIP3 with subnanomolar inhibitory potency (IC50 = 0.3 nM), displayed high selectivity across the kinome and showed activity in vivo. Surprisingly, blockade of RIP3 enzymatic activity in the absence of caspase inhibition induced apoptotic cell death. In depth studies gave hints that RIP3 kinase inhibitors induced a conformational change in the RIP3 kinase domain which in turn induced RIP3 to recruit RIP1 via RIP homotypic interaction motif (RHIM) binding and trigger Casp8-dependent apoptosis, recapitulating the aforementioned genetic studies. With these results in hand, a number of different RIP3 mutants were generated that were viable, demonstrating that the role of RIP3 in necroptosis can be dissected from its role in apoptosis. Thus, RIP3 inhibition can be a strategy to treat inflammatory diseases with the caveat that an enzyme inhibitor needs to avoid certain conformational changes in the kinase domain. IDE Inhibitor. Insulin-degrading enzyme (IDE), a zinc protease that is involved in the degradation of insulin, has for decades been speculated to be a target for the treatment of diabetes type 2.90 Genetic knockout of IDE elevated insulin levels and at the same time impaired glucose tolerance. Selective IDE inhibitors with cellular activity were lacking for a long time. Macrocycle 20 inhibited IDE with nanomolar potency (IC50: 50 nM) and displayed excellent selectivity versus a panel of metalloproteases.53 The compounds’ selectivity could be explained by its binding to a substrate binding site distal from the catalytic center. In an animal model of type-2 diabetes, 20 increased insulin levels in agreement with genetic studies. It also delivered a plausible explanation for the worsened glucose tolerance, namely impaired degradation of glucagon and amylin. Compound 20 validated IDE as a target for therapeutic intervention and gave insight into how inhibition of IDE might be used in treatment of type-2 diabetes.

molecule phosphatase inhibitors has been notoriously difficult since phosphatases display high homology in the catalytic center which recognizes highly polar substrates. Development of allosteric phosphatase inhibitors is a promising strategy to circumvent the problems associated with competitive phosphatase inhibitors, namely hard-to-attain isoform selectivity and the requirement for a polar, phosphate-mimetic moiety that impairs cellular availability. The phosphatase wild-type p53-induced phosphatase 1 (Wip1, PP2Cδ, PPM1D) is strongly linked to malignant diseases.80−82 The Wip1-encoding gene PPM1D is amplified in many tumors and Wip1 negatively regulates several tumor suppressor pathways, e.g., the p38 MAPK pathway and p53.83−85 Thus, Wip1 is an attractive, though elusive target for drug development in oncology. In the first literaturedocumented head-to-head comparison of two compound screening methodologiesbiochemical HTS and selection of a DELidentified a class of peptidic small molecule Wip1 inhibitors that were devoid of a phosphate-mimetic structure.71 The DEL screen and subsequent structure optimization for in vivo studies yielded GSK2830371 (17, Figure 5). The compound inhibited Wip1-catalyzed dephosphorylation of phospho-p53 in vitro in the low nanomolar range with a clear allosteric mode of action. It was selective versus a panel of enzymes, among them several phosphatases. Photolabeling experiments indicated a binding site distal from the catalytic center in a subdomain called flap. The flap domain is likely involved in substrate phosphoprotein binding. The inhibitor increased levels of phosphorylated p53 and p38 MAPK in several tumor cell lines with wild-type p53. Intriguingly, besides inhibition of catalysis, 17 also decreased cellular Wip1 levels, an effect that could be reversed by a proteasome inhibitor. Binding of 17 to Wip1 probably promotes protein ubiquitination as an additional mode of action. Consistent with its pharmacological mode of action 17 reduced growth of several tumor cell lines, suggesting inhibition of Wip1 by small molecules as a therapeutic strategy in oncology. Thus, 17 is a valuable tool compound to investigate Wip1 in disease models. Beyond that it demonstrated the flap domain to be a ligandable region that might be exploited for development of selective inhibitors of other type 2C serine/threonine phosphatases, too. PDE12 Inhibitor. The interferon-induced oligoadenylate synthetase (OAS)/RNase L system is an important pathway in the innate immune system. OAS synthesizes 4−10mer oligoadenylates with 2′-5′-linkage (2−5A) which bind to and activate RNase L, the effector protein that degrades cellular and viral RNAs. The levels of 2−5A are controlled by certain viral nucleases and at least three host cell enzymes, among them phosphodiesterase 12 (PDE12). Inhibition of 2−5A-mediated RNase L activity is associated with impaired immune response to viral infections, while the impact of dysregulated RNase L activity on oncogenic processes is more complex, i.e., both a tumor-suppressive and a tumorigenic role has been described depending on the cancer type.86 Tool compounds to investigate the contribution of PDE12 to the (dys-)regulation of the OAS/ RNase L system in disease models are scarce. PDE12, being an oligonucleotide-binding protein, represents a challenging target for development of small molecule inhibitors. Biochemical assay systems for PDE12, being based on chromatographic methods, are hardly suitable for high throughput screening of very large compound libraries.87 Thus, a selection-based screening approach is highly attractive to screen for inhibitors of PDE12. Selection of a benzimidazole-based DEL yielded 18 (Figure 5), which inhibited PDE12 with nanomolar potency in

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CONCLUSION AND OUTLOOK Advanced DNA-encoded libraries have delivered several promising starting points for probe or drug development. DEL hits proved the ligandability of challenging targets such as allosteric binding sites, protein−protein interaction surfaces, and oligonucleotide binding sites. Probes developed from DEL hits enabled biologists to investigate phenotypes through chemical biology studies. Certainly, ongoing screening activities will yield further interesting bioactive compounds that can be H


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ACS Chemical Biology taken up by biologists. Here, the development of further assay designs that dispense with the immobilization of purified proteins could open the way to screening further protein classes, e.g., membrane proteins91 or protein complexes.61 The possibility to replace the DNA-tag by other structures as shown, e.g., by small molecule dye conjugates50 could represent an intriguing opportunity for strategies such as PROTAC,92 or design of chemically synthesized antibodies.93 However, library design is a critical issue in the field as it directly impacts the probability to identify a “hit” in a screen. Chemical methods used to synthesize DELs are still restricted to mainly appendage reactions. Thus, development of synthesis methodology for DELs is highly relevant, and an opportunity for organic chemists to advance the technology. Here, reactions that yield (hetero)cyclic scaffold structures would be of high interest. Such structures are well represented among bioactive compounds.94,95 In summary, DNA-encoded libraries are nowadays firmly embedded in the arsenal of screening technologies. They appear to be particularly well suited to academic research as they grant affordable access to screening of chemical space.

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Scaffold: A scaffold is a central element in a compound which serves as a vector projecting appended building blocks

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

The authors would like to thank J. Hall (ETH Zurich) for fruitful discussions and critical reading of the manuscript and T. Zilch who kindly provided the photo of the Omega Nebula (Messier 17), taken in August 2009 from Dortmund, which we used for the abstract picture. This work was supported by the German Federal Ministry of Education and Research (BMBF) Grant 131605. Notes

The authors declare no competing financial interest.

■

KEYWORDS DNA-encoded compound libraries: DNA-encoded compound libraries (DELs) are libraries of chemically synthesized compounds that are conjugated with DNA sequences which serve as bar codes identifying the compound Chemical space: Chemical space is the entity of all molecules accessible through chemical reactions Combinatorial chemistry: Combinatorial chemistry is a synthesis strategy. It incorporates procedures which allow for geometric growth of compound numbers DNA-compatible synthesis methodology: DNA-compatible synthesis methodology is the entity of preparative organic reactions that do not compromise the integrity of DNA Chemical Biology probes: Chemical biology probes are bioactive compounds with a defined mode of action that can be used to investigate biological systems Selection assay: The selection assay is a method that uses affinity as the sole determinant to identify from a pool of compounds a compound that binds to a target molecule Hit: A hit is a molecule that was identified by a screening procedure, e.g. by selection Library diversity: The diversity of a library is defined by the chemotypes it encompasses. The chemotypes consist of scaffold structures and appended building blocks. Diversity can be assessed by chemoinformatic means, e.g. by Tanimoto fingerprint analysis I


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Chemical Biology Probes from Advanced DNA-encoded Libraries

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